US20030027287A1 - Recombinant oleandolide polyketide synthase - Google Patents

Recombinant oleandolide polyketide synthase Download PDF

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US20030027287A1
US20030027287A1 US09/808,880 US80888001A US2003027287A1 US 20030027287 A1 US20030027287 A1 US 20030027287A1 US 80888001 A US80888001 A US 80888001A US 2003027287 A1 US2003027287 A1 US 2003027287A1
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pks
oleandolide
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Mary Betlach
Sanjay Shah
Robert McDaniel
Li Tang
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

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  • the present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology.
  • the invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.
  • Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds.
  • PKS polyketide synthase
  • Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin.
  • Modular PKS enzymes for 14-membered polyketides are encoded by PKS genes that often consist of three or more open reading frames (ORFs).
  • Each ORF of a modular PKS can comprise one, two, or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or “domains.”
  • modules of ketosynthase activity
  • These large multifunctional enzymes catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying ⁇ -carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
  • This plasmid-based genetic system for DEBS overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a “clean” host background.
  • This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
  • the resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide.
  • the technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
  • Oleandomycin is an antibacterial polyketide (described in U.S. Pat. No. 2,757,123, incorporated herein by reference) produced by a modular PKS in Streptomyces antibioticus. Oleandomycin has the structure shown below, with the conventional numbering scheme and stereochemical representation.
  • the macrolide product of the PKS, 8,8a-deoxyoleandolide also referred to herein simply as oleandolide (although oleandolide in other contexts refers to the epoxidated aglycone), is further modified by epoxidation (at C-8 and C-8a) and glycosylation (an oleandrose at C-3 and a desosamine at C-5) to yield oleandomycin.
  • the present invention provides recombinant methods and materials for expressing PKS enzymes derived in whole and in part from the oleandolide PKS in recombinant host cells.
  • the invention also provides the polyketides produced by such PKS enzymes.
  • the invention provides in recombinant form all of the genes for the proteins that constitute the complete PKS that ultimately results, in Streptomyces antibioticus, in the production of oleandolide, which is further glycosylated and epoxidated to form oleandomycin.
  • the invention is directed to recombinant materials comprising nucleic acids with nucleotide sequences encoding at least one domain, module, or protein encoded by an oleandolide PKS gene.
  • the DNA compounds of the invention comprise a coding sequence for at least one and preferably two or more of the domains of the loading module and extender modules 1 through 4, inclusive, of 8,8a-deoxyoleandolide synthase.
  • the invention provides a recombinant expression vector that comprises a heterologous promoter positioned to drive expression of one or more of the oleandolide PKS genes.
  • the promoter is derived from another PKS gene.
  • the invention provides recombinant host cells comprising the vector that produces oleandolide.
  • the host cell is Streptomyces lividans or S. coelicolor.
  • the invention provides a recombinant expression vector that comprises a promoter positioned to drive expression of a hybrid PKS comprising all or part of the oleandolide PKS and at least a part of a second PKS.
  • the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide.
  • the host cell is Streptomyces lividans or S. coelicolor.
  • the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention.
  • the resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation.
  • Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit.
  • the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the oleandolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, deletion, insertion, or replacement.
  • the thus modified oleandolide PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the oleandolide PKS.
  • portions of the oleandolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof.
  • the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the oleandolide PKS.
  • each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the oleandolide PKS.
  • the modular PKS is identical to that found in the PKS that produces oleandolide and is identifiable as such.
  • the derived portion can be prepared synthetically or directly from DNA derived from organisms that produce oleandolide.
  • the invention provides methods to screen the resulting polyketide and antibiotic libraries.
  • the invention also provides novel polyketides, motilides, antibiotics, and other useful compounds derived therefrom.
  • the compounds of the invention can also be used in the manufacture of another compound.
  • the compounds of the invention are formulated as antibiotics in a mixture or solution for administration to an animal or human.
  • FIG. 1 shows restriction site and function maps of the insert DNA in cosmids pKOS055-1 and pKOS055-5 of the invention.
  • Various restriction sites XhoI, ClaI, EcoRI
  • Italicized restriction sites in the Figure indicate that not all of such sites are shown; the EcoRI sites shown are derived from the cosmid DNA into which the PKS gene segments were inserted.
  • the location of the coding sequences for modules 1-6 of oleandolide PKS is indicated by brackets with labels underneath the brackets (i.e., mod. 2 is module 2).
  • the sizes (in kilobase (kb) pairs) of various portions of the inserts are also shown.
  • the open reading frames for the oleAI (oleA1), oleAII (oleA2), and oleAIII (oleA3) genes are shown as arrows pointing in the direction of transcription.
  • FIG. 2 shows a function map of the oleandomycin gene cluster.
  • the various open reading frames of the genes (oleI, oleN2, oleR, oleAI, etc.) are shown as arrows pointing in the direction of transcription. Directly beneath, a line indicates the size in base pairs (bp) of the gene cluster.
  • the bar with alphanumeric identifiers under the size indicator line references Genbank accession numbers providing the nucleotide sequence of the indicated region, which sequence information is incorporated herein by reference. The cross-hatched portion of this bar indicates the region of the gene cluster for which sequence information is provided herein.
  • the oleandolide PKS proteins are shown as arrow bars, with the location of the modules of the PKS shown below, and with the various domains of the modules shown below the modules.
  • FIG. 3 shows a restriction site and function map of plasmid pKOSO39-110, described in Example 3, below, which is an expression vector that can integrate (phiC3 1 based attachment and integration functions) into the chromosome of Streptomyces and other host cells and contains the ermE* promoter positioned to drive expression of the oleAI gene.
  • FIG. 4 shows a restriction site and function map of plasmid pKOS039-130, described in Example 4, below, which is an expression vector that replicates (SCP2* origin of replication) in Streptomyces host cells and contains the actI promoter and actII-ORF4 activator positioned to drive expression of the oleAI, oleAII, and oleAIII genes.
  • FIG. 5 shows a restriction site and function map of plasmid pKOS039-133, described in Example 5, below, which is an expression vector that can integrate (phiC31 based attachment and integration functions) into the chromosome of Streptomyces and other host cells and contains the actI promoter and actII-0RF4 activator positioned to drive expression of the oleAIII gene.
  • the present invention provides useful compounds and methods for producing polyketides in recombinant host cells.
  • the term recombinant refers to a compound or composition produced by human intervention.
  • the invention provides recombinant DNA compounds encoding all or a portion of the oleandolide PKS.
  • the invention provides recombinant expression vectors useful in producing the oleandolide PKS and hybrid PKSs composed of a portion of the oleandolide PKS in recombinant host cells.
  • the invention provides the polyketides produced by the recombinant PKS as well as those derived therefrom by chemical processes and/or by treatment with polyketide modification enzymes.
  • the oleandolide synthase gene like other PKS genes, is composed of coding sequences organized in a loading module, a number of extender modules, and a thioesterase domain. As described more fully below, each of these domains and modules is a polypeptide with one or more specific functions.
  • the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module.
  • the building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, 2-hydroxymalonyl, 2-methylmalonyl, and 2-ethylmalonyl CoA.
  • acylthioesters catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks.
  • Each module is responsible for binding a building block, performing one or more functions, and transferring the resulting compound to the next module.
  • the next module is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete. At that point, an enzymatic thioesterase activity cleaves the polyketide from the PKS.
  • Such modular organization is characteristic of the class of PKS enzymes that synthesize complex polyketides and is well known in the art.
  • the polyketide known as 6-deoxyerythronolide B (6-dEB) is a classic example of this type of complex polyketide.
  • the genes, known as eryAI, eryAII, and eryAIII also referred to herein as the DEBS genes, for the proteins, known as DEBS1, DEBS2, and DEBS3, that comprise the 6-dEB synthase), that code for the multi-subunit protein known as DEBS that synthesizes 6-dEB are described in U.S. Pat. No. 5,824,513, incorporated herein by reference.
  • Recombinant methods for manipulating modular PKS genes are described in U.S. Pat. Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and 97/02358, each of which is incorporated herein by reference.
  • the loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain.
  • Each extender module of DEBS like those of other modular PKS enzymes, contains a ketosynthase (KS), AT, and ACP domains, and zero, one, two, or three domains for enzymatic activities that modify the beta-carbon of the growing polyketide chain.
  • a module can also contain domains for other enzymatic activities, such as, for example, a methyltransferase activity.
  • the releasing domain contains a thioesterase and, often, a cyclase activity.
  • the AT domain of the loading module recognizes a particular acyl-CoA (for DEBS this is usually propionyl but sometimes butyryl or acetyl) and transfers it as a thiol ester to the ACP of the loading module.
  • the AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and 2-hydroxymalonyl) and transfers it to the ACP of that module to form a thioester.
  • the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module 1 possesses an acyl-KS and a malonyl (or substituted malonyl) ACP.
  • the acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension).
  • the growing polyketide chain is transferred from the ACP to the KS of the next module, and the process continues.
  • modules may contain a ketodreductase (KR) that reduces the keto group to an alcohol.
  • KR ketodreductase
  • Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond. Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function. As noted above, modules may contain additional enzymatic activities as well.
  • DH dehydratase
  • ER enoylreductase
  • a polyketide chain traverses the final extender module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs.
  • the polyketide is cleaved from the enzyme and cyclyzed.
  • the resulting polyketide can be modified further by tailoring or polyketide modification enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule.
  • PKS enzymes incorporate a building block that is derived from an amino acid.
  • PKS enzymes for such polyketides include an activity that functions as an amino acid ligase or as a non-ribosomal peptide synthetase (NRPS).
  • NRPS non-ribosomal peptide synthetase
  • KS Q This inactivated KS is in most instances called KS Q , where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for activity.
  • the oleandolide PKS loading module contains a KS Q .
  • modules that include a methyltransferase activity; modules can also include an epimerase activity.
  • the components of a PKS are described further below in specific reference to the oleandolide PKS and the various recombinant and hybrid PKSs provided by the invention.
  • the oleandolide PKS was isolated and cloned by the following procedure. Genomic DNA was isolated from an oleandomycin producing strain of Streptomyces antibioticus (ATCC 11891), partially digested with a restriction enzyme, and cloned into a commercially available cosmid vector to produce a genomic library. This library was then introduced into E coli and probed with a DNA fragment generated from S. antibioticus DNA using primers complementary to sequences of KS domains encoding extender modules 5 and 6 of the oleandolide PKS. Several colonies that hybridized to the probe were pooled, replated, and probed again, resulting in the identification of a set of cosmids.
  • cosmids were isolated and transformed into a commercially available E. coli strain. Plasmid DNA was isolated and analyzed by DNA sequence analysis and restriction enzyme digestion, which revealed that the desired DNA had been isolated and that the entire PKS gene cluster was contained in overlapping segments on two of the cosmids identified.
  • FIGS. 1 and 2 show that the complete oleandolide PKS gene cluster is contained within the insert DNA of cosmids pKOS055-1 (insert size of ⁇ 43 kb) and pKOS055-5 (insert size of ⁇ 47 kb).
  • cosmid pKOS055-1 is available under accession no. ATCC 203798
  • cosmid pKOS055-5 is available under accession no. ATCC 203799.
  • Various additional reagents of the invention can be isolated from these cosmids. DNA sequence analysis was also performed on the various subclones of the invention, as described herein.
  • DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the invention.
  • the native DNA sequence encoding the oleandolide PKS of Streptomyces antibioticus is shown herein merely to illustrate a preferred embodiment of the invention, and the invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
  • the present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences encoded by the DNA sequences shown herein merely illustrate preferred embodiments of the invention.
  • the recombinant nucleic acids, proteins, and peptides of the invention are many and diverse. To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following description of the various regions of the oleandolide PKS and corresponding coding sequences is provided. To facilitate description of the invention, reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa. Also, unless otherwise indicated, reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than Streptomyces antibioticus. In addition, reference to a PKS or its coding sequence includes reference to any portion thereof
  • the invention provides DNA molecules in isolated (i.e., not pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form.
  • These DNA molecules comprise one or more sequences that encode one or more domains (or fragments of such domains) of one or more modules in one or more of the ORFs of the oleandolide PKS gene cluster. Examples of such domains include the KS, AT, DH, KR, ER, ACP, and TE domains of at least one of the 6 extender modules and loading module encoded by the 3 ORFs of the oleandomycin PKS genes.
  • the DNA molecule comprises an ORF other than or in addition to the ORFB described in Swan et al., supra; which corresponds to the oleAIII gene ORF herein, the module is a module other than or in addition to extender module 5 and/or module 6 of ORFB; and the domain is a domain other than or in addition to a domain of module 5 and/or module 6 of ORFB or the ACP domain of module 4 of ORFA.
  • the DNA molecule is a recombinant DNA expression vector or plasmid. Such vectors can either replicate in the cytoplasm of the host cell or integrate into the chromosomal DNA of the host cell.
  • the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host cells with increasing numbers of cell divisions).
  • the oleandolide PKS also known as 8, 8a-deoxyoleandolide synthase, is encoded by three ORFs (oleAI, oleAII, and oleAIII).
  • Each ORF encodes 2 extender modules of the PKS; the first ORF also encodes the loading module.
  • Each module is composed of at least a KS, an AT, and an ACP domain. The locations of the various encoding regions of these ORFs are shown in FIG. 2 and described with reference to the sequence information below.
  • ORF1 encodes 8, 8a-deoxyoleandolide synthase I and begins at nucleotide 5772 and ends at nucleotide 18224 in the sequence below.
  • ORF1 encodes a loading module (encoded by nucleotides 5799-8873), composed of a KS Q domain (encoded by nucleotides 5799-7055), a malonyl-specific AT domain (encoded by nucleotides 7458-8563), and an ACP domain (encoded by nucleotides 8634-8873).
  • ORF1 also encodes extender module 1 (encoded by nucleotides 8955-13349), composed of a KS domain (KS1, encoded by nucleotides 8955-10205), an AT domain (AT1, encoded by nucleotides 10512-11549), a KR domain (KR1, encoded by nucleotides 12258-12818), and an ACP domain (ACP1, encoded by nucleotides 13092-13349), and extender module 2 (encoded by nucleotides 13407-17966), composed of a KS domain (KS2, encoded by nucleotides 13407-14690), an AT domain (AT2, encoded by nucleotides 14997-16031), a KR domain (KR2, encoded by nucleotides 16872-17423), and an ACP domain (ACP2, encoded by nucleotides 17709-17996).
  • extender module 1 encoded by nucleotides 8955-13349
  • ORF2 encodes 8, 8a-deoxyoleandolide synthase 2 and begins at nucleotide 18267 and ends at nucleotide 29717 in the sequence below.
  • ORF2 encodes extender module 3 (encoded by nucleotides 18357-22985), composed of a KS domain (KS3, encoded by nucleotides 18357-19643), an AT domain (AT3, encoded by nucleotides 19965-20999), an inactive KR domain (KR3, encoded by nucleotides 21897-22449), and an ACP domain (ACP3, encoded by nucleotides 22728-22985), and extender module 4 (encoded by nucleotides 23046-29396), composed of a KS domain (KS4, encoded by nucleotides 23046-24329), an AT domain (AT4, encoded by nucleotides 24645-25682), a DH domain (DH4, encoded by nucleotides 257
  • ORF3 encodes 8, 8a-deoxyoleandolide synthase 3 and begins at nucleotide 29787 and ends at nucleotide 40346 in the sequence below. This sequence has been previously reported by Swan et al., supra.
  • ORF3 encodes extender module 5 (encoded by nucleotides 29886-34478), composed of a KS domain (KS5, encoded by nucleotides 29886-31184), an AT domain (AT5, encoded by nucleotides 31494-32531), a KR domain (KR5, encoded by nucleotides 33384-33935), and an ACP domain (ACP5, encoded by nucleotides 34221-34478), and extender module 6 (encoded by nucleotides 34845-39440), composed of a KS domain (KS6, encoded by nucleotides 34845-36131), an AT domain (AT6, encoded by nucleotides 36447-37484), a KR domain (KR6, encoded by nucleotides 38352-38903), and an ACP domain (ACP6, encoded by nucleotides 39183-39440). ORF3 also encodes a TE domain at nucleotides 39657-4034
  • the DNA sequence below also includes the sequences of a number of the tailoring enzyme genes in the oleandomycin gene cluster, including oleI (nucleotides 152-1426), oleN2 (nucleotides 1528-2637), oleR (nucleotides 2658-4967), oleP1 (nucleotides 40625-41830), oleG1 (nucleotides 41878-43158), oleG2 (nucleotides 43163-44443), oleM1 (nucleotides 44433-45173), oleY (nucleotides 45251-46411), oleP (nucleotides 46491-47714), and oleB (nucleotides 47808-49517).
  • oleI nucleotides 152-1426
  • oleN2 nucleotides 1528-2637
  • oleR nucleot
  • the above DNA sequence encodes the following 8,8a-deoxyoleandolide synthase proteins: 8,8a-deoxyoleandolide synthase 1: 1 MHVPGEENGH SIAIVGIACR LPGSATPQEF WRLLADSADA LDEPPAGRFP TGSLSSPPAP 61 RGGFLDSIDT FDADFFNISP REAGXTLDPQQ RLALELGWEA LEDAGIVPPH LRGTRTSVFM 121 GAMWDDYAHL AHARGEAALT RHSLTGTHRG MIANRLSYAL GLQGPSLTVD TGQSSSLAAV 181 HMACESLARG ESDLALVGGV NLVLDPAGTT GVERFGALSP DGRCYTFDSR ANGYARGEGG 241 VVVVLKPTHR ALADGDTVYC EILGSALNND GATEGLTVPS ARAQADVLRQ AWERARVAPT 301 DVQYVELHGT GTPAGDPVEA EGLGTALGTA RPAE
  • the recombinant DNA compounds of the invention that encode the oleandolide PKS proteins or portions thereof are useful in a variety of applications. While many of these applications relate to the heterologous expression of the oleandolide PKS or the construction of hybrid PKS enzymes, many useful applications involve the natural oleandomycin producer Streptomyces antibioticus.
  • the recombinant DNA compounds of the invention can disrupt the oleAI, oleAII, or oleAIII genes by homologous recombination in Streptomyces antibioticus.
  • the resulting host cell is a preferred host cell for making polyketides modified by oxidation, hydroxylation, and glycosylation in a manner similar to oleandomycin, because the genes that encode the proteins that perform these reactions are present in the host cell.
  • Such a host cell also does not naturally produce any oleandomycin that could interfere with production or purification of the polyketide of interest.
  • One illustrative recombinant host cell provided by the present invention expresses a recombinant oleandolide PKS in which the module 1 KS domain is inactivated by deletion or other mutation.
  • the inactivation is mediated by a change in the KS domain that renders it incapable of binding substrate (the KS1 o mutation).
  • this inactivation is rendered by a mutation in the codon for the active site cysteine that changes the codon to another codon, such as an alanine codon.
  • Such constructs are especially useful when placed in translational reading frame with extender modules 1 and 2 of an oleandolide or the corresponding modules of another PKS.
  • KS1 o constructs of the invention are useful in the production of 13-substituted-oleandomycin compounds in Streptomyces antibioticus host cells.
  • Preferred compounds of the invention include those compounds in which the substituent at the 13-position is propyl, vinyl, propargyl, other lower alkyl, and substituted alkyl.
  • the compounds of the invention can also be used to construct recombinant host cells of the invention in which coding sequences for one or more domains or modules of the oleandolide PKS have been deleted by homologous recombination with the Streptomyces antibioticus chromosomal DNA.
  • coding sequences for one or more domains or modules of the oleandolide PKS have been deleted by homologous recombination with the Streptomyces antibioticus chromosomal DNA.
  • Those of skill in the art will appreciate that such compounds are characterized by their homology with the chromosomal DNA and not by encoding a functional protein due to their intended function of deleting or otherwise altering portions of chromosomal DNA.
  • the compounds of the present invention include not only those DNA compounds that encode functional proteins but also those DNA compounds that are complementary or identical to any portion of the oleandolide PKS genes.
  • the invention provides a variety of modified Streptomyces antibioticus host cells in which one or more of the genes in the oleandolide PKS gene cluster have been mutated or disrupted. These cells are especially useful when it is desired to replace the disrupted function with a gene product expressed by a recombinant DNA expression vector. While such expression vectors of the invention are described in more detail in the following Section, those of skill in the art will appreciate that the vectors have application to S. antibioticus as well. Such S. antibioticus host cells can be preferred host cells for expressing oleandolide derivatives of the invention.
  • Particularly preferred host cells of this type include those in which the coding sequence for the loading module has been mutated or disrupted, those in which one or more of any of the PKS gene ORFs has been mutated or disrupted, and/or those in which the genes for one or more oleandolide modification enzymes (glycosylation, epoxidation) have been mutated or disrupted.
  • the present invention provides many useful compounds having application to, and recombinant host cells derived from, Streptomyces antibioticus, many important applications of the present invention relate to the heterologous expression of all or a portion of the oleandolide PKS genes in cells other than S. antibioticus, as described in the following Section.
  • the invention provides methods for the heterologous expression of one or more of the oleandolide PKS genes and recombinant DNA expression vectors useful in the method.
  • any host cell other than Streptomyces antibioticus is a heterologous host cell.
  • recombinant expression vectors that include such nucleic acids.
  • expression vector refers to a nucleic acid that can be introduced into a host cell or cell-free transcription and translation system.
  • An expression vector can be maintained permanently or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm.
  • An expression vector also comprises a promoter that drives expression of an RNA, which is translated into a polypeptide in the cell or cell extract.
  • the expression vector also typically contains a ribosome-binding site sequence positioned upstream of the start codon of the coding sequence of the gene to be expressed.
  • Other elements such as enhancers, secretion signal sequences, transcription termination sequences, and one or more marker genes by which host cells containing the vector can be identified and/or selected, may also be present in an expression vector. Selectable markers, i.e., genes that confer antibiotic resistance or sensitivity, are preferred and confer a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
  • an expression vector can vary widely, depending on the intended use of the vector and especially the host cell(s) in which the vector is intended to replicate or drive expression.
  • Expression vector components suitable for the expression of genes and maintenance of vectors in E. coli, yeast, Streptomyces, and other commonly used cells are widely known and commercially available.
  • suitable promoters for inclusion in the expression vectors of the invention include those that function in eucaryotic or procaryotic host cells. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host cell or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus.
  • promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters.
  • synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433), can also be used.
  • recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of the oleandolide PKS coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells.
  • the host cells are modified by transformation with the recombinant DNA expression vectors of the invention to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.
  • the resulting host cells of the invention are useful in methods to produce PKS and post-PKS tailoring (modification) enzymes as well as polyketides and antibiotics and other useful compounds derived therefrom.
  • Preferred host cells for purposes of selecting vector components for expression vectors of the present invention include fungal host cells such as yeast and procaryotic host cells such as E. coli and Streptomyces, but mammalian cell cultures can also be used.
  • yeasts, plants, or mammalian cells that ordinarily do not produce modular polyketide synthase enzymes it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality. Provision of such enzymes is described, for example, in PCT publication Nos. WO 97/13845 and 98/27203, each of which is incorporated herein by reference.
  • Particularly preferred host cells for purposes of the present invention are Streptomyces and Saccharopolyspora host cells, as discussed in greater detail below.
  • the expression vectors of the invention are used to construct a heterologous recombinant Streptomyces host cell that expresses a recombinant PKS of the invention.
  • Streptomyces is a convenient host for expressing polyketides, because polyketides are naturally produced in certain Streptomyces species, and Streptomyces cells generally produce the precursors needed to form the desired polyketide.
  • a Streptomyces host cell produces any portion of a PKS enzyme or produces a polyketide-modifying enzyme
  • the recombinant vector need drive expression of only those genes constituting the remainder of the desired PKS enzyme or other polyketide-modifying enzymes.
  • such a vector may comprise only a single ORF, with the desired remainder of the polypeptides constituting the PKS provided by the genes on the host cell chromosomal DNA.
  • a Streptomyces or other host cell ordinarily produces polyketides, it may be desirable to modify the host so as to prevent the production of endogenous polyketides prior to its use to express a recombinant PKS of the invention.
  • modified hosts include S. coelicolor CH999 and similarly modified S. lividans described in U.S. Pat. No. 5,672,491, and PCT publication Nos. WO 95/08548 and WO 96/40968, incorporated herein by reference.
  • these hosts may not be necessary to provide enzymatic activities for all of the desired post-translational modifications of the enzymes that make up the recombinantly produced PKS, because the host naturally expresses such enzymes.
  • these hosts generally contain holo-ACP synthases that provide the pantotheinyl residue needed for functionality of the PKS.
  • the invention provides a wide variety of expression vectors for use in Streptomyces.
  • the replicating expression vectors of the present invention include, for example and without limitation, those that comprise an origin of replication from a low copy number vector, such as SCP2* (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol.
  • SCP2* see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et
  • High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes.
  • E. coli origin of replication such as from pUC, p1P, p1I, and pBR.
  • E. coli origin of replication such as from pUC, p1P, p1I, and pBR.
  • the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra).
  • plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211 all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed for purposes of the present invention.
  • the Streptomyces recombinant expression vectors of the invention typically comprise one or more selectable markers, including antibiotic resistance conferring genes selected from the group consisting of the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes.
  • antibiotic resistance conferring genes selected from the group consisting of the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to
  • Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and S. lividans K4-114 and K4-155 host cells, which have been modified so as not to produce the polyketide actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. No. 08/828,898, filed Mar. 31, 1997, and Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference. These vectors are particularly preferred in that they contain promoters compatible with numerous and diverse Streptomyces spp.
  • Particularly useful promoters for Streptomyces host cells include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including promoters from aromatic (Type II) PKS gene clusters.
  • Type II PKS gene cluster promoters are act gene promoters and tcm gene promoters;
  • Type I PKS gene cluster promoter are the spiramycin PKS and DEBS genes promoter.
  • the present invention also provides the oleandolide PKS gene promoter in recombinant form.
  • the promoter for the oleA genes is located upstream of the oleAI gene on cosmid pKOS055-5 of the invention.
  • This promoter is contained within an ⁇ 1 kb segment upstream of the oleAI coding sequence and can be used to drive expression of the oleandolide PKS or any other coding sequence of interest in host cells in which the promoter functions, particularly S. antibioticus and generally any Streptomyces species.
  • particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium.
  • Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage.
  • the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention.
  • the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter.
  • one or more activator genes can also be employed to activate initiation of transcription at promoter sequences.
  • Activator genes in addition to the actII-ORF4 gene described above include dnrI, redD, and ptpA genes (see U.S. patent application Ser. No. 09/181,833, supra).
  • the oleandolide PKS genes were placed on a recombinant expression vector that was transferred to the non-macrolide producing host Streptomyces lividans K4-114, as described in Example 4. Transformation of S. lividans K4-114 (strain K4-155 can also be used) with this expression vector resulted in a strain which produced detectable amounts of 8,8a-deoxyoleandolide as determined by analysis of extracts by LC/MS.
  • the present invention also provides recombinant DNA compounds in which the encoded oleandolide module 1 KS domain is inactivated or absent altogether.
  • Example 4 describes the introduction into Streptomyces lividans of a recombinant expression vector of the invention that encodes an oleandolide PKS with a KS1 o domain.
  • the resulting host cells can be fed or supplied with N-acylcysteamine thioesters of precursor molecules to prepare oleandolide derivatives.
  • Such cells of the invention are especially useful in the production of 13-substituted-6-deoxyerythronolide B compounds in recombinant host cells.
  • Preferred compounds of the invention include those compounds in which the substituent at the 13-position is propyl, vinyl, propargyl, other lower alkyl, and substituted alkyl.
  • the unmodified polyketides, called macrolide aglycones, produced in S. lividans K4-114 or K4-155 can be hydroxylated and glycosylated by adding them to the fermentation of a strain, such as, for example, S. antibioticus or Saccharopolyspora erythraea, that contains the requisite modification enzymes.
  • Saccharopolyspora erythraea can convert 6-dEB and oleandolide to a variety of useful compounds.
  • the erythronolide 6-dEB is converted by the eryF gene 2product to erythronolide B, which is, in turn, glycosylated by the eryB gene product to obtain 3-O-mycarosylerythronolide B, which contains L-mycarose at C-3.
  • the enzyme eryC gene product then converts this compound to erythromycin D by glycosylation with D-desosamine at C-5.
  • Erythromycin D differs from 6-dEB through glycosylation and by the addition of a hydroxyl group at C-6.
  • Erythromycin D can be converted to erythromycin B in a reaction catalyzed by the eryG gene product by methylating the L-mycarose residue at C-3.
  • Erythromcyin D is converted to erythromycin C by the addition of a hydroxyl group at C-12 in a reaction catalyzed by the eryK gene product.
  • Erythromycin A is obtained from erythromycin C by methylation of the mycarose residue in a reaction catalyzed by the eryG gene product.
  • the unmodified oleandolide compounds provided by the present invention can be provided to cultures of Saccharopolyspora erythraea and converted to the corresponding derivatives of erythromycins A, B, C, and D in accordance with the procedure provided in Example 6, below.
  • S. erythraea eryA mutant that is unable to produce 6-dEB but can still carry out the desired conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433).
  • mutant strains such as eryB, eryC, eryG, and/or eryK mutants, or mutant strains having mutations in multiple genes, to accumulate a preferred compound.
  • the conversion can also be carried out in large fermentors for commercial production.
  • Streptomyces venezuelae which produces picromycin, contains enzymes that can transfer a desosaminyl group to the C-5 hydroxyl and a hydroxyl group to the C-12 position.
  • S. venezuelae contains a glucosylation activity that glucosylates the 2′-hydroxyl group of the desosamine sugar.
  • S. narbonensis contains the same modification enzymes as S. venezuelae, except the C-12 hydroxylase.
  • the present invention provides the compounds produced by hydroxylation and glycosylation of the macrolide aglycones of the invention by action of the enzymes endogenous to S. narbonensis and S. venezuelae.
  • M. megalomicea produces megalomicin and contains enzymes that hydroxylate the C-6 and C-12 positions and glycosylate the C-3 hydroxyl with mycarose, the C-5 hydroxyl with desosamine, and the C-6 hydroxyl with megosamine (also known as rhodosamine), as well as acylating various positions.
  • compounds of the invention produced by treatment with M. megalomicea enzymes can have antiparasitic activity as well.
  • ftadiae contains enzymes that glycosylate the C-5 hydroxyl with mycaminose and then the 4′-hydroxyl of mycaminose with mycarose, forming a disaccharide.
  • S. thermotolerans contains the same activities as well as acylation activities.
  • the present invention provides the compounds produced by hydroxylation and glycosylation of the macrolide aglycones of the invention by action of the enzymes endogenous to S. antibioticus, M. megalomicea, S. fradiae, and S. thermotolerans.
  • the present invention also provides methods and genetic constructs for producing the glycosylated and/or hydroxylated compounds of the invention directly in the host cell of interest.
  • the recombinant genes of the invention which include recombinant oleAI, oleAII, and oleAIII genes with one or more deletions and/or insertions, including replacements of an oleA gene fragment with a gene fragment from a heterologous PKS gene (as discussed in the next Section), can be included on expression vectors suitable for expression of the encoded gene products in Saccharopolyspora erythraea, Streptomyces antibioticus, S. venezuelae, S.
  • erythromycin high-producing strains of S. erythraea have been developed, and in a preferred embodiment, the oleandolide PKS genes are introduced into such strains (or erythromycin non-producing mutants thereof) to provide the corresponding modified oleandolide compounds in high yields.
  • additional recombinant gene products can be expressed in the host cell to improve production of a desired polyketide.
  • certain recombinant PKS proteins of the invention may produce a polyketide other than or in addition to the predicted polyketide, because the polyketide is cleaved from the PKS by the thioesterase (TE) domain in module 6 prior to processing by other domains on the PKS, in particular, any KR, DH, and/or ER domains in module 6.
  • the production of the predicted polyketide can be increased in such instances by deleting the TE domain coding sequences from the gene and, optionally, expressing the TE domain as a separate protein.
  • the present invention provides methods, expression vectors, and recombinant host cells that enable the production of oleandolide and hydroxylated and glycosylated derivatives of oleandolide in heterologous host cells.
  • the present invention also provides methods for making a wide variety of polyketides derived in part from the oleandolide PKS, as described in the following Section.
  • the present invention provides recombinant DNA compounds encoding each of the domains of each of the modules of the oleandolide PKS.
  • the availability of these compounds permits their use in recombinant procedures for production of desired portions of the oleandolide PKS fused to or expressed in conjunction with all or a portion of a heterologous PKS.
  • the resulting hybrid PKS can then be expressed in a host cell to produce a desired polyketide.
  • a portion of the oleandolide PKS coding sequence that encodes a particular activity can be isolated and manipulated, for example, to replace the corresponding region in a different modular PKS.
  • coding sequences for individual modules of the PKS can be ligated into suitable expression systems and used to produce the portion of the protein encoded.
  • the resulting protein can be isolated and purified or can may be employed in situ to effect polyketide synthesis.
  • suitable control sequences such as promoters, termination sequences, enhancers, and the like are ligated to the nucleotide sequence encoding the desired protein in the construction of the expression vector, as described in the preceding Section.
  • the invention thus provides hybrid PKS enzymes and the corresponding recombinant DNA compounds that encode those hybrid PKS enzymes.
  • a hybrid PKS is a recombinant PKS that comprises all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a first PKS and all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a second PKS.
  • the first PKS is most but not all of the oleandolide PKS
  • the second PKS is only a portion or all of a non-oleandolide PKS.
  • An illustrative example of such a hybrid PKS includes an oleandolide PKS in which the oleandolide PKS loading module has been replaced with a loading module of another PKS.
  • Another example of such a hybrid PKS is an oleandolide PKS in which the AT domain of extender module 3 is replaced with an AT domain that binds only malonyl CoA.
  • the first PKS is most but not all of a non-oleandolide PKS, and the second PKS is only a portion or all of the oleandolide PKS.
  • hybrid PKS includes a rapamycin PKS in which an AT specific for malonyl CoA is replaced with the AT from the oleandolide PKS specific for methylmalonyl CoA.
  • Other illustrative hybrid PKSs of the invention are described below.
  • the oleandolide PKS is comprised of a loading module, six extender modules composed of a KS, AT, ACP, and KR, DH, and ER domains, and a thioesterase domain.
  • the DNA compounds of the invention that encode these domains individually or in combination are useful in the construction of the hybrid PKS encoding DNA compounds of the invention.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS protein or portion thereof.
  • the resulting construct, in which the coding sequence for the loading module of the heterologous PKS is replaced by that for the coding sequence of the oleandolide PKS loading module provides a novel PKS.
  • Examples include the 6-deoxyerythronolide B, rapamycin, FK-506, FK-520, rifamycin, and avermectin PKS protein coding sequences.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion of the loading module coding sequence is utilized in conduction with a heterologous coding sequence.
  • the invention provides, for example, replacing the malonyl CoA (acetyl CoA) specific AT with a propionyl CoA (methylmalonyl), butyryl CoA (ethylmalonyl), or other CoA specific AT.
  • the KS Q and/or ACP can be replaced by another inactivated KS and/or another ACP.
  • the KS Q and AT of the loading module can be replaced by an AT of a loading module such as that of DEBS.
  • the resulting heterologous loading module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS first extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the first extender module of the oleandolide PKS or the latter is merely added to coding sequences for modules of the heterologous PKS provides a novel PKS coding sequence.
  • a DNA compound comprising a sequence that encodes the first extender module of the oleandolide PKS is inserted into a DNA compound that comprises coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion or all of the first extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting (which includes inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a gene for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis.
  • the resulting heterologous first extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • deletion of the KR domain of module 1 or insertion of a DH domain or DH and KR domains into module 1 will prevent the typical cyclization of the polyketide at the hydroxyl group created by the KR if such hybrid module is employed as a first extender module in a hybrid PKS or is otherwise involved in producing a portion of the polyketide at which cyclization is to occur.
  • Such deletions or insertions can be useful, however, to create linear molecules or to induce cyclization at another site in the molecule.
  • the invention also provides recombinant PKSs and recombinant DNA compounds and vectors that encode a PKS protein in which the KS domain of the first extender module has been inactivated.
  • Such constructs are especially useful when placed in translational reading frame with the remaining modules and domains of an oleandolide or oleandolide derivative PKS, a hybrid PKS, or a heterologous PKS.
  • the utility of these constructs is that host cells expressing, or cell free extracts containing, the PKS encoded thereby can be fed or supplied with N-acylcysteamine thioesters of precursor molecules to prepare oleandolide derivative compounds. See U.S. patent application Serial No. 60/117,384, filed Jan. 27, 1999, and PCT publication Nos. WO 99/03986 and 97/02358, each of which is incorporated herein by reference.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS second extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the second extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the second extender module of the oleandolide PKS is inserted into a DNA compound that comprises the coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion or all of the second extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting (or inactivating) the KR; replacing the KR with a KR, a KR and a DH, or a KR, DH, and ER; and/or inserting a DH or a DH and an ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis.
  • the resulting heterologous second extender module coding sequence can be utilized in conjunction with a coding sequence from a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS third extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the third extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the third extender module of the oleandolide PKS is inserted into a DNA compound that comprises coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion or all of the third extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the inactive KR; and/or replacing the KR with an active KR, or a KR and DH, or a KR, DH, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a gene for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis.
  • the resulting heterologous third extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS fourth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fourth extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the fourth extender module of the oleandolide PKS is inserted into a DNA compound that comprises coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion of the fourth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting or inactivating any one, two, or all three of the ER, DIL, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS (except for the DH and ER domains), from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis.
  • the resulting heterologous fourth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS fifth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the fifth extender module of the oleandolide PKS is inserted into a DNA compound that comprises the coding sequence for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion or all of the fifth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting (or inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis.
  • the resulting heterologous fifth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • a DNA compound comprising a sequence that encodes the oleandolide PKS sixth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS.
  • the resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
  • a DNA compound comprising a sequence that encodes the sixth extender module of the oleandolide PKS is inserted into a DNA compound that comprises the coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative.
  • a portion or all of the sixth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module.
  • the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting or inactivating the KR or replacing the KR with another KR, a KR and DH, or a KR, DH, and an ER; and/or inserting a DH or a DH and ER.
  • the KS and/or ACP can be replaced with another KS and/or ACP.
  • the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis.
  • the resulting heterologous sixth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • the sixth extender module of the oleandolide PKS is followed by a thioesterase domain. This domain is important in the cyclization of the polyketide and its cleavage from the PKS.
  • the present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the oleandolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the oleandolide synthase thioesterase.
  • a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth (or other final) extender module coding sequence in recombinant DNA compounds of the invention or the oleandolide PKS thioesterase can be similarly fused to a heterologous PKS.
  • Recombinant DNA compounds encoding this thioesterase domain are useful in constructing DNA compounds that encode the oleandolide PKS, a PKS that produces an oleandolide derivative, and a PKS that produces a polyketide other than oleandolide or an oleandolide derivative.
  • hybrid modules of the invention are incorporated into a PKS to provide a hybrid PKS of the invention.
  • a hybrid PKS of the invention can result not only:
  • heterologous module means two modules are adjacent to one another that are not adjacent to one another in naturally occurring PKS enzymes
  • coding sequences to produce a hybrid coding sequence contained in a PKS gene whose product is incorporated into a PKS
  • hybrid PKS comprising fused modules results from fusion of the loading module of either DEBS or the narbonolide PKS (see PCT patent application No. US99/11814, incorporated herein by reference) with extender modules 1 and 2 of the oleandolide PKS to produce a hybrid oleAI gene.
  • hybrid PKS comprising a hybrid module
  • a hybrid PKS comprising a hybrid module
  • the resulting hybrid PKS of the invention produces 3-deoxy-3-oxo-8,8a-deoxyoleandolide (3-keto-oleandolide). This compound is useful in the production of 14-desmethyl ketolides, compounds with potent anti-bacterial activity.
  • This compound can also be prepared by a recombinant oleandolide derivative PKS of the invention in which the KR domain of module 6 of the oleandolide PKS has been deleted or replaced with an inactive KR domain.
  • the invention provides hybrid PKSs in which not only the above changes have been made but also the AT domain of module 6 has been replaced with a malonyl-specific AT. These hybrid PKSs produce 2-desmethyl-3-deoxy-3-oxo-8,8a-deoxyoleandolide, a useful intermediate in the preparation of 2,14-didesmethyl ketolides, compounds with potent antibiotic activity.
  • hybrid PKS includes the hybrid PKS of the invention resulting only from the latter change in the hybrid PKS just described.
  • co-expression of the oleAI and oleAII genes with a hybrid oleAIII gene in which the AT domain of module 6 has been replaced by a malonyl-specific AT results in the expression of a hybrid PKS that produces 2-desmethyl-8,8a-deoxyoleandolide in recombinant host cells.
  • This compound is a useful intermediate for making 2,14-didesmethyl erythromycins in recombinant host cells of the invention.
  • hybrid PKSs described above are composed primarily of oleandolide PKS proteins
  • present invention provides many different hybrid PKSs, including those composed of only a small portion of the oleandolide PKS.
  • the present invention provides a hybrid PKS in which a hybrid oleAI gene that encodes the oleandolide loading module fused to extender modules 1 and 2 of DEBS is coexpressed with the eryAII and eryAIII genes.
  • the resulting hybrid PKS produces 8,8a-deoxyoleandolide.
  • the resulting recombinant host cell of the invention produces 14-desmethyl erythromycins.
