EP1465908A2 - Compositions et methodes d'hydroxylation d'epothilones - Google Patents

Compositions et methodes d'hydroxylation d'epothilones

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Publication number
EP1465908A2
EP1465908A2 EP02792416A EP02792416A EP1465908A2 EP 1465908 A2 EP1465908 A2 EP 1465908A2 EP 02792416 A EP02792416 A EP 02792416A EP 02792416 A EP02792416 A EP 02792416A EP 1465908 A2 EP1465908 A2 EP 1465908A2
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EP
European Patent Office
Prior art keywords
epothilone
seq
nucleic acid
acid sequence
hydroxylase
Prior art date
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EP02792416A
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German (de)
English (en)
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EP1465908A4 (fr
Inventor
Jonathan David Basch
Shu-Jen Chiang
Suo-Win Liu
Akbar Nayeem
Yuhua Sun
You Li
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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Publication of EP1465908A2 publication Critical patent/EP1465908A2/fr
Publication of EP1465908A4 publication Critical patent/EP1465908A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to isolated nucleic acids sequences and polypeptides encoded thereby for epothilone B hydroxylase and mutants and variants thereof, and a ferredoxin located downstream from the epothilone B hydroxylase gene.
  • the present invention also relates to recombinant microorganisms expressing epothilone B hydroxylase or a mutant or variant thereof and/or ferredoxin which are capable of hydroxylating small organic molecule compounds, such as epothilones, having a terminal alkyl group to produce compounds having a terminal hydroxyalkyl group.
  • compositions and methods of the present invention are useful in preparation of epothilones having a variety of utilities in the pharmaceutical field.
  • a novel epothilone analog produced using a mutant of epothilone B hydroxylase of the present invention is also described.
  • Epothilones are macrolide compounds that find utility in the pharmaceutical field.
  • epothilones A and B having the structures:
  • TAXOL ® paclitaxel
  • Epothilones A and B are natural anticancer agents produced by Sorangium cellulosum that were first isolated and characterized by Hofle et al., DE 4138042; WO 93/10121; Angew. Chem. Int. Ed. Engl. Vol. 35, Nol3/14, 1567-1569 (1996); and Antibiot.. Vol. 49, No. 6, 560-563 (1996). Subsequently, the total syntheses of epothilones A and B have been published by Balog et al., Angew. Chem. Int. Ed. Engl.. Vol. 35, No. 23/24, 2801-2803, 1996; Meng et al, J. Am. Chem. Soc. Vol.
  • WO 98/25929 disclosed the methods for chemical synthesis of epothilone A, epothilone B, analogs of epothilone and libraries of epothilone analogs.
  • Figure 1 provides a diagram of the biotransformation as described in WO 00/39276 of epothilone B to epothilone F in Actinomycetes species strain SC15847 (ATCC PT-1043), subsequently identified as Amycolatopsis orientalis.
  • Cytochrome P450 enzymes are found in prokaryotes and eukaryotic cells and have in common a heme binding domain which can be distinguished by an absorbance peak at 450 nm when complexed with carbon monoxide. Cytochrome P450 enzymes perform a broad spectrum of oxidative reactions on primarily hydrophobic substrates including aromatic and benzylic rings, and alkanes. In prokaryotes they are found as detoxifying systems and as a first enzymatic step in metabolizing substrates such as toluene, benzene and camphor. Cytochrome P450 genes have also been found in biosynthetic pathways of secondary metabolites such as nikkomycin in Streptomyces tendae (Bruntner, C.
  • the cytochrome P450 systems in prokaryotes are composed of three proteins; a ferredoxin NADH or NADPH dependent reductase, an iron-sulfur ferredoxin and the cytochrome P450 enzyme (Lewis, D.F., Hlavica, P., 2000, Biochim. Biophys. Acta., 1460: 353-374). Electrons are transferred from ferredoxin reductase to the ferredoxin and finally to the cytochrome P450 enzyme for the splitting of molecular oxygen.
