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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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)

Definitions

• the present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. More specifically, it relates to narbonolides and derivatives thereof.
• 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 is a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Tetracycline, erythromycin,
• FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds.
• FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds.
• PKS 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.
• PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). 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.
• Modular PKSs are responsible for producing a large number of 12, 14, and 16- membered macrolide antibiotics including methymycin, erythromycin, narbomycin, picromycin, and tylosin. 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 ⁇ - carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
• the modular PKS are generally encoded in multiple ORFs. Each ORF typically comprises two or more "modules" of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three or more enzymatic activities or "domains.”
• the present invention provides methods and reagents relating to the modular PKS gene cluster for the polyketide antibiotics known as narbomycin and picromycin.
• Narbomycin is produced in Streptomyces narbonensis, and both narbomycin and picromycin are produced in S. venezuelae. These species are unique among macrolide producing organisms in that they produce, in addition to the 14-membered macrolides narbomycin and picromycin (picromycin is shown in Figure 1, compound 1), the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in Figure 1, compound 2). Narbomycin differs from picromycin only by lacking the hydroxyl at position 12. Based on the structural similarities between picromycin and methymycin, it was speculated that methymycin would result from premature cyclization of a hexaketide intermediate in the picromycin pathway.
• narbonolide Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosaminyl transferase to produce narbomycin. In Streptomyces venezuelae, narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase. (See Figure 1)
• the macrolide product of the narbonolide PKS is further modified by hydroxylation and glycosylation.
• Figure 1 also shows the metabolic relationships of the compounds discussed above.
• the present invention provides recombinant methods and materials for expressing PKSs derived in whole and in part from the narbonolide PKS and other genes involved in narbomycin and picromycin biosynthesis in recombinant host cells.
• the invention also provides the polyketides derived from the narbonolide PKS.
• the invention provides the complete PKS gene cluster that ultimately results, in Streptomyces venezuelae, in the production of picromycin.
• the ketolide product of this PKS is narbonolide. Narbonolide is glycosylated to obtain narbomycin and then hydroxy lated at C12 to obtain picromycin.
• the enzymes responsible for the glycosylation and hydroxylation are also provided in recombinant form by the invention.
• the invention is directed to recombinant materials that contain nucleotide sequences encoding at least one domain, module, or protein encoded by a narbonolide PKS gene.
• the recombinant materials may be "isolated.”
• the invention also provides recombinant materials useful for conversion of ketolides to antibiotics. These materials include recombinant DNA compounds that encode the C12hydroxylase (fhspicK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the beta- glucosidase enzyme involved in picromycin biosynthesis in S.
• the invention provides a recombinant expression system that comprises a heterologous promoter positioned to drive expression of the narbonolide PKS, including a "hybrid" narbonolide PKS..
• the promoter is derived from a PKS gene.
• the invention provides recombinant host cells comprising the vector that produces narbonolide.
• the host cell is Streptomyces lividans or S. coelicolor.
• the invention provides a recombinant expression system that comprises the desosamine biosynthetic genes as well as the desosaminyl transferase gene.
• the invention provides recombinant host cells comprising a vector that produces the desosamine biosynthetic gene products and desosaminyl transferase gene product.
• the host cell is Streptomyces lividans or S. coelicolor.
• the invention provides a method for desosaminylating polyketide compounds in recombinant host cells, which method comprises expressing the PKS for the polyketide and the desosaminyl transferase and desosamine biosynthetic genes in a host cell.
• the host cell expresses a beta-glucosidase gene as well. This preferred method is especially advantageous when producing desosaminylated polyketides in Streptomyces host cells, because such host cells typically glucosylate desosamine residues of polyketides, which can decrease desired activity, such as antibiotic activity.
• beta-glucosidase the glucose residue is removed from the polyketide.
• the invention provides a recombinant expression system that comprises a promoter positioned to drive expression of a "hybrid" PKS comprising all or part of the narbonolide PKS and at least a part of a second PKS, or comprising a narbonolide PKS modified by deletions, insertions and/or substitutions.
• 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 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 narbonolide 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 narbonolide PKS.
• the derived portion can be prepared synthetically or directly from DNA derived from organisms that produce narbonolide.
• the invention provides methods to screen the resulting polyketide and antibiotic libraries.
• the invention also provides novel polyketides and antibiotics or other useful compounds derived therefrom.
• the compounds of the invention can be used in the manufacture of another compound.
• the antibiotic compounds of the invention are formulated in a mixture or solution for administration to an animal or human.
• Figure 1 shows the structures of picromycin (compound 1), methymycin (compound 2), and the ketolide HMR 3004 (compound 3) and the relationship of several compounds related to picromycin.
• Part B shows the organization of the narbonolide PKS genes on the chromosome of Streptomyces venezuelae, including the location of the various module encoding sequences (the loading module domains are identified as sKS*, sAT, and sACP), as well as t epicB thioesterase gene and two desosamine biosynthesis genes (picCII and picCIII).