  • the hybrid PKS of the invention composed of the oleAI and eryAII and eryAIII gene products. This construct is also useful in expressing 14-desmethyl erythromycins in Saccharopolyspora erythraea host cells, as described in Example 3, below.
  • the S. erythraea host cells are eryAI mutants that do not produce 6-deoxyerythronolide B.
  • hybrid PKS of the invention composed of the products of the picAI and picAII genes (the two proteins that comprise the loading module and extender modules 1-4, inclusive, of the narbonolide PKS) and the oleAIII gene.
  • the resulting hybrid PKS produces the macrolide aglycone 3-hydroxy-narbonolide in Streptomyces lividans host cells and the corresponding erythromycins in Saccharopolyspora erythraea host cells.
  • This hybrid PKS of the invention is described in Example 5, below.
  • hybrid PKS enzymes of the invention can be expressed in a host cell that also expresses a functional oleP gene product. Such expression provides the compounds of the invention in which the C-8-C-8a epoxide is present.
  • hybrid PKSs of the invention certain general methods may be helpful. For example, it is often beneficial to retain the framework of the module to be altered to make the hybrid PKS. Thus, if one desires to add DH and ER functionalities to a module, it is often preferred to replace the KR domain of the original module with a KR, DH, and ER domain-containing segment from another module, instead of merely inserting DH and ER domains.
  • the hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes. Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene.
  • the invention provides a hybrid PKS that contains the naturally occurring loading module and thioesterase domain as well as extender modules one, two, four, and six of the oleandolide PKS and further contains hybrid or heterologous extender modules three and five.
  • Hybrid or heterologous extender modules three and five contain AT domains specific for malonyl CoA and derived from, for example, the rapamycin PKS genes.
  • the invention also provides libraries of PKS genes, PKS proteins, and ultimately, of polyketides, that are constructed by generating modifications in the oleandolide PKS so that the protein complexes produced have altered activities in one or more respects and thus produce polyketides other than the oleandolide natural product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method.
  • Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method.
  • a modular PKS “derived from” the oleandolide or other naturally occurring PKS includes a modular PKS (or its corresponding encoding gene(s)) that retains the scaffolding of the utilized portion of the naturally occurring gene. Not all modules need be included in the constructs; the constructs can include a loading module and six, fewer than six, or more than six extender modules.
  • the constructs can include a loading module and six, fewer than six, or more than six extender modules.
  • at least one enzymatic activity is mutated, deleted, replaced, or inserted so as to alter the activity of the resulting PKS relative to the original PKS. Alteration results when these activities are deleted or are replaced by a different version of the activity, or simply mutated in such a way that a polyketide other than the natural product results from these collective activities.
  • the origin of the replacement activity may come from a corresponding activity in a different naturally occurring PKS or from a different region of the oleandolide PKS.
  • Any or all of the oleandolide PKS genes may be included in the derivative or portions of any of these may be included, but the scaffolding of the PKS protein is retained in whatever derivative is constructed.
  • the derivative preferably contains a thioesterase activity from the oleandolide or another PKS.
  • a PKS derived from the oleandolide PKS includes a PKS that contains the scaffolding of all or a portion of the oleandolide PKS.
  • the derived PKS also contains at least two extender modules that are functional, preferably three extender modules, and more preferably four or more extender modules, and most preferably six extender modules.
  • the derived PKS also contains mutations, deletions, insertions, or replacements of one or more of the activities of the functional modules of the oleandolide PKS so that the nature of the resulting polyketide is altered at both the protein and DNA sequence levels.
  • Particular preferred embodiments include those wherein a KS, AT, or ACP domain has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS. Also preferred are derivatives where at least one non-condensation cycle enzymatic activity (KR, DH, or ER) has been deleted or added or wherein any of these activities has been mutated so as to change the structure of the polyketide synthesized by the PKS.
  • KR non-condensation cycle enzymatic activity
  • a PKS derived from the oleandolide PKS are functional non-oleandolide PKS modules or their encoding genes wherein at least one portion, or two or more portions, of the oleandolide PKS activities have been inserted.
  • exemplary is the use of the oleandolide AT for extender module 2, which accepts a methylmalonyl CoA extender unit rather than malonyl CoA, to replace a malonyl specific AT in another PKS.
  • Other examples include insertion of portions of non-condensation cycle enzymatic activities or other regions of oleandolide synthase activity into a heterologous PKS at both the DNA and protein levels.
  • the polyketide chain length is determined by the number of modules in the PKS, and the present invention includes hybrid PKSs that contain a loading module and 6, as well as fewer or more than 6, extender modules.
  • the nature of the carbon skeleton of the PKS is determined by the specificities of the acyl transferases that determine the nature of the extender units at each position, e.g., malonyl, methylmalonyl, ethylmalonyl, or other substituted malonyl.
  • the loading module specificity also has an effect on the resulting carbon skeleton of the polyketide.
  • the loading module may use a different starter unit, such as priopionyl, butyryl, and the like.
  • a different starter unit such as priopionyl, butyryl, and the like.
  • another method for varying loading module specificity involves inactivating the KS activity in extender module 1 (KS1) and providing alternative substrates, called diketides, that are chemically synthesized analogs of extender module 1 diketide products, for extender module 2.
  • KS1 extender module 1
  • diketides that are chemically synthesized analogs of extender module 1 diketide products
  • the stereochemistry of the resulting polyketide is a function of three aspects of the synthase.
  • the first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase, as the dehydratase would abolish chirality.
  • the specificity of the ketoreductase may determine the chirality of any beta-OH.
  • the enoylreductase specificity for substituted malonyls as extender units may influence the stereochemistry when there is a complete KR/DH/ER available.
  • the modular PKS systems generally and the oleandolide PKS system particularly permit a wide range of polyketides to be synthesized.
  • the modular PKS systems accept a wider range of starter units, including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl).
  • Certain modular PKSs have relaxed specificity for their starter units (Kao et al., 1994, Science, supra). Modular PKSs also exhibit considerable variety with regard to the choice of extender units in each condensation cycle.
  • the degree of beta-ketoreduction following a condensation reaction has also been shown to be altered by genetic manipulation (Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc. Natl. Acad Sci. USA 90: 7119-7123).
  • the size of the polyketide product can be varied by designing mutants with the appropriate number of modules (Kao et al., 1994, J. Am. Chem. Soc. 116:11612-11613).
  • modular PKS enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner.
  • the polyketides, antibiotics, and other compounds produced by the methods of the invention are typically single stereoisomeric forms.
  • hybrid PKSs are most often produced by “mixing and matching” portions of PKS coding sequences
  • mutations in DNA encoding a PKS can also be used to introduce, alter, or delete an activity in the encoded polypeptide. Mutations can be made to the native sequences using conventional techniques.
  • the substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes.
  • Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. See, e.g., Kunkel, 1985, Proc. Natl. Acad. Sci.
  • the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence, at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See Zoller and Smith, 1983, Methods Enzymol. 100:468.
  • Primer extension is effected using DNA polymerase, the product cloned, and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected.
  • Identification can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., 1982, Proc. Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used to effect the desired mutations.
  • Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can also be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants, or by damaging plasmid DNA in vitro with chemicals.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like.
  • plasmid DNA or DNA fragments are treated with chemical mutagens, transformed into E. coli and propagated as a pool or library of mutant plasmids.
  • regions encoding enzymatic activity i.e., regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS, can be recovered, for example, using PCR techniques with appropriate primers.
  • corresponding activity encoding regions is meant those regions encoding the same general type of activity.
  • a KR activity encoded at one location of a gene cluster “corresponds” to a KR encoding activity in another location in the gene cluster or in a different gene cluster.
  • a complete reductase cycle could be considered corresponding.
  • KR/DH/ER can correspond to a KR alone.
  • replacement of a particular target region in a host PKS is to be made, this replacement can be conducted in vitro using suitable restriction enzymes.
  • the replacement can also be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene in a donor plasmid and a receptor region in a recipient plasmid.
  • Such systems advantageously involving plasmids of differing temperature sensitivities are described, for example, in PCT publication No. WO 96/40968, incorporated herein by reference.
  • the vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes can be chosen to contain control sequences operably linked to the resulting coding sequences in a manner such that expression of the coding sequences can be effected in an appropriate host.
  • nucleotide sequences are inserted into appropriate expression vectors. This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into expression vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies.
  • the invention provides a variety of recombinant DNA compounds in which the various coding sequences for the domains and modules of the oleandolide PKS are flanked by non-naturally occurring restriction enzyme recognition sites.
  • the various PKS nucleotide sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter.
  • the PKS subunit encoding regions can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunit encoding sequences so that hybrid PKSs can be generated.
  • the design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR.
  • the expression vectors containing nucleotide sequences encoding a variety of PKS enzymes for the production of different polyketides are then transformed into the appropriate host cells to construct the library.
  • a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected to identify successful transformants.
  • Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide.
  • the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library.
  • a variety of strategies are available to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide.
  • the number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, and more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products.
  • the number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length enables the production of quite large libraries.
  • Methods for introducing the recombinant vectors of the invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl 2 or agents such as other divalent cations, lipofection, DMSO, PEG, protoplast transformation, infection, transfection, and electroporation.
  • the polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.
  • the libraries of the invention can thus be considered at four levels: (1) a multiplicity of colonies each with a different PKS encoding sequence; (2) the proteins produced from the coding sequences; (3) the polyketides produced from the proteins assembled into a functional PKS; and (4) antibiotics or compounds with other desired activities derived from the polyketides.
  • Combination libraries can also be constructed wherein members of a library derived, for example, from the oleandolide PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS or DEBS.
  • Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of polyketides.
  • Polyketides that are secreted into the media or have been otherwise isolated can be screened for binding to desired targets, such as receptors, signaling proteins, and the like.
  • the supernatantsper se can be used for screening, or partial or complete purification of the polyketides can first be effected.
  • screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art.
  • individual polyketide members of the library can be tested against a desired target.
  • screens wherein the biological response of the target is measured can more readily be included.
  • Antibiotic activity can be verified using typical screening assays such as those set forth in Lehrer et al., 1991, J. Immunol. Meth. 137:167-173, incorporated herein by reference, and in Example 7, below.
  • the invention provides methods for the preparation of a large number of polyketides. These polyketides are useful intermediates in formation of compounds with antibiotic or other activity through hydroxylation and glycosylation reactions as described above. In general, the polyketide products of the PKS must be further modified, typically by hydroxylation and glycosylation, to exhibit antibiotic activity. Hydroxylation results in the novel polyketides of the invention that contain hydroxyl groups at C-6, which can be accomplished using the hydroxylase encoded by the eryF gene, and/or C-12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene. Also, the present invention provides the oleP gene in recombinant form, which can be used to express the oleP gene product in any host cell.
  • a host cell such as a Streptomyces host cell or a Saccharopolyspora erythraea host cell modified to express the oleP gene thus can be used to produce polyketides comprising the C-8-C-8a epoxide present in oleandomycin.
  • the invention provides such modified polyketides.
  • the presence of hydroxyl groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart.
  • glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in PCT publication No. WO 98/49315, incorporated herein by reference.
  • glycosylation with desosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention.
  • the approaches to effecting glycosylation mirror those described above with respect to hydroxylation.
  • the purified enzymes, isolated from native sources or recombinantly produced may be used in vitro.
  • glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosyl transferases.
  • synthetic chemical methods may be employed.
  • the antibiotic modular polyketides may contain any of a number of different sugars, although D-desosamine, or a close analog thereof, is most common. Erythromycin, picromycin, narbomycin, and methymycin contain desosamine. Erythromycin also contains L-cladinose (3-0-methyl mycarose). Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose. 2-acetyl-1-bromodesosamine has been used as a donor to glycosylate polyketides by Masamune et al., 1975, J. Am. Chem. Soc. 97: 3512-3513.
  • Glycosylation can also be effected using the polyketide aglycones as starting materials and using Saccharopolyspora erythraea, Streptomyces venezuelae or other host cells to make the conversion, preferably using mutants unable to synthesize macrolides, as discussed in the preceding Section.
  • polyketides can be produced by the hybrid PKS enzymes of the invention. These polyketides are useful as antibiotics and as intermediates in the synthesis of other useful compounds, as described in the following section.
  • the methods and recombinant DNA compounds of the invention are useful in the production of polyketides.
  • the invention provides methods for making antibiotic compounds related in structure to oleandomycin and erythromycin, both potent antibiotic compounds.
  • the invention also provides novel ketolide compounds, polyketide compounds with potent antibiotic activity of significant interest due to activity against antibiotic resistant strains of bacteria. See Griesgraber et al., 1996, J. Antibiot. 49: 465-477, incorporated herein by reference. Most if not all of the ketolides prepared to date are synthesized using erythromycin A, a derivative of 6-dEB, as an intermediate. While the invention provides hybrid PKSs that produce a polyketide different in structure from 6-dEB, the invention also provides methods for making intermediates useful in preparing traditional, 6-dEB- and erythromycin-derived ketolide compounds.
  • 6-dEB in part differs from oleandolide in that it comprises a 13-ethyl instead of a 13-methyl group
  • the novel hybrid PKS genes of the invention based on the oleandolide PKS provide many novel ketolides that differ from the known ketolides only in that they have a 13-methyl instead of 13-ethyl group.
  • the invention provides the 13-methyl analogues of the ketolides and intermediates and precursor compounds described in, for example, Griesgraber et al., supra; Agouridas et al., 1998, J. Med Chem. 41: 4080-4100, U.S. Pat. Nos.
  • the hybrid PKS genes of the invention can be expressed in a host cell that contains the desosamine biosynthetic genes and desosaminyl transferase gene as well as the required hydroxylase gene(s), which may be either pick (for the C-12 position) or eryK (for the C-12 position) and/or eryF (for the C-6 position).
  • the resulting compounds have antibiotic activity but can be further modified, as described in the patent publications referenced above, to yield a desired compound with improved or otherwise desired properties.
  • the aglycone compounds can be produced in the recombinant host cell, and the desired glycosylation and hydroxylation steps carried out in vitro or in vivo, in the latter case by supplying the converting cell with the aglycone.
  • the compounds of the invention are thus optionally glycosylated forms of the polyketide set forth in formula (1) below which are hydroxylated at either the C-6 or the C-12 or both.
  • the compounds of formula (1) can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described. These polyketides have the formula:
  • R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C;
  • each of R 1 -R 6 is independently H or alkyl (1-4C) wherein any alkyl at R 1 may optionally be substituted;
  • each of X 1 -X 5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11;
  • At least two of R 1 -R 6 are alkyl (1-4C).
  • glycosylated forms of the foregoing are also preferred; glycoside residues can be attached at C-3, C-5, and/or C-6; the epoxidated forms are also included, i.e., and epoxide at C-8-C-8a.
  • Saccharopolyspora erythraea can convert oleandolide and 6-dEB to a variety of useful compounds.
  • the compounds provided by the present invention can be provided to cultures of Saccharopolyspora erythraea and converted to the corresponding derivatives of erythromycins A, B, C, and D in accordance with the procedure provided in Example 6, below. To ensure that only the desired compound is produced, one can use an S.
  • erythraea eryA mutant that is unable to produce 6-dEB but can still carry out the desired conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433). Also, one can employ other mutant strains, such as eryB, eryC, eryG, and/or eryK mutants, or mutant strains having mutations in multiple genes, to accumulate a preferred compound. The conversion can also be carried out in large fermentors for commercial production. Each of the erythromycins A, B, C, and D has antibiotic activity, although erythromycin A has the highest antibiotic activity. Moreover, each of these compounds can form, under treatment with mild acid, a C-6 to C-9 hemiketal with motilide activity.
  • erythromycins B, C, and D are preferred, as the presence of a C-12 hydroxyl allows the formation of an inactive compound that has a hemiketal formed between C-9 and C-12.
  • the present invention provides the compounds produced by hydroxylation and glycosylation of the compounds of the invention by action of the enzymes endogenous to Saccharopolyspora erythraea and mutant strains of S. erythraea.
  • Such compounds are useful as antibiotics or as motilides directly or after chemical modification.
  • the compounds of the invention can be used directly without further chemical modification.
  • Erythromycins A, B, C, and D all have antibiotic activity, and the corresponding compounds of the invention that result from the compounds being modified by Saccharopolyspora erythraea also have antibiotic activity.
  • These compounds can be chemically modified, however, to provide other compounds of the invention with potent antibiotic activity.
  • alkylation of erythromycin at the C-6 hydroxyl can be used to produce potent antibiotics (clarithromycin is C-6-O-methyl), and other useful modifications are described in, for example, Griesgraber et al., 1996, J. Antibiot. 49: 465-477, Agouridas et al., 1998, J. Med. Chem. 41: 4080-4100, U.S. Pat. Nos.
  • the compounds of the invention can be used directly without further chemical modification.
  • Erythromycin and certain erythromycin analogs are potent agonists of the motilin receptor that can be used clinically as prokinetic agents to induce phase III of migrating motor complexes, to increase esophageal peristalsis and LES pressure in patients with GERD, to accelerate gastric emptying in patients with gastric paresis, and to stimulate gall bladder contractions in patients after gallstone removal and in diabetics with autonomic neuropathy. See Peeters, 1999, Motilide Web Site, http://www.med.kuleuven.
  • the corresponding compounds of the invention that result from the compounds of the invention being modified by Saccharopolyspora erythraea also have motilide activity, particularly after conversion, which can also occur in vivo, to the C-6 to C-9 hemiketal by treatment with mild acid.
  • Compounds lacking the C-12 hydroxyl are especially preferred for use as motilin agonists.
  • These compounds can also be further chemically modified, however, to provide other compounds of the invention with potent motilide activity.
  • the organisms can be mutants unable to produce the polyketide normally produced in that organism, the fermentation can be carried out on plates or in large fermentors, and the compounds produced can be chemically altered after fermentation.
  • Saccharopolyspora erythraea, Streptomyces venezuelae, S. narbonensis, S. antibioticus, Micromonospora megalomicea, S. fradiae, and S. thermotolerans can also be used.
  • the present invention provides the compounds produced by hydroxylation and glycosylation by action of the enzymes endogenous to S. erythraea, S. venezuelae, S. narbonensis, S. antibioticus, M. megalomicea, S. fradiae, and S. thermotolerans.
  • the present invention also provides methods and genetic constructs for producing the glycosylated and/or hydroxylated compounds of the invention directly in the host cell of interest.
  • the recombinant genes of the invention which include recombinant oleAI, oleAII, and oleAIII genes with one or more deletions and/or insertions, including replacements of an oleA gene fragment with a gene fragment from a heterologous PKS gene, can be included on expression vectors suitable for expression of the encoded gene products in Saccharopolyspora erythraea, Micromonospora megalomicea, Streptomyces antibioticus, S. venezuelae, S. narbonensis, S. ftadiae, and S. thermotolerans.
  • the compounds of the invention can be produced by growing and fermenting the host cells of the invention under conditions known in the art for the production of other polyketides.
  • the compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures.
  • the compounds can be readily formulated to provide the pharmaceutical compositions of the invention.
  • the pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application.
  • the active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form.
  • auxiliary stabilizing, thickening, and coloring agents and perfumes may be used.
  • the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat. No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference.
  • Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference.
  • Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference.
  • the active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.
  • a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles.
  • parenteral includes subcutaneous injections, and intravenous, intramuscular, and intrasternal injection or infusion techniques.
  • Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day.
  • the dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient).
  • the compounds of the invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition.
  • Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient.
  • the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight.
  • the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.
  • the compounds of the invention can be used as single therapeutic agents or in combination with other therapeutic agents.
  • Drugs that can be usefully combined with compounds of the invention include one or more antibiotic or motilide agents.
  • Streptomyces coelicolor CH999 described in WO 95/08548, published Mar. 30, 1995, or S. lividans K4-114 or K4-155, described in Ziermann and Betlach, Jan. 1999, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, incorporated herein by reference, was used as an expression host.
  • DNA manipulations were performed in Escherichia coli XL1-Blue, available from Stratagene.
  • E. coli MC1061 is also suitable for use as a host for plasmid manipulation. Plasmids were passaged through E.
  • E. coli ET12567 (dam dcm hsdS Cm 1 ) (MacNeil, 1988, J. Bacteriol. 170: 5607, incorporated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor or Saccharopolyspora erythraea.
  • E. coli strains were grown under standard conditions.
  • S. coelicolor strains were grown on R2YE agar plates (Hopwood et al., Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985, incorporated herein by reference).
  • plasmid pRM5 Many of the expression vectors of the invention illustrated in the examples are derived from plasmid pRM5, described in WO 95/08548, incorporated herein by reference.
  • This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters, the actI and actIII promoters, to allow for bidirectional cloning, the gene encoding the actII-ORF4 activator which induces transcription from act promoters during the transition from growth phase to stationary phase, and appropriate marker genes.
  • Engineered restriction sites in the plasmid facilitate the combinatorial construction of PKS gene clusters starting from cassettes encoding individual domains of naturally occurring PKSs.
  • plasmid pRM5 When plasmid pRM5 is used for expression of a PKS, all relevant biosynthetic genes can be plasmid-borne and therefore amenable to facile manipulation and mutagenesis in E. coli. This plasmid is also suitable for use in Streptomyces host cells. Streptomyces is genetically and physiologically well characterized and expresses the ancillary activities required for in vivo production of most polyketides. Plasmid pRM5 utilizes the act promoter for PKS gene expression, so polyketides are produced in a secondary metabolite-like manner, thereby alleviating the toxic effects of synthesizing potentially bioactive compounds in vivo.
  • PCR Polymerase chain reaction
  • Pfu polymerase (Stratagene; Taq polymerase from Perkin Elmer Cetus can also be used) under conditions recommended by the enzyme manufacturer.
  • Standard in vitro techniques were used for DNA manipulations (Sambrook et al. Molecular Cloning: A Laboratory Manual (Current Edition)).
  • E. coli was transformed using standard calcium chloride-based methods; a Bio-Rad E. coli pulsing apparatus and protocols provided by Bio-Rad could also be used.
  • S. coelicolor was transformed by standard procedures (Hopwood et al. Genetic manipulation of Streptomyces. A laboratory manual.
  • transformants were selected using 1 mL of a 1.5 mg/mL thiostrepton overlay, 1 mL of a 2 mg/mL apramycin overlay, or both.
  • Genomic DNA (100 ⁇ g) was isolated from an oleandomycin producing strain of Streptomyces antibioticus (ATCC 11891) using standard procedures. The genomic DNA was partially digested with restriction enzyme Sau3A1 to generate fragments ⁇ 40 kbp in length, which were cloned into the commercially available SupercosTM cosmid vector that had been digested with restriction enzymes XbaI and Bam-HI to produce a genomic library. SuperCosITM (Stratagene) DNA cosmid arms were prepared as directed by the manufacturer. A cosmid library was prepared by ligating 2.5 ⁇ g of the digested genomic DNA with 1.5 ⁇ g of cosmid arms in a 20 ⁇ L reaction. One microliter of the ligation mixture was propagated in E. coli XL1-Blue MR (Stratagene) using a GigapackIII XL packaging extract kit (Stratagene).
  • This library was then probed with a radioactively-labeled probe generated by PCR from Streptomyces antibioticus DNA using primers complementary to known sequences of KS domains hypothesized to originate from extender modules 5 and 6 of the oleandolide PKS. This probing identified about 30 different colonies, which were pooled, replated, and probed again, resulting in the identification of 9 cosmids. These latter cosmids were isolated and transformed into the commercially available E. coli strain XL-1 Blue. Plasmid DNA was isolated and analyzed by restriction enzyme digestion, which revealed that the entire PKS gene cluster was contained in overlapping segments on two of the cosmids identified. DNA sequence analysis using the T3 primer showed that the desired DNA had been isolated.
  • FIG. 1 shows that the complete oleandolide PKS gene cluster is contained within the insert DNA of cosmids pKOS055-1 (insert size of ⁇ 43 kb) and pKOS055-5 (insert size of ⁇ 47 kb).
  • This Example describes the construction of an expression vector, plasmid pKOS039-110, that can integrate into the chromosome of Saccharopolyspora erythraea due to the phage phiC31 attachment and integration functions present on the plasmid and drive expression of the oleAI gene product under the control of the ermE* promoter.
  • a restriction site and function map of plasmid pKOS039-110 is shown in FIG. 3 of the accompanying drawings.
  • the expression of the oleAI gene product in a host cell that naturally produces the eryA gene products results in the formation of a functional hybrid PKS of the present invention composed of the oleAI, eryAII, and eryAIII gene products and the concomitant production of 13-methyl erythromycins. While the specific plasmids and vectors utilized in the construction are described herein, those of skill in the art will recognize that equivalent expression vectors of the invention can be readily constructed from publicly available materials and the oleA gene containing cosmids of the present invention deposited with the ATCC.
  • Plasmid pKOS039-98 is a cloning vector that contains convenient restriction sites that was constructed by inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for PacI, a Shine-Dalgarno sequence, and restriction enzyme recognition sites for NdeI, BglII, and HindIII, into a pUC19 derivative, called pKOS24-47.
  • Plasmid pKOS039-98 (see PCT patent application No. WO US99/11814, incorporated herein by reference) was digested with restriction enzymes PacI and EcoRI and ligated to a polylinker composed of the oligonucleotides N39-51 and N39-52 having the following sequence:
  • N39-51 5′-TAAGGAGGACCATATGCATCGCTCGAGTCTAGACCTAGG-3′N39-52: 5′-AATTCCTAGGTCTAGACTCGAGCGATGCATATGGTCCTCC-TTAAT-3′, which thus includes the following restriction enzyme recognition sites in the order shown: NdeI-NsiI-XhoI-XbaI-EcoRI, to yield plasmid pKOS039-105.
  • Plasmid pKOS039-105 was digested with restriction enzymes NsiI and EcoRI, and the resulting large fragment ligated to the 15.2 kb NsiI-EcoRI, restriction fragment of cosmid pKOS055-5 containing the oleAI gene to yield plasmid pKOS039-116.
  • Plasmid pKOS039-116 was digested with restriction enzymes NdeI and EcoRI, and the resulting 15.2 kb fragment containing the oleAI gene was isolated and ligated to the 6 kb NdeI-EcoRI restriction fragment of plasmid pKOS039-134B to yield plasmid pKOSO39-110 (FIG. 3).
  • Plasmid pKOS039-134B is a derivative of pKOS039-104 described in PCT patent application No. WO US99/11814, supra, prepared by digesting the latter with restriction enzyme BglII and ligating the ⁇ 10.5 kb fragment to get pKOS39-104B.
  • Plasmid pKOS39-104B was digested with restriction enzyme PacI and partially digested with restriction enzyme XbaI. The ⁇ 7.4 kb fragment was ligated with PCR61A+62 fragment treated with restriction enzymes PacI and AvrII. The PCR61A+62 fragment was generated using the PCR primers:
  • N39-61A 5′-TTCCTAGGCTAGCCCGACCCGAGCACGCGCCGGCA-3′; and N39-62, 5′-CCTTAATTAAGGATCCTACCAACCGGCACGATTGTGCC-3′,
  • Plasmid pKOS039-110 DNA was passed through E. coli ET cells to obtain non-methylated DNA, which was then used to transform Saccharopolyspora erythraea cells, which contain a mutation in the eryAI coding sequence for the KS domain of module 1 of DEBS that renders the PKS non-functional.
  • the resulting transformants produced detectable amounts of 14-desmethyl erythromycins.
  • This Example describes the construction of an expression vector, plasmid pKOS039-130, that has an SCP2* origin of replication and so can replicate in Streptomyces host cells and drive expression of the oleAI, oleAII, and oleAIII gene products under the control of the actI promoter and actII-ORF4 activator.
  • a restriction site and function map of plasmid pKOS039-130 is shown in FIG. 4 of the accompanying drawings.
  • oleA gene products in this host cell results in the formation of a functional oleandolide PKS composed of the oleAI, oleAII, and oleAIII gene products and the concomitant production of 8,8a-deoxyoleandolide. While the specific plasmids and vectors utilized in the construction are described herein, those of skill in the art will recognize that equivalent expression vectors of the invention can be readily constructed from publicly available materials and the oleA gene containing cosmids of the present invention deposited with the ATCC.
  • the 14 kb EcoRI-EcoRV and 5.4 kb EcoRV-PstI restriction fragments of cosmid pKOS055-1 were ligated with pLitmus28 digested with EcoRI and PstI to yield plasmid pKOS039-115.
  • the 19.5 kb SpeI-XbaI restriction fragment from plasmid pKOS039-115 was inserted into pKOSO39-73, a derivative of plasmid pRM5, to yield plasmid pKOS039-129.
  • the 15.2 kb PacI-EcoRI restriction fragment of plasmid pKOS039-110 was inserted into pKOS039-129 by replacing the 22 kb PacI-EcoRI restriction fragment to yield plasmid pKOS038-174.
  • the 19 kb EcoRI restriction fragment from plasmid pKOS039-129 was then inserted into pKOS038-174 to yield plasmid pKOS039 -130 (FIG. 4), which was used to transform Streptomyces lividans K4-114 (K4-155 could also be used).
  • the resulting transformants produced 8,8a-deoxyoleandolide.
  • the invention provides a recombinant oleAI gene in which the coding sequence for the KS domain of module 1 has been mutated to change the active site cysteine to another amino acid (the KS1 o mutation).
  • Recombinant PKS enzymes comprising this gene product do not produce a polyketide unless provided with diketide (or triketide) compounds that can bind to the KS2 or KS3 domain, where they are then processed to form a polyketide comprising the diketide (or triketide).
  • This recombinant oleAI gene can be used together with the oleAII and oleAII genes to make a recombinant oleandolide PKS or can be used with modified forms of those genes or other naturally occurring or recombinant PKS genes to make a hybrid PKS.
  • primers were used to amplify template DNA prepared from pKOS039-106.
  • the amplification product of primers N39-47 and N39-48 was digested with restriction enzymes EcoRI and NheI, and the amplification product of primers N39-49 and N39-50 was digested with restriction enzymes NheI and HindIII, and the resulting restriction fragments were ligated to EcoRI and HindIII-digested plasmid pLitmus28 to yield plasmid pKOS038-179.
  • the 1.5 kb BsrGI-BbvCI restriction fragment of plasmid pKOS038-179 was inserted into plasmid pKOS039-106 to yield pKOS098-2.
  • the 7 kb NsiI-XhoI restriction fragment of plasmid pKOS098-2 and the 8 kb XhoI-EcoRI restriction fragments of plasmid pKOS039-107 are then used to replace the 15.2 kb NsiI-EcoRI restriction fragment of plasmid pKOS039-110 to yield the desired expression vector, pKOS039-110-KS1 o , which comprises the oleAI KS1 o gene under the control of the ermE* promoter.
  • the oleAI KS1 o gene can be isolated as a PacI-EcoRI restriction fragment from plasmid pKOSO39-110-KS1 o , which is then used to construct an expression vector analogous to the expression vector plasmid pKOS039-130 in the same manner in which the latter vector was constructed.
  • the resulting expression vector can be used in Streptomyces lividans, S. coelicolor, and other compatible host cells to make polyketides by diketide feeding as described in PCT patent publication No. WO 99/03986, incorporated herein by reference.
  • This Example describes the construction of an expression vector, plasmid pKOS039-133, that can integrate into the chromosome of Streptomyces due to the phage phiC31 attachment and integration functions present on the plasmid and drive expression of the oIeAIII gene product under the control of the actI promoter and actII-ORF4 activator.
  • a restriction site and function map of plasmid pKOS039-133 is shown in FIG. 5 of the accompanying drawings.
  • This plasmid was introduced into S. lividans host cells together with a plasmid, pKOS039-83, that drives expression of the narbonolide PKS genes picAI and picAII (see PCT patent application No.
  • WO US99/11814 The expression of the oleAIII and picAI and picAII gene products in a host cell results in the formation of a functional hybrid PKS of the present invention composed of the oleAIII, picAI, and picAII gene products and the concomitant production of 3-hydroxy-narbonolide. While the specific plasmids and vectors utilized in the construction are described herein, those of skill in the art will recognize that equivalent expression vectors of the invention can be readily constructed from publicly available materials and the oleA gene containing cosmids of the present invention deposited with the ATCC.
  • oligonucleotides were prepared for the insertion of the oleAIII gene into pSET152 derivative plasmid pKOS039-42: N39-59, 5′-AATTCATATGGCTGAGGCGGAGAAGCTGCGCGAATACCTGTGG; and N39-60, 5′-CGCGCCACAGGTATTCGCGCAGCTTCTCCGCCTCAGCCATATG
  • Plasmid pKOS039-115 was digested with restriction enzymes EcoRI and AscI to give the ⁇ 13.8 kb restriction fragment, which was inserted with the linker N39-59/N39-60 to yield plasmid pKOS039-132.
  • Plasmid pKOS039-132 was digested with restriction enzymes NdeI and XbaI to give the ⁇ 10.8 kb restriction fragment, which was ligated to the ⁇ 9 kb NdeI-SpeI restriction fragment of plasmid pKOS039-42 to yield plasmid pKOS039-133 (FIG. 5).
  • Plasmid pKOS039-133 and pKOS039-83 were co-transformed into Streptomyces lividans K4-114 (K4-155 can also be used; see Ziermann and Betlach, 1999, Biotechniques 26, 106-110, and U.S. patent application Ser. No.09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference). Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics.
  • the strains were grown in liquid R5 medium (with 20 ⁇ g/mL thiostrepton, see Hopwood el al., Genetic Manipulation of Streptomyces: A Laboratory Manual; John Innes Foundation: Norwich, UK, 1985, incorporated herein by reference) for growth/seed and production cultures at 30° C. Analysis of extracts by LC/MS established the identity of the polyketide as the expected compound, 3-hydroxynarbonolide.
  • a sample of an oleandolide ( ⁇ 50 to 100 mg) is dissolved in 0.6 mL of ethanol and diluted to 3 mL with sterile water. This solution is used to overlay a three day old culture of Saccharopolyspora erythraea WHM 34 (an eryA mutant) grown on a 100 mm R2YE agar plate at 30° C. After drying, the plate is incubated at 30° C. for four days. The agar is chopped and then extracted three times with 100 nL portions of 1% triethylamine in ethyl acetate. The extracts are combined and evaporated.
  • the crude product is purified by preparative HPLC (C-18 reversed phase, water-acetonitrile gradient containing 1% acetic acid). Fractions are analyzed by mass spectrometry, and those containing pure compound are pooled, neutralized with triethylamine, and evaporated to a syrup. The syrup is dissolved in water and extracted three times with equal volumes of ethyl acetate. The organic extracts are combined, washed once with saturated aqueous NaHCO 3 , dried over Na 2 SO 4 , filtered, and evaporated to yield ⁇ 0.15 mg of product.
  • the product is a glycosylated and hydroxylated oleandolide corresponding to erythromycin A, B, C, and D but differing therefrom as the oleandolide provided differed from 6-dEB.
  • Antibacterial activity is determined using either disk diffusion assays with Bacillus cereus as the test organism or by measurement of minimum inhibitory concentrations (MIC) in liquid culture against sensitive and resistant strains of Staphylococcus pneumoniae.
  • MIC minimum inhibitory concentrations

Abstract

Recombinant DNA compounds that encode all or a portion of the oleandolide polyketide synthase are used to express recombinant polyketide synthase genes in host cells for the production of oleandolide, oleandolide derivatives, and polyketides that are useful as antibiotics and motilides.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application claims priority under 35 U.S.C. §119(e) to U.S. provisional application Serial Nos. 60/120,254, filed Feb. 16, 1999; and No. 60/106,100, filed Oct. 29, 1998, each of which is incorporated herein by reference.[0001]
    • FIELD OF THE INVENTION
  • The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology. [0002]
    • BACKGROUND OF THE INVENTION
  • Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, [0003] Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed Engl. 34(8): 881-888, each of which is incorporated herein by reference.
  • Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes. [0004]
  • Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Modular PKS enzymes for 14-membered polyketides are encoded by PKS genes that often consist of three or more open reading frames (ORFs). Each ORF of a modular PKS can comprise one, two, or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or “domains.” These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D. [0005] The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
  • During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based [0006] Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262:1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a “clean” host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).
  • The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, [0007] Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
  • Oleandomycin is an antibacterial polyketide (described in U.S. Pat. No. 2,757,123, incorporated herein by reference) produced by a modular PKS in [0008] Streptomyces antibioticus. Oleandomycin has the structure shown below, with the conventional numbering scheme and stereochemical representation.
    Figure US20030027287A1-20030206-C00001
  • As is the case for certain other macrolide antibiotics, the macrolide product of the PKS, 8,8a-deoxyoleandolide, also referred to herein simply as oleandolide (although oleandolide in other contexts refers to the epoxidated aglycone), is further modified by epoxidation (at C-8 and C-8a) and glycosylation (an oleandrose at C-3 and a desosamine at C-5) to yield oleandomycin. [0009]
  • The reference Swan et al., 1994, entitled “Characterisation of a [0010] Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence,” Mol. Gen. Genet. 242: 358-362, incorporated herein by reference, describes the DNA sequence of the coding region of a gene designated ORFB hypothesized to encode modules 5 and 6 and a fragment of a gene designated ORFA hypothesized to contain the ACP domain of module 4 of the oleandolide PKS. The reference Quiros et al., 1998, entitled “Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus,” Mol. Microbiol. 28(6): 1177-1185, incorporated herein by reference, describes genes and gene products involved in oleandomycin modification during its biosynthesis. In particular, the reference describes a glycosyltransferase involved in rendering oleandomycin non-toxic to the producer cell and a glycosidase that reactivates oleandomycin after the glycosylated form is excreted from the cell. See also Olano et al., August 1998, “Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308, and PCT patent publication No. 99/05283, incorporated herein by reference. While a number of semi-synthetic oleandomycin derivatives have been described, see U.S. Pat. Nos. 4,085,119; 4,090,017; 4,125,705; 4,133,950; 4,140,848; 4,166,901; 4,336,368; and 5,268,462, incorporated herein by reference, the number and diversity of such derivatives have been limited due to the inability to manipulate the PKS genes.
  • Genetic systems that allow rapid engineering of the oleandolide PKS would be valuable for creating novel compounds for pharmaceutical, agricultural, and veterinary applications. The production of such compounds could be accomplished if the heterologous expression of the oleandolide PKS in [0011] Streptomyces coelicolor and S. lividans and other host cells were possible. The present invention meets these and other needs.
    • SUMMARY OF THE INVENTION
  • The present invention provides recombinant methods and materials for expressing PKS enzymes derived in whole and in part from the oleandolide PKS in recombinant host cells. The invention also provides the polyketides produced by such PKS enzymes. The invention provides in recombinant form all of the genes for the proteins that constitute the complete PKS that ultimately results, in [0012] Streptomyces antibioticus, in the production of oleandolide, which is further glycosylated and epoxidated to form oleandomycin. Thus, in one embodiment, the invention is directed to recombinant materials comprising nucleic acids with nucleotide sequences encoding at least one domain, module, or protein encoded by an oleandolide PKS gene. In one preferred embodiment of the invention, the DNA compounds of the invention comprise a coding sequence for at least one and preferably two or more of the domains of the loading module and extender modules 1 through 4, inclusive, of 8,8a-deoxyoleandolide synthase.
  • In one embodiment, the invention provides a recombinant expression vector that comprises a heterologous promoter positioned to drive expression of one or more of the oleandolide PKS genes. In a preferred embodiment, the promoter is derived from another PKS gene. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces oleandolide. In a preferred embodiment, the host cell is [0013] Streptomyces lividans or S. coelicolor.
  • In another embodiment, the invention provides a recombinant expression vector that comprises a promoter positioned to drive expression of a hybrid PKS comprising all or part of the oleandolide PKS and at least a part of a second PKS. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide. In a preferred embodiment, the host cell is [0014] Streptomyces lividans or S. coelicolor.
  • In a related embodiment, the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention. The resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation. Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit. [0015]
  • In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the oleandolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, deletion, insertion, or replacement. The thus modified oleandolide PKS encoding nucleotide sequence can then be expressed in a suitable host cell and the cell employed to produce a polyketide different from that produced by the oleandolide PKS. In addition, portions of the oleandolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof. [0016]
  • In another related embodiment, the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the oleandolide PKS. Thus, at least a portion of the modular PKS is identical to that found in the PKS that produces oleandolide and is identifiable as such. The derived portion can be prepared synthetically or directly from DNA derived from organisms that produce oleandolide. In addition, the invention provides methods to screen the resulting polyketide and antibiotic libraries. [0017]
  • The invention also provides novel polyketides, motilides, antibiotics, and other useful compounds derived therefrom. The compounds of the invention can also be used in the manufacture of another compound. In a preferred embodiment, the compounds of the invention are formulated as antibiotics in a mixture or solution for administration to an animal or human. [0018]
  • These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.[0019]
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows restriction site and function maps of the insert DNA in cosmids pKOS055-1 and pKOS055-5 of the invention. Various restriction sites (XhoI, ClaI, EcoRI) are also shown. Italicized restriction sites in the Figure indicate that not all of such sites are shown; the EcoRI sites shown are derived from the cosmid DNA into which the PKS gene segments were inserted. The location of the coding sequences for modules 1-6 of oleandolide PKS is indicated by brackets with labels underneath the brackets (i.e., mod. 2 is module 2). The sizes (in kilobase (kb) pairs) of various portions of the inserts are also shown. The open reading frames for the oleAI (oleA1), oleAII (oleA2), and oleAIII (oleA3) genes are shown as arrows pointing in the direction of transcription. [0020]
  • FIG. 2 shows a function map of the oleandomycin gene cluster. In the top half of the Figure, the various open reading frames of the genes (oleI, oleN2, oleR, oleAI, etc.) are shown as arrows pointing in the direction of transcription. Directly beneath, a line indicates the size in base pairs (bp) of the gene cluster. The bar with alphanumeric identifiers under the size indicator line references Genbank accession numbers providing the nucleotide sequence of the indicated region, which sequence information is incorporated herein by reference. The cross-hatched portion of this bar indicates the region of the gene cluster for which sequence information is provided herein. In the bottom half of the Figure, the oleandolide PKS proteins are shown as arrow bars, with the location of the modules of the PKS shown below, and with the various domains of the modules shown below the modules. [0021]
  • FIG. 3 shows a restriction site and function map of plasmid pKOSO39-110, described in Example 3, below, which is an expression vector that can integrate ([0022] phiC3 1 based attachment and integration functions) into the chromosome of Streptomyces and other host cells and contains the ermE* promoter positioned to drive expression of the oleAI gene.