  • An object of the present invention is to provide isolated nucleic acid sequences encoding epothilone B hydroxylase and variants or mutants thereof and isolated nucleic acid sequences encoding ferredoxin or variants or mutants thereof.
  • Another object of the present invention is to provide isolated polypeptides comprising amino acid sequences of epothilone B hydroxylase and variants or mutants thereof and isolated polypeptides comprising amino acid sequences of ferredoxin and variants or mutants thereof.
  • Another object of the present invention is to provide structure coordinates of the homology model of the epothilone B hydroxylase.
  • the structure coordinates are listed in Appendix 1.
  • This model of the present invention provides a means for designing modulators of a biological function of epothilone B hydroxylase as well as additional mutants of epothilone B hydroxylase with altered specificities.
  • Another object of the present invention is to provide vectors comprising nucleic acid sequences encoding epothilone B hydroxylase or a variant or mutant thereof and/or ferredoxin or a variant or mutant thereof.
  • these vectors further comprise a nucleic acid sequence encoding ferredoxin.
  • Another object of the present invention is to provide host cells comprising a vector containing a nucleic acid sequence encoding epothilone B hydroxylase or a variant or mutant thereof and/or ferredoxin or a variant or mutant thereof.
  • Another object of the present invention is to provide a method for producing recombinant microorganisms that are capable of hydroxylating compounds, and in particular epothilones, having a terminal alkyl group to produce compounds having a terminal hydroxyalkyl group.
  • Another object of the present invention is to provide microorganisms produced recombinantly which are capable of hydroxylating compounds, and in particular epothilones, having a terminal alkyl group to produce compounds having a terminal hydroxyalkyl group.
  • Another object of the present invention is to provide methods for hydroxylating compounds in these recombinant microorganisms.
  • the present invention provides a method for the preparation of hydroxyalkyl-bearing epothilones, which compounds find utility as antitumor agents and as starting materials in the preparation of other epothilone analogs.
  • Yet another object of the present invention is to provide a compound of Formula A:
  • 24-OH epothilone B or 24-OH EpoB compositions and methods for production of compositions comprising the compound of Formula A.
  • Figure 1 provides a schematic of the biotransformation as set forth in WO 00/39276, U.S. Application Serial No. 09/468,854, filed December 21, 1999, of epothilone B to epothilone F by Amycolatopsis orientalis strain SC15847 (PTA1043).
  • Figure 2 shows the nucleic acid sequence alignments of SEQ ED NO: 5 through SEQ ID NO:22 used to design the PCR primers for cloning of the nucleic acid sequence encoding epothilone B hydroxylase.
  • Figure 3 shows the sequence alignment between epothilone B hydroxylase (SEQ ID NO:2) and EryF (PDB code IJIN chain A; SEQ ID NO:76).
  • SEQ ID NO:2 epothilone B hydroxylase
  • PDB code IJIN chain A SEQ ID NO:76
  • the asterisks indicate sequence identities, the colons (:) similar residues.
  • Figure 4 provides a homology model of epothilone B hydroxylase based upon sequence alignment with EryF as shown in Figure 3.
  • Figure 5 shows an energy plot of the epothilone B hydroxylase model (indicated by dashed line) relative to EryF (PDB code IJIN; indicated by solid line).
  • An averaging window size of 51 residues was used, i.e., the energy at a given residue position is calculated as the average of the energies of the 51 residues in the sequence that lie with the given residue at the central positions.
  • the present invention relates to isolated nucleic acid sequences and polypeptides and methods for obtaining compounds with desired substituents at a terminal carbon position.
  • the present invention provides compositions and methods for the preparation of hydroxyalkyl-bearing epothilones, which compounds find utility as antitumor agents and as starting materials in the preparation of other epothilone analogs.
  • epothilone denotes compounds containing an epothilone core and a side chain group as defined herein.