• Part C shows the engineering of the S. venezuelae host of the invention in which the picAI gene has been deleted.
• ACP is acyl carrier protein
• AT is acyltransf erase
• DH dehydratase
• ER is enoylreductase
• KR ketoreductase
• KS is ketosynthase
• TE is thioesterase.
• 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 narbonolide PKS.
• the invention also provides recombinant DNA compounds encoding the enzymes that catalyze the further modification of the ketolides produced by the narbonolide PKS.
• the invention provides recombinant expression vectors useful in producing the narbonolide PKS and hybrid PKSs composed of a portion of the narbonolide PKS in recombinant host cells.
• 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 or dimethyltransferase activity.
• the releasing domain contains a thioesterase and, often, a cyclase activity.
• 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 enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule.
• 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 narbonolide PKS loading module contains a KS Q .
• modules that include a methyltransferase or dimethyltransferase activity; modules can also include an epimerase activity.
• narbonolide related polyketides in Streptomyces venezuelae and S. narbonensis.
• the narbonolide PKS produces two polyketide products, narbonolide and 10- deoxymethynolide.
• Narbonolide is the polyketide product of all six extender modules of the narbonolide PKS.
• 10-deoxymethynolide is the polyketide product of only the first five extender modules of the narbonolide PKS.
• These two polyketides are desosaminylated to yield narbomycin and YC17, respectively.
• the narbonolide PKS is composed of a loading module, six extender modules, and two thioesterase domains one of which is on a separate protein.
• Figure 4, part B shows the organization of the narbonolide PKS genes on the Streptomyces venezuelae chromosome, as well as the location of the module encoding sequences in those genes, and the various domains within those modules.
• the loading module is not numbered, and its domains are indicated as sKS*, sAT, and ACP. Also shown in the Figure, part A, are the structures of picromycin and methymycin. The loading and six extender modules and the thioesterase domain of the narbonolide
• Figure 2 shows a restriction site and function map of pKOS023-27, which contains the complete coding sequence for the four proteins that constitute narbonolide PKS and four additional ORFs.
• One of these additional ORFs encodes the picB gene product, the type II thioesterase mentioned above.
• PICB shows a high degree of similarity to other type II thioesterases, with an identity of 51 %, 49%, 45% and 40% as compared to those of Amycolatopsis mediterranae, S. griseus, S. fradiae and Saccharopolyspora erythraea, respectively.
• the three additional ORFs in the cosmid pKOS023-27 insert DNA sequence, from the picCII, picCIII, and picCVI, genes, are involved in desosamine biosynthesis and transfer and described in the following section.
• 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 shown merely illustrate preferred embodiments of the invention.
• the present invention provides recombinant DNA compounds that encode the loading module of the narbonolide PKS and useful portions thereof. These recombinant DNA compounds are useful in the construction of PKS coding sequences that encode all or a portion of the narbonolide PKS and in the construction of hybrid PKS encoding DNA compounds of the invention, as described in the section concerning hybrid PKSs below.
• reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa.
• reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than Streptomyces venezuelae.
• reference to a PKS or its coding sequence includes reference to any portion thereof.
• the present invention provides recombinant DNA compounds that encode one or more of the domains of each of the six extender modules (modules 1 - 6, inclusive) of the narbonolide PKS.
• Modules 1 and 5 of the narbonolide PKS are functionally similar.
• Each of these extender modules contains a KS domain, an AT domain specific for methylmalonyl Co A, a KR domain, and an ACP domain.
• Module 2 of the narbonolide PKS contains a KS domain, an AT domain specific for malonyl CoA, a KR domain, a DH domain, and an ACP domain.
• Module 3 differs from extender modules 1 and 5 only in that it contains an inactive ketoreductase domain.
• the deletion When a deletion is within a module, the deletion typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains.
• Contig 002 from cosmid pKOS023-26 contains 5970 nucleotides and the following ORFs: from nucleotide 995 to 1 is an ORF of picCIV that encodes a partial sequence of an amino transferase-dehydrase; from nucleotides 1356 to 2606 is an ORF of picK that encodes a cytochrome P450 hydroxylase; and from nucleotides 2739 to 5525 is ORF12, which encodes a transcriptional activator. (SEQ ID NO:21)
• Contig 003 from cosmid pKOS023-26 contains 3292 nucleotides and the following ORFs: from nucleotide 104 to 982 is ORF13, which encodes dNDP glucose synthase
• Contig 004 from cosmid pKOS023-26 contains 1693 nucleotides and the following ORFs: from nucleotide 1692 to 694 is ORF 15, which encodes a part of S-adenosylmethionine synthetase; and from nucleotide 692 to 1 is ORF 16, which encodes a part of a protein homologous to the M. tuberculosis cbhK gene. (SEQ ID NO:23)
• Contig 005 from cosmid pKOS023-26 contains 1565 nucleotides and contains the ORF of the picCV gene that encodes PICCV, involved in desosamine biosynthesis. (SEQ ID NO:24)
• the recombinant desosamine biosynthesis and transfer and beta-glucosidase genes and proteins provided by the invention are useful in the production of glycosylated polyketides in a variety of host cells, as described in Section IV below.