  • FIG. 4 shows a restriction site and function map of plasmid pKOS039-130, described in Example 4, below, which is an expression vector that replicates (SCP2* origin of replication) in Streptomyces host cells and contains the actI promoter and actII-ORF4 activator positioned to drive expression of the oleAI, oleAII, and oleAIII genes. [0023]
  • FIG. 5 shows a restriction site and function map of plasmid pKOS039-133, described in Example 5, below, which is an expression vector that can integrate (phiC31 based attachment and integration functions) into the chromosome of Streptomyces and other host cells and contains the actI promoter and actII-0RF4 activator positioned to drive expression of the oleAIII gene. [0024]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides useful compounds and methods for producing polyketides in recombinant host cells. As used herein, the term recombinant refers to a compound or composition produced by human intervention. The invention provides recombinant DNA compounds encoding all or a portion of the oleandolide PKS. The invention provides recombinant expression vectors useful in producing the oleandolide PKS and hybrid PKSs composed of a portion of the oleandolide PKS in recombinant host cells. The invention provides the polyketides produced by the recombinant PKS as well as those derived therefrom by chemical processes and/or by treatment with polyketide modification enzymes. [0025]
  • To appreciate the many and diverse benefits and applications of the invention, the description of the invention below is organized as follows. In Section I, the recombinant oleandolide PKS provided by the invention is described. In Section II, methods for heterologous expression of the oleandolide PKS and oleandolide modification enzymes provided by the invention are described. In Section III, the hybrid PKS genes provided by the invention and the polyketides produced thereby are described. In Section IV, the polyketide compounds provided by the invention and pharmaceutical compositions of those compounds are described. The detailed description is followed by a variety of working examples illustrating the invention. [0026]
  • The oleandolide synthase gene, like other PKS genes, is composed of coding sequences organized in a loading module, a number of extender modules, and a thioesterase domain. As described more fully below, each of these domains and modules is a polypeptide with one or more specific functions. Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, 2-hydroxymalonyl, 2-methylmalonyl, and 2-ethylmalonyl CoA. Other building blocks include amino acid like acylthioesters. PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks. Each module is responsible for binding a building block, performing one or more functions, and transferring the resulting compound to the next module. The next module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete. At that point, an enzymatic thioesterase activity cleaves the polyketide from the PKS. [0027]
  • Such modular organization is characteristic of the class of PKS enzymes that synthesize complex polyketides and is well known in the art. The polyketide known as 6-deoxyerythronolide B (6-dEB) is a classic example of this type of complex polyketide. The genes, known as eryAI, eryAII, and eryAIII (also referred to herein as the DEBS genes, for the proteins, known as DEBS1, DEBS2, and DEBS3, that comprise the 6-dEB synthase), that code for the multi-subunit protein known as DEBS that synthesizes 6-dEB are described in U.S. Pat. No. 5,824,513, incorporated herein by reference. Recombinant methods for manipulating modular PKS genes are described in U.S. Pat. Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. 98/49315 and 97/02358, each of which is incorporated herein by reference. [0028]
  • The loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain. Each extender module of DEBS, like those of other modular PKS enzymes, contains a ketosynthase (KS), AT, and ACP domains, and zero, one, two, or three domains for enzymatic activities that modify the beta-carbon of the growing polyketide chain. A module can also contain domains for other enzymatic activities, such as, for example, a methyltransferase activity. Finally, the releasing domain contains a thioesterase and, often, a cyclase activity. [0029]
  • The AT domain of the loading module recognizes a particular acyl-CoA (for DEBS this is usually propionyl but sometimes butyryl or acetyl) and transfers it as a thiol ester to the ACP of the loading module. Concurrently, the AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, i.e., methylmalonyl, ethylmalonyl, and 2-hydroxymalonyl) and transfers it to the ACP of that module to form a thioester. Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, [0030] extender module 1 possesses an acyl-KS and a malonyl (or substituted malonyl) ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension). The growing polyketide chain is transferred from the ACP to the KS of the next module, and the process continues.
  • The polyketide chain, growing by two carbons each module, is sequentially passed as a covalently bound thiol ester from module to module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Most commonly, however, additional enzymatic activities modify the beta keto group of each two-carbon unit just after it has been added to the growing polyketide chain but before it is transferred to the next module. Thus, in addition to the minimal module containing KS, AT, and ACP domains necessary to form the carbon-carbon bond, modules may contain a ketodreductase (KR) that reduces the keto group to an alcohol. Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond. Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function. As noted above, modules may contain additional enzymatic activities as well. [0031]
  • Once a polyketide chain traverses the final extender module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs. Here, the polyketide is cleaved from the enzyme and cyclyzed. The resulting polyketide can be modified further by tailoring or polyketide modification enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule. [0032]
  • While the above description applies generally to modular PKS enzymes, there are a number of variations that exist in nature. For example, some polyketides, such as epothilone, incorporate a building block that is derived from an amino acid. PKS enzymes for such polyketides include an activity that functions as an amino acid ligase or as a non-ribosomal peptide synthetase (NRPS). Another example of a variation, which is actually found more often than the two domain loading module construct found in DEBS, occurs when the loading module of the PKS is not composed of an AT and an ACP but instead utilizes an inactivated KS, an AT, and an ACP. This inactivated KS is in most instances called KS[0033] Q, where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for activity. For example, the oleandolide PKS loading module contains a KSQ. Yet another example of a variation has been mentioned above in the context of modules that include a methyltransferase activity; modules can also include an epimerase activity. The components of a PKS are described further below in specific reference to the oleandolide PKS and the various recombinant and hybrid PKSs provided by the invention.
  • Section I: The Oleandolide PKS [0034]
  • The oleandolide PKS was isolated and cloned by the following procedure. Genomic DNA was isolated from an oleandomycin producing strain of [0035] Streptomyces antibioticus (ATCC 11891), partially digested with a restriction enzyme, and cloned into a commercially available cosmid vector to produce a genomic library. This library was then introduced into E coli and probed with a DNA fragment generated from S. antibioticus DNA using primers complementary to sequences of KS domains encoding extender modules 5 and 6 of the oleandolide PKS. Several colonies that hybridized to the probe were pooled, replated, and probed again, resulting in the identification of a set of cosmids. These latter cosmids were isolated and transformed into a commercially available E. coli strain. Plasmid DNA was isolated and analyzed by DNA sequence analysis and restriction enzyme digestion, which revealed that the desired DNA had been isolated and that the entire PKS gene cluster was contained in overlapping segments on two of the cosmids identified.
  • Further analysis of these cosmids and subclones prepared from the cosmids facilitated the identification of the location of various oleandolide PKS genes and ORFs, as well as the modules and domains in the PKS proteins encoded by those ORFs. The location of these genes and modules is shown on FIGS. 1 and 2. FIG. 1 shows that the complete oleandolide PKS gene cluster is contained within the insert DNA of cosmids pKOS055-1 (insert size of ˜43 kb) and pKOS055-5 (insert size of ˜47 kb). Each of these cosmids has been deposited with the American Type Culture Collection in accordance with the terms of the Budapest Treaty (cosmid pKOS055-1 is available under accession no. ATCC 203798; cosmid pKOS055-5 is available under accession no. ATCC 203799). Various additional reagents of the invention can be isolated from these cosmids. DNA sequence analysis was also performed on the various subclones of the invention, as described herein. [0036]
  • Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the invention. The native DNA sequence encoding the oleandolide PKS of [0037] Streptomyces antibioticus is shown herein merely to illustrate a preferred embodiment of the invention, and the invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences encoded by the DNA sequences shown herein merely illustrate preferred embodiments of the invention.
  • The recombinant nucleic acids, proteins, and peptides of the invention are many and diverse. To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following description of the various regions of the oleandolide PKS and corresponding coding sequences is provided. To facilitate description of the invention, reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa. Also, unless otherwise indicated, reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than [0038] Streptomyces antibioticus. In addition, reference to a PKS or its coding sequence includes reference to any portion thereof
  • Thus, the invention provides DNA molecules in isolated (i.e., not pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form. These DNA molecules comprise one or more sequences that encode one or more domains (or fragments of such domains) of one or more modules in one or more of the ORFs of the oleandolide PKS gene cluster. Examples of such domains include the KS, AT, DH, KR, ER, ACP, and TE domains of at least one of the 6 extender modules and loading module encoded by the 3 ORFs of the oleandomycin PKS genes. [0039]
  • In one embodiment, the DNA molecule comprises an ORF other than or in addition to the ORFB described in Swan et al., supra; which corresponds to the oleAIII gene ORF herein, the module is a module other than or in addition to [0040] extender module 5 and/or module 6 of ORFB; and the domain is a domain other than or in addition to a domain of module 5 and/or module 6 of ORFB or the ACP domain of module 4 of ORFA. In an especially preferred embodiment, the DNA molecule is a recombinant DNA expression vector or plasmid. Such vectors can either replicate in the cytoplasm of the host cell or integrate into the chromosomal DNA of the host cell. In either case, the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host cells with increasing numbers of cell divisions).
  • The oleandolide PKS, also known as 8, 8a-deoxyoleandolide synthase, is encoded by three ORFs (oleAI, oleAII, and oleAIII). Each ORF encodes 2 extender modules of the PKS; the first ORF also encodes the loading module. Each module is composed of at least a KS, an AT, and an ACP domain. The locations of the various encoding regions of these ORFs are shown in FIG. 2 and described with reference to the sequence information below. [0041]
  • ORF1 encodes 8, 8a-deoxyoleandolide synthase I and begins at nucleotide 5772 and ends at nucleotide 18224 in the sequence below. ORF1 encodes a loading module (encoded by nucleotides 5799-8873), composed of a KS[0042] Q domain (encoded by nucleotides 5799-7055), a malonyl-specific AT domain (encoded by nucleotides 7458-8563), and an ACP domain (encoded by nucleotides 8634-8873). ORF1 also encodes extender module 1 (encoded by nucleotides 8955-13349), composed of a KS domain (KS1, encoded by nucleotides 8955-10205), an AT domain (AT1, encoded by nucleotides 10512-11549), a KR domain (KR1, encoded by nucleotides 12258-12818), and an ACP domain (ACP1, encoded by nucleotides 13092-13349), and extender module 2 (encoded by nucleotides 13407-17966), composed of a KS domain (KS2, encoded by nucleotides 13407-14690), an AT domain (AT2, encoded by nucleotides 14997-16031), a KR domain (KR2, encoded by nucleotides 16872-17423), and an ACP domain (ACP2, encoded by nucleotides 17709-17996).
  • ORF2 encodes 8, 8a-[0043] deoxyoleandolide synthase 2 and begins at nucleotide 18267 and ends at nucleotide 29717 in the sequence below. ORF2 encodes extender module 3 (encoded by nucleotides 18357-22985), composed of a KS domain (KS3, encoded by nucleotides 18357-19643), an AT domain (AT3, encoded by nucleotides 19965-20999), an inactive KR domain (KR3, encoded by nucleotides 21897-22449), and an ACP domain (ACP3, encoded by nucleotides 22728-22985), and extender module 4 (encoded by nucleotides 23046-29396), composed of a KS domain (KS4, encoded by nucleotides 23046-24329), an AT domain (AT4, encoded by nucleotides 24645-25682), a DH domain (DH4, encoded by nucleotides 25719-26256), an ER domain (ER4, encoded by nucleotides 27429-28301), a KR domain (KR4, encoded by nucleotides 28314-28862), and an ACP domain (ACP4, encoded by nucleotides 29147-29396).
  • ORF3 encodes 8, 8a-[0044] deoxyoleandolide synthase 3 and begins at nucleotide 29787 and ends at nucleotide 40346 in the sequence below. This sequence has been previously reported by Swan et al., supra. ORF3 encodes extender module 5 (encoded by nucleotides 29886-34478), composed of a KS domain (KS5, encoded by nucleotides 29886-31184), an AT domain (AT5, encoded by nucleotides 31494-32531), a KR domain (KR5, encoded by nucleotides 33384-33935), and an ACP domain (ACP5, encoded by nucleotides 34221-34478), and extender module 6 (encoded by nucleotides 34845-39440), composed of a KS domain (KS6, encoded by nucleotides 34845-36131), an AT domain (AT6, encoded by nucleotides 36447-37484), a KR domain (KR6, encoded by nucleotides 38352-38903), and an ACP domain (ACP6, encoded by nucleotides 39183-39440). ORF3 also encodes a TE domain at nucleotides 39657-40343.
  • The DNA sequence below also includes the sequences of a number of the tailoring enzyme genes in the oleandomycin gene cluster, including oleI (nucleotides 152-1426), oleN2 (nucleotides 1528-2637), oleR (nucleotides 2658-4967), oleP1 (nucleotides 40625-41830), oleG1 (nucleotides 41878-43158), oleG2 (nucleotides 43163-44443), oleM1 (nucleotides 44433-45173), oleY (nucleotides 45251-46411), oleP (nucleotides 46491-47714), and oleB (nucleotides 47808-49517). [0045]
  • The sequence of the portion of the oleandomycin gene cluster described above follows: [0046]
    1 GCATGCCCGC CCGCAACACC GGCTCCCGTA ACGGGGCGAG CCGGTGGTCA TCCATCAGTT
    61 TCCTTCCGCC CGGCCCGTGT CAGGCCCGTG TGCGCATACC GCCGTACGGC TGCGCCGGTC
    121 CCCCGCGGAA CACCTCACCG GAGTGAGATC CATGACGAGC GAGCACCGCT CTGCCTCCGT
    181 GACACCCCGT CACATCTCCT TCTTCAACAT CCCCGGCCAC GGCCACGTGA ACCCGTCACT
    241 CGGCATTGTC CAGGGACTTG TCGCGCGCGG CCAACGGGTC AGCTACGGCA TTACCGACGA
    301 GTTCGGCGCA CAGGTCAAGG CGGGCCGCGC GACGGCCGTT GTGTACGGCT TCATTCTGCC
    361 GGAGGAGTTC AACCCCGAGG AGTTGTTGGC CGAGGACCAG GGTTCCCGAT GGGCCTGTTC
    421 CTTGGCGGAG GCGTTCCGGG TCTTGCCGCA GCTGAGGACG GCTACGCCGA CGACCGGCCG
    481 GGACCTGATC GTCTACGACA TCGCCTCCTG GCCCGCCCCG GTGCTCGGCC GGAAGTGGGA
    541 CATCCCCTTC GTCCAGCTCT CCCCGACCTC CGTCGCCTAC GAGGGCTTCG AGGAGGACGT
    601 ACCCGCGGTG CAGGACCCCA CGGCCGACCG CGGCGAGGAG GCCGCCGCCC CCGCGGGGAC
    661 CGGGGACGCC GAGGAGGGTG CCGACGCCGA GGACGGCCTG GTGCGCTTCT TCACCCGGCT
    721 CTCGGCCTTC CTGGAGGAGC ACGGGGTGGA CACCCCGGCC ACCGAGTTCC TCATCGCGCC
    781 CAACCGCTGC ATCGTCGGCT GCCGCGCACC TTCCCAGATC AAGGGCGACA CGGTCGGCGA
    841 CAACTACACC TTCGTCGGTC CCACCTACGG CGACCGGTCC CACCAGGGCA CCTGGGAAGG
    901 CCCCGGGCAC GGGCGTCCGG TGCTGCTGAT CGCCCTGGGC TCGGCGTTCA CCGACCACCT
    961 CGACTTCTAC CGCACCTGCC TGTCCGCCGT CGACGGCCTG GACTGGCACG TGGTGCTCTC
    1021 CGTGGGCCGC TTCGTCGACC CCGCGGACCT CGGCGAGGTC CCGCCGAACG TCGAGGTGCA
    1081 CCAGTGGGTG CCGCAGCTCG ACATCCTGAC CAAAGCCTCC GCGTTCATCA CGCACGCGGG
    1141 CATGGGCAGC ACCATGGAGG CCCTGTCGAA CGCGGTGCCC ATGGTCGCGG TGCCGCAGAT
    1201 CGCGGAGCAG ACGATGAACG CCGAGCGGAT CGTCGAGCTG GGCCTCGGCC GGCACATCCC
    1261 GCGGGACCAG GTCACGGCCG AGAAGCTGCG CGAGGCCGTG CTCGCCGTCG CCTCCGACCC
    1321 CGGTGTCGCC GAACGGCTCG CGGCCGTCCG GCAGGAGATC CGTGAGGCGG GCGGCGCCCG
    1381 GGCGGCCGCC GACATCCTGG AGGGCATCCT CGCCGAAGCA GGCTGACCGC CCCTGCCTGA
    1441 CGGTGCGCGG GCCGCCCGGC CCGCCGCGTG AGAGTCGGCC CCCGTACCCG ACGACGGGTA
    1501 CGGGGGCCGA CGCGCGCGGG CCCGGACTCA GCAGGCGGCC ACCGCGCCCC GTACCGCCTC
    1561 GATCACCGCC TTGACGGCGT CGTCGGACAG GTGCGGGCCT ATGGGCAGGC TCAGCACCTC
    1621 CCGGGCGAGC CGCTCCGCCA CGGGCTGTGC GCGGGCGGCC TGCCGGCTGC CGGCGTACGC
    1681 CTCCGACCGG TGCACCGGCA CCGGGTAGTG GATCAGCGTC TCGACGCCGG CTGCCGCCAG
    1741 CCGCTCCCGC AGCGCGGACC GGTCCGCGGA ACGAATCACG AACAGGTGCC ACACGGGGTC
    1801 CGCCCACGGC GCCGGCCTCG GCAGCACGAT CCCGTCCAGG CCGGCGAGCC CGTCGAGATA
    1861 GCGCGCCGCC ACCGCGGCCC GGCGCTCGGG TCCCAGCCGT CCCAGGTGGG CGAGCTTGAC
    1921 CCGCAGAACG GCCGCTTGCA GCTCGTCCAG CCGGAAGTTG GTGGCCCGGA CCTCGTGCCG
    1981 GTACTTCTCC CGCGACCCGT AGTTGCGCAG CAGCCGCACC CGCTCCGCCA GCTCCGCGTC
    2041 GTCCGTCACC ACGGCGCCGC CGTCACCGAA GCCGCCCAGG TTCTTGCCCG GGTAGAAGCT
    2101 GAAGGCGGTG GTGGACCACG CGCCCACCCG CCGGCCGTAC GCCTGCGCAC CGTGCGCCTG
    2161 GGCGGCGTCC TCCAGGATCC GCACGCCGTG CCGCTCGGCG ACCTCGGACA ACGCCGCCAG
    2221 GTCCGCCGGA TGCCCGTACA GGTGCACCGG GAGGATCACC CGGGTGCGGG AGGTGATCGC
    2281 AGCCTCGACG CGCTCCGGGT CCAGGGTGAA CGTCGCAGGC TCCGGTTCCA CCGCGACGGG
    2341 CTCCGCACCC GTCGCCGAGA CGGCGAGCCA GGTCGCGGCG AAGGTGTGCG CCGGGACGAT
    2401 CACCTCGTCA CCCGGCCCGA TGTCCATGGC GCGCAGCGCC AGTTCCAGGG CGTCGCACCC
    2461 GCTGCCCACC GCCACGCAGT GCCGGGCCCC GCAGTAGGCG GCCCACTCCG TCTCGAACGC
    2521 GGCGAGTTCG GGGCCCAGGA GGTAGCGCCC GGAGTCCAGG ACGCGGCCGG TCGCGGCGTC
    2581 GATGTCGTGC TTGAGCTCCA GGTAGGCGGC CCGGAGGTCC AGGAACGGAA CGTCCATGCG
    2641 TCCTCCGTGG GAGCTGCTCA CGGCGCCGTG GCGCTGAGCG GGAGACGGCC GAGGGACGGG
    2701 CCCACCATGA CCTGCCGTCC GGGTCCGGTC ACCCAGGTGT GGGCCCCGCT GTCCCAGTTC
    2761 TGGASGGCCC TGCGCTCGAC GTGCAGGGTC AGCCTCCTGC TCTCGCCCGG CCGCAGCTCG
    2821 ACCTTCCCGT AGGCCGCCAG GGCACGCTTG GCCTGCGCCA CCCGCACGTG CGGGGACGGC
    2881 CCCACGTAGA CCTGCGGGAC CTCCTTGCCG GTGCGCGTAC CGGTGTTGCG CAGCGTGAAG
    2941 CAGACGTCGA GCCCGCCGTC CGCCGTCGCC GTCACCTTCA GGTCCCGGTA GTCGAAGGAG
    3001 GTGTAGCACA ACCCGTGGCC GAAGGAGAAC AGCGGCTGGA CGCCCTGCTG TTCGTACCAG
    3061 CGGTAGCCGG AGTAGATGCC CTCGGAGTAG TCCAGTTGGT CATCGACTCC CGGGTAGCGC
    3121 CTGGCGTCCC CGGCGAACGG CGTCTGCCCC TCGTCGGCCG GGAAGGTCTG GGTCAGCCGG
    3181 CCTCCTGGGT CGGCGTCGCC GAACAGCAGG GCGGTGGTCG CCTCGGCGCC GGCCTGGCCC
    3241 GGGTACCACA TGGTGAGCAC CGCGGCGGTC TTCCTCAGCC AGGGCATGGT GAGGGAGGAG
    3301 CCCGTGTTGA GCACCACCAC GGTCCGTGGG TTGACCGCGG CCACGGCGCT GATCAGGTCG
    3361 TCCTGGCGGC CGGGCAGGGA CAGCGACGTG CGGTCCCCGT CCTCCGAGCC GTCGTCGTAC
    3421 GCGAAGACGA CCGCGGTCCT CGCCGTCCGC GCGATCGACA CGGCCCGGTC GATCGCCTCC
    3481 TGGGCGGCCT GCGGAGTGAC CCACGTCAGC TCGAAGGTCA TGGGCGACTT CGCCAGGGCC
    3541 GCGCCGGTGA TGCGCAGCTT GTGCGTTCCG GCCGCCAGCC GCATGGGGCG GCTGCTGACG
    3601 TCGCCGTAGA CCCAGGGCCG ACGGCCGAAC GGCTCCTGGC CGTCGAGTTC GACGTAGGCG
    3661 TTGCCGCCCT GCGCGCGGGC CGCGATGCGG TAGCTGCCGG TGACCGGCAC GGTGATGGTG
    3721 CCGTCGTAGA GGACACCGCC CCCACCGGCG GGGAACACCT CGCCCGAGGG GCGGGGCCGC
    3781 GGAAGAGGGG CGGACTGCGG AACGGGAACC CCGACCGTCT CCTCACCGGT GCTGTAGCGC
    3841 ACGGTGCTGC CGGCGCCGGC CCGTTCGCGG ATGGTGTCCA GAGGGGCGGA CGCGCCGTCC
    3901 GGCACGATGT ACGAACTGCC CAGCCCGGTC ACCTTCGGGA CCTTGGCGGT GGGGCCGATC
    3961 ACGGCGATGT CCGCCGCCGT CTCCGTGGTC AGGGGAAGGG TGGCGCCCTC GTTGCGCAGC
    4021 AGGACCGCGC CGTCCTCGGC GACCTGGCGC GCGACCTTCA AGCCGCCCGC GAGGTCGCGC
    4081 GCCGGGCGGG CGGGCGGATC CTCGTCCAGC AGCCGGAACC GGGCCATCTG CGACACGATG
    4141 CGGGTGACGG CCTCGTCGAG GGCCGACTCG GGGATGCGTC CCTCCCGGAT CGCCGTCTTG
    4201 AGCGGGTCGC CGAAGAACTT GCCGCCGGGT ATCGGCTCGC CCGGGGCGGG TTCGTGGTCC
    4261 AGCTCGATGC CGAGTTCCTG GTCGAGCCCC TTGGTGAGGG CGTCCGTGCT CTGCGTCGCC
    4321 AGCCAGTCCG AGGTCACCCA GCCACGGAAC TTCCACTGCT CCTTGAGGAC CTTGTTCAGC
    4381 AGTTCGTCAC TGCCGCAGGC CGGCTGGCCG TTGACCTTGT TGTAGGCGCA CATCACCGAG
    4441 CCGGTTCCGG CAGCCACGGC GCTCTCGAAA CCGGGCAGTT CCCGCTCGCG CAACGTCTGT
    4501 TCGTCGACGT TCACGTTAAC GCTGAAACGA TTCTTCTCCT GGTTGTTCGC CGCGTAGTGC
    4561 TTGGTGGCGG CGATCAGCCC CTGACTCTGG ATGCCCTTGA TCTCCGCGGC GGCCATCCGC
    4621 GAGGTGACCA GGGGGTCCTC GCTGAACGTC TCGAAGTTCC GCCCGGCGTA CGGCACGCGT
    4681 ATGGAGTTCA CCATCGGCGC GAACACCACG TCCTGCCCGA AGGCGCGCCC CTCCCGGCCG
    4741 ATCACCGCCC CGTAGGACCG CGCCAGGCCG TCGTCGAAGG TGGAGGCCAG CGCCACGGGA
    4801 GCGGGCAGCG CGAGGGACGG CCGGTGGATC GTGATTCCGG CGGGACCGTC GGTGGCCCGC
    4861 ATCTCGGGTA TGCCGAGGCG GGGAACGCCC GGCAGGTACA CCTTTGCCGA CTCATCGCTC
    4921 GTGTGATAGC TCCAGTGCAC GAACGACAGC TTTTCTTCCA GGGTCATCCG AGCCGTCAGA
    4981 AGACGAGCCG TTTCCCACGG ATCGCCCGAT TCGGCGACGG ACGGAACAGA GGGGAGCAGG
    5041 GCGAGACCGA GGGCCAGGCC GAGAGTACCC GCGGAGGTCC GTGGCGGGAC CGGACTCCTG
    5101 CGCTGCGCAC GGCCGCCGAG ACGTAACCGA AGTGATCTCA AAAGGCTTCC AAATCCTCCG
    5161 CGCCCTCGTG CTGCGAGGCG CATGAAATGG GCGGTTGTCG CGACCACAGT GCACCGTCAC
    5221 CGAAGCCGGA GCAATGCCCG TGAATAAGGT CGCGCCCTTC CGTGGATGAT CTCCGCACGA
    5281 GATCATGCCC AGCTCAAGTG ATGGTCATGC ACGTACCAAG AAGGGGCTTG CCTGGGGGGC
    5341 GTGAGCTGAT CTAGCGTTGC CGCACGACGA CGAGTCGTGA GCGAGGCGAA CGCTCTGCCG
    5401 CTCAGGGGGT GAACAGACGG CAGCCCGGAC GTTCGACGAG GGTCAAGCGG AACGCAGGCG
    5461 ACAGGACGCG GCCACCCTCC GAGGCACCCG TGCCGACCAT CCTCGCAGGT CCTTCGCCAT
    5521 GCCCGTCGCA ACTCTCCGAT CGCTGCCGCC GATGGCGACA GCCCGGCACC GAGGCCCCTG
    5581 GACCAGGAGG CGAAGCGAGG GCCGGCCGCG ATGCACGAAT CGGACCCAGG CGAACACCGG
    5641 CACATCCACC CCGGCGCGTG CGGTACGGGC CGCGCCCGAT GACGGGCGAA CGACGACCGA
    5701 AAAGCAGACC CCTTGATTCG CTTCCATGGT TGTGGCAGCC GCGGGGAGCG TCGGCAGAGA
    5761 GGTGGGAAAC CATGCATGTC CCCGGCGAGG AAAACGGGCA TTCCATTGCC ATTGTCGGAA
    5821 TTGCGTGCCG ACTGCCGGGC TCTGCCACCC CCCAGGAGTT CTGGAGACTC CTGGCCGACT
    5881 CCGCAGACGC ATTGGACGAG CCCCCCGCCG GCCGTTTCCC GACCGGCTCA TTATCCTCGC
    5941 CCCCCGCTCC GCGCGGCGGA TTCCTCGACA GCATCGACAC TTTCGACGCG GATTTCTTCA
    6001 ACATCTCGCC CAGAGAAGCC GGTGTCCTCG ACCCCCAGCA ACGCCTCGCG CTGGAACTCG
    6061 GCTGGGAGGC GCTGGAAGAC GCCGGAATCG TCCCGCGACA CCTCAGGGGA ACCCGCACCT
    6121 CGGTCTTCAT GGGCGCCATG TGGGACGACT ACGCGCACCT GGCGCACGCA CGGGGAGAAG
    6181 CCGCCCTCAC CCGGCATTCC CTGACGGGAA CGCACCGCGG CATGATCGCC AACCGGCTCT
    6241 CCTACGCCCT GGGCCTCCAA GGCCCCAGCC TCACCGTCGA CACCGGACAA TCCTCCTCCC
    6301 TCGCCGCCGT GCACATGGCC TGCGAGAGCC TGGCCCGCGG CGAATCCGAC CTGGCCCTCG
    6361 TCGGCGGCGT CAACCTCGTC CTCGATCCGG CCGGCACGAC CGGCGTCGAG AGGTTCGGAG
    6421 CACTCTCACC GGACGGCAGG TGCTACACCT TCGACTCCCG GGGGAACGGC TACGCCCGAG
    6481 GAGAGGGCGG CGTCGTAGTC GTCCTCAAGC CCACCCACCG CGCGCTCGCG GAGGGTGACA
    6541 CCGTCTACTG CGAGATCCTG GGCAGCGCCC TCAACAACGA CGGCGCCACG GAAGGCCTCA
    6601 CCGTCCCCAG CGCCCGCGCC GAGGCGGACG TCCTGCGACA GGGATGGGAA CGGGCACGCG
    6661 TGGCCCCGAC GGACGTCCAG TACGTGGAAC TGCACGGAAC CGGCACACCG GCCGGCGACC
    6721 CCGTCGAGGC CGAGGGCCTC GGCACCGCGC TCGGCACCGC ACGCCCGGCC GAGGCGCCGC
    6781 TCCTGGTCGG CTCGGTCAAG ACGAACATCG GTCACCTCGA AGGCGCGGCA GGCATCGCGG
    6841 GCCTCCTGAA GACGGTCCTG AGCATCAAGA ACCGGCACCT CCCGGCAAGC CTGAACTTCA
    6901 CCTCGCCCAA CCCCCGCATC GACCTCGACG CCCTGCGCCT GGGCGTCCAC ACCGCGTACG
    6961 GCCCCTGGCC GAGCCCCGAC CGGCCGCTCG TGGCGGGCGT CTCCTCCTTC GGCATGGGCG
    7021 GGACGAACTG CCACGTCGTC CTGTCCGAGT TACGGAACGC GGGAGGCGAC GGCGCCGGAA
    7081 AAGGGCCGTA CACCGGCACG GAAGACCGGC TCGGCGCCAC GGAGGCGGAG AAGAGGCCGG
    7141 ACCCGGCAAC CGGAAACGGT CCTGATCCCG CCCAGGACAC CCACCGCTAC CCGCCGCTGA
    7201 TCCTGTCCGC CCGCAGCGAC GCGGCCCTGC GCGCACAGGC GGAACGGCTC CGCCACCACC
    7261 TGGAACACAG CCCCGGACAG CGCGTGCGGG ACACCGCCTA CAGCCTGGGG ACCCGGCGCC
    7321 AGGTCTTCGA GCGGCACGCG GTGGTCACCG GACACGACCG CGAGGACCTG CTCAACGGCC
    7381 TGCGTGACCT GGAGAACGGC CTCCCGGCCC CCCAGGTCCT GCTCGGCCGC ACGCCCACCC
    7441 CCGAACCGGG CGGCCTCGCC TTCCTCTTCT CCGGGCAGGG CAGOCAGGAG CCCGGCATGG
    7501 GCAAGCGACT CCACCAGGTG TTCCCCGGCT TCCGGGACGC CCTGGACGAG GTCTGCGCCG
    7561 AACTCGACAC CCACCTCGGC CGACTCCTCG GCCCCGAGGC CGGCCCGCCC CTGCGCGACG
    7621 TGATGTTCGC CGAGCGGGGC ACGGCGCACA GCGCCCTGCT CTCCGAGACC CACTACACCC
    7681 AGGCCGCCCT CTTCGCCCTG GAAACCGCCC TCTTCCGCCT CCTGGTCCAG TGGGGCCTGA
    7741 AACCCGACCA CCTCGCAGGC CACTCCGTCG GCGAGATCGC GGCCGCCCAC GCAGCAGGCA
    7801 TCCTCGACCT GTCCGACGCG GCCGAACTCG TGGCCACCCG CGGCGCGTTG ATGCGTTCCC
    7861 TGCCCGGCGG CGGCGTCATG CTCTCGGTCC AGGCACCCGA GTCCGAGGTC GCACCCCTGG
    7921 TGCTCGGCCG TGAGGCCCAC GTCGGCCTGG CCGCCGTGAA CGGCCCCGAC GCGGTGGTCG
    7981 TGTCCGGCGA GCGCGGCCAC GTCGCCGCCA TCGAACAGAT CCTCCGGGAC AGGGGCCGCA
    8041 AAAGCCGGTA CCTGCGCGTC AGCCACGCCT TCCACTCCCC GCTCATGGAA CCGGTGCTGG
    8101 AGGAGTTCGC CGAAGCCGTC GCCGGCCTGA CCTTCCGGGC ACCGACCACA CCCCTCGTCT
    8161 CCAACCTCAC CGGCGCACCA GTCGACGACC GGACCATGGC CACGCCCGCC TACTGGGTCC
    8221 GGCACGTCCG GGAAGCGGTC CGCTTCGGCG ACGGCATCCG GGCACTCGGG AAACTGGGCA
    8281 CCGGCAGCTT CCTGGAAGTC GGGCCGGACG GCGTCCTCAC CGCCATGGCG CGCGCATGCG
    8341 TCACCGCCGC CCCGGAGCCC GGCCACCGCG GCGAACAGGG CGCCGATGCC GACGCCCACA
    8401 CCGCGTTGCT GCTGCCCGCC CTGCGCCGAG GACGGGACGA GGCGCGATCG CTCACCGAGG
    8461 CCGTGGCACG GCTCCACCTG CACGGCGTGC CGATGGACTG GACCTCCGTC CTCGGCGGCG
    8521 ACGTGAGCCG GGTCCCCCTC CCGACGTACG GCTTCCAACG CGAATCCCAC TGGCTGCCGT
    8581 CCGGAGAGGC TCACCCGCGA CCGGCGGACG AGACCGAATC CGGCACGGGA CGGACCGAGG
    8641 CGTCCCCGCC GCGGCCGCAC GACGTCCTGC ACCTCGTGCG CTCCCACGCG GCGGCTGTGC
    8701 TCGGACATTC CCGGGCCGAG CGGATCGACC CCGACCGCGC GTTCCGCGAC CTCGGCTTCG
    8761 ACTCGCTGAC GGCGCTGGAA CTGCGGGACC GGCTCGACAC CGCACTCGGC CTCCGCCTGC
    8821 CCAGCAGCGT GCTCTTCGAC CACCCGAGCC CCGGCGCACT GGCACGCTTC CTCCAGGGCG
    8881 AGGACAGGAG GCGCCCCGAA CCAGGGAAGA CGAACGGCAC GCGCGCCACG GAGCCAGGCC
    8941 CGGACCCGGA CGACGAGCCG ATCGCCATCG TCGGCATGGC GTGCCGCTTC CCGGGTGGCG
    9001 TGACCTCTCC GGAGGACCTG TGGCGCCTGC TCGCCGCAGG CGAGGACGCG GTGTCCGGCT
    9061 TCCCCACGGA CCGGGGCTGG AACGTCACTG ACTCCGCCAC GCGCCGCGGA GGCTTCCTGT
    9121 ACGACGCCGG CGAGTTCGAT GCCGCCTTCT TCGGTATCTC GCCGCGTGAG GCGTTGGTGA
    9181 TGGACCCGCA GCAGCGGTTG CTGCTGGAGA CGTCCTGGGA GGCCCTCGAA CGCGCGGGCG
    9241 TGAGCCCCGG CAGTCTGCGC GGCAGCGACA CGGCCGTGTA CATCGGAGCC ACAGCGCAGG
    9301 ACTACGGCCC CCGACTGCAC GAGTCGGACG ACGACTCGGG CGGCTACGTC CTGACCGGCA
    9361 ATACCGCCAG CGTGGCCTCC GGCCGCATCG CCTACTCCCT CGGTCTGGAG GGGCCTGCGG
    9421 TCACGGTGGA CACGGCGTGT TCGTCGTCGC TGGTGGCACT GCACCTGGCG GTGCAGGCGC
    9481 TGCGCCGTGG CGAGTGCTCA CTGGCATTGG CCGGCGGAGC CACGGTGATG CCTTCGCCCG
    9541 GCATGTTCGT GGAGTTCTCA CGGCAAGGGG GCCTCTCCGA GGACGGCCGC TGCAAGGCGT
    9601 TCGCCGCGAC GGCGGACGGC ACCGGCTGGG CCGAGGGTGT GGGTGTGTTG TTGGTGGAGC
    9661 GGTTGTCGGA TGCGCGGCGG TTGGGTCATC GGGTGTTGGC GGTGGTGCGG GGGAGTGCGG
    9721 TCAATCAGGA TGGTGCGTCG AATGGGTTGA CGGCGCCGAA TGGTCCGTCG CAGCAGCGGG
    9781 TGATCCGTGG GGCGTTGGCT GACGCGGGTC TGGTTCCTGC TGATGTGGAT GTGGTGGAGG
    9841 CGCATGGTAC GGGGACGCGG TTGGGTGATC CGATCGAGGC TCAGGCGTTG TTGGCGACGT
    9901 ATGGGCAGGG GCGTGCGGGT GGGCGTCCGG TGGTGTTGGG GTCGGTGAAG TCGAACATCG
    9961 GTCATACGCA GGCGGCGGCT GGTGTGGCTG GTGTGATGAA GATGGTGCTG GCGCTGGGGC
    10021 GGGGTGTGGT GCCGAAGACG TTGCATGTGG ATGAGCCGTC TGCGCATGTG GACTGGTCGG
    10081 CTGGTGAGGT GGAGTTGGCG GTTGAGGCGG TGCCGTGGTC GCGGGGTGGG CGGGTGCGGC
    10141 GGGCTGGTGT GTCGTCGTTC GGGATCAGTG GCACGAATGC GCATGTGATC GTGGAGGAGG
    10201 CGCCTGCGGA GCCGGAGCCG GAGCCGGAGC GGGGTCCGGG CTCTGTTGTG GGTGTGGTGC
    10261 CGTGGGTGGT GTCCGGGCGG GATGCGGGGG CGTTGCGTGA GCAGGCGGCA CGCTTGGCTG
    10321 CGCACGTGTC GGGTGTAAGT GCGGTCGATG TGGGCTGGTC GTTGGTGGCC ACGAGGTCGG
    10381 TGTTCGAGCA CCGGGCGGTG ATGGTCGGCA GTGAACTCGA TGCCATGGCG GAGTCGTTGG
    10441 CCGGCTTCGC TGCGGGTGGG GTTGTGCCGG GGGTGGTGTC GGGTGTGGCT CCGGCTGAGG
    10501 GTCGTCGTGT GGTGTTCGTC TTTCCTGGTC AGGGTTCGCA GTGGGTGGGG ATGGCGGCTG
    10561 GGTTGCTGGA TGCGTGCCCG GTGTTCGCGG AGGCGGTGGC GGAGTGCGCT GCGGTGCTGG
    10621 ACCCGTTGAC CGGTTGGTCG CTGGTCGAGG TGTTGCGCGG TGGTGGTGAG GCTGTTCTTG
    10681 GGCGGGTTGA TGTGGTGCAG CCGGCGTTGT GGGCGGTGAT GGTGTCACTG GCCCGGACCT
    10741 GGCGGTATTA CGGTGTGGAG CCTGCTGCGG TTGTGGGGCA TTCGCAGGGT GAGATTGCTG
    10801 CGGCTTGTGT GGCTGGGGGG TTGAGTCTGG CCGATGGTGC GCGGGTGGTG GTGTTGCGGA
    10861 GCCGGGCGAT CGCCCGGATC GCTGGTGGGG GCGGCATGGT CTCCGTCAGC CTGCCGGCCG
    10921 GCCGTGTCCG CACCATGCTG GAGGAGTTCG ACGGCAGGGT TTCCGTTGCG GCGGTCAACG
    10981 GTCCGTCCTC GACCGTGGTG TCGGGTGACG TCCAGGCCCT GGATGAGTTG TTGGCCGGTT
    11041 GTGAGCGGGA GGGTGTCCGG GCTCGTCGTG TCCCGGTGGA CTATGCCTCC CACTCCGCGC
    11101 AGATGGACCA GTTACGCGAT GATCTGCTGG AAGCGCTGGC GACGATCGTC CCTACATCGG
    11161 CGAACGTACC GTTCTTCTCG ACGGTGACGG CGGACTGGCT GGACACGACC GCTCTGGATG
    11221 CGGGGTACTG GTTCACGAAT CTGCGGGAGA CGGTCCGGTT CCAAGAAGCC GTCGAAGGGC
    11281 TCGTGGCTCA GGGGATGGGC GCGTTCGTCG AGTGCAGCCC GCACCCCGTC CTCGTCCCGG
    11341 GCATCACAGA AACACTCGAC ACCTTCGACG CCGACGCTGT CGCACTGTCG TCGCTGCGGC
    11401 GTGACGAAGG CGGCCTGGAT CGGTTCCTCA CGTCCCTCGC GGAAGCCTTC GTCCAGGGCG
    11461 TCCCGGTCGA CTGGTCCCGC GCCTTCGAGG GTGCGAGCCC CCGCACCGTC GACCTGCCCA
    11521 CCTACCCCTT CCAACGGCAA CGCTACTGGC TGCTCGACAA GGCGGCGCAA CGGGAACGCG
    11581 AGCGGCTGGA GGACTGGCGC TACCACGTCG AGTGGCGCCC CGTCACGACA CGACCTTCCG
    11641 CACGGCTGTC CGGTGTCTGG GCCGTGGCGA TTCCGGCACG TCTGGCCCGT GACTCACTGT
    11701 TGGTCGGCGC CATCGACGCA CTGGAGCGAG GCGGCGCCCG TGCCGTGCCC GTGGTGGTCG
    11761 ATGAGCGGGA CCACGACCGG CAAGCGCTGG TCGAGGCTCT GCGGAACGGG CTGGGCGACG
    11821 ACGACCTCGC CGGTGTGCTC TCCCTTTTGG CCCTCGACGA AGCCCCGCAC GGTGACCACC
    11881 CCGACGTGCC CGTCGGCATG GCCGCTTCGC TGGCGCTCGT GCAGGCGATG GCCGACGCCG
    11941 CGGCCGAGGT GCCCGTATGG TTCGCGACCC GAGGCGCCGT AGCGGCACTG CCCGGTGAGT
    12001 CACCGGAGCG ACCCAGGCAG GCGCTGCTCT GGGGACTGGG ACGGGTCGTC GCCCTGGAAC
    12061 AGCCGCAGAT ATGGGGCGGG TTGGTCGACC TCCCGCAACA CCTGGACGAG GACGCGGGCC
    12121 GACGGCTGGT CGATGTCGTG GGCGGCCTGG CGGACGAGGA CCAGCTTGCC GTACGGGCCT
    12181 CCTCCGTCCT CGCCCGACGC CTCGTTCGTA CGCCGGGTCA CCGTATGTCG AGCCAGGCGG
    12241 GCGGGCGCGA GTGGTCGCCC AGCGGCACGG TCCTGGTGAC CGGAGGCACC GGGGCGCTGG
    12301 GCGCGCACGT CGCCCGCTGG CTGGCCGGCA AGGGCGCCGA GCACCTGGTA CTCATCAGCC
    12361 GTCGCGGAGC GGACGCAGCC GGGGCCGCTG CCCTTCGGGA CAGCCTCACG GACATGGGTG
    12421 TCCGGGTGAC CCTGGCCGCG TGCGATGCAG CGGACCGGCA CGCACTGGAG ACGCTCCTCG
    12481 ACTCGCTGCG CACGGATCCG GCGCAGCTGA CGGCCGTCAT CCACGCCGCG GGTGCTCTGG
    12541 ACGACGGCAT GACGACGGTG CTCACACCGG AGCAGATGAA CAACGCCCTG CGAGCGAAAG
    12601 TCACGGCCAC CGTCAACCTG CACGAACTGA CCCGGGACCT CGACCTCTCG GCCTTCGTAC
    12661 TGTTCTCGTC CATCTCCGCC ACCCTGGGAA TCCCCGGGCA GGCCAACTAC GCGCCGGGAA
    12721 ACTCGTTCTT GGACGCCTTC GCGGAATGGC GCAGGGCTCA GGGGCTCGTG GCGACCTCCA
    12781 TCGCCTGGGG ACCGTGGTCC GGCGGCACCG GCATGGCACA TGAAGGGTCG GTGGGCGAAC
    12841 GGCTCCAGCG GCACGGTGTA CTCGCCATGG AACCCGCGGC GGCCATCGCT GCGCTCGACC
    12901 ACACGCTGGC GAGCGACGAA ACCGCAGTGG CCGTGGCCGA CATCGACTGG AGCCGGTTCT
    12961 TCCTGGCGTA CACAGCACTG CGGGCACGGC CCTTGATCGG AGAGATACCC GAGGCACGCC
    13021 CCATGCTGGA GTCCGGCTCA GGCCCCGGCG ACCTCGAGCC GGACCGTGCC GAACCCGAGC
    13081 TTGCCGTGCG TCTCGCGGGC CTCACCGCGG TCGAGCAGGA ACGTCTTCTG GTGCAGGTCG
    13141 TGAGGGAGCA GGCCGCCGTC GTCCTCGGAC ATTCCGGCGC CGAGGCGGTG GCTCCGGACC
    13201 GCGCGTTCAA GGATCTCGGA TTCGACTCGC TGACCTCGGT CGAACTGCGC AACCGGCTGA
    13261 ACACCGCCAC CGGCCTCAGA CTGCCCGTGA CGGCCGTCTT CGACTACGCG AGGCCCGCGG
    13321 CGCTGGCCGG CCATCTGCGC TCCAGGCTGA TCGACGACGA TGGTGACCAC GGTGCCTTGC
    13381 CCGGCGTGGA GAAGCACGCG ATCGACGAGC CGATCGCGAT CGTGGGAATG GCATGCCGCT
    13441 TCCCGGGAGG CATCGCTTCC CCGGAGGATC TGTGGGACGT GCTCACCGCT GGTGAGGACG
    13501 TTGTCTCCGG ACTGCCGCAG AACCGCGGGT GGGACTTGGG GCGCCTGTAC GATCCCGATC
    13561 CGGACCGGGC CGGTACGTCA TACATGCGTG AGGGTGCTTT CCTGCACGAG GCGGGGGAGT
    13621 TCGACGCGGC CTTCTTCGGT ATCTCGCCGC GTGAGGCGTT GGCGATGGAC CCGCAGCAGC
    13681 GGTTGCTGCT GGAGACGTCC TGGGAGGCCC TCGAACGGGC CGGCATCACT CCTTCCAAGC
    13741 TGGCGGGCAG TCCGACCGGT GTGTTCTTCG GCATGTCGAA CCAGGACTAC GCCGCCCAGG
    13801 CGGGCGACGT GCCGTCCGAG CTGGAGGGCT ACCTGCTCAC CGGCTCCATC TCCAGCGTCG
    13861 CTTCGGGGCG TGTTGCTTAC ACGTTCGGTC TTGAGGGGCC TGCGGTGACG GTGGATACGG
    13921 CGTGTTCGTC GTCGTTGGTG GCGTTGCATC TGGCGGTGCA GGGGTTGCGG CGGGGTGAGT
    13981 GTTCGCTTGC GTTGGTGGGT GGGGTGACGG TGATGTCGTC GCCGGTGACG TTGACGACGT
    14041 TCAGTCGGCA GCGGGGTTTG TCGGTGGATG GGCGGTGCAA GGCGTTCGCG GCTTCGGCGG
    14101 ATGGTTTTGG TGCTGCCGAG GGTGTGGGTG TGTTGTTGGT GGAGCGGTTG TCGGATGCGC
    14161 GGCGGTTGGG TCATCGGGTG TTGGCGGTGG TGCGGGGGAG TGCGGTCAAT CAGGATGGTG
    14221 CGTCCAATGG TCTGGCGGCG CCGAATGGTC CGTCGCAGCA GCGGGTGATC CGTGCGGCGT
    14281 TGGCTGACGC GGGTCTGGCT CCTGCCGATG TGGATGTGGT GGAGGCGCAT GGCACGGGGA
    14341 CGCGGTTGGG TGATCCGATC GAGGCTCAGG CGTTGCTGGC GACGTATGGG CAGGGTCGTA
    14401 CCAGTGGGCG TCCGGTGTGG CTGGGGTCGG TGAAGTCGAA CATCGGGCAT ACGCAGGCCG
    14461 CGGCCGGTGT GGCTGGTGTG ATGAAGATGG TGCTGGCGTT GGGTCGGGGT GTGGTGCCGA
    14521 AGACGTTGCA TGTGGATGAG CCGTCACCGC ATGTGGACTG GTCGGCTGGT GAGGTGGAGT
    14581 TGGCGGTTGA GGCGGTGCCG TGGTCGCGGG GTGGGCGGGT GCGGCGGGCT GGTGTGTCGT
    14641 CGTTCGGGAT CAGCGGCACG AATGCGCATG TGATCGTGGA GGAGGCGCCT GCGGAGCCTT
    14701 CGGTGGAGGA GGGTCCGGGC TCCGTTGTGG GTGTGGTGCC GTGGGTGGTG TCCGGGCGGG
    14761 ATGCGGGGGC GTTGCGTGCA CAGGCGGCAC GCTTGGCTGC GCACGTGTCG AGCACGGGTG
    14821 CGGGTGTGGT TGATGTGGGC TGGTCGTTGG TGGCCACGAG GTCGGTGTTC GAGCACCGGG
    14881 CGGTAATGGT CGGCACTGAT CTTGATTCCA TGGCGGGGTC GTTGGCCGGC TTCGCTGCGG
    14941 GTGGTGTTGT GCCGGGGGTG GTGTCGGGTG TGGCTCCGGC TGAGGGCCGT CGTGTGGTGT
    15001 TCGTCTTTCC TGGTCAGGGT TCGCAGTGGG TGGGGATGGC GGCTGGGTTG CTGGATGCGT
    15061 GTCCGGTGTT CGCGGAGGCG GTGGCGGAGT GTGCCGCGGT GCTGGACCGG TTGACCGGTT
    15121 GGTCGCTGGT CGAGGTGTTG CGTGGTGGTG AGGCTGTTCT TGGGCGGGTT GATGTGGTGC
    15181 AGCCGGCGTT GTGGGCGGTG ATGGTGTCAC TGGCTCGGAC CTGGCGGTAT TACGGTGTGG
    15241 AGCCTGCTGC GGTTGTGGGG CATTCGCAGG GTGAGATTGC TGCGGCTTGT GTGGCTGGGG
    15301 GGTTGAGTCT GGCCGATGGT GCGCGGGTGG TGGTGTTGCG GAGTCGGGCG ATCGCCCGGA
    15361 TCGCTGGTGG GGGCGGCATG GTCTCGGTCG GTCTTTCAGC TGAGCGTGTC CGCACCATGC
    15421 TCGACACCTA CGGCGGCAGG GTTTCCGTCG CGGCGGTCAA TGGCCCGTCC TCGACCGTGG
    15481 TGTCCGGTGA CGCCCAGGCC CTGGATGAGT TGTTGGCCGG TTGTGAGCGG GAGGGTGTCC
    15541 GGGCTCGTCG TGTCCCGGTG GACTATGCCT CCCACTCCGC GCAGATGGAC CAGTTACGCG
    15601 ATGAGTTGCT GGAGGCGCTG GCGGACGTCA CTCCGCAGGA CTCCAGTGTT CCGTTTTTCT
    15661 CGACGGTGAC GGCGGACTGG CTGGACACGA CCGCTCTGGA TGCGGGGTAC TGGTTCACGA
    15721 ATCTGCGGGA GACGGTCCGG TTCCAGGAAG CCGTTGAAGG GCTTGTGGCT CAGGGGATGG
    15781 GCGCGTTCGT CGAGTGCAGC CCGCACCCTG TCCTCGTCCC GGGCATCACA GAAACACTCG
    15841 ACACCTTCGA CGCCGACGCT GTCGCACTGT CGTCGCTGCG GCGTGACGAA GGCGGCCTGG
    15901 ATCGGTTCCT CACGTCCCTC GCGGAAGCCT TCGTCCAAGG CGTTCCCGTC GACTGGACCC
    15961 ATGCCTTCGA GGGTGGACGC CCGCGCTTCG TCGACCTGCC CACCTATGCC TTCCAGCGAC
    16021 AGCGCTACTG GCTGCACGAA GAGCCGCTGC AAGAGCCGGT CGATGAGGCG TGGGATGCCG
    16081 AGTTCTGGTC TGTGGTCGAA CGCGGCGATG CCACAGCCGT GTCCGACTTG CTGAGCACGG
    16141 ACGCCGAGGC TTTGCACACG GTGTTGCCGG CTTTGTCGTC GTGGCGGCGG CGTCGGGTGG
    16201 AGCATCGACG GCTTCAGGAC TGGCGTTACC GGGTGGAGTG GAAGCCTTTC CCGGCCGCGC
    16261 TTGATGAGGT GCTCGGTGGT GGCTGGTTGT TCGTGGTGCC GCGGGGCTTG GCGGATGATG
    16321 GTGTGGTTGC GCGGGTGGTG GCTGCCGTCA CGGCGCGGGG TGGCGAGGTC AGTGTCGTGG
    16381 AGCTCGATCC GACCCGTCCT GACCGCCGGG CTTATGCGGA GGCTGTCGCG GGCCGTGGTG
    16441 TGAGCGGGGT CGTGTCGTTC TTGTCCTGGG ATGATCGGCG GCACTCGGAG CATTCTGTTG
    16501 TTCCCGCCGG TCTTGCCGCG TCGCTGGTGT TGGCGCAGGC GTTGGTTGAT CTTGGCCGGG
    16561 TTGGTGAGGG GCCGCGGTTG TGGCTGGTGA CGCGOGGTGC GGTGGTTGCT GGTCCTTCGG
    16621 ATGCCGGTGT GGTGATTGAT CCGGTGCAGG CGCAGGTGTG GGGTTTCGGG CGTGTTCTGG
    16681 GTCTGGAGCA TCCCGAGTTG TGGGGTGGGC TGGTGGACCT GCCGGTGGGG GTTGATGAGG
    16741 AGGTGTGCCG GCGGTTCGTG GGTGTTGTGG CGTCGGCTGG TTTTGAGGAT CAGGTGGCGG
    16801 TGCGTGGTTC GGGTGTGTGG GTGCGTCGTC TGGTGCGTGC TGTGGTGGAT GGTGGTGGGG
    16861 GTGGTTGGCG GCCGCGTGGG ACGGTGTTGG TCACGGGTGG TCTTGGTGGT TTGGGTGCGC
    16921 ATACGGCCCG GTGGTTGGTG GGTGGTGGGG CGGATCATGT GGTTCTTGTG AGCCGTCGTG
    16981 GTGGCAGTGC GCCTGGTGCT GGGGATCTGG TGCGGGAGCT GGAGGGCTTG GGCGGGGCTC
    17041 GGGTGTCGGT GCGGGCCTGT GATGTGGCTG ATCGTGTGGC GTTGCGGGCG TTGTTGTCGG
    17101 ATCTGGGTGA GCCGGTGACG GCGGTGTTCC ATGCGGCTGG TGTTCCTCAG TCGACGCCTT
    17161 TGGCGGAGAT CTCTGTCCAG GAGGCGGCTG ATGTGATGGC GGCCAAGGTG GCGGGTGCGG
    17221 TGAATCTGGG TGAGTTGGTG GATCCCTGTG GTCTGGAGGC GTTTGTGTTG TTCTCCTCCA
    17281 ATGCCGGTGT GTGGGGCAGT GGGGGGCAGG CGGTGTATGC GGCGGCGAAT GCGTTTCTTG
    17341 ATGCGTTGGC GGTGCGTCGT CGGGGTGTTG GTCTGCCGGC CACGAGTGTG GCGTGGGGGA
    17401 TGTGGGCTGG TGAGGGGATG GCGTCGGTGG GTGGTGCGGC GCGGGAGTTG TCCCGTCGGG
    17461 GGGTGCGGGC GATGGATCCC GAGCGTGCTG TGGCGGTGAT GGCTGATGCG GTGGGTCGTG
    17521 GTGAGGCGTT CGTCGCGGTC GCTGATGTGG ACTGGGAACG TTTCGTCACC GGTTTCGCTT
    17581 CTGCCCGTCC CCGTCCGTTG ATCAGTGACC TGCCGGAGGT GCGTGCTGTT GTGGAGGGCC
    17641 AGGTCCAGGG CCGGGGCCAG GGGTTGGGCT TGGTCGGTGA GGAGGAGTCG TCGGGGTGGT
    17701 TGAAGCGGTT GTCGGGGTTG TCTCGTGTGC GGCAGGAGGA GGAGTTGGTG GAGTTGGTCC
    17761 GTGCTCAGGC TGCCGTTGTT CTCGGGCATG GTTCCGCGCA GGACGTCCCG GCTGAGCGGG
    17821 CGTTCAAGGA GTTGGGTTTT GATTCCCTCA CTGCTGTCGA GCTACGCAAC GGGCTGGCCG
    17881 CGGCCACCGG GATCCGGCTG CCGGCCACCA TGGCATTCGA TCATCCCACC GCCACCGCCA
    17941 TCGCACGCTT CCTGCAATCC GAACTCGTGG GAAGTGACGA CCCGCTGACG CTCATGCGGT
    18001 CGGCGATCGA CCAGTTGGAG ACCGGTCTGG CTCTGCTGGA ATCGGACGAA GAAGCTCGCT
    18061 CGGAAATCAC GAAGCGATTG AACATTCTTC TGCCCCGCTT CGGAAGCGGA GGCAGTTCGA
    18121 GAGGCAGGGA AGCAGGACAA GACGCAGGCG AACATCAGGA TGTCGAGGAC GCCACCATCG
    18181 ATGAGCTATT CGAGGTGCTC GACAACGAAC TCGGCAATTC CTGAAAACCT GTCCGACTGC
    18241 TACCGCGACC TTGACCGGAG AACGCTGTGA CGAACGACGA AAAGATCGTC GAGTATCTCA
    18301 AGCGCGCGAC CGTGGACCTG CGCAAGGCCC GGCACCGCAT CTGGGAGCTG GAGGACGAGC
    18361 CCATCGCGAT CACGTCGATG GCCTGCCACT TCCCGGGCGG GATCGAGAGT CCGGAGCAGC
    18421 TGTGGGAACT CCTGTCCGCC GGAGGCGAGG TGCTTTCCGA GTTCCCCGAC GACCGCGGCT
    18481 GGGACCTGGA CGAGATCTAC CATCCTGACC CGGAACACAG TGGGACGAGC TACGTCCGTC
    18541 ACGGCGGTTT CCTGGATCAT GCGACGCAGT TCGACACGGA CTTCTTCGGT ATCTCGCCGC
    18601 GTGAGGCGTT GGCGATGGAC CCGCAGCAGC GGTTGCTGCT GGAGACGTCC TGGCAGCTTT
    18661 TCGAGCGCGC AGGAGTCGAT CCCCATACGC TGAAGGGAAG CCGGACCGGA GTATTCGTCG
    18721 GCGCCGCACA CATGGGTTAT GCGGACAGGG TGGACACTCC GCCGGCGGAG GCCGAGGGCT
    18781 ACCTGCTGAC AGGGAACGCC TCGGCCGTTG TCTCCGGGCG TATTTCCTAC ACCTTCGGCC
    18841 TTGAGGGGCC TGCGGTGACG GTGGACACGG CGTGCTCGTC GTCGCTGGTG GCGCTGCACC
    18901 TGGCGGTGCA GGCGCTGCGC CGTGGCGAGT GCTCGCTGGC GGTCGTCGGT GGTGTGGCCG
    18961 TCATGTCGGA CCCGAAGGTC TTCGTCGAGT TCAGCCGGCA GCGCGGACTG GCCAGGGACG
    19021 GCCGGTCCAA GGCTTTTGCG GCGTCAGCGG ATGGTTTCGG CTTCGCCGAG GGAGTTTCGC
    19081 TGCTCTTGCT GGAGCGGTTG TCGGATGCGC GGCGGTTGGG TCATCGGGTG TTGGCGGTGG
    19141 TGCGGGGGAG TGCGGTCAAT CAGGATGGTG CGTCCAATGG TCTGGCGGCG CCGAATGGTC
    19201 CGTCGCAGCA GCGGGTGATT CGTGCGGCGT TGGCTGACGC GGGTCTGGCT CCTGCCGATG
    19261 TGGATGTGGT GGAGGCGCAT GGTACGGGGA CGCGGTTGGG TGATCCGATC GAGGCTCAGG
    19321 CGTTGCTGGC GACGTATGGG CAGGGGCGTA CCAGTGGGCG TCCGGTGTGG CTGGGGTCGG
    19381 TGAAGTCGAA CATCGGTCAT ACGCAGGCGG CGGCCGGTGT GGCTGGTGTG ATGAAGATGG
    19441 TGCTGGCTCT GGAGCGGGGT GTGGTGCCGA AGACGTTGCA CGTGGATGAG CCGTCTCCGC
    19501 ATGTGGACTG GTCGACCGGT GCGGTGGAGT TGCTGACTGA AGAGCGGCCG TGGGTGCCGG
    19561 AGGCTGAGCG TCTTCGTCGG GCAGGCATTT CCGCCTTCGG TGTCAGTGGC ACGAATGCGC
    19621 ATGTGATCGT GGAGGAGGCA CCTGCGGAAC CGGAACCGGA GCCGGAGCCG GGAACTCGTG
    19681 TGGTTGCTGC CGGTGATCTG GTGGTGCCGT GGGTGGTGTC CGGGCGGGAT GCGGGGGCGT
    19741 TGCGTGCACA GGCGGCACGC TTGGCTGCGC ATGTGTCGAG CACGGGTGCG GGTGTGGTTG
    19801 ATGTGGGCTG GTCGTTGGTG GCCACGAGGT CGGTGTTCGA GCACCGGGCG GTGATGGTCG
    19861 GCACTGATCT TGATTCCATG GCGGGGTCGT TGGCCGGGTT TGCTGCGGGT GGGGTTGTGC
    19921 CGGGGGTGGT GTCGGGTGTG GCTCCGGCTG AGGGTCGTCG TGTGGTGTTC GTCTTTCCTG
    19981 GTCAGGGTTC GCAGTGGGTG GGGATGGCGG CTGGGTTGCT GGATGCGTGT CCGGTGTTCG
    20041 CGGAGGCGGT GGCGGAGTGT GCCGCGGTGC TGGACCCGTT GACCGGTTGG TCGCTGGTCG
    20101 AGGTGTTGCG CGGTGGTGAG GCTGTTCTTG GGCGGGTTGA TGTGGTGCAG CCGGCGTTGT
    20161 GGGCGGTGAT GGTGTCACTG GCTCGGACCT GGCGGTATTA CGGTGTGGAG CCTGCTGCGG
    20221 TTGTGGGGCA TTCGCAGGGT GAGATTGCTG CGGCTTGTGT GGCTGGGGGG TTGAGTCTGG
    20281 CCGATGGTGC GCGGGTGGTG GTGTTGCGGA GCCGGGCGAT CGCCCGGATC GCCGGTGGGG
    20341 GCGGCATGGT CTCCGTCAGT CTCCCGGCCG GCCGTGTCCG CACCATGCTC GACACCTACG
    20401 GCGGCCGGTT GTCGGTGGCT GCGGTCAACG GCCCGTCCTC GACCGTGGTG TCCGGTGACG
    20461 CCCAGGCCCT GGATGAGTTG TTGGCCGGCT GTGAGCGGGA GGGGGTCCGG GCTCGTCGTG
    20521 TCCCGGTGGA CTATGCCTCC CACTCCGCGC AGATGGACCA GTTACGCGAT GAGCTGCTGG
    20581 AAGCGCTGGC GGACATCACT CCGCAACACT CCAGCGTTCC GTTCTTCTCG ACGGTGACGG
    20641 CGGACTGGCT GGACACGACC GCTCTGGATG CGGGGTACTG GTTCACGAAT CTGCGGGAGA
    20701 CGGTCCGGTT CCAGGAAGCC GTCGAAGGGC TTGTGGCTCA GGGGATGGGC GCGTTCGTCG
    20761 AGTGCAGCCC ACACCCCGTC CTCGTCCCCG GTATCGAGCA GACCCTCGAC ACCGTGGAAG
    20821 CCGATGCTGT GGCGCTGGGT TCGCTACGGC GTGATGAGGG CGGCCTGGGA CGGTTCCTCA
    20881 CGTCCCTCGC GGAAGCCTTC GTCCAGGGCG TCCCGGTCGA CTGGTCCCGC ACCTTCGAGG
    20941 GTGCGAGCCC CCGCACCGTC GACCTGCCCA CCTATCCCTT CCAACGGCAA CGTTTCTGGT
    21001 TGGAGGGATC CCCGGCGTTG TCTTCGAACG GCGTCGAGGG TGAGGCGGAC GTCGCGTTCT
    21061 GGGATGCGGT CGAGCGCGAG GACTCGGCGG TTGTAGCCGA GGAGTTGGGG ATCGACGCCA
    21121 AGGCTCTGCA CATGACATTG CCGGCCTTGT CGTCGTGGCG GCGGCGTGAG CGGCAGCGTC
    21181 GGAAGGTGCA GCGCTGGCGT TACCGGGTGG AGTGGAAGCG TCTCCCGAAT TCGCGGGCAC
    21241 AGGAGTCGCT GCAGGGCGGC TGGTTGCTCG TCGTCCCGCA GGGCCGTGCC GGCGATGTCC
    21301 GCGTCACTCA GTCGGTGGCG GAGGTGGCGG CCAAGGGTGG TGAAGCCACG GTCCTGGAGG
    21361 TCGACGCCCT GCATCCCGAC CGCGCAGCAT ACGCCGAGGC CCTCACCCGG TGGCCGGGTG
    21421 TGCGGGGTGT GGTGTCGTTC CTGGCGTGGG AGGAGCAGGC CCTTGCCGAA CACCCCGTTC
    21481 TGTCTGCGGG TCTGGCGGCA TCGCTGGCGT TGGCCCAGGC GTTGATCGAT GTCGGCGGGT
    21541 CCGGTGAGTC GGCGCCGCGT CTGTGGCTGG TCACGGAAGC TGCCGTCGTG ATCGGTGCTG
    21601 CCGACACCGG TGCGGTGATC GACCCCGTAC ACGCGCAGCT GTGGGGCTTC GGCCGTGTCC
    21661 TTGCTCTGGA ACACCCCGAA TTGTGGGGCG GGCTGATCGA CCTGCCCGCT GTGGCAGGCG
    21721 AGCCTGGTTC GATTACCGAC CACGCGCATG CCGACCTACT GGCCACGGTC CTGGCCACGA
    21781 TGGTGCAGGC TGCTGCCCGA GGCGAGGACC AGGTCGCGGT CCGGACGACC GGTACTTACG
    21841 TACCCAGGCT GGTGCGTTCA GGCGGCAGTG CACACTCGGG TGCGCGGAGG TGGCAGCCGC
    21901 GCGACACCGT ACTGGTCACC GGCGGGATGG GACCGCTGAC CGCCCACATC GTCCGTTGGC
    21961 TGGCTGACAA CGGTGCCGAC CAGGTAGTAC TCCTGGGAGG TCAGGGAGCA GACGGCGAGG
    22021 CCGAGGCGCT GAGGGCCGAG TTCGACGGGC ACACGACGAA GATCGAACTC GCGGACGTGG
    22081 ACACCGAGGA CAGCGACGCG CTGCGGTCCT TGCTCGACCG CACGACCGGC GAACACCCGC
    22141 TGCGCGCGGT CATCCATGCG CCGACCGTGG TCGAGTTCGC CTCGGTGGCC GAGTCGGACC
    22201 TGGTGCGATT CGCCCGCACC ATCAGCAGCA AGATCGCCGG CGTCGAGCAG CTCGACGAGG
    22261 TGCTGAGCGG CATCGACACG GCGCACGACG TGGTCTTCTT CTCCTCCGTC GCGGGCGTCT
    22321 GGGGAAGCGC GGGGCAGAGC GCCTACGCGG CGGGCAACGC CTTCCTCGAC GCCGTCGCCC
    22381 AGCACCGCCG TCTGCGCGGA CTGCCCGGTA CGTCGGTGGC CTGGACTCCG TGGGACGACG
    22441 ATCGATCCCT TGCCTCCCTC GGTGACTCGT ACCTCGACCG ACGAGGACTG CGAGCACTGT
    22501 CCATACCCGG CGCGCTCGCC TCCCTCCAGG AAGTGCTCGA CCAGGACGAG GTCCACGCCG
    22561 TGGTGGCGGA TGTCGACTGG GAGCGGTTCT ACGCCGGCTT CAGTGCCGTC CGGCGCACTT
    22621 CCTTCTTCGA CGACGTGCAC GACGCCCACC GGCCGGCCCT GTCCACGGCT GCGACCAACG
    22681 ACGGACAGGC CCGGGACGAG GACGGCGGTA CGGAACTCGT ACGACGTCTG CGTCCGCTGA
    22741 CCGAGACGGA GCAACAGCGA GAGCTCGTGT CGCTCGTCCA GAGTGAAGTC GCTGCCGTCC
    22801 TAGGCCACTC CTCCACCGAC GCGGTCCAGC CACAGCGCGC GTTCCGAGAG ATCGGGTTCG
    22861 ACTCACTGAC AGCGGTCCAG CTCCGGAACC GGCTTACGGC CACCACGGGC ATGCGCCTTC
    22921 CGACAACGCT GGTCTTCGAC TACCCGACCA CCAACGGACT CGCCGAGTAC CTGCGCTCCG
    22981 AACTGTTCGG TGTGTCCGGC GCACCAGCTG ACCTCTCCGT CGTCCGGAAC GCGGATGAGG
    23041 AGGACGACCC CGTCGTCATC GTGGGGATGG CCTGCCGGTT CCCGGGCGGG ATCGATACGC
    23101 CGGAAGCCTT CTGGAAGCTG CTCGAAGCGG GCGGCGATGT CATCTCCGAA CTTCCGGCCA
    23161 ACCGCGGCTG GGACATGGAG CGACTCCTGA ACCCGGACCC CGAGGCGAAG GGCACCAGCG
    23221 CCACACGCTA CGGCGGTTTC CTCTACGACG CCGGGGAGTT CGACGCCGCC TTCTTCGGTA
    23281 TCTCGCCGCG TGAGGCGTTG GCGATGGACC CGCAGCAACG GCTGCTGCTG GAAACCGTCT
    23341 GGGAGCTCAT CGAGAGCGCC GGCGTGGCGC CCGACTCGCT CCACCGGAGC CGGACCGGCA
    23401 CGTTCATCGG CAGCAACGGC CAGTTCTACG CACCGCTGCT GTGGAACTCC GGCGGTGATC
    23461 TGGAGGGCTA CCAAGGCGTG GGCAACGCCG GCAGCGTCAT GTCCGGCCGC GTCGCCTACT
    23521 CCCTCGGTCT TGAGGGGCCT GCGGTGACGG TGGATACGGC GTGTTCGTCG TCGCTGGTGG
    23581 CACTGCACCT GGCGGTGCAG GCGCTGCGCC GTGGCGAGTG CTCACTCGCC ATAGCCGGCG
    23641 GTGTGACGGT GATGTCCACA CCGGACAGCT TCGTTGAGTT CTCACGGCAA CAGGGCCTTT
    23701 CCGAGGACGG CCGTTGCAAG GCGTTCGCGA GCACAGCCGA TGGTTTCGGC CTCGCCGAGG
    23761 GCGTTTCGGC GCTGTTGGTG GAGCGGTTGT CGGATGCGCG GCGGTTGGGT CATCGGGTGT
    23821 TGGCGGTGGT GCGGGGGAGT GCGGTCAATC AGGATGGTGC GTCGAATGGG TTGACGGCGC
    23881 CGAATGGTCC GTCGCAGCAG CGGGTGATTC GTGCGGCGTT GGCTGACGCG GGTCTGGCTC
    23941 CTGCTGATGT GGATGTGGTG GAGGCGCATG GTACGGGGAC GCGGTTGGGT GATCCGATCG
    24001 AGGCTCAGGC GTTGTTGGCG ACGTATGGGC AGGGTCGTGC GGGTGGGCGT CCGGTGGTGT
    24061 TGGGGTCGGT GAAGTCGAAC ATCGGGCATA CGCAGGCGGC GGCTGGCGTG GCTGGTGTGA
    24121 TGAAGATGGT GCTGGCGCTG GAGCGGGGTG TGGTGCCGAA GACGTTGCAT GTGGATGAGC
    24181 CGTCACCGCA TGTGGACTGG TCGGCTGGTG AGGTGGAGTT GGCGGTTGAG GCGGTGCCGT
    24241 GGTCGCGGGG TGGGCGGGTG CGGCGGGCTG GTGTGTCGTC GTTCGGGATC AGTGGCACGA
    24301 ATGCGCATGT GATTGTGGAG GAGGCGCCTG CGGAGCCGGA GCCGGAGCCG GGAACTCGTG
    24361 TGGTTGCTGC TGGTGATCTG GTGGTGCCGT GGGTGGTGTC CGGGCGGGAT GCGGGGGCGT
    24421 TGCGTGAGCA GGCGGCCCGG TTGGCTGCGC ACGTGTCGAG CACGGGTGCG GGTGTGGTTG
    24481 ATGTGGGGTG GTCGTTGGTG GCCACGAGGT CGGTGTTCGA GCACCGGGCG GTGATGGTCG
    24541 GCAGTGAACT CGATTCCATG GCGGAGTCGT TGGCTGGCTT CGCTGCGGGT GGGGTTGTGC
    24601 CGGGGGTGGT GTCGGGTGTG GCTCCGGCTG AGGGTCGTCG TGTGGTGTTC GTCTTTCCTG
    24661 GTCAGGGTTC GCAGTGGGTG GGGATGGCGG CTGGGTTGCT GGATGCGTGT CCGGTGTTCG
    24721 CGGAGGCGGT GGCGGAGTGT GCCGCGGTGC TGGATCCGGT GACGGGTTGG TCGCTGGTCG
    24781 AGGTGTTGCG CGGTGGTGGT GAGGCTGTTC TTGGGCGGGT TGATGTGGTG CAGCCGGCGT
    24841 TGTGGGCGGT GATGGTGTCA CTGGCCCGGA CCTGGCGGTA TTACGGTGTG GAGCCTGCTG
    24901 CGGTTGTGGG GCATTCGCAG GGTGAGATCG CTGCGGCTTG TGTGGCTGGG GGGTTGAGTC
    24961 TGGCCGATGG TGCGCGGGTG GTGGTGTTGC GGAGCCGGGC GATCGCCCGG ATCGCTGGTG
    25021 GGGGCGGCAT GGTCTCGGTC GGTCTTTCAG CTGAGCGTGT CCGCACCATG CTCGACACCT
    25081 ACGGTGGCCG GGTTTCGGTC GCGGCGGTCA ATGGCCCGTC CTCGACCGTC GTGTCCGGTG
    25141 ACGTCCAGGC CCTGGATGAG TTGTTGGCCG GTTGTGAGCG GGAGGGTGTC CGGGCTCGTC
    25201 GTGTCCCGGT GGACTATGCC TCCCACTCCG CGCAGATGGA CCAGTTACGC GATGAGCTGC
    25261 TGGAAGCGCT GGCGGACATC ACTCCGCAAC ATTCCAGTGT TCCGTTCTTC TCGACGGTGA