• the recombinant PicK enzyme of the invention hydroxylates narbomycin at the C 12 position and YC-17 at either the CIO or C12 position. Hydroxylation of these compounds at the respective positions increases the antibiotic activity of the compound relative to the unhydroxylated compound. Hydroxylation can be achieved by a number of methods. First, the hydroxylation may be performed in vitro using purified hydroxylase, or the relevant hydroxylase can be produced recombinantly and utilized directly in the cell that produces it. Thus, hydroxylation may be effected by supplying the nonhydroxylated precursor to a cell that expresses the hydroxylase.
• coli origin of replication such as from pUC, plP, pll, and pBR.
• phage phiC31 and its derivative KC515 can be employed (see Hopwood et al, supra).
• plasmid pS ⁇ T152, plasmid pSAM, plasmids pSElOl and pSE211 can be employed.
• 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 carboxyglycolyl 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 narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, 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 narbonolide, a narbonolide derivative, or another polyketide.
• telomere sequences for the modules of the heterologous PKS are useful for a variety of applications.
• a DNA compound comprising a sequence that encodes the narbonolide 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 narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
• the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, 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 narbonolide, a narbonolide derivative, or another polyketide.
• 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 carboxyglycolyl CoA specific AT; deleting any one, two, or all three of the ER, DH, 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.
• a DNA compound comprising a sequence that encodes the fifth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.
• the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, 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 narbonolide, a narbonolide derivative, or another polyketide.
• a DNA compound comprising a sequence that encodes the narbonolide 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 narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS provides a novel PKS.
• 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 carboxyglycolyl CoA specific AT; and/or inserting a KR, a KR and DH, or a KR, 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 narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, 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 narbonolide, a narbonolide derivative, or another polyketide.
• the sixth extender module of the narbonolide 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 narbonolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the narbonolide synthase thioesterase.
• a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth extender module coding sequence in recombinant DNA compounds of the invention.
• Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode the narbonolide PKS, a PKS that produces a narbonolide derivative, and a PKS that produces a polyketide other than narbonolide or a narbonolide derivative.
• Streptomyces hygroscopicus analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.
• 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 narbonolide PKS that contains the naturally occurring loading module and thioesterase domain as well as extender modules one, two, four, and six of the narbonolide 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.
• a modular PKS “derived from” the narbonolide or other naturally occurring PKS is a subset of the “hybrid” PKS family and 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.
• 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.
• a PKS "derived from” the narbonolide PKS includes a PKS that contains the scaffolding of all or a portion of the narbonolide 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 narbonolide PKS so that the nature of the resulting polyketide is altered. This definition applies both at the protein and DNA sequence levels.
• Particular preferred embodiments include those wherein a KS, AT, KR, DH, or ER 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.
• a PKS derived from the narbonolide PKS are functional PKS modules or their encoding genes wherein at least one portion, preferably two portions, of the narbonolide PKS activities have been inserted.
• exemplary is the use of the narbonolide AT for extender module 2 which accepts a malonyl CoA extender unit rather than methylmalonyl CoA to replace a methylmalonyl specific AT in a PKS.
• Other examples include insertion of portions of non-condensation cycle enzymatic activities or other regions of narbonolide synthase activity into a heterologous PKS.
• the derived from definition applies to the PKS at both the genetic and protein levels.
• the polyketide chain length is determined by the number of modules in the PKS.
• 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 acetyl, butyryl, and the like.
• KS1 extender module 1
• diketides that are chemically synthesized analogs of extender module 1 diketide products
• extender module 2 extender module 2
• KS 1 activity was inactivated through mutation.
• 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.
• 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 result when there is a complete KR/DH/ER available.
• the modular PKS systems permit a wide range of polyketides to be synthesized.
• a wider range of starter units including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl) are found in various macrocyclic polyketides.
• aliphatic monomers acetyl, propionyl, butyryl, isovaleryl, etc.
• aromatics aminohydroxybenzoyl
• alicyclics cyclohexanoyl
• heterocyclics thiazolyl
• 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).
• these enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner.
• the polyketides and antibiotics produced by the methods of the invention are typically single stereoisomeric forms.
• the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers.
• the combinatorial potential within modular PKS pathways based on any naturally occurring modular, such as the narbonolide, PKS scaffold is virtually unlimited.
• the combinatorial potential is increased even further when one considers that mutations in DNA encoding a polypeptide can 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 chemicals, 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/ ⁇ R corresponds to KR alone.
• 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.
• 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 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 is quite large.