    25321 CGGCGGACTG GCTGGACACG ACCGCTCTGG ATGCGGGGTA CTGGTTCACG AATCTGCGGG
    25381 AGACGGTCCG GTTCCAGGAA GCCGTCGAAG GGCTCGTGGC TCAGGGGATG GGCGCGTTCG
    25441 TCGAGTGCAG CCCGCACCCC GTCCTCGTCC CCGGTATCGA GCAGACCCTC GACGCCCTCG
    25501 ACCAGAACGC CGCCGTACTC GGCTCCCTGC GGCGTGACGA AGGCGGCCTG GACCGACTCC
    25561 TCACATCCCT CGCGGAAGCC TTCGTCCAAG GCGTTCCCGT CGACTGGACC CACGCCTTCG
    25621 AAGGCATGAC CCCCCGCACC GTCGACCTGC CCACCTACCC CTTCCAACGA CAGCACTACT
    25681 GGCCCAAGCC CGCACCGGCC CCCGGCGCGA ACCTGGGCGA CGTGGCGTCC GTGGGCCTCA
    25741 CCGCGGCCGG CCACCCCCTT CTGGGCGCGG TCGTGGAGAT GCCCGACTCC GACGGGTTGG
    25801 TGCTCACCGG GCAGATCTCC CTGCGGACCC ATCCCTGGCT CGCCGACCAC GAGGTGCTCG
    25861 GATCGGTGCT CCTGCCGGGC ACCGCGTTCG TCGAGCTTGC CGTCCAGGCC GCCGACCGCG
    25921 CCGGTTACGA CGTACTGGAC GAGCTGACGC TGGAGGCGCC CCTCGTGCTC CCCGACAGGG
    25981 GCGGCATCCA GGTGCGTCTG GCCCTCGGGC CGTCCGAGGC AGACGGACGC CGGTCCCTCC
    26041 AGCTGCACAG CAGGCCGGAG GAGGCTGCCG GGTTCCACCG CTGGACGAGG CACGCGAGTG
    26101 GATTCGTCGT TCCCGGCGGT ACCGGGGCGG CGCGGCCCAC CGAGCCGGCC GGCGTGTGGC
    26161 CGCCCGCAGG TGCCGAGCCG GTCGCTCTCG CATCGGACCG GTACGCCCGG CTCGTCGAGC
    26221 GCGGCTACAC CTACGGCCCC TCCTTCCAGG GGCTGCACAC CGCATGCCGC CACGGGGACG
    26281 ACGTGTACGC GGAAGTGGCG CTGCCAGAAG GAACACCGGC CGACGGCTAC GCCCTGCATC
    26341 CGGCCCTGCT GGACGCGGCG GTCCAGGCCG TCGGACTCGG CTCGTTCGTC GAGGATCCCG
    26401 GCCAGGTGTA CCTGCCGTTC CTCTGGAGCG ACGTGACGCT GCACGCGACC GGGGCCACGT
    26461 CCCTGCGGGT GAGGGTTTCA CCGGCCGGTC CCGACACCGT TGCGCTGGCC CTCGCCGACC
    26521 CGGCCGGGGC GCCGGTGGCC ACGGTGGGCG CCCTCCGTCT GCGTACGACG TCCGCGGCGC
    26581 AGCTCGCCCG TGCGCGCGGG AGCGCGGAAC ACGCGATGTT CCGCGTGGAG TGGGTGGAGG
    26641 AGGGCTCGGC CGCGGACCGG TGCCGGGGCG GCGCGGGCGG GACGACGTAC GAGGGGGAAC
    26701 GCGCCGCCGA GGCCGGGGCC GCCGCTGGTA CCTGGGCCGT ACTCGGCCCC CGGGTGCCGG
    26761 CCGCCGTCCG GACGATGGGC GTGGATGTCG TCACCGCCCT CGACACGCCG GACCACCCCG
    26821 CGGACCCGCA GAGCCTCGCG GACCTGCCGG CGCTCGGGGA CACCGTTCCC GACGTGGTCG
    26881 TCGTGACCAG CCTCCTGAGC CTCGCCTCCG GAGCGGATTC CCCCCTAGGG AACCGGCCCC
    26941 GGCCGACCGC CGCCGAGCAG GACACCGCCG CCACGGTCGC CGGCGTCCAC AGCGCACTCC
    27001 ACGCGGCCCT GGACCTGGTG CAGGCATGGC TGGCCGACGA ACGCCACACC GCCTCCCGGC
    27061 TGGTGCTCGT CACCCGGCAC GCGATGACCG TCGCCGAGTC CGACCCCGAG CCTGACCTGC
    27121 TCCTCGCCCC GGTGTGGGGA CTCGTGCGGT CCGCCCAGGC CGAGAACCCC GGCCGCTTCG
    27181 TGCTCGCCGA CATCGACGGC GACGAGGCAT CCTGGGATGC TCTGCCCCGA GCCGTCGCCT
    27241 CGGCCGCATC GGAGGTGGCG ATACGGGCCG GCGCCGTGTA CGTACCGCGG CTGGCCCGCG
    27301 CCACGGACGA GGGACTGGTC GTGGCCGACG AGGCTGCGGG GCCCTGGCGG CTGGACGTCA
    27361 CGGAAGCGGG CACCCTGGCG AACCTCGCCC TGGTGCCGTG CCCGGACGCC TCCCGCCCGC
    27421 TGGGCCCCGA CGAGGTACGG ATCGCCGTCC GTGCCGCCGG GGTCAACTTC CGGGACGTCC
    27481 TCCTGGCCCT GGGCATGTAC CCGGACGAGG GGCTCATGGG CGCGGAGGCG GCGGGCGTCG
    27541 TCACCGAGGT CGGCGGGGGC GTCACGACGC TCGCGCCAGG TGACCGGGTG ATGGGCCTGG
    27601 TGACCGGTGG ATTCGGGCCG GTGGCCGTGA CGCACCACCG GATGCTCGTA CGGATGCCGC
    27661 GTGGCTGGTC CTTCGCCGAG GCCGCGTCGG TGCCGGTGGC GTTCCTGACC GCGTACTACG
    27721 CCCTGCACGA CCTGGCAGGC CTGCGCGGCG GCGAGTCGGT GCTGGTGCAC TCCGCTGCGG
    27781 GCGGTGTCGG CATGGCGGCC GTGCAGTTGG CACGGCACTG GGATGCCGAG GTGTTCGGCA
    27841 CCGCGAGCAA GGGCAAGTGG GACGTTCTCG CGGCGCAGGG CCTCGACGAG GAGCACATCG
    27901 GCTCGTCCAG GACGACCGAG TTCGAGCAGC GCTTCCGCGC GACCAGTGGT GGGCGCGGGA
    27961 TCGATGTCGT CCTGAATGCC CTCTCGGGTG ACTTCGTCGA CGCCTCGGCG CGTCTCCTGC
    28021 GCGAGGGCGG CCGGTTCGTC GAGATGGGCA AGACCGACAT CCGTACCGAC CTCGGCGTCG
    28081 TCGGGGCGGA CGGCGTCCCG GACATCCGGT ACGTCGCCTT CGACCTCGCC GAGGCGGGTG
    28141 CCGAGCGGAT CGGGCAGATG CTCGACGAGA TCATGGCGCT CTTCGACGCC GGTGTCCTGC
    28201 GGTTGCCGCC GTTGCGCGCC TGGCCGGTGC GGCGCGCCCA CGAGGCACTG AGGTTCGTCA
    28261 GCCAGGCACG TCATGTGGGC AAGGTCGTCC TCACCGTCCC GGCCGCGCTC GACGCCGAGG
    28321 GAACCGTGCT GATCACCGGG GCGGGCACGC TGGGAGCCCT GGTCGCCCGC CACCTCGTCA
    28381 CCGAGCACGA CGTCCGCCGG CTGCTGCTGG TCAGCCGCAG CGGCGTCGCC CCCGACCTGG
    28441 CGGCCGAACT CGGTGCGCTG GGCGCCGAGG TCACGGTGGC GGCCTGCGAC GTCGCCAACC
    28501 GCAAGGCGCT CAAGGGCCTC CTGGAGGACA TACCGCCCGA GCATCCGGTC ACGGGCATCG
    28561 TTCACACGGC CGGCGTGCTC GACGACGGTG TGGTGTCCGG GCTCACCCCT GAACGGGTGG
    28621 ACACCGTCCT CAAACCCAAG GTGGACGCGG CCCTGACCCT GGAGTCAGTG ATCGGCGAAC
    28681 TGGACCTCGA CCCGGCCCTG TTCGTGATCT TCTCATCGGC AGCGAGCATG CTGGGCGGGC
    28741 CCGGCCAGGG CAGTTACGCC GCGGCCAATC AGTTCCTGGA CACCCTCGCC CGACACCGGG
    28801 CGCGCCCCGG GCTCACCTCC GTGTCACTCG GCTGGGGGCT GTGGCACGAG GCCAGCGGTC
    28861 TCACCGGCGG CCTGGCCGAC ATCGACCGTG ACCGGATGAG CCGGGCGGGG ATCGCGCCCA
    28921 TGCCGACCGA CGAGGCCCTG CACCTGTTCG ACAGGGCAAC GGAACTCGGC GATCCGGTAC
    28981 TCCTGCCGAT GCGCCTGAAC GAGGCCGCGC TGGAGGACCG GGCCGCGGAC GGAACACTGC
    29041 CGCCGCTGCT GAGTGGTCTG GTCCGGGTGC GGCACAGGCC GTCGGCGCGG GCAGGTACCG
    29101 CGACCGCCGC CCCCGCCACC GGCCCCGAGG CGTTCGCCCG GGAGCTGGCG GCGGCACCGG
    29161 ACCCACGTCG TGCCCTGCGC GACCTCGTCC GCGGCCACGT CGCCCTGGTG CTCGGACACA
    29221 GTGGCCCCGA GGCCATCGAC GCCGAACAGG CCTTCCGGGA CATCGGTTTC GACTCCCTGA
    29281 CCGCAGTCGA ACTCAGAAAC CGGCTGAACG CCGAGACCGG CCTCCGCTTG CCCGGCACGC
    29341 TCGTGTTCGA CTACCCCAAC CCGAGCGCGC TCGCCGATCA CCTGCTCGAA CTCCTCGCTC
    29401 CCGCGACACA ACCCACCGCA GCCCCGCTGC TCGCCGAACT GGAACGGGTG GAACAACTCC
    29461 TGTCTGCGGC CGCGTCACCC GGCGGACCGG CATCCGCGGT GGACGAGGAG ACGCGCACGC
    29521 TCATCGCCAC ACGGCTGGCC ACCCTTGCCT CGCAGTGGAC ACACCTCCCG GTCGGTTCGC
    29581 CGGGCAACGC GGACAACCGC AGCGGCCCCG GCGAGTCCGG GCAGGCCCAG GAATCCGGAG
    29641 CAACCGGGGA GCACACGGCG GCGTGGACGT CGGACGACGA TCTCTTCGCC TTCCTCGACA
    29701 AGCGGTTGGA GACGTGATGG CCGCCGGCCG AGTCAGCGAG TCCTTTCGTC CTTCTGCTGG
    29761 GGAAAACGAC GCACCGGGAG GTTTTGGTGG CTGAGGCGGA GAAGCTGCGC GAATACCTGT
    29821 GGCGCGCCAC GACCGAACTC AAGGAGGTCA GCGATCGACT CCGCGAGACC GAGGAACGGG
    29881 CCCGAGAGCC GATCGCCATC GTGGGAATGA GCTGCCGGTT CCCCGGCGGC GGCGACGCCA
    29941 CCGTCAACAC GCCCGAACAG TTCTGGGACC TGCTGAACAG CGGCGGTGAC GGCATCGCGG
    30001 GTCTACCCGA GGACCGCGGG TGGGACTTGG GGCGCCTGTA CGATCCCGAT CCGGACCGGG
    30061 CCGGTACGTC GTACGTGCGT GAGGGCGGTT TCCTGTACGA CTCGGGGGAG TTCGACGCCG
    30121 CCTTCTTCGG GATCTCGCCG CGTGAGGCGT TGGCGATGGA CCCGCAGCAG CGGTTGCTGC
    30181 TGGAGACGTC CTGGGAGGCA TTCGAGAGCG CCGGTATCAA GCGCGCCGCT CTGAGAGGCA
    30241 GCGACACCGG CGTGTACATC GGCGCGTGGA GCACCGGCTA TGCCGGCAGC CCCTACCGCC
    30301 TGGTCGAAGG CCTGGAAGGC CAGCTCGCCA TCGGCACCAC ACTAGGGGCC GCTTCGGGGC
    30361 GTGTTGCTTA CACGTTCGGT CTTGAGGGGC CTGCGGTGAC GGTGGATACG GCGTGTTCGT
    30421 CGTCGTTGGT GGCGTTGCAT CTGGCGGTGC AGGGGTTGCG GCGGGGTGAG TGTTCGCTGG
    30481 CGTTGGTGGG TGGGGTGACG GTGATGTCGT CGCCGGTGAC GTTGACGACG TTCAGTCGGC
    30541 AGCGGGGTTT GTCGGTGGAT GGGCGGTGCA AGGCGTTCCC GGCTTCGGCG GATGGTTTTG
    30601 GTGCTGCCGA GGGTGTGGGT GTGTTGTTGG TGGAGCGGTT GTCGGATGCG CGGCGGTTGG
    30661 GTCATCGGGT GTTGGCGGTG GTGCGGGGGA GTGCGGTCAA TCAGGATGGT GCGTCGAATG
    30721 GGTTGACGGC GCCGAATGGT CCGTCGCAGC AGCGGGTGAT CCGTGCGGCG TTGGCTGACG
    30781 CGGGTCTGGC TCCTGCTGAT GTGGATGTGG TGGAGGCGCA TGGTACGGGG ACGCGGTTGG
    30841 GTGATCCGAT CGAGGCTCAG GCGTTGTTGG CGACGTATGG GCAGGGGCGT GCGGGTGGGC
    30901 GTCCGGTGTG GCTGGGGTCG GTGAAGTCGA ACATCGGGCA TACGCAGGCG GCGGCCGGTG
    30961 TGGCTGGTGT GATGAAGATG GTGCTGGCGC TGGGGCGGGG TGTGGTGCCG AAGACGTTGC
    31021 ATGTGGATGA GCCGTCACCG CACGTGGACT GGTCGGCCGG TGCGGTGGAG TTGCTGACTG
    31081 AAGAGCGGCC GTGGGAGCCG GAGGCTGAGC GTCTTCGTCG GGCAGGCATC TCCGCCTTCG
    31141 GTGTCAGTGG CACGAACGCG CATGTGATCG TGGAGGAGGC GCCTGCGGAA CCGGAGCCGG
    31201 AGCCGGGAAC TCGTGTGGTT GCTGCCGGTG ATCTGGTGGT GCCGTGGGTG GTGTCCGGGC
    31261 GGGATGCGAG GGCGTTGCGT GCACAGGCGG CACGCTTGGC TGCGCACGTG TCGGGTGTAA
    31321 GTGCGGTCGA TGTGGGCTGG TCATTGGTGG CCACGAGGTC GGTGTTCGAG CACCGGGCTG
    31381 TTGCGATCGG CAGTGAACTC GACTCCATGG CGGGTTCGTT GGCCGGCTTC GCTGCGGGTG
    31441 GGGTGGTGCC GGGGGTGGTG TCGGGTGTGG CTCCGGCTGA GGGTCGTCGT GTGGTGTTCG
    31501 TCTTTCCTGG TCAGGGTTCG CAGTGGGTGG GGATGGCGGC TGGGTTGCTG GATGCGTGTC
    31561 CGGTGTTCGC GGAGGCGGTG GCGGAGTGCG CTGCGGTGCT GGATCCGGTG ACGGGTTGGT
    31621 CGCTGGTCGA GGTGTTGCAG GGCAGGGACG CGACTGTTCT TGGGCGGGTT GATGTGGTGC
    31681 AGCCGGCGTT GTGGGCGGTG ATGGTGTCAC TGGCTCGGAC CTGGCGGTAT TACGGTGTGG
    31741 AGCCTGCTGC GGTTGTGGGG CATTCGCAGG GTGAGATTGC TGCGGCTTGT GTGGCTGGGG
    31801 GGTTGAGTCT GGCCGATGGT GCGCGGGTGG TGGTGTTGCG GAGCCGGGCG ATCGCCCGGA
    31861 TCGCTGGTGG GGGCGGCATG GTCTCCGTCA GCCTGCCGGC CGGCCGTGTC CGCACCATGC
    31921 TGGAGGAGTT CGACGGCCGG TTGTCGGTGG CTGCGGTCAA TGGCCCGTCC TCGACCGTGG
    31981 TGTCCGGTGA CGTCCAGGCC CTGGATGAGT TGTTGGCCGG TTGTGAGCGG GAGGGTGTCC
    32041 GGGCTCGTCG TGTCCCGGTG GACTATGCTT CCCACTCCGC GCAGATGGAC CAGTTACGCG
    32101 ATGAGCTGCT GGAGGCGCTG GCGGACATCA CTCCGCAGGA CTCCAGTGTT CCGTTTTTCT
    32161 CGACGGTGAC GGCGGACTGG CTGGGCACGA CTGCCCTGGG TGCGGGGTAC TGGTTCACGA
    32221 ATCTGCGGGA GACGGTCCGG TTCCAGGAAG CCGTCGAAGG GCTTGTGGCT CAGGGGATGG
    32281 GCGCGTTCGT CGAGTGCAGC CCGCACCCCG TCCTCGTCCC CGGTATCGAG CAGACCCTCG
    32341 ACGCCCTCGA CCAGAATGCC GCCGTATTCG GCTCGCTGCG GCGTGACGAA GGCGGCCTGG
    32401 ACCGGTTTCT CACGTCCCTC GCGGAAGCCT TCGTCCAGGG CGTTCCCGTC GACTGGTCCC
    32461 GCGCCTTCGA AGGCGTGACC CCTCGCACCG TCGACCTGCC CACCTACCCC TTCCAACGAC
    32521 AGCACTACTG GTTGATGGCG GAAGAGGCAC CGGTCTCTCA GCCCCCTCAC TCGGAGAACA
    32581 GCTTCTGGTC GGTAGTGGCC GATGCGGATG CCGAGGCTGC TGCTGAACTT CTGGGTGTCG
    32641 ATGTAGAGGC AGTCGAGGCT GTAATGCCGG CGTTGTCTTC GTGGCACCGG CAGAGCCAAC
    32701 TTCGTGCCGA AGTCAACCAG TGGCGCTACG ACGTTGCGTG GAAGCGTCTG ACCACCGGGG
    32761 CGCTGCCCGA AAAGCCGGGC AACTGGCTCG TCGTGACTCC AGCAGGAACC GACACCACGT
    32821 TCGCTGAGTC GTTGGCGAGG ACGGCAGCCG CAGAACTGGG CGTATCCGTC AGCTTTGCGC
    32881 AGGTGGACAC TGCTCATCCT GACCGGTCGC AATACGCGCA TGCGCTGCGT CAAGCCCTGA
    32941 CCGGCCCGGA GAACGTCGAT CACCTCGTGT CCTTGCTGGC CCTGGACCAG GCCACTGACG
    33001 ACCTCGCCGC CGCACCTTCC TGTCTTGCCG CGTCGCTGGT GTTGGCGCAG GCGTTGGTTG
    33061 ATCTTGGCCG GGTTGGTGAG GGGCCGCGGT TGTGGCTGGT GACGCGGGGT GCGGTGGTTG
    33121 CTGGTCCTTC GGATGCCGGT GCGGTGATTG ATCCGGTACA GGCGCAGGTG TGGGGTTTCG
    33181 GGCGTGTTCT GGGTCTGGAG CATCCCGAGT TGTGGGGTGG GCTGATCGAC CTGCCGGTGG
    33241 GGGTTGATGA GGAGGTGTGC CGGCGGTTCG TGGGTGTTGT GGCGTCGGCT GGTTTTGAGG
    33301 ATCAGGTGGC GGTGCGTGGT TCGGGTGTGT GGGTGCGTCG TCTGGTGCGT GCTGTGGTGG
    33361 ATGGTGGTGG GGGTGGTTGG CGGCCGCGTG GGACGGTGTT GGTCACGGGT GGTCTTGGTG
    33421 GTTTGGGTGC GCATACGGCC CGGTGGTTGG TGGGTGGTGG GGCGGATCAT GTGGTTCTTG
    33481 TGAGCCGTCG TGGTGGCAGT GCGCCTGGTG CTGGGGATCT GGTGCGGGAG CTGGAGGGGT
    33541 TGGGCGGGGC TCGGGTGTCG GTGCGGGCCT GTGATGTGGC TGATCGTGTG GCGTTGCGGG
    33601 CGTTGTTGTC GGATCTGGGT GACCCGGTGA CGGCGGTGTT CCATGCGGCT GGTGTTCCTC
    33661 AGTCGACGCC TTTGGCGGAG ATCTCTGTCC AGGAGGCGGC TGATGTGATG GCGGCCAAGG
    33721 TGGCGGGTGC GGTGAATCTG GGTGAGTTGG TGGATCCCTG TGGTCTGGAG GCGTTTGTGT
    33781 TGTTCTCCTC CAATGCCGGT GTGTGGGGCA GTGGGGGGCA GGCGGTGTAT GCGGCGGCGA
    33841 ATGCGTTTCT TGATGCGTTG GCGGTGCGTC GTCGGGGTGT TGGTCTGCCG GCCACGAGTG
    33901 TGGCGTGGGG GATGTGGGCT GGTGAGGGGA TGGCGTCGGT GGGTGGTGCG GCGCGGGAGT
    33961 TGTCCCGTCG GGGGGTGCGG GCGATGGATC CCGAGCGTGC TGTGGCGGTG ATGGCTGATG
    34021 CGGTGGGTCG TGGTGAGCCG TTCGTCGCGG TCGCTGATGT GGACTGGGAA CGTTTCGTCA
    34081 CCGGTTTCGC TTCTGCCCGT CCCCGTCCGT TGATCAGTGA CCTGCCGGAG GTGCGTGCTG
    34141 TTGTGGAGGG CCAGGTCCAG GGCCGGGGCC AGGGGTTGGG CTTGGTCGGT GAGGAGGAGT
    34201 CGTCGGGGTG GTTGAAGCGG TTGTCGGGGT TGTCTCGTGT GCGGCAGGAG GAGGAGTTGG
    34261 TGGAGTTGGT CCGTGCTCAG GCTGCCGTTG TTCTCGGGCA TGGTTCCGCG CAGGACGTCC
    34321 CGGCTGAGCG GGCGTTCAAG GAGTTGGGTT TTGATTCCCT CACTGCTGTC GAGCTACGCA
    34381 ACGGGCTGGC CGCGGCCACC GGGATCCGGC TGCCGGCCAC CATGGCATTC GATCATCCCA
    34441 ACGCCACCGC CATCGCACGC TTCCTGCAGT CTCAGCTCCT TCCTGACGCC GAGAGCGAGT
    34501 CGGCCGTGCC GTCTTCACCG GAAGACGAGG TCCGCCAGGC ATTGGCGTCC CTTTCCCTGG
    34561 ACCAGCTGAA AGGCGCTGGG CTTCTTGACC CACTGCTCGC TCTGACACGC CTCCGGGAGA
    34621 TCAACAGCAC GGTGCAGAAC CCTGAGCCGA CCACCGAATC GATCGACGAG ATGGATGGCG
    34681 AGACGTGCTG CGCCTGGCGC TCGGCGAAAT CGACGGCTGA GCCACTGACC ACTGGAGCTG
    34741 ACATGCCTGA CCCCACCGCC AAATATGTGG AAGCGCTCCG TGCGTCGCTC AAGGAGAACG
    34801 AACGCCTGCG CCAACAGAAT CACTCGCTTC TCGCCGCCTC CCGTGAAGCG ATCGCCATCA
    34861 CGGCGATGAG CTGCCGTTTC GGCGGGGGCA TCGACTCGCC CGAAGATCTC TGGCGCTTCC
    34921 TGGCCGAAGG CCGCGACGCG GTGGCGGGGC TTCCCGAGGA CCGCGGGTGG GATCTGGATG
    34981 CCTTGTATCA CCCGGACCCG GAGAACCCCG GCACCACGTA CGTCCGGGAA GGCGCGTTCC
    35041 GGTACGACGC AGCCCAGTTC GATGCGGGGT TCTTCGGGAT TTCGCCGCGT GAGGCGTTGG
    35101 CGATGGACCC GCAGCAGCGG TTGCTGCTGG AGACATCCTG GGAGCTTTTC GAGCGTGCCG
    35161 ATATCGATCC GTACACAGTC AGGGGAACGG CGACGGGGAT ATTCATCGGA GCCGGACATC
    35221 AGGGCTATAG TCCCGACCCC AAGAGGGCTC CGGAGAGCGT GGCGGGTTAC CTGCTGACGG
    35281 GAACGGCATC GGCCGTGCTG TCCGGGCGTA TTTCCTACAC GTTCGGTCTT GAGGGGCCTG
    35341 CGGTCACGGT GGACACGGCG TGTTCGTCAT CGCTGGTGGC ACTGCACCTG GCGGTGCAGG
    35401 CGCTGCGCCG GGGCGAGTGC TCACTCGCCA TAGCCGGCGG TGTGGCCGTC ATGTCGACCC
    35461 CGGATGCCTT CGTGGAGTTC AGCCGCCAAC AGGGCATGGC AAGAGACGGC CGATGTAAGG
    35521 CATTCGCCGC GGCAGCGGAC GGTATGGGAT GGGGCGAGGG AGTTTCGCTG CTCTTGCTGG
    35581 AGCGGTTGTC GGATGCGCGG CGGTTGGGTC ATCGGGTGTT GGCGGTGGTG CGGGGGAGTG
    35641 CGGTCAATCA GGATGGTGCG TCGAATGGCC TGGCGGCGCC GAATGGTCCG TCGCAGCAGC
    35701 GGGTGATTCG TGCGGCGTTG GCTGACGCGG GTCTGGCTCC TGCCGATGTG GATGTGGTGG
    35761 AGGCGCATGG TACGGGGACG CGGTTGGGTG ATCCGATCGA GGCTCAGGCG TTGCTGGCGA
    35821 CGTATGGGCA GGGGCGTGCG GGTGGGCGTC CGGTGTGGCT GGGGTCGGTG AAGTCGAACA
    35881 TCGGGCATAC GCAGGCGGCG GCTGGTGTGG CTGGTGTGAT GAAGATGGTG CTGGCGTTGG
    35941 GGCGGGGTGT GGTGCCGAAG ACGTTGCATG TGGATGAGCC GTCACCGCAC GTGGACTGGT
    36001 CGGCCGGTGC GGTGGAGTTG CTGACTGAAG AGCGGCCGTG GGAGCCGGAG GCTGAGCGTC
    36061 TTCGTCGGGC AGGCATCTCC GCCTTCGGTG TCAGTGGCAC GAACGCGCAT GTGATCGTGG
    36121 AGGAGGCGCC TGCGGAACCG GAGCCGGAGC CGGGAACTCG TGTGGTTGCT GCCGGTGATC
    36181 TGGTGGTGCC GTGGGTGGTG TCCGGGCGGG ATGTGGGGGC GTTGCGTGAG CAGGCGGCAC
    36241 GCTTGGCTGC GCACGTGTCG AGCACGGGTG CGGGTGTGGT TGATGTGGGC TGGTCGTTGG
    36301 TGGCCACGAG GTCGGTGTTC GAGCACCGGG CGGTGATGGT CGGCACTGAT CTTGATTCCA
    36361 TGGCGGGGTC GTTGGCCGGG TTTGCTGCGG GTGGTGTCGT CCCCGGGGTG GTGTCGGGTG
    36421 TGGCGCCGGC TGAGGGTCGT CGTGTGGTGT TCGTCTTTCC TGGTCAGGGT TCGCAGTGGG
    36481 TGGGGATGGC GGCTGGGTTG CTGGATGCGT GCCCGGTGTT CGCGGAGGCG GTGGCGGAGT
    36541 GTGCCGCGGT GCTGGATCCG GTGACGGGTT GGTCGCTGGT CGAGGTGTTG CAGGGCAGGG
    36601 ACGCGACTGT TCTTGGGCGG GTTGATGTGG TGCAGCCGGC GTTGTGGGCG GTGATGGTGT
    36661 CACTGGCTCG GACCTGGCGG TATTACGGTG TGGAGCCTGC TGCGGTTGTG GGGCATTCGC
    36721 AGGGTGAGAT TGCTGCGGCT TGTGTGGCTG GGGGGTTGAG TCTGGCCGAT GGTGCGCGGG
    36781 TGGTGGTGTT GCGGAGCCGG GCGATCGCCC GGATCGCTGG TGGGGGCGGC ATGGTCTCCG
    36841 TCAGTCTCCC GGCCGGCCGT GTCCGCACCA TGCTCGACAC CTACGGCGGC CGGGTTTCGG
    36901 TCGCGGCGGT CAACGGTCCG TCCTCGACCG TGGTGTCCGG TGACGTCCAG GCCCTTGATG
    36961 AGTTGTTGGC CGGTTGTGAG CGGGAGGGTG TCCGGGCTCG TCGTGTCCCG GTGGACTATG
    37021 CCTCCCACTC CGCGCAGATG GACCAGTTAC GCGATGAGCT GCTGGAGGCG CTGGCGGACA
    37081 TCACTCCGCA GGACTCCAGT GTTCCGTTCT TCTCGACGGT GACGGCGGAC TGGCTGGACA
    37141 CGACCGCTCT GGATGCGGGG TACTGGTTCA CGAATCTGCG GGAGACGGTC CGGTTCCAGG
    37201 AAGCCGTCGA AGGGCTTGTG GCTCAGGGGA TGGGCGCGTT CGTCGAGTGC AGCCCGCACC
    37261 CCGTCCTCGT CCCCGGTATC GAGCAGACCC TCGACGCCCT CGACCAGAAT GCCGCCGTAC
    37321 TCGGCTCGCT GCGGCGTGAC GAAGGCGGCC TGGACCGACT TCTCACATCC CTCGCGGAAG
    37381 CCTTCGTCCA AGGCGTTCCC GTCGATTGGA CCCACGCCTT CGAGGGCGTG ACCCCTCGCA
    37441 CCGTCGACCT GCCCACCTAC CCCTTCCAAC GGCAACGTTT CTGGTTGGAC GGTTCGCCGG
    37501 CATCGTCTGC GAATGGCGTT GACGGTGAGG CGGACGCCAT GATCTGGGAC GCGGTCGAGC
    37561 GTGAGGACTC GGTCGCTGTA GCCGAGGAGT TGGGGATCGA CGCCGAGGCT TTGCACACGG
    37621 TGTTGCCGGC CTTGTCGTCG TGGCGGCGGC GTCGGGTGGA GCATCGACGG CTTCAGGACT
    37681 GGCGTTACCG GGTGGAGTGG AAGCCTTTCC CGGCCGCGCT TGATGAGGTG CTCGGTGGTG
    37741 GCTGGTTGTT CGTGGTGCCG CGGGGCTTGG CGGATGATGG TGTGGTTGCG CGGGTGGTGG
    37801 CTGCCGTCAC GGCGCGGGGT GGCGAGGTCA GTGTCGTGGA GCTCGATCCG ACCCGTCCTG
    37861 ACCGCCGGGC TTATGCGGAG GCTGTCGCGG GCCGTGGTGT GAGCGGGGTC GTGTCGTTCT
    37921 TGTCCTGGGA TGATCGGCGG CACTCGGAGC ATCCTGTTGT TCCCGCCGGT CTTGCCGCGT
    37981 CGCTGGTGTT GGCGCAGGCG TTGGTTGATC TTGGCCGGGT TGGTGAGGGG CCGCGGTTGT
    38041 GGCTGGTGAC GCGGGATGCG GTGGTCGCTG GTCCTTCGGA TGCCGGTGCG GTGATTGATC
    38101 CGGTACAGGC GCAGGTGTGG GGTTTCGGGC GTGTTCTGGG TCTGGAGCAT CCCGAGTTGT
    38161 GGGGTGGGCT GATCGACCTG CCCGTGGAGG CGCCCGAACC TGGCTCGACG TGCGACCACA
    38221 CGTATGCCGA CCTGCTCGCC ACGGTTGTGG CGTCGGCTGG TTTTGAGGAT CAGGTGGCGG
    38281 TGCGTGGTTC GGGTGTGTGG GTGCGTCGTC TGGTGCGTGC TGTGGTGGAT GGTGGTGGGG
    38341 GTGGTTGGCG GCCGCGTGGG ACGGTGTTGG TCACGGGTGG TCTTGGTGGT TTGGGTGCGC
    38401 ATACGGCCCG GTGGTTGGTG GGTGGTGGGG CGGATCATGT GGTGCTTGTG AGCCGTCGTG
    38461 GTGGCAGTGC GCCTGGTGCT GGGGATCTGG TGCGGGAGCT GGAGGGGTTG GGCGGGGCTC
    38521 GGGTGTCGGT GCGGGCCTGT GATGTGGCTG ATCGTGTGGC GTTGCGGGCG TTGTTGTCGG
    38581 ATCTGGGTGA GCCGGTGACG GCGGTGTTCC ATGCGGCTGG TGTTCCTCAG TCGACGCCTT
    38641 TGGCGGAGAT CTCTGTCCAG GAGGCGGCTG ATGTGATGGC GGCCAAGGTG GCGGGTGCGG
    38701 TGAATCTGGG TGAGTTGGTG GATCCCTGTG GTCTGGAGGC GTTTGTGTTG TTCTCCTCCA
    38761 ATGCCGGTGT GTGGGGCAGT GGGGGGCAGG CGGTGTATGC GGCGGCGAAT GCGTTTCTTG
    38821 ATGCGTTGGC GGTGCGTCGT CGGGGTGTTG GTCTGCCGGC GACGAGTGTG GCGTGGGGGA
    38881 TGTGGGCTGG TGAGGGGATG GCGTCGGTGG GTGGTGCGGC GCGGGAGTTG TCCCGTCGGG
    38941 GGGTGCGGGC GATGGATCCC GAGCGTGCTG TGGCGGTGAT GGCTGATGCG GTGGGGCGTG
    39001 GTGAGGCGTT CGTCGCGGTC GCCGATGTGG ACTGGGAACG TTTCGTCACC GGTTTCGCCT
    39061 CTGCCCGTCC CCGTCCGTTG ATCAGCGACC TCCCGGAGGT CCGTACCGCC CTGCGGAACC
    39121 AGGAGCAGGA GCAACTCCAC GCCCCCGTCC CCGAGGACCG ATCGGCACAG CTTCTGCGGC
    39181 GGCTGTCCAT GCTGTCTCCC GCCGGACGGG AAGCCGAACT GGTGAAGCTC GTCCGTACCG
    39241 AGGCAGCCGC TGTTCTGGGG CACGGCTCCG CGCAGGACGT CCCGGCCGAG CGGGCGTTCA
    39301 AGGAGCTGGG CTTCGACTCC CTCACCGCTG TTCAGCTACG CAACAGACTG GCCGCCGCCA
    39361 CCGGCACCAG GCTCCCCGCC AGCGCCGTCT TCGACCACCC CCACGCTGCG GCTCTCGCCA
    39421 GGTGGCTGCT CGCGGGGATG CGGCATGCCG ACGGTGGACA CGGTGGTGGG CACGCCGGTG
    39481 GACCCGGGCC GGACGCCGAC GAAGGTCGGT CGGCCGGCGC TGGTCACAGC GGAATGCTGG
    39541 CCGATCTGTA CCGGCGTTCC GCCGAGTTGG GCCGGAGCCG GGAGTTCATC GGGCTGCTGG
    39601 CCGACACCGC GGCCTTCCGC CCGGTGTTCC ACGGGCCGGC GGACCTCGAC GCGCCGTTGG
    39661 AGGCCGTTCC GCTGGCGGAC GGGGTGCGCA AACCGCAGTT GATCTGTTGC AGCGGGACCG
    39721 CGCCGGTCGG CGGGCCGCAC GAGTTCGCGC GCCTGGCTTC GTTCTTCCGC GGCACTCGTG
    39781 CGGTCTCGGC GCTTCCGCTG CCCGGCTACC TGCCCGGTGA GCAGTTGCCC GCGGACCTCG
    39841 ACGCCGTGCT CGCCGCGCAG GCCGAGGCGG TCGAGAAGCA GACCGGGGGT GCGCCGTTCG
    39901 TCCTGGTCGG CTACTCGGCG GGCGGACTGA TGGCCCACGC ACTGGCCTGC CACCTGGCCG
    39961 GGCGCGGCAC ACCGCCGAGC GGTGAGGTGC TGGTGGACGT CTATCCGCCG GGCCGGCAGG
    40021 AACCGGTGTT CGGCTGGCAG AAGGAGCTCA CCGAGGGCAT GTTCGCCCAG GACTTCGTGC
    40081 CCATGGACGA TACGCGGCTG ACGGCCCTCG GCACGTACGA CCGTCTCATG GGCGAGTGGC
    40141 GGCCGGCGCC CTCCGGACTG CCCACCCTCC TGATCCGGGC CACCGAACCC ATGGCGGAGT
    40201 GGACCGGGGC CATCGACTCG CGGGCCTCCT GGGAGTACGA CCACACCGCC GTCGACATGC
    40261 CGGGGAACCA CTTCACGATC ATGCGCGAGC ACGCGGAGGA CGCGGCCCGG CACATCGACG
    40321 TCTGGCTGAA GGGGCTCACC CCCTGACACC TGCCCGCACC CTGTGACTCC TGCCCGTACC
    40381 GGCGTCCCGG TCCTCCCGAC CCGCGTGCGC AACGGACGAG TCGCTCAGGA GGTCCCCATC
    40441 GGCATGCCCC GCTTTCCTCC CCCTCTCCGA ACGCATCGAC GACCCGATCC CCCTCAGGGA
    40501 CCGGTGAAGG AGCGTGTTGC ACTCATGCAG GACATGCAAG GCGTACAGCC CGAACCAGCC
    40561 AGTGTCGAAC ACGCGGCGGA CGCAGCTCGA ACAGAGCGAA CGGCGCACGG AAGCCGCCCA
    40621 GGAGATGGAG GACAGCGAAC TGGGGCGCCG CCTGCAGATG CTCCGCGGCA TGCAGTGGGT
    40681 CTTCGGCGCC AACGGCGATC CGTACGCCCG GCTGCTGTGT GGCATGGAGG ATGACCCGTC
    40741 ACCTTTCTAC GACGCGATAC GGACCCTGGG CGAGCTGCAC CGGAGCAGGA CCGGAGCCTG
    40801 GGTCACCGCC GACCCCGGGC TCGGGGGCCG CATCCTCGCC GACCGGAAGG CTCGGTGCCC
    40861 GGAAGGCTCG TGGCCGGTGC GGGCGAAGAC CGACGGGCTG GAGCAGTACG TGCTGCCCGG
    40921 GCACCAGGCG TTCCTGCGGC TGGAGCGCGA GGAGGCCGAG CGACTGCGGG AGGTCGCGGC
    40981 GCCGGTGCTG GGGGCCGCGG CGGTCGACGC GTGGCGCCCG CTGATCGACG AGGTCTGCGG
    41041 GGGGCTCGCG AAGGGGCTGC CGGACACGTT CGACCTGGTC GAGGAGTACG CGGGGCTGGT
    41101 GGCGGTCGAG GTGCTGGCGC GGATCTGGGG CGTCCCGGAG GAGGACCGCG CCCGGTTCGG
    41161 GCGTGACTGC CGGGCGCTCG CTCCCCCGCT GGACAGCCTC CTGTGTCCCC AGCAGTTGGC
    41221 GCTGAGCAAG GACATGGCGT CCGCCCTGGA GGACCTGCGT CTCCTCTTCG ACGGCCTCGA
    41281 CGCGACGCCG CGCCTCGCCG GCCCCGCCGA CGGTGACGGA ACGGCCGTGG CCATGCTCAC
    41341 CGTTCTGCTC TGCACGGAGC CGGTGACCAC GGCGATCGGG AACACCGTGC TCGGGCTCCT
    41401 TCCCGGGCAG TGGCCCGTGC CCTGCACCGG CCGGGTGGCT GCCGGGCAGG TTGCCGGGCA
    41461 GGCGCTGCAC CGGGCGGTGT CGTACCGTAT CGCGACGCGG TTCGCCCGGG AGGACCTGGA
    41521 GTTGGCGGGC TGCGAGGTCA AGTCCGGTGA CGAGGTGGTG GTCCTGGCCG GAGCGATCGG
    41581 CCGGAACGGA CCGTCCGCAG CCGCCCCGCC TGCCCCACCG GGCCCAGCGG CCCCGCCCGC
    41641 CCCGTCGGTC TTCGGTGCCG CCGCCTTCGA GAACGCGCTG GCCGAACCCC TCGTCCGGGC
    41701 TGTGACGGGA GCGGCCCTCC AGGCCCTCGC GGAGGGGCCC CCCCGGCTGA CGGCGGCGGG
    41761 ACCCGTCGTA CGACGGCGGC GTTCCCCTGT CGTCGGCGGG CTGCACCGGG CTCCGGTGGC
    41821 CGCCGCATGA GCATCGCGTC GAACGGCGCG CGCTCGGCCC CCCGCCGGCC CCTGCGCGTG
    41881 ATGATGACCA CCTTCGCGGC CAACACGCAC TTCCAGCCGC TGGTTCCCCT GGCCTGGGCA
    41941 CTGCGGACAG CCGGGCACGA GGTGCGCGTG GTGAGCCAGC CCTCGCTGAG CGACGTGGTG
    42001 ACGCAGGCGG GGCTCACCTC GGTCCCGGTG GGCACCGAGG CTCCGGTCGA GCAGTTCGCG
    42061 GCGACCTGGG GCGACGATGC CTACATCGGC GTCAACAGCA TCGACTTCAC CGGCAACGAC
    42121 CCCGGCCTGT GGACGTGGCC GTACCTCCTG GGCATGGAGA CCATGCTGGT GCCGGCCTTC
    42181 TACGAGTTGC TGAACAACGA GTCCTTCGTG GACGGCGTAG TCGAGTTCGC CCGTGACTGG
    42241 CGGCCCGACC TGGTGATCTG GGAGCCGCTG ACGTTCGCCG GCGCGGTGGC GGCGCGCGTC
    42301 ACCGGCGCGG CCCACGCCCG GCTGCCGTGG GGGCAGGAGA TCACCCTGCG CGGGCGGCAG
    42361 GCGTTCCTCG CCGAGCGTGC CCTGCAACCG TTCGAGCACC GGGAGGATCC CACGGCCGAG
    42421 TGGCTGGGCC GCATGCTCGA CCGGTACGGC TGCTCGTTCG ACGAGGAGAT GGTCACCGGG
    42481 CAGTGGACCA TCGACACGCT GCCGCGCAGC ATGCGGCTGG AGCTGTCCGA GGAGCTGCGC
    42541 ACCCTGGACA TGCGGTACGT GCCGTACAAC GGACCGGCGG TCGTACCCCC CTGGGTGTGG
    42601 GAACCGTGCG AGCGGCCCCG CGTCTGTCTG ACGATCGGCA CCTCCCAGCG TGACTCCGGC
    42661 CGGGACCATG TCCCCCTCGA CCACCTGCTC GACTCCCTCG CCGACGTGGA CGCGGAGATC
    42721 GTGGCCACGC TCGACACCAC CCAGCAGGAG CGCCTGCGGG GCGCGGCCCC CGGCAACGTC
    42781 CGGCTGGTGG ACTTCGTCCC GCTGCACGCG CTGATGCCGA CCTGCTCGGC GATCGTGCAC
    42841 CACGGTGGTC CGGGCACGTG GTCGACGGCG GCGCTCCACG GCGTCCCGCA GATCATCCTG
    42901 GACACCTCGT GGGACACACC GGTGCGGGCG CAGCGCATGC AGCAACTCGG GGCGGGCCTG
    42961 TCGATGCCGG TGGGGGAACT GGGCGTCGAG GCGCTGCGGG ACCGGGTCCT GCGGCTGCTG
    43021 GGGGAGCCGG AGTTCCGCGC GGGCGCCGAG CGGATCCGGG CCGAGATGCT CGCGATGCCC
    43081 GCCCCCGGTG ACGTCGTACC GGACCTGGAA CGACTCACCG CGGAGCATGC CACCGGCGCG
    43141 ATGGCGGGAA GGCGGTGAGA CGATGCGCGT ACTGCTGACC TGCTTCGCCA ACGACACCCA
    43201 CTTCCACGGG CTGGTGCCGC TGGCGTGGGC GCTGCGGGCC GCCGGGCACG AAGTCCGCGT
    43261 GGCCAGTCAG CCCGCCCTGT CCGACACGAT CACCCAAGCG GGACTGACCG CGGTGCCCGT
    43321 GGGCCGGGAC ACCGCCTTCC TGGAGCTGAT GGGGGAGATC GGCGCGGACG TCCAGAAGTA
    43381 CTCCACCGGC ATCGACCTGG GCGTCCGCGC GGAGCTGACG AGCTGGGAGT ACCTGCTCGG
    43441 CATGCACACG ACCCTGGTGC CCACGTTCTA CTCGCTGGTC AACGACGAGC CGTTCGTCGA
    43501 CGGGCTCGTC GCGCTGACCC GGGCCTGGCG GCCCGACCTC ATCCTGTGGG AGCACTTCAG
    43561 CTTCGCCGGG GCGTTGGCGG CGCGGGCCAC CGGCACGCCC CACGCCCGCG TGCTGTGGGG
    43621 GTCGGACCTC ATCGTCCGGT TCCGCCGGGA CTTCCTCGCG GAGCGGGCGA ACCGGCCCGC
    43681 CGAGCACCGC GAGGACCCCA TGGCGGAGTG GCTGGGCTGG GCGGCCGAAC GGCTGGGCTC
    43741 CACCTTCGAC GAGGAGCTGG TGACCGGGCA GTGGACGATC GACCCGCTGC CGCGGAGCAT
    43801 GCGGCTGCCC ACCGGGACGA CGACGGTGCC GATGCGGTAC GTGCCGTACA ACGGGCGGGC
    43861 CGTGGTCCCC GCATGGGTCC GGCAGCGTGC GCGGCGGCCC CGGATCTGCC TGACGCTCGG
    43921 TGTGTCGGCC CGGCAGACCC TGGGCGACGG CGTGTCGCTG GCGGAGGTGC TGGCCGCGCT