• 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 CaC12 or agents such as other divalent cations, Hpofection, DMSO, 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) colonies that contain the proteins that are members of the PKS library produced by the coding sequences; (3) the polyketides produced; 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 narbonolide 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.
• the polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like.
• desired targets such as receptors, signaling proteins, and the like.
• the supematants per 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. 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, J Immunol. Meth. 137:167-173, incorporated herein by reference, and in the examples 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 C6, which can be accomplished using the hydroxylase encoded by the eryF gene, and/or C12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene. The presence of hydroxyl groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart.
• Gycosylation is important in conferring antibiotic activity to a polyketide as well.
• 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.
• 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 glycosylases.
• 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-O-methyl mycarose).
• Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose.
• 2-acetyl-l-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 or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.
• Saccharopolyspora erythraea or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.
• a portion of the narbonolide PKS gene was fused to the DEBS genes.
• the 12-membered intermediate can be formed by other recombinant PKS enzymes, see Kao et al, 1995, supra, the PIC TE domain appears incapable of forcing premature cyclization of the hexaketide intermediate generated by DEBS. This result, along with others reported herein, suggests that protein interactions between the narbonolide PKS modules play a role in formation of the 12 and 14-membered macrolides.
• a variant of the first construct hybrid PKS was constructed that contained an inactivated DEBS1 extender module 1 KS domain.
• host cells containing the resultant hybrid PKS were supplied the appropriate diketide precursor, the desired 13-desethyl-13- propyl compounds were obtained, as described in the examples below.
• hybrid PKSs of the invention were made by coexpressing fhe pic Al and picAII genes with genes encoding DEBS3 or DEBS3 variants. These constructs illustrate the method of the invention in which a hybrid PKS is produced from coexpression of PKS genes unmodified at the modular or domain level.
• the eryAIII gene was coexpressed with the picAI and picAII genes, and the hybrid PKS produced 10-desmethyl- 10,1 l-anhydro-6-deoxyerythronolide B in Streptomyces lividans.
• Such a hybrid PKS could also be constructed in accordance with the method of the invention by transformation of S. venezuelae with an expression vector that produces the eryAIII gene product, DEBS3.
• the S. venezuelae host cell has been modified to inactivate the picAIII gene.
• the DEB S3 gene was a variant that had an inactive KR in extender module 5.
• the hybrid PKS produced 5,6-dideoxy-5-oxo-10-desmethyl-10,l 1- anhydroerythronolide B in Streptomyces lividans.
• the DEB S3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS.
• This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,ll- anhydroerythronolide B and 5,6-dideoxy-4,5-anhydro-10-desmethyl-10,l l- anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH and KR domains functioned only inefficiently in this construct.
• the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH, KR, and ER domains of extender module 1 of the rapamycin PKS.
• This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,l 1- anhydroerythronolide B as well as 5,6-dideoxy-10-desmethyl-10,l l-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH, KR, and ER domains functioned only inefficiently in this construct.
• the DEB S3 gene was a variant in which the KR domain of extender module 6 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS.
• This construct produced 3,6-dideoxy-2,3-anhydro-10-desmethyl-10,l 1- anhydroerythronolide B in Streptomyces lividans.
• the DEB S3 gene was a variant in which the AT domain of extender module 6 was replaced by the AT domain of extender module 2 of the rapamycin PKS.
• This construct produced 2, 10-didesmethyl- 10,11 -anhydro-6-deoxyerythronolide B in Streptomyces lividans.
• PKSs illustrate the wide variety of polyketides that can be produced by the methods and compounds 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 ketolides, polyketide compounds with significant antibiotic activity. 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-derived ketolide compounds.
• 6-dEB in part differs from narbonolide in that it comprises a 10-methyl group
• the novel hybrid PKS genes of the invention based on the narbonolide PKS provide many novel ketolides that differ from the known ketolides only in that they lack a 10-methyl group.
• the invention provides the 10-desmethyl 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. Patent Nos.
• a hybrid PKS of the invention that produces 10-methyl narbonolide is constructed by substituting the malonyl-specific AT domain of the narbonolide PKS extender module 2 with a methylmalonyl specific AT domain from a heterologous PKS.
• a hybrid narbonolide PKS in which the AT of extender module 2 was replaced with the AT from DEBS extender module 2 was constructed using boundaries described in PCT publication No. WO 98/49315, incorporated herein by reference. However, when the hybrid PKS expression vector was introduced into Streptomyces venezuelae, detectable quantities of 10- methyl picromycin were not produced.
• an AT domain from a module other than DEBS extender module 2 is preferred.
• DEBS extender module 2 or another methylmalonyl specific AT but utilize instead different boundaries than those used for the substitution described above.
• the extent of hybrid modules engineered need not be limited to module 2 to make 10-methyl narbonolide.