    43981 GGGCGACGTG GACGCGGAGA TCGTGGCCAC GCTGGACGCC TCCCAGCGCA AGCTCCTGGG
    44041 GCCGGTGCCG GACAACGTCC GGCTGGTGGA CTTCGTGCCC CTGCACGCCC TGATGCCGAC
    44101 CTGTTCGGCG ATCGTGCACC ACGGCGGCGC CGGTACCTGG CTGACGGCCG CCGTCCACGG
    44161 CGTCCCGCAG ATCGTCCTCG GTGACCTCTG GGACAACCTG CTGCGCGCCC GGCAGACACA
    44221 GGCCGCGGGC GCGGGCCTGT TCATCCATCC GTCCGAGGTC ACCGCGGCCG GGCTCGGTGA
    44281 GGGCGTGCGC CGGGTGCTGA CGGACCCTTC CATCCGGGCC GCCGCACAGC GCGTCCGGGA
    44341 CGAGATGAAT GCAGAGCCGA CGCCGGGCGA GGTCGTCACG GTGCTGGAGC GGCTCGCCGC
    44401 GAGCGGCGGA CGCGGACGAG GAGGCGGGAA CCATGCGGGC TGACACGGAG CCGACCACCG
    44461 GGTACGAGGA CGAGTTCGCC GAGATCTACG ACGCCGTGTA CCGGGGCCGG GGCAAGGACT
    44521 ACGCCGGCGA GGCGAAGGAC GTGGCGGACC TCGTGCGCGA CCGGGTGCCG GACGCGTCCT
    44581 CCCTCCTGGA CGTGGCCTGC GGCACGGGCG CGCACCTGCG GCACTTCGCC ACGCTCTTCG
    44641 ACGACGCCCG CGGTCTCGAA CTGTCCGCGP GCATGCTGGA CATCGCCCGC TCCCGCATGC
    44701 CGGGCGTGCC GCTGCACCAA GGGGACATGC GATCCTTCGA CCTGGGGCCA CGCGTCTCCG
    44761 CGGTCACCTG CATGTTCAGC TCCGTCGGCC ACCTGGCCAC CACCGCCGAA CTCGACGCGA
    44821 CGCTGCGGTG CTTCGCCCGG CACACCCGGC CCGGCGGCGT GGCCGTCATC GAACCGTGGT
    44881 GGTTCCCGGA GACCTTCACC GACGGCTACG TGGCGGGTGA CATCGTACGC GTCGACGGCC
    44941 GGACCATCTC CCGGGTGTCC CACTCGGTAC GGGACGGCGG CGCCACCCGC ATGGAGATCC
    45001 ACTACGTGAT CGCCGACGCC GAGCACGGTC CCCGGCACCT GGTCGAGCAC CACCGCATCA
    45061 CGCTGTTCCC GCGGCATGCG TACACGGCCG CGTACGAGAA GGCGGGCTAC ACCGTCGAGT
    45121 ACCTCGACCG CGGGCCCTCG GGCCGGGGGC TGTTCGTCGG CACCCGGACG TGAACCCGCC
    45181 CGCGCACCGC CCGATCACCC TGCTCAACGC CGTTCACACG GATCACCGGA CCACGCGAAG
    45241 GACCTTTCAC ATGTCGTACG ACGACCACGC GGTGCTGGAA GGGATACTGC GGTGCGCCGG
    45301 AGGTGACGAG CGCTTCCTGC TGAACACCGT CGAGGAATGG GGAGCCGCCG AGATCACCGC
    45361 GGCGCTCGTG GACGAGTTGC TGTTCCGCTG CGAGATCCCG CAGGTGGGCG GTGAGCCGTT
    45421 CATCGGCCTG GACGTCCTGC ACGGCGCCGA CCGGATCAGC CATGTGCTGC AGGTGACGGA
    45481 CGGCAAGCCG GTCACGTCGG CGGAACCGGC CGGCCAGGAA CTGGGCGGCC GTACCTGGAG
    45541 TTCACGCTCA GCGACCCTCC TGCGGGAGCT GTTCGGCCCG CCGTCCGGCC GCACCGCGGG
    45601 GGGCTTCGGC GTCTCCTTCC TGCCCGACCT GCGCGGCCCG CGGACCATGG AGGGCGCGGC
    45661 CCTGGCCGCC CGCGCCACCA ACGTGGTGCT GCACGCGACG ACCAACGAGA CGCCCCCACT
    45721 GGACCGGCTG GCCCTGCGCT ACGAGTCCGA CAAGTGGGGC GGCGTCCACT GGTTCACCGG
    45781 CCACTACGAC CGGCACCTGC GGGCCGTGCG CGACCAGGCG GTGCGGATCC TGGAGATCGG
    45841 CATCGGCGGC TACGACGACC TGCTGCCGAG CGGCGCCTCA CTGAAGATGT GGAAGCGCTA
    45901 CTTCCCGCGC GGCCTGGTCT TCGGCGTGGA CATCTTCGAC AGTCGGCGTG CGACCAGCCG
    45961 CGTGTCAAGA CGCTCCGCGG CCCGGCAGGA CGACCCGGAG TTCATGCGCC GCGTCGCCGA
    46021 GGAGCACGGG CCGTTCGACG TCATCATCGA CGACGGCAGC CACATCAACG CACACATGCG
    46081 GACGTCGTTC TCGGTGATGT TCCCCCACCT GCGCAACGGC GGCTTCTACG TCATCGAGGA
    46141 CACCTTCACC TCCTACTGGC CCGGGTACGG AGGGCCATCC GGAGCCCGGT GCCCGTCCGG
    46201 AACAACCGCG CTGGAGATGG TCAAGGGACT GATCGACTCG GTGCACTACG AGGAGCGGCC
    46261 GGACGGCGCG GCCACGGCCG ACTACATCGC CAGGAACCTC GTCGGGCTGC ACGCCTACCA
    46321 AACGACCTCG TCTTCCTCGA GAAGGGCGAT CAACAAGGAG GGCGGCATCC CCCACACCGT
    46381 GCCCCGGGAG CCGTTCTGGA ACGACAACTA GCCACGGCCG CAACCAGAGC GGGAAACCGC
    46441 ACCACTGTCC GCGCCACCTC GGAACCACCT CCAGCAAAGG ACACACCGCT GTGACCGATA
    46501 CGCACACCGG ACCGACACCG GCCGACGCGG TACCCGCCTA CCCGTTCAGC CTGCCGCACG
    46561 CCCTGGACCT CGACCCGCAC TACGCCGAAC TCCGCCGCGA CGAACCCGTC TCCAGGGTGC
    46621 GCCTGCCCTA CGGCGAGGGC ACGGCCTGGC TGGTCACCCG CATGTCCGAC GCCCGTATCG
    46681 TTCTGGGCGA CTCCCGCTTC AGCACCGCGG CCGCCACCGA TCCCGCCACC CCCCGGATGT
    46741 TCCCCACCCC GCCCGAGCCG GACGGCGTCC TCGCCCAGGA CCCGCCGGAC CACACCCGGC
    46801 TGCGGCGGCT GGTGGGCAAG GCCTTCACGG CACGCCGGGT GGAGGAGATG CGGCCCCGTG
    46861 TCCGCTCCCT CGTCGACTCC CTGCTCGACG ACATGGTGGC GCACGGTTCA CCCGCCGACC
    46921 TGGTCGAGTT CCTCGCCGTT CCCTTCCCCG TCGCGGTCAT CTGCGAACTG CTCGGCGTGC
    46981 CCTTGGAGGA CCGCGACCTG TTCCGGACCT TCTCCGACGC CATGCTCTCC TCGACCCGGC
    47041 TCACCGCCGC GGAGATACAG CGGGTCCAGC AGGACTTCAT GGTCTACATG GACGGCCTGG
    47101 TCGCCCAGCG CCGCGACGCC CCCACCGAGG ACCTGCTCGG CGCCCTCGCC CTCGCCACCG
    47161 ACAACGACGA CCACCTGACC AAGGGCGAGA TCGTCAACAT GGGGGTGAGC CTGCTCATCG
    47221 CGGGCCACGA GACGTCGGTC AACCAGATCA CCAACCTCGT CCACCTCCTG CTGACCGAGC
    47281 GCAAGCGCTA CGAGTCGCTG GTCGCCGACC CGGCCCTCGT GCCCGCGGCG GTGGAGGAGA
    47341 TGCTGCGGTA CACACCGCTG GTGTCCGCCG GCAGCTTCGT CCGCGTGGCC ACCGAGGACG
    47401 TGGAGCTGAG CACCGTGACC GTGCGGGCCG GGGAGCCCTG CGTCGTCCAC TTCGCGTCGG
    47461 CCAACCGGGA CGAGGAGGTC TTCGACCACG CCGACGAGCT GGACTTCCAC CGTGAGCGCA
    47521 ACCCGCACAT AGCGTTCGGG CACGGAGCGC ACCACTGCAT CGGCGCCCAA CTGGGCCGAC
    47581 TGGAACTCCA GGAGGCCCTG TCCGCCCTCG TCCGGCGCTT CCCCACCCTC GATCTGGCCG
    47641 AGCCGGTCGC GGGACTGAAG TGGAAGCAGG GCATGCTGAT CCGCGGACTG GAACGCCAGA
    47701 TCGTCTCCTG GTGACGGCCG GCCGCCCGGC CGCCCGCCGG GCACCGGCGC CACCAGGGCA
    47761 CCGGCCGGGA CCGCAGACCC GGCCGGTGCC CCTCGCCCGA GGCCGCCTCA CTCCACGAAG
    47821 CGGCCACCCT CCATGTGCAT GCGGCGACCG GTGAACCGCT GCGCGAACAT GCGGTCGTGG
    47881 GAGACCACGA CCAGTGCGCC CCGGTAGTGC GCCAGCGCCT CCTCCAGGTC CTCCACGAGC
    47941 GCGGGCGACA GGTGGTTCGT CGGCTCGTCG AGCAGCAGCA GGTCCGCCGG GTCGCGCAGC
    48001 AGACGGGCCA GGGCCAGCCG CCTCAACTGC CCGGTGGACA GGTCTCCCAC CGCGGTGCCC
    48061 AGCGCCGAGG GCCGGAAGAG CCCGAATCCC AGGAGCGCGC CCCGGTGTTC CTCCGCGATG
    48121 CCGGGCAGCC CCGCCGCGAA GGCCGCCAGC AGGCTCTGCT GCCGGTCGGT GATCTCCGTC
    48181 TCCTGCGGCA GCCAGCCGAT GCGCTCCGGG CGCTCGCACT CGCCCTGATC GGGCGCCAGG
    48241 TCACCGGCCA GCACGCGCAG CAGGGTGCTC TTGCCCGCGC CGTTGTGCCC CGTGATCAGG
    48301 ATGCGCTCAC CGGGGTCGAC GGTGAAGGAC GGGACGTCGA GCCGCGTGCC GACGGTGACC
    48361 TTGTACAGCT CGGCGAGTGC CCCGCCGCGC CCGACCGTGC CGCCACCCTC CACCCGGGCC
    48421 CGGAAACGCA TGGGTTGAGG GGGCCGCGGC ACCGGGTTCT CCTCCAGCCG GCGGACCCGC
    48481 TCCTTGGCGT TGCGGACCCG CGCGGAGATC TGCTTCTCCA CGTTGCGCTG GTGGCGCTGG
    48541 TTCGACCGCT CGGTGTTGCG CCGCGGGCCG GTGGCCAGGT GGTCGGCGGC GCTGCGGGCC
    48601 AGTTCCCGCT GGCGTGCCAG GTCCTCCAGC CAGTCCTGGT AAGCCTGCTC CCAGCGGCGC
    48661 CGCGCGGCCG CCTTGGCTTG CAGGTATCCC GCGTAACCGC CGCCGTGCCG GTTGACGGTG
    48721 CGCCGCTCGC CGTCCACCTC CCACAGGGCG GTGGCCACGC GCTCCAGGAA GACCCGGTCG
    48781 TGCGAGACGA CCAGCACGCT GCCGCGGTGG GCCCGCAGGC GCTCCTCCAG CCACTCCAGC
    48841 GCCCCGACGT CGAGGTGGTT GGTGGGTTCG TCGAGCAGCA TCAGCTGCGG GGACGCGGCC
    48901 AGCAGGCAGG CCAGGTTGAG ACGCGCCTGC TCACCTCCGC AGAGGCTGCC GAGCCGCCGG
    48961 TCGCCCGTGA TGCCCGCCAG ACCGAGGCCG TGCATCGCCG CGTCGACACG GGCGTCCGCC
    49021 GCGTAGCCGT CGCGGGCCTC GAACGCCTCC AGCAGGTCGC CGTAGGCGCC GAGCAGGCCC
    49081 TCCAGCTCCT CGGGCTCCGC CCCGGCCAGC GCCTGCTCCG CCTCACGCAA CCCCCGCTCC
    49141 AGGGAGCGCA GTTCGGCGAG GGCGTGGTCG ATGGCGTCCT GAACGGTGTC CTCCGGGGGC
    49201 AGGTCCGGTG TCTGGGGGAG GTAGCCGCAG CCGCCGGGAG CCCGGACGAG GACCTGGCCA
    49261 CCGTCCGGGC GGTCCACGCC GGCGAGCATG CGGAGCAGGG TCGACTTGCC CGATCCGTTC
    49321 TCACCGATGA TGCCGACGCG CTCGCCGAGT GCCACCGACT GGTTGACGCC GTCCAACAGC
    49381 GGCCGTCCGC CGGGTGCCCG GACGACGTCG TCGAGGACGA CCTGGAAGGA ACCGGTCTCA
    49441 GGTTGCGTGG GAAGGAGCTT TTCCGGCGTG CCGGTGAGCG CCGCGGCGCC GGTATCGGAA
    49501 CGGTGTGCGT TCTGCATGGG TGATCCGCCA TTCGGAGAAA AAGAGGCAGT GTGGCCAAAA
    49561 GGGAGCGGCC CACGGCAGAC GGCGGAAGAA GAGAACGCCT CGGCGAACGC GGCGCACCCG
    49621 ACGGTGCGCA GCGCGAAAAA AGGGAGGCGA AGAAGCGAGC CGGAGGCGTC GCGATCAGCG
    49681 GCGGGAGAAG CCGCGTCACC GTCCTGCCGG GAACCCTCGA CGGCGCCGGA GCGGCAACCG
    49741 CGTACGCGGT GCTCCTCGGC GCCCGGACTC CCGTGGCGGT ATCAGAGGAA GTAGTAACTG
    49801 ACCACGTCGG CACGATAGCA GAGCAGACGG AGCCGGCAGG GGGTCGCGAG GTGCGATGGC
    49861 TGAATGTGTG CCACGCTTCG GATTTTTTGC TCGCGGGACG ACGAGGCCGT GTGCGAACGT
    49921 GTCCCGGGCA GTCGTTCGTC AGCGGGAGGT TCATATGCAG GACAACCAGG GTGGATCCGG
    49981 AGCCGAGTCC GAGACCGGGA CCGAGAGCGA CGTCAAGCGG AAGTTCCGGG AGGCACTGGA
    50041 GCGCAAGAAG CTCCTCAGCC GGGAACGCCG GGCGCACGAG GACGCTCGTT CCAAGGTGAA
    50101 CGGAACGTCC CGCAATGGCG CCAGGAAGGC GAATTTCCGC CGCAAGGCCG GGTGACACCG
    50161 ACCGCTGCGC ACACCCGTGC CCCACAGCTC GACTCCGCTG CGACAGGGGC CTGCCCGCGC
    50221 CGGGGAACCG GCCCGGGCAG GTGTAGGGTG GCGGGCATGT ATCCAGGTGT CGGTTCCCTG
    50281 AAGCTCCGCC GCCGCGCCTG ACGGTGCGGC CCTGAACTCT CGTTTCGCGT GCCCACCGTC
    50341 GCGGTGTCAG TGCCGGGCGG CTGTTTCGTG CTGCCCGGTT CCGGAGCGAA CCTGTGGAGC
    50401 ACACCGTGGG CGCATTCCCC GCAAGGCCGG CCTGAGGCCG CGACCGATAC ACGAGTTCAC
    50461 CGATGCGAGC GAGGGCCGCC GCCGCGCCGG TGGCGACGAC CACCCCTTCC GCACCGGCCC
    50521 CGACGCCCTC TCGCCGGCGC CGCTCCCGGC CCCGGCCGGC GGCGCCACCC GGGTACGCCG
    50581 CTCCCGCGGC CCCGGCGGCG CGTGCCGCGC ACAGGCCGTA CCGGCCGGCC GTTCGGCCGG
    50641 TGGACCTCTG CGCCCTGCCG TCCCCGCGGC AACGTCGCCG GACACGGACA CCGCCCCTCG
    50701 GCCGCCGGCC GCCGTCACCA CCCCGGGGCG CCGGCGTCTC GCCGCTCTCG CGCCGGCCCC
    50761 GTCCACGACC GCTCCCGTGC CTGCCGGAAG GGCCGACTCA TGACCGAGCG ACACCTCCCC
    50821 GCCGTCCTCG CGCCCCTCGG CCGGCCGGGC TACCGCCGCC TCTTCGCCGC CATGGTCCTC
    50881 GCCCTCTTCG GGTACGGCGG GTGGACCATC TACCTCGCGC TCCAGGCGCT GGAGCTC
  • The above DNA sequence encodes the following 8,8a-deoxyoleandolide synthase proteins: [0047]
    8,8a-deoxyoleandolide synthase 1:
    1 MHVPGEENGH SIAIVGIACR LPGSATPQEF WRLLADSADA LDEPPAGRFP TGSLSSPPAP
    61 RGGFLDSIDT FDADFFNISP REAGXTLDPQQ RLALELGWEA LEDAGIVPPH LRGTRTSVFM
    121 GAMWDDYAHL AHARGEAALT RHSLTGTHRG MIANRLSYAL GLQGPSLTVD TGQSSSLAAV
    181 HMACESLARG ESDLALVGGV NLVLDPAGTT GVERFGALSP DGRCYTFDSR ANGYARGEGG
    241 VVVVLKPTHR ALADGDTVYC EILGSALNND GATEGLTVPS ARAQADVLRQ AWERARVAPT
    301 DVQYVELHGT GTPAGDPVEA EGLGTALGTA RPAEAPLLVG SVKTNIGHLE GAAGIAGLLK
    361 TVLSIKNRHL PASLNFTSPN PRIDLDALRL RVHTAYGPWP SPDRPLVAGV SSFGMGGTNC
    421 HVVLSELRNA GGDGAGKGPY TGTEDRLGAT EAEKRPDPAT GNGPDPAQDT HRYPPLILSA
    481 RSDAALPAQA ERLPHHLEHS PGQRLRDTAY SLATRRQVFE RHAVVTGHDR EDLLNGLRDL
    541 ENGLPAPQVL LGRTPTPEPG GLAFLFSGQG SQQPGMGKRL HQVFPGFRDA LDEVCAELDT
    601 HLGRLLGPEA GPPLRDVMFA ERGTAHSALL SETHYTQAAL FALETALFRL LVQWGLKPDH
    661 LAGHSVGEIA AAHAAGILDL SDAAELVATR GALMRSLPGG GVMLSVQAPE SEVAPLLLGR
    721 EAHVGLAAVN GPDAVVVSGE RGHVAAIEQI LRDRGRKSRY LRVSHAFNSP LMEPVLEEFA
    781 EAVAGLTFRA PTTPLVSNLT GAPVDDRTMA TPAYWVRHVR EAVRFGDGIR ALGKLGTGSF
    841 LEVGPDGVLT AMARACVTAA PEPGHPGEQG ADADAHTALL LPALRRGRDE ARSLTEAVAR
    901 LHLHGVPMDW TSVLGGDVSR VPLPTYAFQR ESHWLPSGEA HPRPADDTES GTGRTEASPP
    961 RPHDVLHLVR SHAAAVLGHS RAERIDPDRA FRDLGFDSLT ALELRDPLDT ALGLRLPSSV
    1021 LFDHPSPGAL ARFLQGDDTR RPEPGKTNGT PATEPGPDFD DEPIAIVGMA CRFPGGVTSP
    1081 EDLWRLLAAG EDAVSGFPTD RGWNVTDSAT RRGGFLYDAG EFDAAFFGIS PREALVMDPQ
    1141 QRLLLETSWE ALEPAGVSPG SLRGSDTAVY IGATAQDYGP RLHESDDDSG GYVLTGNTAS
    1201 VASGRIAYSL GLEGPAVTVD TACSSSLVAL HLAVQALRRG ECSLALAGGA TVMPSPGHEV
    1261 EFSRQGGLSE DGRCKAFATT ADGTGWAEGV GVLLVERLSD ARRLGNRVLA VVRGSAVNQD
    1321 GASNGLTAPN GPSQQRVIRA ALADAGLVPA DVDVVEAHGT GTRLGDPIEA QALLATYGQG
    1381 RAGGRPVVLG SVKSNIGHTQ AAAGVAGVMK MVLALGRGVV PKTLHVDEPS AHVDWSAGEV
    1441 ELAVEAVPWS RGGRVRRAGV SSFGISGTNA HVIVEEAPAE PEPEPERGPG SWGVVPWV
    1501 SGRDAGALRE QAARLAAHVS GVSAVDVGWS LVATRSVFEH RAVMVGSELD AMAESLAGFA
    1561 AGGVVPGVVS GVAPAEGRRV VFVFPGQGSQ WVGNAAGLLD ACPVFAEAVA ECAAVLDPLT
    1621 GWSLVEVLRG GGEAVLGRVD VVQPALWAVM VSLARTWRYY GVEPAAVVGH SQGEIAAACV
    1681 AGGLSLADGA RVVVLRSRAI APJAGGGGMV SVSLPAGRVR TMLEEFDGRV SVAAVNGPSS
    1741 TVVSGDVQAL DELLAGGERE GVRARRVPVD YASHSAQMDQ LRDDLLEALA TIVPTSANVP
    1801 FFSTVTADWL DTTALDAGYW FTNLRETVRF QEAVEGLVAQ GMGAFIECSP HPVLVPGITE
    1861 TLDTFDADAV ALSSLRRDEG GLDRFLTSLA EAFVQGVPVD WSRAFEGASP RTVDLPTYPF
    1921 QRQRYWLLDK AAQRERERLE DWRYHVEWRP VTTRPSARLS GVWAVAIPAP LARDSLLVGA
    1981 IDALERGGAR AVPVVVDERD NDRQALVEAL RNGLGDDDLA GVLSLLALDE APHGDHPDVP
    2041 VGMAASLALV QAMADAAAEV PVWFATRGAV AALPGESPER PRQALLWGLG RVVALEQPQI
    2101 WGGLVDLPQH LDEDAGRRLV DVVGGLADED QLAVRASSVL ARRLVRTPGH RMSSQAGGRE
    2161 WSPSGTVLVT GGTGALGABW ARWLAGK&AE HLVLISRRGA DAAGAAALRD SLTDMGVRVT
    2221 LAACDAADRH ALETLLDSLR TDPAQLTAVI HAAGALDDGM TTVLTPEQMN NALRAKVTAT
    2281 VNLHELTRDL DLSAFVLFSS ISATLGIPGQ ANYAPGNSFL DAFAEWRPAQ GLVATSIAWG
    2341 PWSGGTGMAH EGSVGERLQR HGVLAMERAA AIAALDHTLA SDETAVAVAD IDWSRFFLAY
    2401 TALRARPLIG EIPEARRNLE SGSGPGDLEP DRAEPELAVR LAGLTAVEQE RLLVQLVREQ
    2461 AAVVLGHSGA EAVAPDRAFK DLGFDSLTSV ELRNRLNTAT GLRLPVTAVF DYARPAALAG
    2521 HLRSRLIDDD GDHGALPGVE KHAIDEPIAI VGMACRFPGG IASPEDLWDV LTAGEDVVSG
    2581 LFQNRGWDLG RLYDPDPDRA GTSYMREGAF LHEAGEFDAA FFGISPREAL AMDPQQRLLL
    2641 ETSWEALERA GITPSKLAGS PTGVFFGMSN QDYAAQAGDV PSELEGYLLT GSISSVASGR
    2701 VAYTFGLEGP AVTVDTACSS SLVALHLAVQ GLRRGECSLA LVGGVTVMSS PVTLTTFSRQ
    2761 RGLSVDGRCK AFAASADGFG AAEGVGVLLV ERLSDARPLG HRVLAVVRGS AVNQDGASNG
    2821 LAAPNGPSQQ RVIRAALADA GLAPADVDVV EAHGTGTRLG DPIEAQALLA TYGQGRTSGR
    2881 PVWLGSVKSN IGHTQAAAGV AGVMKMVLAL GRGVVPKTLH VDEPSPHVDW SAGEVELAVE
    2941 AVPWSRGGRV RRAGVSSFGI SGTNAHVIVE EAPAEPSVEE GPGSVVGVVP WVVSGRDAGA
    3001 LRAQAARLAA HVSSTGAGVV DVGWSLVATR SVFEHRAVMV GTDLDSNAGS LAGFAAGGVV
    3061 PGVVSGVAPA EGRRVVFVFP GQGSQWVGMA AGLLDACPVF AEAVAECAAV LDRLTGWSLV
    3121 EVLRGGEAVL GRVDVVQPAL WAVMVSLART WRYYGVEPAA VVGHSQGEIA AACVAGGLSL
    3181 ADGARVVVLR SRAIAPJAGG GGMVSVGLSA ERVRTMLDTY GGRVSVAAVN GPSSTVVSGD
    3241 AQALDELLAG CEREGVRARR VPVDYASHSA QMDQLRDELL EALADVIPOD SSVPFFSTVT
    3301 ADWLDTTALD AGYWFTNLRE TVRFQEAVEG LVAQGMGAFV ECSPHPVLVP GITETLDTFD
    3361 ADAVALSSLR RDEGGLDRFL TSLAEAFVQG VPVDWTHAFE GGRPRFVDLP TYAFQRQRYW
    3421 LHEEPLQEPV DEAWDAEFWS VVERGDATAV SDLLSTDAEA LHTVLPALSS WRRPRVEHRR
    3481 LQDWRYRVEW KPFPAALDEV LGGGWLFVVP RGLADDGVVA RVVAAVTARG GEVSVVELDP
    3541 TRPDRRAYAE AVAGRGVSGV VSFLSWDDRP HSEHSVVPAG LAASLVLAQA LVDLGRVGEG
    3601 PRLWLVTRGA VVAGPSDAGV VIDPVQAQVW GFGRVLGLEH PELWGGLVDL PVGVDEEVCR
    3661 RBVGVVASAG FEDQVAVRGS GVWVRRLVRA VVDGGGGGWR PRGTVLVTGG LGGLGAHTAR
    3721 WLVGGGADHV VLVSRRGGSA PGAGDLVREL EGLGGARVSV PACDVADRVA LRALLSDLGE
    3781 PVTAVFHAAG VPQSTPLAEI SVQEAADVMA AKVAGAVNLG ELVDPCGLEA FVLFSSNAGV
    3841 WGSGGQAVYA AADAFLDALA VRRRGVGLPA TSVAWGMWAG EGMASVGGAA RELSPRGVRA
    3901 MDPERAVAVM ADAVGRGEAF VAVADVDWER FVTGFASARP RPLISDLPEV RAVVEGQVQG
    3961 RGQGLGLV&E EESSGWLKRL SGLSRVRQEE ELVELVRAQA AVVLGHGSAQ DVPAERAFKE
    4021 LGFDSLTAVE LRNGLAAATG IRLPATMAFD HPTATAIARF LQSELVGSDD PLTLMRSAID
    4081 QLETGLALLE SDEEARSEIT KRLNILLPRF GSGGSSPGRE AGQDAGEHQD VEDATIDELF
    4141 EVLDNELGNS
    8,8a-deoxyoleandolide synthase 2:
    1 VINDEKIVEY LKRATVDLRK ARERIWELED EPIAITSMAC HFPGGIESPE QLWELLSAGG
    61 EVLSEFPDDR GWDLDEIYHP DPEHSGTSYV RHGGFLDHAT QFDTDFFGIS PREALPIMDPQ
    121 QRLLLETSWQ LFERAGVDPH TLKGSRTGVF VGAAHMGYAD RVDTPPAEAE GYLLTGNASA
    181 VVSGRISYTF GLEGPAVTVD TACSSSLVAL HLAVQALRRG ECSLAVVGGV AVMSDPKVFV
    241 EFSRQRGLAR DGRSKAFAAS ADGFGFAEGV SLLLLERLSD ARRLGHRVLA VVRGSAVNQD
    301 GASNGLAAPN GPSQQRVIRA ALADAGLAFA DVDVVFAHGT GTRLGDPIEA QALLATYGQG
    361 RTSGRPVWLG SVKSNIGHTQ AAAGVAGVNK MVLALERGVV PKTLHVDEPS PHVDWSTGAV
    421 ELLTEERPWE PEAERLRRAG ISAFGVSGTN AHVIVEEAPA EPEPEPEPGT RVVAAGDLVV
    481 PWVVSGRDAG ALRAQAARLA AHVSSTGAGV VDVGWSLVAT RSVFEHRAVM VGTDLDSMAG
    541 SLAGFAAGGV VPGVVSGVAP AEGRRVVFVF PGQGSQWVGM AAGLLDACPV FAEAVAECAA
    601 VLDPLTGWSL VEVLRGGEAV LGRVDVVQPA LWAVMVSLAR TWRYYGVEPA AVVGHSQGEI
    661 AAACVAGGLS LADGARVVVL RSRAIARIAG GGGMVSVSLP AGRVRTMLDT YGGRLSVAAV
    721 NGPSSTVVSG DAQALDELLA GCEREGVRAR RVPVDYASNS AQMDQLRDEL LEALADITPQ
    781 HSSVPFFSTV TADWLDTTAL DAGYWFTNLR ETVRFQEAVE GLVAQGMGAF VECSPHPVLV
    841 PGIEQTLDTV EADAVALGSL RRDEGGLGRF LTSLAFAFVQ GVPVDWSRTF EGASPRTVDL
    901 PTYPFQRQRF WLEGSPALSS NGVEGEADVA FWDAVEREDS AVVAEELGID AKAlHMTLPA
    961 LSSWRRRERQ RRKVQRWRYR VEWKRLPNSR AQESLQGGWL LVVPQGRAGD VRVTQSVAEV
    1021 AAKGGEATVL EVDALHPDRA AYAEALTRWP GVRGVVSFLA WEEQALANHP VLSAGLAASL
    1081 AIAQALIDVG GSGESAPRLW LVTEAAVVIG AADTGAVIDP VHAQLWGFGR VLALEHPELW
    1141 GGLIDLPAVA GEPGSITDHA HADLLATVLA TMVQAAARGE DQVAVRTTGT YVPRLVRSGG
    1201 SAHSGARRWQ PRDTVLVTGG MGPLTAHIVR WLADNGADQV VLLGGQGADG EAEALRAEFD
    1261 GHTTKIELAD VDTEDSDALR SLLDRTTGEH PLRAVIHAPT VVEFASVAES DLVRFARTIS
    1321 SKIAGVEQLD EVLSGIDTAH DVVFFSSVAG VWGSAGQSAY AAGNAFLDAV AQHRRLRGLP
    1381 GTSVAWTPWD DDRSLASLGD SYLDRRGLRA LSIPGALASL QEVLDQDEVH AVVADVDWER
    1441 FYAGFSAVPR TSFFDDVHDA HRPALSTAAT NDGQAPDEDG GTELVRRLRP LTETEQQPEL
    1501 VSLVQSEVAA VLGNSSTDAV QPQRAFREIG FDSLTAVQLR NRLTATTGMR LPTTLVFDYP
    1561 TTNGLAEYLR SELFGVSGAP ADLSVVRNAD EEDDPVVIVG MACRFPGGID TPEAFWKLLE
    1621 AGGDVISELP ANRGWDMERL LNPDPFAKGT SATRYGGFLY DAGEFDAAFF GISPREALAM
    1681 DPQQRLLLET VWELIE5AGV APDSLHRSRT GTFIGSNGQF YAPLLWNSGG DLEGYQGVGN
    1741 AGSVMSGRVA YSLGLEGPAV TVDTACSSSL VALHLAVQAL RRGECSLAIA GGVTVMSTPD
    1801 SFVEFSRQQG LSEDGRCKAF ASTADGFGLA EGVSALLVER LSDARPLGHR VLAVVRGSAV
    1861 NQDGASNGLT APNGPSQQRV IRAALADAGL APADVDVVEA HGTGTRLGDP IEAQALLATY
    1921 GQGRAGGRPV VLGSVKSNIG HTQAAAGVAG VMKMVLALER GVVPKTLHVD EPSPHVDWSA
    1981 GEVELAVEAV PWSRGGRVRR AGVSSFGISG TNAHVIVEEA PAEPEPEPGT RVVAAGDLVV
    2041 PWVVSGRDAG ALPEQAAPLA AHVSSTGAGV VDVGWSLVAT RSVFEHRAVM VGSELDSMAE
    2101 SLAGFAAGGV VPGVVSGVAP AEGRRVVFVF PGQGSQWVGM AAGLLDACPV FAEAVAECAA
    2161 VLDPVTGWSL VEVLRGGGEA VLGRVDVVQP ALWAVMVSLA RTWRYYGVEP AAVVGHSQGE
    2221 IAAACVAGGL SLADGARVVV LRSRAIARIA GGGGMVSVGL SAERVRTMLD TYGGRVSVAA
    2281 VNGPSSTVVS GDVQALDELL AGCEREGVRA RRVPVDYASH SAQMDQLRDE LLEALADITP
    2341 QHSSVPFFST VTADWLDTTA LDAGYWFTNL RETVRFQEAV EGLVAQGNGA FVECSPHPVL
    2401 VPGIEQTLDA LDQNAAVLGS LRRDEGGLDP LLTSLAEAFV QGVPVDWTHA FEGMTPRTVD
    2461 LPTYPFQRQH YWPKPAPAPG ANLGDVASVG LTAAGHPLLG AVVEMPDSDG LVLTGQISLR
    2521 THFWLADHEV LGSVLLPGTA FVELAVQAAD RAGYDVLDEL TLEAPLVLPD RGGIQVRLAL
    2581 GPSEADGRRS LQLHSRPEEA AGFHRWTRHA SGFVVPGGTG AAPPTEPAGV WPPAGAEPVA
    2641 LASDPYARLV ERGYTYGPSF QGLHTAWRHG DDVYAEVALP EGTPADGYAL HPALLDAAVQ
    2701 AVGLGSFVED PGQVYLPFLW SDVTLHATGA TSLRVRVSPA GPDTVALAIA DPAGAPVATV
    2761 GALRLRTTSA AQLARARGSA EHAMFRVEWV EEGSAADRCR GGAGGTTYEG ERAAEAGAAA
    2821 GTWAVLGPRV PAAVRTMGVD VVTALDTPDH PADPQSLADL AALGDTVPDV VVVTSLLSLA
    2881 SGADSPLGNR PRPTAAEQDT AATVAGVHSA LHAALDLVQA WLADERHTAS RLVLVTRHAM
    2941 TVAESDPEPD LLLAPVWGLV RSAQAENPGR FVLADIDGDE ASWDALPRAV ASAASEVAIR
    3001 AGAVYVPRLA RATDEGLVVA DEAAGPWRLD VTEAGTLANL ALVPCPDASR PLGPDEVRIA
    3061 VRAAGVNFRD VLLALGMYPD EGLMGAEAAG VVTEVGGGVT TLAPGDRVMG LVTGGFGPVA
    3121 VTHHPMLVPM PRGWSFAEAA SVPVAFLTAY YALHDLAGLP GGESVLVHSA AGGVGMAAVQ
    3181 LAPHWDAEVF GTASKGKWDV LAAQGLDEEH IGSSRTTEFE QRFRATSGGR GIDVVLNALS
    3241 GDFVDASARL LREGGRFVEM GKTDIRTDLG VVGADGVPDI RYVAFDLAEA GAERIGQKLD
    3301 EIMALFDAGV LRLPPLRAWP VRRAHEALRF VSQARHVGKV VLTVPAALDA EGTVLITGAG
    3361 TLGALVABHL VTENDVRRLL LVSRSGVAPD LAABLGALGA EVTVAACDVA NRKALKALLE
    3421 DIPPEHPVTG IVNTAGVLDD GVVSGLTPER VDTVLKPKVD AALTLESVIG ELDLDPALFV
    3481 IFSSAASMLG GPGQGSYAAA NQFLDTLAPH RARRGLTSVS LGWGLWHEAS GLTGGLADID
    3541 RDPMSRAGIA PNPTDEALHL FDRATELGDP VLLPMRLNEA ALEDRAADGT LPPLLSGLVR
    3601 VRHRPSARAG TATAAPATGP EAFARELAAA PDPRRALRDL VRGHVALVLG HSGPEAIDAE
    3661 QAFRDIGFDS LTAVELRNRL NAETGLRLPG TLVFDYPNPS ALADHLLELL APATQPTAAP
    3721 LLABLERVEQ LLSAAASPGG PASAVDEETR TLIATRLATL ASQWTHLPVG SPGNADNRSG
    3781 PGESGQAQES GATGEHTAAW TSDDDLFAFL DKRLET
    8,8a-deoxyoleandolide synthase 3.:
    1 VAEAEKLREY LWRATTELKE VSDRLRETEE RAREPIAIVG MSCRFPGGGD ATVNTPEQFW
    61 DLLNSGGDGI AGLPEDRGWD LGRLYDPDFD RAGTSYVREG GFLYDSGEFD AAFFGISPRE
    121 ALANDFQQRL LLETSWEAFE SAGIKRAALR GSDTGVYIGA WSTGYAGSPY RLVEGLEGQL
    181 AIGTTLGAAS GRVAYTFGLE GPAVTVDTAC SSSLVALHLA VQGLRPGECS LALVGGVTVM
    241 SSPVTLTTFS RQRGLSVDGR CKAFPASADG FGAAEGVGVL LVERLSDARR LGNRVLAVVR
    301 GSAVNQDGAS NGLTAPNGPS QQRVIPAALA DAGLAPADVD VVEAHGTGTR LGDPIEAQAL
    361 LATYGQGRAG GRPVWLGSVK SNIGNTQAAA GVAGVMKMVL ALGRGVVPKT LHVDEPSPHV
    421 DWSAGAVELL TEERPWEPEA ERLRRAGISA FGVSGTNAHV IVEEAPAEPE PEPGTRVVAA
    481 GDLVVPWVVS GRDARALRAQ AAPLAAHVSG VSAVDVGWSL VATRSVFEHR AVAIGSELDS
    541 MAGSLAGFAA GGVVPGVVSG VAPAEGRRVV VFPGQGSQVV VGNAAGLLDA CPVFAEAVAE
    601 CAAVLDPVTG WSLVEVLQGR DATVLGRVDV VQPALWAVMV SLARTWRYYG VEPAAVVGHS
    661 QGEIAAACVA GGLSLADGAR VVVLRSRAIA RIAGGGGMVS VSLPAGRVRT MLEEFDGRLS
    721 VAAVNGPSST VVSGDVQALD ELIAGCEREG VRAPRVPVDY ASHSAQMDQL RDELLEALAF
    781 ITPQDSSVPF FSTVTADWLG TTALGAGYWF TNLRETVRFQ EAVEGLVAQG MGAFVECSPH
    841 PVLVPGIEQT LDALDQNAAV FGSLRRDEGG LDRFLTSLAE AFVQGVPVDW SRAFEGVTPR
    901 TVDLPTYPFQ RQHYWLMAEE APVSQPPHSE NSFWSVVADA DAEAAAELLG VDVEAVEAVM
    961 PALSSWHRQS QLRAEVNQWR YDVAQKRLTT GALPEKPGNW LVVTPAGTDT TFAESLARTA
    1021 AAELGVSVSF AQVDTAHPDR SQYAHALRQA LTGPENVDHL VSLLALDQAT DDLAAAPSCL
    1081 AASLVLAQAL VDLGRVGEGP RLWLVTRGAV VAGPSDAGAV IDPVQAQVWG FGRVLGLEHP
    1141 ELWGGLIDLP VGVDEEVCRR FVGVVASAGF EDQVAVRGSG VWVRRLVRAV VDGGGGGWRP
    1201 RGTVLVTGGL GGLGAHTARW LVGGGADHVV LVSRRGGSAP GAGDLVRELE GLGGARVSVR
    1261 ACDVADRVAL RSLLSDLGEP VTAVFHAAGV PQSTPLAEIS VQEAADVMAA KVAGAVNLGE
    1321 LVDPCGLEAF VLFSSNAGVW GSGGQAVYAA ANAFLDALAV RRRGVGLPAT SVAWGMWAGE
    1381 GMASVGGAAP ELSRRGVRAM DPERAVAVMA DAVGRGFAEV AVADVDWERF VTGFASARPR
    1441 PLISDLPEVR AVVEGQVQGR GQGLGLVGEE ESSGWLKRLS GLSRVRQEEE LVELVRAQAA
    1501 VVLGHGSAQD VPAERAFKEL GFDSLTAVEL RNGLAAATGI RLPATMAFDH PNATAIARFL
    1561 QSQLLPDAES ESAVPSSPED EVRQALASLS LDQLKGAGLL DPLLALTRLR EINSTVQNPE
    1621 PTTESIDEMD GETCCAWRSA KSTAEPLTTG ADMPDPTAKY VEALRASLKE NERLRQQNHS
    1681 LLAASREAIA ITANSCRFGG GIDSPEDLWR FLAEGPDAVA GLPEDRGWDL DALYHPDPEN
    1741 PGTTYVREGA FRYDAAQFDA GFFGISPREA LAMDPQQRLL LETSWELFER ADIDPYTVRG
    1801 TATGIFIGAG HQGYGPDPKR APESVAGYLL TGTASAVLSG RISYTFGLEG PAVTVDTACS
    1861 SSLVALHIAV QALRRGECSL AIAGGVAVMS TPDAFVEFSR QQGMARDGRC KAFAAAADGM
    1921 GWGEGVSLLL LERLSDARRL GHRVLAVVRG SAVNQDGASN GLAAPNGPSQ QRVIRAAIAD
    1981 AGLAPADVDV VEAHGTGTRL GDPIFAQALL ATYGQGRAGG RPVWLGSVKS NIGHTQAAAG
    2041 VAGVMKMVLA LGRGVVPKTL HVDEPSPHVD WSAGAVELLT EERPWEPEAE RLRRAGISAF
    2101 GVSGTNABVI VEEAPAEPEP EPGTRVVAAG DLVVPWVVSG PDVGALREQA ARLAAHVSST
    2161 GAGVVDVGWS LVATRSVFEH FAVMVGTDLD SMAGSLAGFA AGGVVPGVVS GVAPAEGRRV
    2221 VFVFPGQGSQ VNGMAAGLLD ACPVFAEAVA ECAAVLDPVT GWSLVEVLQG RDATVLGRVD
    2281 VVQPALWAVM VSLARTWRYY GVEPAAVVGH SQGEIAAACV AGGLSLADGA RVVVLRSRAI
    2341 ARIAGGGGMV SVSLPAGRVR TMLDTYGGRV SVAAVNGPSS TVVSGDVQAL DELLACCERE
    2401 GVRARRVPVD YASHSAQMDQ LRDELLEAIA DITPQDSSVP FFSTVTADWL DTTALDAGYW
    2461 FTNLRETVRF QEAVEGLVAQ GMGAFJECSP HFVLVPGIEQ TLDALDQNAA VLGSLRRDEG
    2521 GLDRLLTSLA EAFVQGVPVD WTHAFEGVTP RTVDLPTYPF QRQRFWLDGS PASSANGVDG
    2581 EADAMIWDAV EREDSVAVAE ELGIDAEALH TVLPALSSWR RRRVEHRRLQ DWRYRVEWKP
    2641 FPAALDEVLG GGWLFVVPRG LADDGVVARV VAAVTARGGE VSVVELDPTR PDRPAYAEAV
    2701 AGRGVSGVVS FLSWDDRRHS EHPVVPAGLA ASLVLAQALV DLGRVGEGPR LWLVTRDAVV
    2761 AGPSDAGAVI DPVQAQVWGF GRVLGLEHPE LWGGLIDLPV EAPEPGSTCD HTYADLLATV
    2821 VASAGFEDQV AVRGSGVWVR RLVRAVVDGG GGGWRPRGTV LVTGGLGGLG AHTARWLVGG
    2881 GADHVVLVSR RGGSAPGAGD LVRELEGLGG ARVSVPACDV ADRVALRALL SDLGEPVTAV
    2941 FHAAGVPQST PLAEISVQEA ADVMAAKVAG AVNLGELVDP CGLEAFVLFS SNAGVWGSGG
    3001 QAVYAAANAF LDALAVRRRG VGLPATSVAW GMWAGEGMAS VGGAARELSR RGVRAMDPER
    3061 AVAVMADAVG RGEAFVAVAD VDWERFVTGF ASARPRPLIS DLPEVRTALR NQEQEQLHAP
    3121 VPEDRSAQLL RRLSMLSPAG REABLVKLVR TEAAAVLGHG SAQDVPAERA FKELGFDSLT
    3181 AVQLRNRLAA ATGTRLPASA VFDHPHAAAL ARWLLAGMRH ADGGMGGGHA GGPGPDADEG
    3241 RSAGAGHSGM LADLYRPSAE LGRSREFIGL LADTAAFRPV FHGPADLDAP LEAVPLADGV
    3301 RKPQLICCSG TAPVGGPHEF ARLASFFRGT RAVSALPLPG YLPGEQLPAD LDAVLAAQAE
    3361 AVEKQTGGAP FVLVGYSAGG LMAHALACHL AGRGTPPSGE VLVDVYPPGR QEPVFGWQKE
    3421 LTEGMFAQDF VPMDDTRLTA LGTYDRLMGE WRPAPSGLPT LLIRATEPMA EWTGAIDWPA
    3481 SWEYDHTAVD MPGNHFTIMR EHAEDAARHI DVWLKGLTP
  • The recombinant DNA compounds of the invention that encode the oleandolide PKS proteins or portions thereof are useful in a variety of applications. While many of these applications relate to the heterologous expression of the oleandolide PKS or the construction of hybrid PKS enzymes, many useful applications involve the natural oleandomycin producer [0048] Streptomyces antibioticus.