• substitution of the KS domain of extender module 3 of the narbonolide PKS with a heterologous domain or module can result in more efficient processing of the intermediate generated by the hybrid extender module 2.
• a heterologous TE domain may be more efficient in cyclizing 10-methyl narbonolide.
• Substitution of the entire extender module 2 of the narbonolide PKS with a module encoding the correct enzymatic activities, i.e., a KS, a methylmalonyl specific AT, a KR, a DH, and an ACP, can also be used to create a hybrid PKS of the invention that produces a 10- methyl ketolide.
• Modules useful for such whole module replacements include extender modules 4 and 10 from the rapamycin PKS, extender modules 1 and 5 from the FK506 PKS, extender module 2 of the tylosin PKS, and extender module 4 of the rifamycin PKS.
• the invention provides many different hybrid PKSs that can be constructed starting from the narbonolide PKS that can be used to produce 10-methyl narbonolide. While 10-methyl narbonolide is referred to in describing these hybrid PKSs, those of skill recognize that the invention also therefore provides the corresponding derivatives produces by glycosylation and hydroxylation. For example, if the hybrid PKS is expressed in Streptomyces narbonensis or S. venezuelae, the compounds produced are 10-methyl narbomycin and picromycin, respectively. Alternatively, the PKS can be expressed in a host cell transformed with the vectors of the invention that encode the desosamine biosynthesis and desosaminyl transferase and picK hydroxylase genes.
• 6-hydroxy ketolides include 3-deoxy-3-oxo erythronolide B, 6-hydroxy narbonolide, and 6- hydroxy- 10-methyl narbonolide.
• the invention provides a method for utilizing EryF to hydroxy late 3 -ketolides that is applicable for the production of any 6- hydroxy-3-ketolide.
• 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 C12 position) or eryK (for the C 12 position) and/or eryF (for the C6 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 (2) below which are hydroxylated at either the C6 or the C 12 or both.
• the compounds of formula (2) can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described.
• -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; with the proviso that: at least two of R'-R 6 are alkyl (1-4C).
• the invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking extender modules 5 and 6 of the narbonolide PKS .
• 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, com starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi- solid, or liquefied 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. Patent 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, inco ⁇ orated 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 intrastemal 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.
• Example 1 General Methodology Bacterial strains, plasmids, and culture conditions, streptomyces coelicolor CH999 described in WO 95/08548, published 30 March 1995, or S. lividans K4-114, described in Ziermann and Betlach, Jan. 99, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, inco ⁇ orated herein by reference, was used as an expression host. DNA manipulations were performed in Escherichia coli XLl-Blue, available from Stratagene. E. coli MCI 061 is also suitable for use as a host for plasmid manipulation. Plasmids were passaged through E.
• E. coli ET12567 (dam dcm hsdS Cmr) (MacNeil, 1988, J. Bacteriol. 170: 5607, inco ⁇ orated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor.
• 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, inco ⁇ orated 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, inco ⁇ orated herein by reference.
• This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters to allow for bidirectional cloning, the gene encoding the actll- 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-bome 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.
• the library was initially screened by direct colony hybridization with a DNA probe specific for ketosynthase domain coding sequences of PKS genes. Colonies were alkaline lysed, and the DNA was crosslinked to the membrane using UV irradiation. After overnight incubation with the probe at 42°C, the membrane was washed twice at 25°C in 2xSSC buffer + 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2xSSC buffer at 55°C. Approximately 30 colonies gave positive hybridization signals with the degenerate probe. Several cosmids were selected and divided into two classes based on restriction digestion patterns. A representative cosmid was selected from each class for further analysis.
• the representative cosmids were designated pKOS023-26 and pKOS023-27. These cosmids were determined by DNA sequencing to comprise the narbonolide PKS genes, the desosamine biosynthesis and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene.
• Cosmid pKOS023-26 was assigned accession number ATCC 203141
• cosmid pKOS023-27 was assigned accession number ATCC 203142.
• narbonolide PKS genes had been cloned and to illustrate how the invention provides methods and reagents for constructing deletion variants of narbonolide PKS genes
• a narbonolide PKS gene was deleted from the chromosome of Streptomyces venezuelae. This deletion is shown schematically in Figure 4, parts B and C.
• the -4.5 kb Hindlll-Spel fragment from plasmid pKOS039-07 was ligated with the 2.5 kb Hindlll-Nhel fragment of integrating vector pS ⁇ T152, available from the NRRL, which contains an E. coli origin of replication and an apramycin resistance-conferring gene to create plasmid pKOS039-16.
• This vector was used to transform S. venezuelae, and apramycin- resistant transformants were selected.
• the selected transformants were grown in TSB liquid medium without antibiotics for three transfers and then plated onto non- selective media to provide single colony isolates.
• the isolated colonies were tested for sensitivity to apramycin, and the apramycin-sensitive colonies were then tested to determine if they produced picromycin.
• the tests performed included a bioassay and LC/MS analysis of the fermentation media. Colonies determined not to produce picromycin (or methymycin or neomethymycin) were then analyzed using PCR to detect an amplification product diagnostic of the deletion. A colony designated K39-03 was identified, providing confirmation that the narbonolide PKS genes had been cloned.
• the cosmids were digested with BamHl endonuclease and electrophoresed on a 1% agarose gel, and the resulting fragments were transferred to a nylon membrane.
• the membrane was incubated with the eryK probe overnight at 42°C, washed twice at 25°C in 2XSSC buffer with 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2XSSC buffer at 50°C.
• Cosmid pKOS023- 26 produced an -3 kb fragment that hybridized with the probe under these conditions. This fragment was subcloned into the PCRscriptTM (Stratagene) cloning vector to yield plasmid pKOS023-28 and sequenced.
• the -1.2 kb gene designated picK above was thus identified.
• the picK gene product is homologous to eryK and other known macrolide cytochrome P450 hydroxylases.
• the narbonolide PKS was transferred to the non-macrolide producing host Streptomyces lividans K4-114 (see Ziermann and Betlach, 1999, Biotechniques 26, 106-110, and U.S. patent application Serial No. 09/181,833, filed 28 Oct. 1998, each of which is inco ⁇ orated herein by reference). This was accomplished by replacing the three DEBS ORFs on a modified version of pCK7 (see Kao et al, 1994, Science 265, 509-512, and U.S. Patent No.
• pCK7'Kan' differs from pCK7 only in that it contains a kanamycin resistance conferring gene inserted at its Hindlll restriction enzyme recognition site. Because the plasmid contains two selectable markers, one can select for both markers and so minimize contamination with cells containing rearranged, undesired vectors.
• Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics.
• the strains were grown in liquid R5 medium for growth/seed and production cultures at 30°C.
• a 2 L shake flask culture of S. lividans K4- 114/pKOS039-86 was grown for 7 days at 30°C.
• the mycelia was filtered, and the aqueous layer was extracted with 2 x 2 L ethyl acetate. The organic layers were combined, dried over MgSO4, filtered, and evaporated to dryness.
• Polyketides were separated from the crude extract by silica gel chromatography (1 :4 to 1 :2 ethyl acetate:hexane gradient) to give an -10 mg mixture of narbonolide and 10-deoxymethynolide, as indicated by LC/MS and 1H NMR. Purification of these two compounds was achieved by HPLC on a C- 18 reverse phase column (20-80% acetonitrile in water over 45 minutes). This procedure yielded -5 mg each of narbonolide and 10-deoxymethynolide. Polyketides produced in the host cells were analyzed by bioassay against Bacillus subtilis and by LC/MS analysis.
• narbonolide in Streptomyces lividans represents the expression of an entire modular polyketide pathway in a heterologous host.
• the combined yields of compounds 4 and 5 are similar to those obtained with expression of DEBS from pCK7 (see Kao et al, 1994, Science 265: 509-512, inco ⁇ orated herein by reference).
• the narbonolide PKS itself possesses an inherent ability to produce both 12 and 14-membered macrolactones without the requirement of additional activities unique to S. venezuelae.
• the existence of a complementary enzyme present in S. lividans that provides this function is possible, it would be unusual to find such a specific enzyme in an organism that does not produce any known macrolide.
• the picB gene was integrated into the chromosome of Streptomyces lividans harboring plasmid pKOS039-86 to yield S. lividans K39-18/pKOS039-86.
• the picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al, 1992, Gene 116, 43, inco ⁇ orated herein by reference) under control of the same promoter (P ⁇ cti) as the PKS on plasmid pKOS039-86.
• Plasmid pKOS039-104 comprises the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene.
• This plasmid was constructed by first inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for Pad, a Shine-Dalgamo sequence, and restriction enzyme recognition sites for Ndel, Bglll, and Hindlll, into a pUC 19 derivative, called pKOS24-47, to yield plasmid pKOS039-98.
• An -0.3 kb PCR fragment comprising the coding sequence for the N-terminus of the desl gene product and an -0.12 kb PCR fragment comprising the coding sequence for the C- terminus of the desR gene product were amplified from cosmid pKOS23-26 (ATCC 203141) and inserted together into pLitmus28 treated with restriction enzymes Nsil and EcoRI to produce plasmid pKOS039-101.
• the -6 kb Sphl-Pstl restriction fragment of pKOS23-26 containing the desl, desll, desIII, desIV, and desV genes was inserted into plasmid pUC19 (Stratagene) to yield plasmid pKOS039-102.
• the -6 kb Sphl-EcoRI restriction fragment from plasmid pKOS039-102 was inserted into pKOS039-101 to produce plasmid pKOS039-103.
• the -6 kb Bglll-Pstl fragment from pKOS23-26 that contains the desR, desVI, desVII, and desVIII genes was inserted into pKOS39-98 to yield pKOS39-100.