  • For example, one can use the recombinant DNA compounds of the invention to disrupt the oleAI, oleAII, or oleAIII genes by homologous recombination in [0049] Streptomyces antibioticus. The resulting host cell is a preferred host cell for making polyketides modified by oxidation, hydroxylation, and glycosylation in a manner similar to oleandomycin, because the genes that encode the proteins that perform these reactions are present in the host cell. Such a host cell also does not naturally produce any oleandomycin that could interfere with production or purification of the polyketide of interest.
  • One illustrative recombinant host cell provided by the present invention expresses a recombinant oleandolide PKS in which the [0050] module 1 KS domain is inactivated by deletion or other mutation. In a preferred embodiment, the inactivation is mediated by a change in the KS domain that renders it incapable of binding substrate (the KS1o mutation). In a particularly preferred embodiment, this inactivation is rendered by a mutation in the codon for the active site cysteine that changes the codon to another codon, such as an alanine codon. Such constructs are especially useful when placed in translational reading frame with extender modules 1 and 2 of an oleandolide or the corresponding modules of another PKS. The utility of these constructs is that host cells expressing, or cell free extracts containing, a PKS comprising the protein encoded thereby can be fed or supplied with N-acylcysteamine thioesters of precursor molecules to prepare a polyketide of interest. See U.S. patent application Serial No. 60/117,384, filed Jan. 27, 1999, and PCT patent publication No. US99/03986, both of which are incorporated herein by reference. Such KS1o constructs of the invention are useful in the production of 13-substituted-oleandomycin compounds in Streptomyces antibioticus host cells. Preferred compounds of the invention include those compounds in which the substituent at the 13-position is propyl, vinyl, propargyl, other lower alkyl, and substituted alkyl.
  • The compounds of the invention can also be used to construct recombinant host cells of the invention in which coding sequences for one or more domains or modules of the oleandolide PKS have been deleted by homologous recombination with the [0051] Streptomyces antibioticus chromosomal DNA. Those of skill in the art will appreciate that such compounds are characterized by their homology with the chromosomal DNA and not by encoding a functional protein due to their intended function of deleting or otherwise altering portions of chromosomal DNA. For this and a variety of other applications, the compounds of the present invention include not only those DNA compounds that encode functional proteins but also those DNA compounds that are complementary or identical to any portion of the oleandolide PKS genes.
  • Thus, the invention provides a variety of modified [0052] Streptomyces antibioticus host cells in which one or more of the genes in the oleandolide PKS gene cluster have been mutated or disrupted. These cells are especially useful when it is desired to replace the disrupted function with a gene product expressed by a recombinant DNA expression vector. While such expression vectors of the invention are described in more detail in the following Section, those of skill in the art will appreciate that the vectors have application to S. antibioticus as well. Such S. antibioticus host cells can be preferred host cells for expressing oleandolide derivatives of the invention. Particularly preferred host cells of this type include those in which the coding sequence for the loading module has been mutated or disrupted, those in which one or more of any of the PKS gene ORFs has been mutated or disrupted, and/or those in which the genes for one or more oleandolide modification enzymes (glycosylation, epoxidation) have been mutated or disrupted.
  • While the present invention provides many useful compounds having application to, and recombinant host cells derived from, [0053] Streptomyces antibioticus, many important applications of the present invention relate to the heterologous expression of all or a portion of the oleandolide PKS genes in cells other than S. antibioticus, as described in the following Section.
  • Section II: Heterologous Expression of the Oleandolide PKS [0054]
  • In one important embodiment, the invention provides methods for the heterologous expression of one or more of the oleandolide PKS genes and recombinant DNA expression vectors useful in the method. For purposes of the invention, any host cell other than [0055] Streptomyces antibioticus is a heterologous host cell. Thus, included within the scope of the invention in addition to isolated nucleic acids encoding domains, modules, or proteins of the oleandolide PKS, are recombinant expression vectors that include such nucleic acids. The term expression vector refers to a nucleic acid that can be introduced into a host cell or cell-free transcription and translation system. An expression vector can be maintained permanently or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm. An expression vector also comprises a promoter that drives expression of an RNA, which is translated into a polypeptide in the cell or cell extract. For efficient translation of RNA into protein, the expression vector also typically contains a ribosome-binding site sequence positioned upstream of the start codon of the coding sequence of the gene to be expressed. Other elements, such as enhancers, secretion signal sequences, transcription termination sequences, and one or more marker genes by which host cells containing the vector can be identified and/or selected, may also be present in an expression vector. Selectable markers, i.e., genes that confer antibiotic resistance or sensitivity, are preferred and confer a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
  • The various components of an expression vector can vary widely, depending on the intended use of the vector and especially the host cell(s) in which the vector is intended to replicate or drive expression. Expression vector components suitable for the expression of genes and maintenance of vectors in [0056] E. coli, yeast, Streptomyces, and other commonly used cells are widely known and commercially available. For example, suitable promoters for inclusion in the expression vectors of the invention include those that function in eucaryotic or procaryotic host cells. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host cell or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can also be used.
  • Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of the oleandolide PKS coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the invention to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome. The resulting host cells of the invention are useful in methods to produce PKS and post-PKS tailoring (modification) enzymes as well as polyketides and antibiotics and other useful compounds derived therefrom. [0057]
  • Preferred host cells for purposes of selecting vector components for expression vectors of the present invention include fungal host cells such as yeast and procaryotic host cells such as [0058] E. coli and Streptomyces, but mammalian cell cultures can also be used. In hosts such as yeasts, plants, or mammalian cells that ordinarily do not produce modular polyketide synthase enzymes, it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality. Provision of such enzymes is described, for example, in PCT publication Nos. WO 97/13845 and 98/27203, each of which is incorporated herein by reference. Particularly preferred host cells for purposes of the present invention are Streptomyces and Saccharopolyspora host cells, as discussed in greater detail below.
  • In a preferred embodiment, the expression vectors of the invention are used to construct a heterologous recombinant Streptomyces host cell that expresses a recombinant PKS of the invention. Streptomyces is a convenient host for expressing polyketides, because polyketides are naturally produced in certain Streptomyces species, and Streptomyces cells generally produce the precursors needed to form the desired polyketide. Those of skill in the art will recognize that, if a Streptomyces host cell produces any portion of a PKS enzyme or produces a polyketide-modifying enzyme, the recombinant vector need drive expression of only those genes constituting the remainder of the desired PKS enzyme or other polyketide-modifying enzymes. Thus, such a vector may comprise only a single ORF, with the desired remainder of the polypeptides constituting the PKS provided by the genes on the host cell chromosomal DNA. If a Streptomyces or other host cell ordinarily produces polyketides, it may be desirable to modify the host so as to prevent the production of endogenous polyketides prior to its use to express a recombinant PKS of the invention. Such modified hosts include [0059] S. coelicolor CH999 and similarly modified S. lividans described in U.S. Pat. No. 5,672,491, and PCT publication Nos. WO 95/08548 and WO 96/40968, incorporated herein by reference. In such hosts, it may not be necessary to provide enzymatic activities for all of the desired post-translational modifications of the enzymes that make up the recombinantly produced PKS, because the host naturally expresses such enzymes. In particular, these hosts generally contain holo-ACP synthases that provide the pantotheinyl residue needed for functionality of the PKS.
  • The invention provides a wide variety of expression vectors for use in Streptomyces. The replicating expression vectors of the present invention include, for example and without limitation, those that comprise an origin of replication from a low copy number vector, such as SCP2* (see Hopwood et al., [0060] Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol. Gen. Genet. 219: 341-348, and Bierman et al., 1992, Gene 116: 43-49, each of which is incorporated herein by reference), or a high copy number vector, such as pIJ101 and pJV1 (see Katz et al., 1983, J Gen. Microbiol. 129: 2703-2714; Vara et al., 1989, J. Bacteriol. 171: 5782-5781; and Servin-Gonzalez, 1993, Plasmid 30: 131-140, each of which is incorporated herein by reference). High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes. For non-replicating and integrating vectors and generally for any vector, it is useful to include at least an E. coli origin of replication, such as from pUC, p1P, p1I, and pBR. For phage based vectors, the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra). Also, plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211, all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed for purposes of the present invention.
  • The Streptomyces recombinant expression vectors of the invention typically comprise one or more selectable markers, including antibiotic resistance conferring genes selected from the group consisting of the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes. Alternatively, several polyketides are naturally colored, and this characteristic can provide a built-in marker for identifying cells. [0061]
  • Preferred Streptomyces host cell/vector combinations of the invention include [0062] S. coelicolor CH999 and S. lividans K4-114 and K4-155 host cells, which have been modified so as not to produce the polyketide actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. No. 08/828,898, filed Mar. 31, 1997, and Ser. No. 09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference. These vectors are particularly preferred in that they contain promoters compatible with numerous and diverse Streptomyces spp. Particularly useful promoters for Streptomyces host cells include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including promoters from aromatic (Type II) PKS gene clusters. Examples of Type II PKS gene cluster promoters are act gene promoters and tcm gene promoters; examples of Type I PKS gene cluster promoter are the spiramycin PKS and DEBS genes promoter. The present invention also provides the oleandolide PKS gene promoter in recombinant form. The promoter for the oleA genes is located upstream of the oleAI gene on cosmid pKOS055-5 of the invention. This promoter is contained within an ˜1 kb segment upstream of the oleAI coding sequence and can be used to drive expression of the oleandolide PKS or any other coding sequence of interest in host cells in which the promoter functions, particularly S. antibioticus and generally any Streptomyces species.
  • As described above, particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium. The promoter contained in the aforementioned plasmid pRM5, i.e., the actI/actIII promoter pair and the actII-ORF4 activator gene, is particularly preferred. Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage. In addition, the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention. In this system, the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter. As noted above, one or more activator genes can also be employed to activate initiation of transcription at promoter sequences. Activator genes in addition to the actII-ORF4 gene described above include dnrI, redD, and ptpA genes (see U.S. patent application Ser. No. 09/181,833, supra). [0063]
  • To provide a preferred host cell and vector for purposes of the invention, the oleandolide PKS genes were placed on a recombinant expression vector that was transferred to the non-macrolide producing host [0064] Streptomyces lividans K4-114, as described in Example 4. Transformation of S. lividans K4-114 (strain K4-155 can also be used) with this expression vector resulted in a strain which produced detectable amounts of 8,8a-deoxyoleandolide as determined by analysis of extracts by LC/MS.
  • Moreover, and as noted in the preceding Section, the present invention also provides recombinant DNA compounds in which the encoded [0065] oleandolide module 1 KS domain is inactivated or absent altogether. Example 4 below describes the introduction into Streptomyces lividans of a recombinant expression vector of the invention that encodes an oleandolide PKS with a KS1o domain. The resulting host cells can be fed or supplied with N-acylcysteamine thioesters of precursor molecules to prepare oleandolide derivatives. Such cells of the invention are especially useful in the production of 13-substituted-6-deoxyerythronolide B compounds in recombinant host cells. Preferred compounds of the invention include those compounds in which the substituent at the 13-position is propyl, vinyl, propargyl, other lower alkyl, and substituted alkyl. The unmodified polyketides, called macrolide aglycones, produced in S. lividans K4-114 or K4-155 can be hydroxylated and glycosylated by adding them to the fermentation of a strain, such as, for example, S. antibioticus or Saccharopolyspora erythraea, that contains the requisite modification enzymes.
  • There are a wide variety of diverse organisms that can modify macrolide aglycones to provide compounds with, or that can be readily modified to have, useful activities. For example, [0066] Saccharopolyspora erythraea can convert 6-dEB and oleandolide to a variety of useful compounds. The erythronolide 6-dEB is converted by the eryF gene 2product to erythronolide B, which is, in turn, glycosylated by the eryB gene product to obtain 3-O-mycarosylerythronolide B, which contains L-mycarose at C-3. The enzyme eryC gene product then converts this compound to erythromycin D by glycosylation with D-desosamine at C-5. Erythromycin D, therefore, differs from 6-dEB through glycosylation and by the addition of a hydroxyl group at C-6. Erythromycin D can be converted to erythromycin B in a reaction catalyzed by the eryG gene product by methylating the L-mycarose residue at C-3. Erythromcyin D is converted to erythromycin C by the addition of a hydroxyl group at C-12 in a reaction catalyzed by the eryK gene product. Erythromycin A is obtained from erythromycin C by methylation of the mycarose residue in a reaction catalyzed by the eryG gene product.
  • The unmodified oleandolide compounds provided by the present invention, such as, for example, the oleandolide produced in [0067] Streptomyces lividans, can be provided to cultures of Saccharopolyspora erythraea and converted to the corresponding derivatives of erythromycins A, B, C, and D in accordance with the procedure provided in Example 6, below. To ensure that only the desired compound is produced, one can use an S. erythraea eryA mutant that is unable to produce 6-dEB but can still carry out the desired conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433). Also, one can employ other mutant strains, such as eryB, eryC, eryG, and/or eryK mutants, or mutant strains having mutations in multiple genes, to accumulate a preferred compound. The conversion can also be carried out in large fermentors for commercial production.
  • Moreover, there are other useful organisms that can be employed to hydroxylate and/or glycosylate the compounds of the invention. As described above, the organisms can be mutants unable to produce the polyketide normally produced in that organism, the fermentation can be carried out on plates or in large fermentors, and the compounds produced can be chemically altered after fermentation. Thus, [0068] Streptomyces venezuelae, which produces picromycin, contains enzymes that can transfer a desosaminyl group to the C-5 hydroxyl and a hydroxyl group to the C-12 position. In addition, S. venezuelae contains a glucosylation activity that glucosylates the 2′-hydroxyl group of the desosamine sugar. This latter modification reduces antibiotic activity, but the glucosyl residue is removed by cellular enzymatic action. Another organism, S. narbonensis, contains the same modification enzymes as S. venezuelae, except the C-12 hydroxylase. Thus, the present invention provides the compounds produced by hydroxylation and glycosylation of the macrolide aglycones of the invention by action of the enzymes endogenous to S. narbonensis and S. venezuelae.
  • Other organisms suitable for making compounds of the invention include [0069] Streptomyces antibioticus (discussed in the preceding Section), Micromonospora megalomicea, S. ftadiae, and S. thermotolerans. M. megalomicea produces megalomicin and contains enzymes that hydroxylate the C-6 and C-12 positions and glycosylate the C-3 hydroxyl with mycarose, the C-5 hydroxyl with desosamine, and the C-6 hydroxyl with megosamine (also known as rhodosamine), as well as acylating various positions. In addition to antibiotic activity, compounds of the invention produced by treatment with M. megalomicea enzymes can have antiparasitic activity as well. S. ftadiae contains enzymes that glycosylate the C-5 hydroxyl with mycaminose and then the 4′-hydroxyl of mycaminose with mycarose, forming a disaccharide. S. thermotolerans contains the same activities as well as acylation activities. Thus, the present invention provides the compounds produced by hydroxylation and glycosylation of the macrolide aglycones of the invention by action of the enzymes endogenous to S. antibioticus, M. megalomicea, S. fradiae, and S. thermotolerans.
  • The present invention also provides methods and genetic constructs for producing the glycosylated and/or hydroxylated compounds of the invention directly in the host cell of interest. Thus, the recombinant genes of the invention, which include recombinant oleAI, oleAII, and oleAIII genes with one or more deletions and/or insertions, including replacements of an oleA gene fragment with a gene fragment from a heterologous PKS gene (as discussed in the next Section), can be included on expression vectors suitable for expression of the encoded gene products in [0070] Saccharopolyspora erythraea, Streptomyces antibioticus, S. venezuelae, S. narbonensis, Micromonospora megalomicea, S. fradiae, and S. thermotolerans. A number of erythromycin high-producing strains of S. erythraea have been developed, and in a preferred embodiment, the oleandolide PKS genes are introduced into such strains (or erythromycin non-producing mutants thereof) to provide the corresponding modified oleandolide compounds in high yields.
  • Moreover, additional recombinant gene products can be expressed in the host cell to improve production of a desired polyketide. As but one non-limiting example, certain recombinant PKS proteins of the invention may produce a polyketide other than or in addition to the predicted polyketide, because the polyketide is cleaved from the PKS by the thioesterase (TE) domain in [0071] module 6 prior to processing by other domains on the PKS, in particular, any KR, DH, and/or ER domains in module 6. The production of the predicted polyketide can be increased in such instances by deleting the TE domain coding sequences from the gene and, optionally, expressing the TE domain as a separate protein. See Gokhale et al., February 1999, “Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase,” Chem. & Biol. 6: 117-125, incorporated herein by reference.
  • Thus, in one important aspect, the present invention provides methods, expression vectors, and recombinant host cells that enable the production of oleandolide and hydroxylated and glycosylated derivatives of oleandolide in heterologous host cells. The present invention also provides methods for making a wide variety of polyketides derived in part from the oleandolide PKS, as described in the following Section. [0072]
  • Section III: Hybrid PKS Genes [0073]
  • The present invention provides recombinant DNA compounds encoding each of the domains of each of the modules of the oleandolide PKS. The availability of these compounds permits their use in recombinant procedures for production of desired portions of the oleandolide PKS fused to or expressed in conjunction with all or a portion of a heterologous PKS. The resulting hybrid PKS can then be expressed in a host cell to produce a desired polyketide. [0074]
  • Thus, in accordance with the methods of the invention, a portion of the oleandolide PKS coding sequence that encodes a particular activity can be isolated and manipulated, for example, to replace the corresponding region in a different modular PKS. In addition, coding sequences for individual modules of the PKS can be ligated into suitable expression systems and used to produce the portion of the protein encoded. The resulting protein can be isolated and purified or can may be employed in situ to effect polyketide synthesis. Depending on the host for the recombinant production of the domain, module, protein, or combination of proteins, suitable control sequences such as promoters, termination sequences, enhancers, and the like are ligated to the nucleotide sequence encoding the desired protein in the construction of the expression vector, as described in the preceding Section. [0075]
  • In one important embodiment, the invention thus provides hybrid PKS enzymes and the corresponding recombinant DNA compounds that encode those hybrid PKS enzymes. For purposes of the invention, a hybrid PKS is a recombinant PKS that comprises all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a first PKS and all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a second PKS. In one preferred embodiment, the first PKS is most but not all of the oleandolide PKS, and the second PKS is only a portion or all of a non-oleandolide PKS. An illustrative example of such a hybrid PKS includes an oleandolide PKS in which the oleandolide PKS loading module has been replaced with a loading module of another PKS. Another example of such a hybrid PKS is an oleandolide PKS in which the AT domain of [0076] extender module 3 is replaced with an AT domain that binds only malonyl CoA. In another preferred embodiment, the first PKS is most but not all of a non-oleandolide PKS, and the second PKS is only a portion or all of the oleandolide PKS. An illustrative example of such a hybrid PKS includes a rapamycin PKS in which an AT specific for malonyl CoA is replaced with the AT from the oleandolide PKS specific for methylmalonyl CoA. Other illustrative hybrid PKSs of the invention are described below.
  • Those of skill in the art will recognize that all or part of either the first or second PKS in a hybrid PKS of the invention need not be isolated from a naturally occurring source. For example, only a small portion of an AT domain determines its specificity. See PCT patent application No. WO US99/15047, and Lau et al., infra, incorporated herein by reference. The state of the art in DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain. Thus, the desired derivative coding sequences can be synthesized using standard solid phase synthesis methods such as those described by Jaye et al., 1984, [0077] J. Biol. Chem. 259: 6331, and instruments for automated synthesis are available commercially from, for example, Applied Biosystems, Inc. For purposes of the invention, such synthetic DNA compounds are deemed to be a portion of a PKS.
  • With this general background regarding hybrid PKSs of the invention, one can better appreciate the benefit provided by the DNA compounds of the invention that encode the individual domains, modules, and proteins that comprise the oleandolide PKS. As described above, the oleandolide PKS is comprised of a loading module, six extender modules composed of a KS, AT, ACP, and KR, DH, and ER domains, and a thioesterase domain. The DNA compounds of the invention that encode these domains individually or in combination are useful in the construction of the hybrid PKS encoding DNA compounds of the invention. [0078]
  • The recombinant DNA compounds of the invention that encode the loading module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS protein or portion thereof. The resulting construct, in which the coding sequence for the loading module of the heterologous PKS is replaced by that for the coding sequence of the oleandolide PKS loading module provides a novel PKS. Examples include the 6-deoxyerythronolide B, rapamycin, FK-506, FK-520, rifamycin, and avermectin PKS protein coding sequences. In another embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0079]
  • In another embodiment, a portion of the loading module coding sequence is utilized in conduction with a heterologous coding sequence. In this embodiment, the invention provides, for example, replacing the malonyl CoA (acetyl CoA) specific AT with a propionyl CoA (methylmalonyl), butyryl CoA (ethylmalonyl), or other CoA specific AT. In addition, the KS[0080] Q and/or ACP can be replaced by another inactivated KS and/or another ACP. Alternatively, the KSQ and AT of the loading module can be replaced by an AT of a loading module such as that of DEBS. The resulting heterologous loading module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide.
  • The recombinant DNA compounds of the invention that encode the first extender module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS first extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the first extender module of the oleandolide PKS or the latter is merely added to coding sequences for modules of the heterologous PKS, provides a novel PKS coding sequence. In another embodiment, a DNA compound comprising a sequence that encodes the first extender module of the oleandolide PKS is inserted into a DNA compound that comprises coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0081]
  • In another embodiment, a portion or all of the first extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting (which includes inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a gene for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis. The resulting heterologous first extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide. [0082]
  • Those of skill in the art will recognize, however, that deletion of the KR domain of [0083] module 1 or insertion of a DH domain or DH and KR domains into module 1 will prevent the typical cyclization of the polyketide at the hydroxyl group created by the KR if such hybrid module is employed as a first extender module in a hybrid PKS or is otherwise involved in producing a portion of the polyketide at which cyclization is to occur. Such deletions or insertions can be useful, however, to create linear molecules or to induce cyclization at another site in the molecule.
  • As noted above, the invention also provides recombinant PKSs and recombinant DNA compounds and vectors that encode a PKS protein in which the KS domain of the first extender module has been inactivated. Such constructs are especially useful when placed in translational reading frame with the remaining modules and domains of an oleandolide or oleandolide derivative PKS, a hybrid PKS, or a heterologous PKS. The utility of these constructs is that host cells expressing, or cell free extracts containing, the PKS encoded thereby can be fed or supplied with N-acylcysteamine thioesters of precursor molecules to prepare oleandolide derivative compounds. See U.S. patent application Serial No. 60/117,384, filed Jan. 27, 1999, and PCT publication Nos. WO 99/03986 and 97/02358, each of which is incorporated herein by reference. [0084]
  • The recombinant DNA compounds of the invention that encode the second extender module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS second extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the second extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the second extender module of the oleandolide PKS is inserted into a DNA compound that comprises the coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0085]
  • In another embodiment, a portion or all of the second extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting (or inactivating) the KR; replacing the KR with a KR, a KR and a DH, or a KR, DH, and ER; and/or inserting a DH or a DH and an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis. The resulting heterologous second extender module coding sequence can be utilized in conjunction with a coding sequence from a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide. [0086]
  • The recombinant DNA compounds of the invention that encode the third extender module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS third extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the third extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the third extender module of the oleandolide PKS is inserted into a DNA compound that comprises coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0087]
  • In another embodiment, a portion or all of the third extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the inactive KR; and/or replacing the KR with an active KR, or a KR and DH, or a KR, DH, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a gene for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis. The resulting heterologous third extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide. [0088]
  • The recombinant DNA compounds of the invention that encode the fourth extender module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS fourth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fourth extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the fourth extender module of the oleandolide PKS is inserted into a DNA compound that comprises coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0089]
  • In another embodiment, a portion of the fourth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting or inactivating any one, two, or all three of the ER, DIL, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS (except for the DH and ER domains), from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis. The resulting heterologous fourth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide. [0090]
  • The recombinant DNA compounds of the invention that encode the fifth extender module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS fifth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the fifth extender module of the oleandolide PKS is inserted into a DNA compound that comprises the coding sequence for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0091]
  • In another embodiment, a portion or all of the fifth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting (or inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis. The resulting heterologous fifth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide. [0092]
  • The recombinant DNA compounds of the invention that encode the sixth extender module of the oleandolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the oleandolide PKS sixth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth extender module of the oleandolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the sixth extender module of the oleandolide PKS is inserted into a DNA compound that comprises the coding sequences for the oleandolide PKS or a recombinant oleandolide PKS that produces an oleandolide derivative. [0093]
  • In another embodiment, a portion or all of the sixth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting or inactivating the KR or replacing the KR with another KR, a KR and DH, or a KR, DH, and an ER; and/or inserting a DH or a DH and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the oleandolide PKS, from a coding sequence for a PKS that produces a polyketide other than oleandolide, or from chemical synthesis. The resulting heterologous sixth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes oleandolide, an oleandolide derivative, or another polyketide. [0094]
  • The sixth extender module of the oleandolide PKS is followed by a thioesterase domain. This domain is important in the cyclization of the polyketide and its cleavage from the PKS. The present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the oleandolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the oleandolide synthase thioesterase. Thus, for example, a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth (or other final) extender module coding sequence in recombinant DNA compounds of the invention or the oleandolide PKS thioesterase can be similarly fused to a heterologous PKS. Recombinant DNA compounds encoding this thioesterase domain are useful in constructing DNA compounds that encode the oleandolide PKS, a PKS that produces an oleandolide derivative, and a PKS that produces a polyketide other than oleandolide or an oleandolide derivative. [0095]
  • Thus, the hybrid modules of the invention are incorporated into a PKS to provide a hybrid PKS of the invention. A hybrid PKS of the invention can result not only: [0096]
  • (i) from fusions of heterologous domain (where heterologous means the domains in that module are from at least two different naturally occurring modules) coding sequences to produce a hybrid module coding sequence contained in a PKS gene whose product is incorporated into a PKS, but also: [0097]
  • (ii) from fusions of heterologous module (where heterologous module means two modules are adjacent to one another that are not adjacent to one another in naturally occurring PKS enzymes) coding sequences to produce a hybrid coding sequence contained in a PKS gene whose product is incorporated into a PKS, [0098]
  • (iii) from expression of one or more oleandolide PKS genes with one or more non-oleandolide PKS genes, including both naturally occurring and recombinant non-oleandolide PKS genes, and [0099]
  • (iv) from combinations of the foregoing. [0100]
  • Various hybrid PKSs of the invention illustrating these various alternatives are described herein. [0101]
  • An example of a hybrid PKS comprising fused modules results from fusion of the loading module of either DEBS or the narbonolide PKS (see PCT patent application No. US99/11814, incorporated herein by reference) with [0102] extender modules 1 and 2 of the oleandolide PKS to produce a hybrid oleAI gene. Co-expression of either one of these two hybrid oleAI genes with the oleAII and oleAIII genes in suitable host cells, such as Streptomcyes lividans, results in expression of a hybrid PKS of the invention that produces 6-deoxyerythronolide B in recombinant host cells. Co-expression of either one of these two hybrid oleAI genes with the eryAII and eryAIII genes similarly results in the production of 6-dEB, while co-expression with the analogous narbonolide PKS genes (picAII and picAIII) results in the production of 3-keto-6-dEB.
  • Another example of a hybrid PKS comprising a hybrid module is prepared by co-expressing the oleAI and oleAII genes with an oleAIII hybrid gene [0103] encoding extender module 5 and the KS and AT of extender module 6 of the oleandolide PKS fused to the ACP of extender module 6 and the TE of the narbonolide PKS. The resulting hybrid PKS of the invention produces 3-deoxy-3-oxo-8,8a-deoxyoleandolide (3-keto-oleandolide). This compound is useful in the production of 14-desmethyl ketolides, compounds with potent anti-bacterial activity. This compound can also be prepared by a recombinant oleandolide derivative PKS of the invention in which the KR domain of module 6 of the oleandolide PKS has been deleted or replaced with an inactive KR domain. Moreover, the invention provides hybrid PKSs in which not only the above changes have been made but also the AT domain of module 6 has been replaced with a malonyl-specific AT. These hybrid PKSs produce 2-desmethyl-3-deoxy-3-oxo-8,8a-deoxyoleandolide, a useful intermediate in the preparation of 2,14-didesmethyl ketolides, compounds with potent antibiotic activity.
  • Another illustrative example of a hybrid PKS includes the hybrid PKS of the invention resulting only from the latter change in the hybrid PKS just described. Thus, co-expression of the oleAI and oleAII genes with a hybrid oleAIII gene in which the AT domain of [0104] module 6 has been replaced by a malonyl-specific AT results in the expression of a hybrid PKS that produces 2-desmethyl-8,8a-deoxyoleandolide in recombinant host cells. This compound is a useful intermediate for making 2,14-didesmethyl erythromycins in recombinant host cells of the invention.
  • While many of the hybrid PKSs described above are composed primarily of oleandolide PKS proteins, those of skill in the art recognize that the present invention provides many different hybrid PKSs, including those composed of only a small portion of the oleandolide PKS. For example, the present invention provides a hybrid PKS in which a hybrid oleAI gene that encodes the oleandolide loading module fused to [0105] extender modules 1 and 2 of DEBS is coexpressed with the eryAII and eryAIII genes. The resulting hybrid PKS produces 8,8a-deoxyoleandolide. When the construct is expressed in Saccharopolyspora erythraea host cells (either via chromosomal integration in the chromosome or via a vector that encodes the hybrid PKS), the resulting recombinant host cell of the invention produces 14-desmethyl erythromycins. Another illustrative example is the hybrid PKS of the invention composed of the oleAI and eryAII and eryAIII gene products. This construct is also useful in expressing 14-desmethyl erythromycins in Saccharopolyspora erythraea host cells, as described in Example 3, below. In a preferred embodiment, the S. erythraea host cells are eryAI mutants that do not produce 6-deoxyerythronolide B.
  • Another example is the hybrid PKS of the invention composed of the products of the picAI and picAII genes (the two proteins that comprise the loading module and extender modules 1-4, inclusive, of the narbonolide PKS) and the oleAIII gene. The resulting hybrid PKS produces the macrolide aglycone 3-hydroxy-narbonolide in [0106] Streptomyces lividans host cells and the corresponding erythromycins in Saccharopolyspora erythraea host cells. This hybrid PKS of the invention is described in Example 5, below.
  • Each of the foregoing hybrid PKS enzymes of the invention, and the hybrid PKS enzymes of the invention generally, can be expressed in a host cell that also expresses a functional oleP gene product. Such expression provides the compounds of the invention in which the C-8-C-8a epoxide is present. [0107]
  • The following Table lists references describing illustrative PKS genes and corresponding enzymes that can be utilized in the construction of the recombinant hybrid PKSs and the corresponding DNA compounds that encode them of the invention. Also presented are various references describing tailoring enzymes and corresponding genes that can be employed in accordance with the methods of the invention. [0108]
  • Avermectin [0109]
  • U.S. Pat. No. 5,252,474 to Merck. [0110]
  • MacNeil et al., 1993, [0111] Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.
  • MacNeil et al., 1992, [0112] Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.
  • Candicidin (FR008) [0113]
  • Hu et al., 1994, [0114] Mol. Microbiol. 14: 163-172.
  • Epothilone [0115]
  • U.S. patent application Serial No. 60/130,560, filed Apr. 22, 1999, and Serial No. 60/122,620, filed Mar. 3, 1999. [0116]
  • Erythromycin [0117]
  • PCT Pub. No. 93/13663 to Abbott. [0118]
  • U.S. Pat. No. 5,824,513 to Abbott. [0119]
  • Donadio et al., 1991, [0120] Science 252:675-9.
  • Cortes et al., Nov. 8, 1990, [0121] Nature 348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea.
  • Glycosylation Enzymes [0122]
  • PCT Pat. App. Pub. No. 97/23630 to Abbott. [0123]
  • FK-506 [0124]
  • Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506, [0125] Eur. J biochem. 256: 528-534.
  • Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506, [0126] Eur. J. Biochem. 244: 74-80.
  • Methyltransferase [0127]
  • U.S. Pat. No. 5,264,355, issued Nov. 23, 1993, Methylating enzyme from Streptomyces MA6858. 31 -O-desmethyl-FK506 methyltransferase. [0128]
  • Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520, [0129] J. Bacteriol. 178: 5243-5248.
  • FK-520 [0130]
  • U.S. patent application Serial No. 60/139,650, filed Jun. 17, 1999, and No. 60/123,810, filed Mar. 11, 1999. See also Nielsen et al., 1991, [0131] Biochem. 30:5789-96 (enzymology of pipecolate incorporation).
  • Lovastatin [0132]
  • U.S. Pat. No. 5,744,350 to Merck. [0133]
  • Narbomycin (and Picromycin) [0134]
  • PCT patent application No. WO US99/11814, filed May 28, 1999. [0135]
  • Nemadectin [0136]
  • MacNeil et al., 1993, supra. [0137]
  • Niddamycin [0138]
  • Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from [0139] Streptomyces caelestis, J. Bacteriol. 179: 7515-7522.
  • Platenolide [0140]
  • EP Pat. App. Pub. No. 791,656 to Lilly. [0141]
  • Rapamycin [0142]
  • Schwecke et al., August 1995, The biosynthetic gene cluster for the polyketide rapamycin, [0143] Proc. Natl. Acad. Sci. USA 92:7839-7843.
  • Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in [0144] Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.
  • Rifamycin [0145]
  • August et al., Feb. 13, 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of [0146] Amycolatopsis mediterranei S669, Chemistry & Biology, 5(2): 69-79.
  • Soraphen [0147]
  • U.S. Pat. No. 5,716,849 to Novartis. [0148]
  • Schupp et al., 1995, [0149] J. Bacteriology 177: 3673-3679. A Sorangium cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.
  • Spiramycin [0150]
  • U.S. Pat. No. 5,098,837 to Lilly. [0151]
  • Activator Gene [0152]
  • U.S. Pat. No. 5,514,544 to Lilly. [0153]
  • Tylosin [0154]
  • EP Pub. No. 791,655 to Lilly. [0155]
  • Kuhstoss et al., 1996, [0156] Gene 183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.
  • U.S. Pat. No. 5,876,991 to Lilly. [0157]
  • Tailoring enzymes [0158]
  • Merson-Davies and Cundliffe, 1994, [0159] Mol. Microbiol 13: 349-355. Analysis of five tylosin biosynthetic genes from the tylBA region of the Streptomyces fradiae genome.
  • As the above Table illustrates, there are a wide variety of PKS genes that serve as readily available sources of DNA and sequence information for use in constructing the hybrid PKS-encoding DNA compounds of the invention. Methods for constructing hybrid PKS-encoding DNA compounds are described without reference to the oleandolide PKS in U.S. Pat. Nos. 5,672,491 and 5,712,146 and PCT publication No. 98/49315, each of which is incorporated herein by reference. [0160]
  • In constructing hybrid PKSs of the invention, certain general methods may be helpful. For example, it is often beneficial to retain the framework of the module to be altered to make the hybrid PKS. Thus, if one desires to add DH and ER functionalities to a module, it is often preferred to replace the KR domain of the original module with a KR, DH, and ER domain-containing segment from another module, instead of merely inserting DH and ER domains. One can alter the stereochemical specificity of a module by replacement of the KS domain with a KS domain from a module that specifies a different stereochemistry. See Lau et al., 1999, “Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units” [0161] Biochemistry 38(5):1643-1651, incorporated herein by reference. One can alter the specificity of an AT domain by changing only a small segment of the domain. See Lau et al., supra. One can also take advantage of known linker regions in PKS proteins to link modules from two different PKSs to create a hybrid PKS. See Gokhale et al., Apr. 16, 1999, “Dissecting and Exploiting Intermodular Communication in Polyketide Synthases”, Science 284: 482-485, incorporated herein by reference.