• the -6 kb Pacl-Pstl restriction fragment of pKOS39-100 and the -6.4 kb Nsil-EcoRI fragment of pKOS39-103 were cloned into pKOS39-44 to yield pKOS39-104.
• plasmid pKOS39-104 drives expression of the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene.
• the glycosylated antibiotic narbomycin was produced in these host cells, and it is believed that YC17 was produced as well.
• these host cells are transformed with vectors that drive expression of fhepicK gene, the antibiotics methymycin, neomethymycin, and picromycin are produced.
• Example 4 Expression Vector for Desosaminyl Transferase While the invention provides expression vectors comprising all of the genes required for desosamine biosynthesis and transfer to a polyketide, the invention also provides expression vectors that encode any subset of those genes or any single gene. As one illustrative example, the invention provides an expression vector for desosaminyl transferase. This vector is useful to desosaminylate polyketides in host cells that produce NDP- desosamine but lack a desosaminyl transferase gene or express a desosaminyl transferase that does not function as efficiently on the polyketide of interest as does the desosaminyl transferase of Streptomyces venezuelae.
• This expression vector was constructed by first amplifying the desosaminyl transferase coding sequence from pKOS023-27 using the primers: N3917: 5'-CCCTGCAGCGGCAAGGAAGGACACGACGCCA-3' (SEQ ID NO:25); and N3918: 5'-AGGTCTAGAGCTCAGTGCCGGGCGTCGGCCGG-3' (SEQ ID NO:26), to give a 1.5 kb product.
• Plasmid pWHMl 104 described in Tang et al, 1996, Molec. Microbiol. 22(5): 801-813, inco ⁇ orated herein by reference, encodes the ermE* promoter.
• Plasmid pKOS039-14 is constructed so that the desosaminyl transferase gene is placed under the control of the ermE* promoter and is suitable for expression of the desosaminyl transferase in Streptomyces, Saccharopolyspora erythraea, and other host cells in which the ermE* promoter functions.
• the picK gene was PCR amplified from plasmid pKOS023-28 using the oligonucleotide primers: N024-36B (forward): 5'-TTGCATGCATATGCGCCGTACCCAGCAGGGAACGACC (SEQ ID NO:27); and N024-37B (reverse):
• Plasmid pKOS023-61 was digested with restriction enzymes Spel and EcoRI, and a short linker fragment encoding 6 histidine residues and a stop codon (composed of oligonucleotides 30-85a: 5'- CTAGTATGCATCATCATCATCATCATTAA-3' (SEQ ID NO:29); and 30-85b: 5'-AATTTTAATGATGATGATGATGATGCATA-3' (SEQ ID NO:30) was inserted to obtain plasmid pKOS023-68. Both plasmid pKOS023-61 and pKOS023-68 produced active PicK enzyme in recombinant E. coli host cells.
• Plasmid pKOS023-61 was transformed into E. coli BL21-DE3. Successful transformants were grown in LB-containing carbenicillin (100 ⁇ g/ml) at 37°C to an OD600 of 0.6. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown for an additional 3 hours before harvesting. The cells were collected by centrifugation and frozen at -80°C. A control culture of BL21-DE3 containing the vector plasmid pET21c (Invitrogen) was prepared in parallel.
• IPTG Isopropyl-beta-D-thiogalactopyranoside
• the frozen BL21-DE3/pKOS023-61 cells were thawed, suspended in 2 ⁇ L of cold cell disruption buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris/HCl, pH 8.0) and sonicated to facilitate lysis. Cellular debris and supernatant were separated by centrifugation and subjected to SDS-PAGE on 10-15% gradient gels, with Coomassie Blue staining, using a Pharmacia Phast Gel Electrophoresis system.
• the soluble crude extract from BL21- DE3/pKOS023-61 contained a Coomassie stained band of Mr ⁇ 46 kDa, which was absent in the control strain BL21-DE3/ ⁇ ET21c.
• the hydroxylase activity of the picK protein was assayed as follows.
• the crude supernatant (20 ⁇ L) was added to a reaction mixture (100 ⁇ L total volume) containing 50 mM Tris/HCl (pH 7.5), 20 ⁇ M spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose- ⁇ phosphate, and 20 nmol of narbomycin.
• the reaction was allowed to proceed for 105 minutes at 30°C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by evaporative light scattering (ELSD) and mass spectrometry.
• the control extract (BL21-DE3/pET21c) was processed identically.
• the conversion of narbomycin to picromycin under these conditions was estimated to be greater than 90% by ELSD peak area.
• the poly-histidine-linked PicK hydroxylase was prepared from pKOS023-68 transformed into E. coli BL21 (DE3) and cultured as described above. The cells were harvested and the PicK protein purified as follows. All purification steps were performed at 4°C. E. coli cell pellets were suspended in 32 ⁇ L of cold binding buffer (20 mM Tris/HCl, pH 8.0, 5 mM imidazole, 500 mM NaCl) per mL of culture and lysed by sonication. For analysis of E.