  • The hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes. Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene. Thus, as one illustrative example, the invention provides a hybrid PKS that contains the naturally occurring loading module and thioesterase domain as well as extender modules one, two, four, and six of the oleandolide PKS and further contains hybrid or heterologous extender modules three and five. Hybrid or heterologous extender modules three and five contain AT domains specific for malonyl CoA and derived from, for example, the rapamycin PKS genes. [0162]
  • To construct a hybrid PKS or oleandolide PKS of the invention, one can employ a technique, described in PCT Pub. No. 98/27203 and U.S. provisional patent application Serial No. 60/129,731, filed Apr. 16, 1999, incorporated herein by reference, in which the large oleandolide PKS gene cluster is divided into two or more, typically three, segments, and each segment is placed on a separate expression vector. In this manner, each of the segments of the gene can be altered, and various altered segments can be combined in a single host cell to provide a recombinant PKS gene of the invention. This technique makes more efficient the construction of large libraries of recombinant PKS genes, vectors for expressing those genes, and host cells comprising those vectors. [0163]
  • The invention also provides libraries of PKS genes, PKS proteins, and ultimately, of polyketides, that are constructed by generating modifications in the oleandolide PKS so that the protein complexes produced have altered activities in one or more respects and thus produce polyketides other than the oleandolide natural product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method. By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities. As will be further described below, the metes and bounds of this embodiment of the invention can be described on the polyketide, protein, and the encoding nucleotide sequence levels. [0164]
  • As described above, a modular PKS “derived from” the oleandolide or other naturally occurring PKS includes a modular PKS (or its corresponding encoding gene(s)) that retains the scaffolding of the utilized portion of the naturally occurring gene. Not all modules need be included in the constructs; the constructs can include a loading module and six, fewer than six, or more than six extender modules. On the constant scaffold, at least one enzymatic activity is mutated, deleted, replaced, or inserted so as to alter the activity of the resulting PKS relative to the original PKS. Alteration results when these activities are deleted or are replaced by a different version of the activity, or simply mutated in such a way that a polyketide other than the natural product results from these collective activities. This occurs because there has been a resulting alteration of the starter unit and/or extender unit, stereochemistry, chain length or cyclization, and/or reductive or dehydration cycle outcome at a corresponding position in the product polyketide. Where a deleted activity is replaced, the origin of the replacement activity may come from a corresponding activity in a different naturally occurring PKS or from a different region of the oleandolide PKS. Any or all of the oleandolide PKS genes may be included in the derivative or portions of any of these may be included, but the scaffolding of the PKS protein is retained in whatever derivative is constructed. The derivative preferably contains a thioesterase activity from the oleandolide or another PKS. [0165]
  • Thus, a PKS derived from the oleandolide PKS includes a PKS that contains the scaffolding of all or a portion of the oleandolide PKS. The derived PKS also contains at least two extender modules that are functional, preferably three extender modules, and more preferably four or more extender modules, and most preferably six extender modules. The derived PKS also contains mutations, deletions, insertions, or replacements of one or more of the activities of the functional modules of the oleandolide PKS so that the nature of the resulting polyketide is altered at both the protein and DNA sequence levels. Particular preferred embodiments include those wherein a KS, AT, or ACP domain has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS. Also preferred are derivatives where at least one non-condensation cycle enzymatic activity (KR, DH, or ER) has been deleted or added or wherein any of these activities has been mutated so as to change the structure of the polyketide synthesized by the PKS. [0166]
  • Conversely, also included within the definition of a PKS derived from the oleandolide PKS are functional non-oleandolide PKS modules or their encoding genes wherein at least one portion, or two or more portions, of the oleandolide PKS activities have been inserted. Exemplary is the use of the oleandolide AT for [0167] extender module 2, which accepts a methylmalonyl CoA extender unit rather than malonyl CoA, to replace a malonyl specific AT in another PKS. Other examples include insertion of portions of non-condensation cycle enzymatic activities or other regions of oleandolide synthase activity into a heterologous PKS at both the DNA and protein levels.
  • Thus, there are at least five degrees of freedom for constructing a hybrid PKS in terms of the polyketide that will be produced. First, the polyketide chain length is determined by the number of modules in the PKS, and the present invention includes hybrid PKSs that contain a loading module and 6, as well as fewer or more than 6, extender modules. Second, the nature of the carbon skeleton of the PKS is determined by the specificities of the acyl transferases that determine the nature of the extender units at each position, e.g., malonyl, methylmalonyl, ethylmalonyl, or other substituted malonyl. Third, the loading module specificity also has an effect on the resulting carbon skeleton of the polyketide. The loading module may use a different starter unit, such as priopionyl, butyryl, and the like. As noted above and in the examples below, another method for varying loading module specificity involves inactivating the KS activity in extender module 1 (KS1) and providing alternative substrates, called diketides, that are chemically synthesized analogs of [0168] extender module 1 diketide products, for extender module 2. This approach was illustrated in PCT publication Nos. 97/02358 and 99/03986, incorporated herein by reference, wherein the KS1 activity was inactivated through mutation. Fourth, the oxidation state at various positions of the polyketide will be determined by the dehydratase and reductase portions of the modules. This will determine the presence and location of ketone and alcohol moieties and C-C double bonds or C-C single bonds in the polyketide. Finally, the stereochemistry of the resulting polyketide is a function of three aspects of the synthase. The first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase, as the dehydratase would abolish chirality. Second, the specificity of the ketoreductase may determine the chirality of any beta-OH. Finally, the enoylreductase specificity for substituted malonyls as extender units may influence the stereochemistry when there is a complete KR/DH/ER available.
  • Thus, the modular PKS systems generally and the oleandolide PKS system particularly permit a wide range of polyketides to be synthesized. As compared to the aromatic PKS systems, the modular PKS systems accept a wider range of starter units, including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl). Certain modular PKSs have relaxed specificity for their starter units (Kao et al., 1994, [0169] Science, supra). Modular PKSs also exhibit considerable variety with regard to the choice of extender units in each condensation cycle. The degree of beta-ketoreduction following a condensation reaction has also been shown to be altered by genetic manipulation (Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc. Natl. Acad Sci. USA 90: 7119-7123). Likewise, the size of the polyketide product can be varied by designing mutants with the appropriate number of modules (Kao et al., 1994, J. Am. Chem. Soc. 116:11612-11613). Lastly, modular PKS enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner. The polyketides, antibiotics, and other compounds produced by the methods of the invention are typically single stereoisomeric forms. Although the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers. Thus, the combinatorial potential within modular PKS pathways based on any naturally occurring modular, such as the oleandolide, PKS scaffold is virtually unlimited.
  • While hybrid PKSs are most often produced by “mixing and matching” portions of PKS coding sequences, mutations in DNA encoding a PKS can also be used to introduce, alter, or delete an activity in the encoded polypeptide. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. See, e.g., Kunkel, 1985, [0170] Proc. Natl. Acad. Sci. USA 82: 448; Geisselsoder et al., 1987, BioTechniques 5:786. Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence, at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See Zoller and Smith, 1983, Methods Enzymol. 100:468. Primer extension is effected using DNA polymerase, the product cloned, and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Identification can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., 1982, Proc. Natl. Acad. Sci. USA 79: 6409. PCR mutagenesis can also be used to effect the desired mutations.
  • Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can also be accomplished by several different techniques known in the art, e.g., by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants, or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA fragments are treated with chemical mutagens, transformed into [0171] E. coli and propagated as a pool or library of mutant plasmids.
  • In constructing a hybrid PKS of the invention, regions encoding enzymatic activity, i.e., regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS, can be recovered, for example, using PCR techniques with appropriate primers. By “corresponding” activity encoding regions is meant those regions encoding the same general type of activity. For example, a KR activity encoded at one location of a gene cluster “corresponds” to a KR encoding activity in another location in the gene cluster or in a different gene cluster. Similarly, a complete reductase cycle could be considered corresponding. For example, KR/DH/ER can correspond to a KR alone. [0172]
  • If replacement of a particular target region in a host PKS is to be made, this replacement can be conducted in vitro using suitable restriction enzymes. The replacement can also be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene in a donor plasmid and a receptor region in a recipient plasmid. Such systems, advantageously involving plasmids of differing temperature sensitivities are described, for example, in PCT publication No. WO 96/40968, incorporated herein by reference. The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes can be chosen to contain control sequences operably linked to the resulting coding sequences in a manner such that expression of the coding sequences can be effected in an appropriate host. [0173]
  • However, simple cloning vectors may be used as well. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into expression vectors, the resulting vectors transformed or transfected into host cells, and the resulting cells plated out into individual colonies. The invention provides a variety of recombinant DNA compounds in which the various coding sequences for the domains and modules of the oleandolide PKS are flanked by non-naturally occurring restriction enzyme recognition sites. [0174]
  • The various PKS nucleotide sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter. The PKS subunit encoding regions can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunit encoding sequences so that hybrid PKSs can be generated. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR. [0175]
  • The expression vectors containing nucleotide sequences encoding a variety of PKS enzymes for the production of different polyketides are then transformed into the appropriate host cells to construct the library. In one straightforward approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected to identify successful transformants. Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide. Typically, there will be duplications in some, most, or all of the colonies; the subset of the transformed colonies that contains a different PKS in each member colony can be considered the library. Alternatively, the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library. A variety of strategies are available to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide. The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, and more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length enables the production of quite large libraries. [0176]
  • Methods for introducing the recombinant vectors of the invention into suitable hosts are known to those of skill in the art and typically include the use of CaCl[0177] 2 or agents such as other divalent cations, lipofection, DMSO, PEG, protoplast transformation, infection, transfection, and electroporation. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.
  • The libraries of the invention can thus be considered at four levels: (1) a multiplicity of colonies each with a different PKS encoding sequence; (2) the proteins produced from the coding sequences; (3) the polyketides produced from the proteins assembled into a functional PKS; and (4) antibiotics or compounds with other desired activities derived from the polyketides. Combination libraries can also be constructed wherein members of a library derived, for example, from the oleandolide PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS or DEBS. [0178]
  • Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of polyketides. Polyketides that are secreted into the media or have been otherwise isolated can be screened for binding to desired targets, such as receptors, signaling proteins, and the like. The supernatantsper se can be used for screening, or partial or complete purification of the polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can more readily be included. Antibiotic activity can be verified using typical screening assays such as those set forth in Lehrer et al., 1991, [0179] J. Immunol. Meth. 137:167-173, incorporated herein by reference, and in Example 7, below.
  • The invention provides methods for the preparation of a large number of polyketides. These polyketides are useful intermediates in formation of compounds with antibiotic or other activity through hydroxylation and glycosylation reactions as described above. In general, the polyketide products of the PKS must be further modified, typically by hydroxylation and glycosylation, to exhibit antibiotic activity. Hydroxylation results in the novel polyketides of the invention that contain hydroxyl groups at C-6, which can be accomplished using the hydroxylase encoded by the eryF gene, and/or C-12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene. Also, the present invention provides the oleP gene in recombinant form, which can be used to express the oleP gene product in any host cell. A host cell, such as a Streptomyces host cell or a [0180] Saccharopolyspora erythraea host cell modified to express the oleP gene thus can be used to produce polyketides comprising the C-8-C-8a epoxide present in oleandomycin. Thus the invention provides such modified polyketides. The presence of hydroxyl groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart.
  • Methods for glycosylating the polyketides are generally known in the art; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in PCT publication No. WO 98/49315, incorporated herein by reference. Preferably, glycosylation with desosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention. In general, the approaches to effecting glycosylation mirror those described above with respect to hydroxylation. The purified enzymes, isolated from native sources or recombinantly produced may be used in vitro. Alternatively and as noted, glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosyl transferases. In addition, synthetic chemical methods may be employed. [0181]
  • The antibiotic modular polyketides may contain any of a number of different sugars, although D-desosamine, or a close analog thereof, is most common. Erythromycin, picromycin, narbomycin, and methymycin contain desosamine. Erythromycin also contains L-cladinose (3-0-methyl mycarose). Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose. 2-acetyl-1-bromodesosamine has been used as a donor to glycosylate polyketides by Masamune et al., 1975, [0182] J. Am. Chem. Soc. 97: 3512-3513. Other, apparently more stable donors include glycosyl fluorides, thioglycosides, and trichloroacetimidates; see Woodward et al., 1981, J. Am. Chem. Soc. 103: 3215; Martin et al., 1997, J. Am. Chem. Soc. 119: 3193; Toshima et al., 1995, J. Am. Chem. Soc. 117: 3717; Matsumoto et al., 1988, Tetrahedron Lett. 29: 3575. Glycosylation can also be effected using the polyketide aglycones as starting materials and using Saccharopolyspora erythraea, Streptomyces venezuelae or other host cells to make the conversion, preferably using mutants unable to synthesize macrolides, as discussed in the preceding Section.
  • Thus, a wide variety of polyketides can be produced by the hybrid PKS enzymes of the invention. These polyketides are useful as antibiotics and as intermediates in the synthesis of other useful compounds, as described in the following section. [0183]
  • Section IV: Compounds [0184]
  • The methods and recombinant DNA compounds of the invention are useful in the production of polyketides. In one important aspect, the invention provides methods for making antibiotic compounds related in structure to oleandomycin and erythromycin, both potent antibiotic compounds. The invention also provides novel ketolide compounds, polyketide compounds with potent antibiotic activity of significant interest due to activity against antibiotic resistant strains of bacteria. See Griesgraber et al., 1996, [0185] J. Antibiot. 49: 465-477, incorporated herein by reference. Most if not all of the ketolides prepared to date are synthesized using erythromycin A, a derivative of 6-dEB, as an intermediate. While the invention provides hybrid PKSs that produce a polyketide different in structure from 6-dEB, the invention also provides methods for making intermediates useful in preparing traditional, 6-dEB- and erythromycin-derived ketolide compounds.
  • Because 6-dEB in part differs from oleandolide in that it comprises a 13-ethyl instead of a 13-methyl group, the novel hybrid PKS genes of the invention based on the oleandolide PKS provide many novel ketolides that differ from the known ketolides only in that they have a 13-methyl instead of 13-ethyl group. Thus, the invention provides the 13-methyl analogues of the ketolides and intermediates and precursor compounds described in, for example, Griesgraber et al., supra; Agouridas et al., 1998, [0186] J. Med Chem. 41: 4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; 5,439,889; and PCT publication Nos. WO 98/09978 and 98/28316, each of which is incorporated herein by reference.
  • As noted above, the hybrid PKS genes of the invention can be expressed in a host cell that contains the desosamine biosynthetic genes and desosaminyl transferase gene as well as the required hydroxylase gene(s), which may be either pick (for the C-12 position) or eryK (for the C-12 position) and/or eryF (for the C-6 position). The resulting compounds have antibiotic activity but can be further modified, as described in the patent publications referenced above, to yield a desired compound with improved or otherwise desired properties. Alternatively, the aglycone compounds can be produced in the recombinant host cell, and the desired glycosylation and hydroxylation steps carried out in vitro or in vivo, in the latter case by supplying the converting cell with the aglycone. [0187]
  • The compounds of the invention are thus optionally glycosylated forms of the polyketide set forth in formula (1) below which are hydroxylated at either the C-6 or the C-12 or both. The compounds of formula (1) can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described. These polyketides have the formula: [0188]
    Figure US20030027287A1-20030206-C00002
  • including the glycosylated and isolated stereoisomeric forms thereof; [0189]
  • wherein [0190]
  • R* is a straight chain, branched or cyclic, saturated or unsaturated substituted or unsubstituted hydrocarbyl of 1-15C; [0191]
  • each of R[0192] 1-R6 is independently H or alkyl (1-4C) wherein any alkyl at R1 may optionally be substituted;
  • each of X[0193] 1-X5 is independently two H, H and OH, or =O; or
  • each of X[0194] 1-X5 is independently H and the compound of formula (2) contains a double-bond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11;
  • with the proviso that: [0195]
  • at least two of R[0196] 1-R6 are alkyl (1-4C).
  • Preferred [0197] compounds comprising formula 2 are those wherein at least three of R1-R5 are alkyl (1-4C), preferably methyl or ethyl; more preferably wherein at least four of R1-R5 are alkyl (1-4C), preferably methyl or ethyl. Also preferred are those wherein X2 is two H, =O, or H and OH, and/or X3 is H, and/or X1 is OH and/or X4 is OH and/or X5 is OH. Also preferred are compounds with variable R* when R1-R5 is methyl, X2 is =O, and X1, X4 and X5 are OH. The glycosylated forms of the foregoing are also preferred; glycoside residues can be attached at C-3, C-5, and/or C-6; the epoxidated forms are also included, i.e., and epoxide at C-8-C-8a.
  • As described above, there are a wide variety of diverse organisms that can modify compounds such as those described herein to provide compounds with or that can be readily modified to have useful activities. For example, [0198] Saccharopolyspora erythraea can convert oleandolide and 6-dEB to a variety of useful compounds. The compounds provided by the present invention can be provided to cultures of Saccharopolyspora erythraea and converted to the corresponding derivatives of erythromycins A, B, C, and D in accordance with the procedure provided in Example 6, below. To ensure that only the desired compound is produced, one can use an S. erythraea eryA mutant that is unable to produce 6-dEB but can still carry out the desired conversions (Weber et al., 1985, J. Bacteriol. 164(1): 425-433). Also, one can employ other mutant strains, such as eryB, eryC, eryG, and/or eryK mutants, or mutant strains having mutations in multiple genes, to accumulate a preferred compound. The conversion can also be carried out in large fermentors for commercial production. Each of the erythromycins A, B, C, and D has antibiotic activity, although erythromycin A has the highest antibiotic activity. Moreover, each of these compounds can form, under treatment with mild acid, a C-6 to C-9 hemiketal with motilide activity. For formation of hemiketals with motilide activity, erythromycins B, C, and D, are preferred, as the presence of a C-12 hydroxyl allows the formation of an inactive compound that has a hemiketal formed between C-9 and C-12.
  • Thus, the present invention provides the compounds produced by hydroxylation and glycosylation of the compounds of the invention by action of the enzymes endogenous to [0199] Saccharopolyspora erythraea and mutant strains of S. erythraea. Such compounds are useful as antibiotics or as motilides directly or after chemical modification. For use as antibiotics, the compounds of the invention can be used directly without further chemical modification. Erythromycins A, B, C, and D all have antibiotic activity, and the corresponding compounds of the invention that result from the compounds being modified by Saccharopolyspora erythraea also have antibiotic activity. These compounds can be chemically modified, however, to provide other compounds of the invention with potent antibiotic activity. For example, alkylation of erythromycin at the C-6 hydroxyl can be used to produce potent antibiotics (clarithromycin is C-6-O-methyl), and other useful modifications are described in, for example, Griesgraber et al., 1996, J. Antibiot. 49: 465-477, Agouridas et al., 1998, J. Med. Chem. 41: 4080-4100, U.S. Pat. Nos. 5,770,579; 5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; and 5,439,889; and PCT publication Nos. WO 98/09978 and 98/28316, each of which is incorporated herein by reference.
  • For use as motilides, the compounds of the invention can be used directly without further chemical modification. Erythromycin and certain erythromycin analogs are potent agonists of the motilin receptor that can be used clinically as prokinetic agents to induce phase III of migrating motor complexes, to increase esophageal peristalsis and LES pressure in patients with GERD, to accelerate gastric emptying in patients with gastric paresis, and to stimulate gall bladder contractions in patients after gallstone removal and in diabetics with autonomic neuropathy. See Peeters, 1999, Motilide Web Site, http://www.med.kuleuven. ac.be/med/gih/motilid.htm, and Omura et al., 1987, Macrolides with gastrointestinal motor stimulating activity, [0200] J. Med. Chem. 30: 1941-3). The corresponding compounds of the invention that result from the compounds of the invention being modified by Saccharopolyspora erythraea also have motilide activity, particularly after conversion, which can also occur in vivo, to the C-6 to C-9 hemiketal by treatment with mild acid. Compounds lacking the C-12 hydroxyl are especially preferred for use as motilin agonists. These compounds can also be further chemically modified, however, to provide other compounds of the invention with potent motilide activity.
  • Moreover, and also as noted above, there are other useful organisms that can be employed to hydroxylate and/or glycosylate the compounds of the invention. As described above, the organisms can be mutants unable to produce the polyketide normally produced in that organism, the fermentation can be carried out on plates or in large fermentors, and the compounds produced can be chemically altered after fermentation. In addition to [0201] Saccharopolyspora erythraea, Streptomyces venezuelae, S. narbonensis, S. antibioticus, Micromonospora megalomicea, S. fradiae, and S. thermotolerans can also be used. In addition to antibiotic activity, compounds of the invention produced by treatment with M. megalomicea enzymes can have antiparasitic activity as well. Thus, the present invention provides the compounds produced by hydroxylation and glycosylation by action of the enzymes endogenous to S. erythraea, S. venezuelae, S. narbonensis, S. antibioticus, M. megalomicea, S. fradiae, and S. thermotolerans.
  • The present invention also provides methods and genetic constructs for producing the glycosylated and/or hydroxylated compounds of the invention directly in the host cell of interest. Thus, the recombinant genes of the invention, which include recombinant oleAI, oleAII, and oleAIII genes with one or more deletions and/or insertions, including replacements of an oleA gene fragment with a gene fragment from a heterologous PKS gene, can be included on expression vectors suitable for expression of the encoded gene products in [0202] Saccharopolyspora erythraea, Micromonospora megalomicea, Streptomyces antibioticus, S. venezuelae, S. narbonensis, S. ftadiae, and S. thermotolerans.
  • Many of the compounds of the invention contain one or more chiral centers, and all of the stereoisomers are included within the scope of the invention, as pure compounds as well as mixtures of stereoisomers. Thus the compounds of the invention may be supplied as a mixture of stereoisomers in any proportion. [0203]
  • The compounds of the invention can be produced by growing and fermenting the host cells of the invention under conditions known in the art for the production of other polyketides. The compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures. The compounds can be readily formulated to provide the pharmaceutical compositions of the invention. The pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. [0204]
  • The carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used. For example, the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat. No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference. [0205]
  • Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, [0206] Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference. Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference. The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.
  • For the treatment of conditions and diseases caused by infection, a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intramuscular, and intrasternal injection or infusion techniques. [0207]
  • Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day. The dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient). In addition, the compounds of the invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals. [0208]
  • The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition. Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient. For external administration, the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight. [0209]
  • It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought. [0210]
  • The compounds of the invention can be used as single therapeutic agents or in combination with other therapeutic agents. Drugs that can be usefully combined with compounds of the invention include one or more antibiotic or motilide agents. [0211]
  • A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims. [0212]
    • EXAMPLE 1 General Methodology
  • Bacterial strains, plasmids, and culture conditions. [0213] Streptomyces coelicolor CH999 described in WO 95/08548, published Mar. 30, 1995, or S. lividans K4-114 or K4-155, described in Ziermann and Betlach, Jan. 1999, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, incorporated herein by reference, was used as an expression host. DNA manipulations were performed in Escherichia coli XL1-Blue, available from Stratagene. E. coli MC1061 is also suitable for use as a host for plasmid manipulation. Plasmids were passaged through E. coli ET12567 (dam dcm hsdS Cm1) (MacNeil, 1988, J. Bacteriol. 170: 5607, incorporated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor or Saccharopolyspora erythraea. E. coli strains were grown under standard conditions. S. coelicolor strains were grown on R2YE agar plates (Hopwood et al., Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985, incorporated herein by reference).
  • Many of the expression vectors of the invention illustrated in the examples are derived from plasmid pRM5, described in WO 95/08548, incorporated herein by reference. This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters, the actI and actIII promoters, to allow for bidirectional cloning, the gene encoding the actII-ORF4 activator which induces transcription from act promoters during the transition from growth phase to stationary phase, and appropriate marker genes. Engineered restriction sites in the plasmid facilitate the combinatorial construction of PKS gene clusters starting from cassettes encoding individual domains of naturally occurring PKSs. When plasmid pRM5 is used for expression of a PKS, all relevant biosynthetic genes can be plasmid-borne and therefore amenable to facile manipulation and mutagenesis in [0214] E. coli. This plasmid is also suitable for use in Streptomyces host cells. Streptomyces is genetically and physiologically well characterized and expresses the ancillary activities required for in vivo production of most polyketides. Plasmid pRM5 utilizes the act promoter for PKS gene expression, so polyketides are produced in a secondary metabolite-like manner, thereby alleviating the toxic effects of synthesizing potentially bioactive compounds in vivo.
  • Manipulation of DNA and organisms. Polymerase chain reaction (PCR) was performed using Pfu polymerase (Stratagene; Taq polymerase from Perkin Elmer Cetus can also be used) under conditions recommended by the enzyme manufacturer. Standard in vitro techniques were used for DNA manipulations (Sambrook et al. [0215] Molecular Cloning: A Laboratory Manual (Current Edition)). E. coli was transformed using standard calcium chloride-based methods; a Bio-Rad E. coli pulsing apparatus and protocols provided by Bio-Rad could also be used. S. coelicolor was transformed by standard procedures (Hopwood et al. Genetic manipulation of Streptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985), and depending on what selectable marker was employed, transformants were selected using 1 mL of a 1.5 mg/mL thiostrepton overlay, 1 mL of a 2 mg/mL apramycin overlay, or both.
    • EXAMPLE 2 Cloning of the Oleandomycin Biosynthetic Gene Cluster from Streptomyces antibioticus
  • Genomic DNA (100 μg) was isolated from an oleandomycin producing strain of [0216] Streptomyces antibioticus (ATCC 11891) using standard procedures. The genomic DNA was partially digested with restriction enzyme Sau3A1 to generate fragments ˜40 kbp in length, which were cloned into the commercially available Supercos™ cosmid vector that had been digested with restriction enzymes XbaI and Bam-HI to produce a genomic library. SuperCosI™ (Stratagene) DNA cosmid arms were prepared as directed by the manufacturer. A cosmid library was prepared by ligating 2.5 μg of the digested genomic DNA with 1.5 μg of cosmid arms in a 20 μL reaction. One microliter of the ligation mixture was propagated in E. coli XL1-Blue MR (Stratagene) using a GigapackIII XL packaging extract kit (Stratagene).
  • This library was then probed with a radioactively-labeled probe generated by PCR from [0217] Streptomyces antibioticus DNA using primers complementary to known sequences of KS domains hypothesized to originate from extender modules 5 and 6 of the oleandolide PKS. This probing identified about 30 different colonies, which were pooled, replated, and probed again, resulting in the identification of 9 cosmids. These latter cosmids were isolated and transformed into the commercially available E. coli strain XL-1 Blue. Plasmid DNA was isolated and analyzed by restriction enzyme digestion, which revealed that the entire PKS gene cluster was contained in overlapping segments on two of the cosmids identified. DNA sequence analysis using the T3 primer showed that the desired DNA had been isolated.
  • Further analysis of these cosmids and subclones prepared from the cosmids facilitated the identification of the location of various oleandolide PKS ORFs, modules in those ORFs, and coding sequences for oleandomycin modification enzymes. The location of these genes and modules is shown on FIG. 1. FIG. 1 shows that the complete oleandolide PKS gene cluster is contained within the insert DNA of cosmids pKOS055-1 (insert size of ˜43 kb) and pKOS055-5 (insert size of ˜47 kb). Each of these cosmids has been deposited with the American Type Culture Collection in accordance with the terms of the Budapest Treaty (cosmid pKOS055-1 is available under accession no. ATCC 203798; cosmid pKOS055-5 is available under accession no. ATCC 203799). Various additional reagents of the invention can therefore be isolated from these cosmids. DNA sequence analysis was also performed on the various subclones of the invention, as described above. [0218]
    • EXAMPLE 3
  • Expression of an Oleandolide/DEBS Hybrid PKS in [0219] Saccharopolyspora eryathraea
  • This Example describes the construction of an expression vector, plasmid pKOS039-110, that can integrate into the chromosome of [0220] Saccharopolyspora erythraea due to the phage phiC31 attachment and integration functions present on the plasmid and drive expression of the oleAI gene product under the control of the ermE* promoter. A restriction site and function map of plasmid pKOS039-110 is shown in FIG. 3 of the accompanying drawings. The expression of the oleAI gene product in a host cell that naturally produces the eryA gene products results in the formation of a functional hybrid PKS of the present invention composed of the oleAI, eryAII, and eryAIII gene products and the concomitant production of 13-methyl erythromycins. While the specific plasmids and vectors utilized in the construction are described herein, those of skill in the art will recognize that equivalent expression vectors of the invention can be readily constructed from publicly available materials and the oleA gene containing cosmids of the present invention deposited with the ATCC.
  • Plasmid pKOS039-98 is a cloning vector that contains convenient restriction sites that was constructed by inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for PacI, a Shine-Dalgarno sequence, and restriction enzyme recognition sites for NdeI, BglII, and HindIII, into a pUC19 derivative, called pKOS24-47. Plasmid pKOS039-98 (see PCT patent application No. WO US99/11814, incorporated herein by reference) was digested with restriction enzymes PacI and EcoRI and ligated to a polylinker composed of the oligonucleotides N39-51 and N39-52 having the following sequence: [0221]
  • N39-51: 5′-TAAGGAGGACCATATGCATCGCTCGAGTCTAGACCTAGG-3′N39-52: 5′-AATTCCTAGGTCTAGACTCGAGCGATGCATATGGTCCTCC-TTAAT-3′, which thus includes the following restriction enzyme recognition sites in the order shown: NdeI-NsiI-XhoI-XbaI-EcoRI, to yield plasmid pKOS039-105. [0222]
  • Plasmid pKOS039-105 was digested with restriction enzymes NsiI and EcoRI, and the resulting large fragment ligated to the 15.2 kb NsiI-EcoRI, restriction fragment of cosmid pKOS055-5 containing the oleAI gene to yield plasmid pKOS039-116. Plasmid pKOS039-116 was digested with restriction enzymes NdeI and EcoRI, and the resulting 15.2 kb fragment containing the oleAI gene was isolated and ligated to the 6 kb NdeI-EcoRI restriction fragment of plasmid pKOS039-134B to yield plasmid pKOSO39-110 (FIG. 3). [0223]
  • Plasmid pKOS039-134B is a derivative of pKOS039-104 described in PCT patent application No. WO US99/11814, supra, prepared by digesting the latter with restriction enzyme BglII and ligating the ˜10.5 kb fragment to get pKOS39-104B. Plasmid pKOS39-104B was digested with restriction enzyme PacI and partially digested with restriction enzyme XbaI. The ˜7.4 kb fragment was ligated with PCR61A+62 fragment treated with restriction enzymes PacI and AvrII. The PCR61A+62 fragment was generated using the PCR primers: [0224]
  • N39-61A, 5′-TTCCTAGGCTAGCCCGACCCGAGCACGCGCCGGCA-3′; and N39-62, 5′-CCTTAATTAAGGATCCTACCAACCGGCACGATTGTGCC-3′, [0225]
  • and the template was pWHM1104 (Tang et al., 1996, [0226] Molecular Microbiology 22(5): 801-813).
  • Plasmid pKOS039-110 DNA was passed through [0227] E. coli ET cells to obtain non-methylated DNA, which was then used to transform Saccharopolyspora erythraea cells, which contain a mutation in the eryAI coding sequence for the KS domain of module 1 of DEBS that renders the PKS non-functional. The resulting transformants produced detectable amounts of 14-desmethyl erythromycins.
    • EXAMPLE 4
  • Heterologous Expression of an Oleandolide PKS in [0228] Streptomyces lividans
  • This Example describes the construction of an expression vector, plasmid pKOS039-130, that has an SCP2* origin of replication and so can replicate in Streptomyces host cells and drive expression of the oleAI, oleAII, and oleAIII gene products under the control of the actI promoter and actII-ORF4 activator. A restriction site and function map of plasmid pKOS039-130 is shown in FIG. 4 of the accompanying drawings. The expression of the oleA gene products in this host cell results in the formation of a functional oleandolide PKS composed of the oleAI, oleAII, and oleAIII gene products and the concomitant production of 8,8a-deoxyoleandolide. While the specific plasmids and vectors utilized in the construction are described herein, those of skill in the art will recognize that equivalent expression vectors of the invention can be readily constructed from publicly available materials and the oleA gene containing cosmids of the present invention deposited with the ATCC. [0229]
  • The 7.2 kb NsiI-XhoI restriction fragment of cosmid pKOS055-5 was cloned into pKOS39-105 to give plasmid pKOS039-106. The 8.0 kb XhoI-PstI restriction fragment of cosmid pKOS055-5 was cloned into commercially available plasmid pLitmus28 to yield plasmid pKOS039-107. The 14 kb EcoRI-EcoRV and 5.4 kb EcoRV-PstI restriction fragments of cosmid pKOS055-1 were ligated with pLitmus28 digested with EcoRI and PstI to yield plasmid pKOS039-115. The 19.5 kb SpeI-XbaI restriction fragment from plasmid pKOS039-115 was inserted into pKOSO39-73, a derivative of plasmid pRM5, to yield plasmid pKOS039-129. The 15.2 kb PacI-EcoRI restriction fragment of plasmid pKOS039-110 was inserted into pKOS039-129 by replacing the 22 kb PacI-EcoRI restriction fragment to yield plasmid pKOS038-174. The 19 kb EcoRI restriction fragment from plasmid pKOS039-129 was then inserted into pKOS038-174 to yield plasmid pKOS039 -130 (FIG. 4), which was used to transform [0230] Streptomyces lividans K4-114 (K4-155 could also be used). The resulting transformants produced 8,8a-deoxyoleandolide.
  • As noted above, the invention provides a recombinant oleAI gene in which the coding sequence for the KS domain of [0231] module 1 has been mutated to change the active site cysteine to another amino acid (the KS1o mutation). Recombinant PKS enzymes comprising this gene product do not produce a polyketide unless provided with diketide (or triketide) compounds that can bind to the KS2 or KS3 domain, where they are then processed to form a polyketide comprising the diketide (or triketide). This recombinant oleAI gene can be used together with the oleAII and oleAII genes to make a recombinant oleandolide PKS or can be used with modified forms of those genes or other naturally occurring or recombinant PKS genes to make a hybrid PKS.
  • To make the KS1[0232] o mutation in oleAI, the following primers were prepared:
    N39-47, 5′-GCGAATTCCCGGGTGGCGTGACCTCT;
    N39-48, 5′-GAGCTAGCCGCCGTGTCCACCGTGACC;
    N39-49, 5′-CGGCTAGCTCGTCGCTGGTGGCACTGCAC; and
    N39-50, 5′-CGAAGCTTGACCAGGAAAGACGAACACC.
  • These primers were used to amplify template DNA prepared from pKOS039-106. The amplification product of primers N39-47 and N39-48 was digested with restriction enzymes EcoRI and NheI, and the amplification product of primers N39-49 and N39-50 was digested with restriction enzymes NheI and HindIII, and the resulting restriction fragments were ligated to EcoRI and HindIII-digested plasmid pLitmus28 to yield plasmid pKOS038-179. The 1.5 kb BsrGI-BbvCI restriction fragment of plasmid pKOS038-179 was inserted into plasmid pKOS039-106 to yield pKOS098-2. The 7 kb NsiI-XhoI restriction fragment of plasmid pKOS098-2 and the 8 kb XhoI-EcoRI restriction fragments of plasmid pKOS039-107 are then used to replace the 15.2 kb NsiI-EcoRI restriction fragment of plasmid pKOS039-110 to yield the desired expression vector, pKOS039-110-KS1[0233] o, which comprises the oleAI KS1o gene under the control of the ermE* promoter.
  • To provide an expression vector of the invention that encodes the complete oleandolide PKS with the recombinant oleAI KS1[0234] o gene product, the oleAI KS1o gene can be isolated as a PacI-EcoRI restriction fragment from plasmid pKOSO39-110-KS1o, which is then used to construct an expression vector analogous to the expression vector plasmid pKOS039-130 in the same manner in which the latter vector was constructed. The resulting expression vector can be used in Streptomyces lividans, S. coelicolor, and other compatible host cells to make polyketides by diketide feeding as described in PCT patent publication No. WO 99/03986, incorporated herein by reference.
    • EXAMPLE 5 Expression of an Oleandomycin/Picromycin Hybrid PKS
  • This Example describes the construction of an expression vector, plasmid pKOS039-133, that can integrate into the chromosome of Streptomyces due to the phage phiC31 attachment and integration functions present on the plasmid and drive expression of the oIeAIII gene product under the control of the actI promoter and actII-ORF4 activator. A restriction site and function map of plasmid pKOS039-133 is shown in FIG. 5 of the accompanying drawings. This plasmid was introduced into [0235] S. lividans host cells together with a plasmid, pKOS039-83, that drives expression of the narbonolide PKS genes picAI and picAII (see PCT patent application No. WO US99/11814, supra). The expression of the oleAIII and picAI and picAII gene products in a host cell results in the formation of a functional hybrid PKS of the present invention composed of the oleAIII, picAI, and picAII gene products and the concomitant production of 3-hydroxy-narbonolide. While the specific plasmids and vectors utilized in the construction are described herein, those of skill in the art will recognize that equivalent expression vectors of the invention can be readily constructed from publicly available materials and the oleA gene containing cosmids of the present invention deposited with the ATCC.
  • Two oligonucleotides were prepared for the insertion of the oleAIII gene into pSET152 derivative plasmid pKOS039-42: [0236]
    N39-59, 5′-AATTCATATGGCTGAGGCGGAGAAGCTGCGCGAATACCTGTGG; and
    N39-60, 5′-CGCGCCACAGGTATTCGCGCAGCTTCTCCGCCTCAGCCATATG
  • Plasmid pKOS039-115 was digested with restriction enzymes EcoRI and AscI to give the ˜13.8 kb restriction fragment, which was inserted with the linker N39-59/N39-60 to yield plasmid pKOS039-132. Plasmid pKOS039-132 was digested with restriction enzymes NdeI and XbaI to give the ˜10.8 kb restriction fragment, which was ligated to the ˜9 kb NdeI-SpeI restriction fragment of plasmid pKOS039-42 to yield plasmid pKOS039-133 (FIG. 5). Plasmid pKOS039-133 and pKOS039-83 were co-transformed into [0237] Streptomyces lividans K4-114 (K4-155 can also be used; see Ziermann and Betlach, 1999, Biotechniques 26, 106-110, and U.S. patent application Ser. No.09/181,833, filed Oct. 28, 1998, each of which is incorporated herein by reference). Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics. The strains were grown in liquid R5 medium (with 20 μg/mL thiostrepton, see Hopwood el al., Genetic Manipulation of Streptomyces: A Laboratory Manual; John Innes Foundation: Norwich, UK, 1985, incorporated herein by reference) for growth/seed and production cultures at 30° C. Analysis of extracts by LC/MS established the identity of the polyketide as the expected compound, 3-hydroxynarbonolide.
    • EXAMPLE 6 Conversion of Erythronolides to Erythromycins
  • A sample of an oleandolide (˜50 to 100 mg) is dissolved in 0.6 mL of ethanol and diluted to 3 mL with sterile water. This solution is used to overlay a three day old culture of [0238] Saccharopolyspora erythraea WHM34 (an eryA mutant) grown on a 100 mm R2YE agar plate at 30° C. After drying, the plate is incubated at 30° C. for four days. The agar is chopped and then extracted three times with 100 nL portions of 1% triethylamine in ethyl acetate. The extracts are combined and evaporated. The crude product is purified by preparative HPLC (C-18 reversed phase, water-acetonitrile gradient containing 1% acetic acid). Fractions are analyzed by mass spectrometry, and those containing pure compound are pooled, neutralized with triethylamine, and evaporated to a syrup. The syrup is dissolved in water and extracted three times with equal volumes of ethyl acetate. The organic extracts are combined, washed once with saturated aqueous NaHCO3, dried over Na2SO4, filtered, and evaporated to yield ˜0.15 mg of product. The product is a glycosylated and hydroxylated oleandolide corresponding to erythromycin A, B, C, and D but differing therefrom as the oleandolide provided differed from 6-dEB.
    • EXAMPLE 7 Measurement of Antibacterial Activity
  • Antibacterial activity is determined using either disk diffusion assays with [0239] Bacillus cereus as the test organism or by measurement of minimum inhibitory concentrations (MIC) in liquid culture against sensitive and resistant strains of Staphylococcus pneumoniae.
  • The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims. [0240]

Claims (20)

1. An isolated recombinant DNA compound that comprises a coding sequence for a domain of a loading module or any one of extender modules one through four, inclusive of an oleandolide polyketide synthase (PKS).
2. The isolated recombinant DNA compound of claim 1, wherein said domain is selected from the group consisting of a thioesterase domain, a KSQ domain, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain and an ER domain.
3. The isolated recombinant DNA compound of claim 2 that comprises the coding sequence for a loading module and extender modules one and two of the oleandolide PKS.
4. The isolated recombinant DNA compound of claim 2 that comprises the coding sequence for the loading module and all six extender modules.
5. An isolated recombinant DNA compound that comprises a coding sequence for a domain of a loading module or any one of extender modules one through six, inclusive of an oleandolide polyketide synthase (PKS) operably linked to a promoter.
6. The isolated recombinant DNA compound of claim 5, wherein said coding sequence encodes a loading module or any one of extender modules one through four, inclusive, of oleandolide PKS.
7. The isolated recombinant DNA compound of claim 5 that is a recombinant DNA expression vector that further comprises an origin of replication or a segment of DNA that enables chromosomal integration.
8. The recombinant DNA expression vector of claim 7 that codes for expression of a PKS in Streptomyces host cells.
9. A recombinant host cell selected from the group consisting of Streptomyces host cells and Saccharopolyspora host cells that comprises a recombinant DNA expression vector of claim 7.
10. The recombinant DNA expression vector of claim 7 that encodes a hybrid PKS comprising at least a portion of an oleandolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than oleandolide.
11. The recombinant DNA compound of claim 10, wherein said second PKS gene is a DEBS gene.
12. The recombinant DNA compound of claim 11, wherein said hybrid PKS comprises a loading module and any one of extender modules one through four, inclusive, of oleandolide PKS and an extender module of DEBS.
13. The recombinant DNA compound of claim 10, wherein said hybrid PKS comprises a loading module and any one of extender modules one through four, inclusive, of oleandolide PKS and an extender module of narbonolide PKS.
14. A recombinant host cell, which in its untransformed state does not produce oleandolide, that comprises a recombinant DNA expression vector of claim 11 and said cell produces a macrolide aglycone synthesized by said hybrid PKS.
15. The recombinant host cell of claim 14 that is Streptomyces lividans.
16. The recombinant host cell of claim 14 that is Saccharopolyspora erythraea.
17. The recombinant host cell of claim 13, wherein said oleandolide PKS has a non-functional KS domain in extender module one.
18. The recombinant host cell of claim 17 that is Streptomyces coelicolor or Streptomyces lividans.
19. The recombinant host cell of claim 17 that is Saccharopolyspora erythraea.
20. A method for producing a polyketide in a cell, which method comprises transforming the cell with a recombinant expression vector that encodes at least a portion of an oleAI, oleAII, or oleAIII gene.
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