• Reactions for kinetic assays (100 ⁇ L) consisted of 50 mM Tris/HCl (pH 7.5), 100 ⁇ M spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 U glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, 20-500 ⁇ M narbomycin substrate, and 50-500 nM of PicK enzyme. The reaction proceeded at 30°C, and samples were withdrawn for analysis at 5, 10, 15, and 90 minutes. Reactions were stopped by heating to 100°C for 1 minute, and denatured protein was removed by centrifugation.
• narbomycin and formation of picromycin were determined by high performance liquid chromatography (HPLC, Beckman C-18 0.46x15 cm column) coupled to atmospheric pressure chemical ionization (APCI) mass spectroscopic detection (Perkin Elmer/Sciex API 100) and evaporative light scattering detection (Alltech 500 ELSD).
• HPLC high performance liquid chromatography
• APCI atmospheric pressure chemical ionization
• Mass spectroscopic detection Perkin Elmer/Sciex API 100
• evaporative light scattering detection Alltech 500 ELSD
• the picK gene was amplified from cosmid pKOS023-26 using the primers: N3903: 5*-TCCTCTAGACGTTTCCGT-3' (SEQ ID NO:31); and N3904: 5'-TGAAGCTTGAATTCAACCGGT-3' (SEQ ID NO:32) to obtain an -1.3 kb product.
• the product was treated with restriction enzymes Xbal and Hindlll and ligated with the 7.6 kb Xba ⁇ -Hindlll restriction fragment of plasmid pWHMl 104 to provide plasmid pKOS039-01, placing fhepicK gene under the control of the ermE* promoter.
• the resulting plasmid was transformed into purified stocks of S. narbonensis by protoplast fusion and electroporation. The transformants were grown in suitable media and shown to convert narbomycin to picromycin at a yield of over 95%.
• Example 7 Construction of a Hybrid DEBS/Narbonolide PKS
• the hybrid PKS contains portions of the narbonolide PKS and portions of rapamycin and/or DEBS PKS.
• the hybrid PKS comprises the narbonolide PKS extender module 6 ACP and thioesterase domains and the DEBS loading module and extender modules 1-5 as well as the KS and AT domains of DEBS extender module 6 (but not the KR domain of extender module 6).
• the hybrid PKS is identical except that the KSl domain is inactivated, i.e., the ketosynthase in extender module 1 is disabled.
• the inactive DEBS KSl domain and its construction are described in detail in PCT publication Nos. WO 97/02358 and WO 99/03986, each of which is inco ⁇ orated herein by reference.
• the primers used in the PCR were:
• N3905 5'-TTTATGCATCCCGCGGGTCCCGGCGAG-3' (SEQ ID NO:33); and N3906: 5'-TCAGAATTCTGTCGGTCACTTGCCCGC-3' (SEQ ID NO:34).
• the 1.6 kb PCR product was digested with Pstl and EcoRI and cloned into the corresponding sites of plasmid pKOSO 15-52 (this plasmid contains the relevant portions of the coding sequence for the DEBS extender module 6) and commercially available plasmid pLitmus 28 to provide plasmids pKOS039-12 and pKOS039-13, respectively.
• Plasmid pJRJ2 is described in PCT publication Nos. WO 99/03986 and WO 97/02358, inco ⁇ orated herein by reference. Plasmids pKOS039-18 and pKOS039-19, respectively, were obtained. These two plasmids were transformed into streptomyces coelicolor CH999 by protoplast fusion.
• the resulting cells were cultured under conditions such that expression of the PKS occurred.
• Cells transformed with plasmid pKOS039-18 produced the expected product 3-deoxy-3-oxo- 6-deoxyerythronolide B.
• cells transformed with plasmid pKOS039-19 were provided (2S,3R)-2-methyl-3-hydroxyhexanoate NACS, 13-desethyl-l 3 -propyl-3-deoxy-3-oxo-6- deoxyerythronolide B was produced.
• Example 8 6-Hydroxylation of 3,6-dideoxy-3-oxoerythronolide B using the eryF hydroxylase
• Certain compounds of the invention can be hydroxylated at the C6 position in a host cell that expresses the eryF gene. These compounds can also be hydroxylated in vitro, as illustrated by this example.
• the 6-hydroxylase encoded by eryF was expressed in E. coli, and partially purified.
• the hydroxylase (100 pmol in 10 ⁇ L) was added to a reaction mixture (100 ⁇ l total volume) containing 50 mM Tris/HCl (pH 7.5), 20 ⁇ M spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, and 10 nmol 6-deoxyerythronolide B.
• the reaction was allowed to proceed for 90 minutes at 30°C.
• hemiketal formation in the above compound and compounds of similar structure.
• Antibacterial activity was 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

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