  • epothilone core denotes a moiety containing the core structure (with the numbering of ring system positions used herein shown):
  • Q is selected from the group consisting of
  • W is O or NR 6 ;
  • X is selected from the group consisting of O, H and OR 7 ;
  • M is O, S, NR 8 , CR 9 R ⁇ 0 ;
  • ⁇ and B 2 are selected from the group consisting of ORn, OCORj 2 ;
  • R 1 -R 5 and R ⁇ 2 -Ri 7 are selected from the group consisting of H, alkyl, substituted alkyl, aryl, and heterocyclo, and wherein Ri and R 2 are alkyl they can be joined to form a cycloalkyl;
  • R 6 is selected from the group consisting of H, alkyl, and substituted alkyl
  • R 7 and Ri 1 are selected from the group consisting of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl and dialkylarylsilyl
  • side chain group refers to substituent G as defined above for
  • Ai and A 2 are independently selected from the group of optionally substituted C]-C 3 alkyl and alkenyl;
  • Q is optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring;
  • n, m, and o are integers independently selected from the group consisting of zero and 1, where at least one of m, n or o is 1.
  • terminal carbon or terminal alkyl group refers to the terminal carbon or terminal methyl group of the moiety either directly bonded to the epothilone core at position 15 or to the terminal carbon or terminal alkyl group of the side chain group bonded at position 15. It is understood that the term “alkyl group” includes alkyl and substituted alkyl as defined herein.
  • alkyl refers to optionally substituted, straight or branched chain saturated hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms.
  • lower alkyl refers to optionally substituted alkyl groups of 1 to 4 carbon atoms.
  • substituted alkyl refers to an alkyl group substituted by, for example, one to four substituents, such as, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, aralkylamino, cycloalkylamino, heterocycloamino, disubstituted amines in which the 2 amino substituents are selected from alkyl, aryl or aralkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, aralkylthio, cycloalkyl
  • isolated polynucleotides that encode epothilone B hydroxylase, an enzyme capable of hydroxylating epothilones having a terminal alkyl group to produce epothilones having a terminal hydroxyalkyl group.
  • isolated polynucleotides that encode a ferredoxin, the gene for which is located downstream from the epothilone B hydroxylase gene.
  • Ferredoxin is a protein of the cytochrome P450 system.
  • polynucleotides as used herein, it is meant to include any form of DNA or RNA such as cDNA or genomic DNA or mRNA, respectively, encoding these enzymes or an active fragment thereof which are obtained by cloning or produced synthetically by well known chemical techniques.
  • DNA may be double- or single- stranded.
  • Single-stranded DNA may comprise the coding or sense strand or the non- coding or antisense strand.
  • polynucleotide also includes polynucleotides exhibiting at least 60% or more, preferably at least 80%, homology to sequences disclosed herein, and which hybridize under stringent conditions to the above-described polynucleotides.
  • stringent conditions means hybridization conditions of 60°C at 2xSSC buffer. More preferred are isolated nucleic acid molecules capable of hybridizing to the nucleic acid sequence set forth in 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72, or 74 or SEQ ID NO: 3, or to the complementary sequence of the nucleic acid sequence set forth in SEQ ID NO:l, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62 ,64, 66, 68, 70, 72 ,or 74 or SEQ ID NO:3, under hybridization conditions of 3X SSC at 65°C for 16 hours, and which are capable of remaining hybridized to the nucleic acid sequence set forth in SEQ ID NO:l, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72 or 74 or SEQ ID NO:3, or
  • a polynucleotide of the present invention comprises the genomic DNA depicted in SEQ ID NO: 1 or a homologous sequence or fragment thereof which encodes a polypeptide having similar activity to that of this epothilone B hydroxylase.
  • a polynucleotide of the present invention may comprise the genomic DNA depicted in SEQ ID NO:3 or a homologous sequence or fragment thereof which encodes a polypeptide having similar activity to this ferredoxin. Due to the degeneracy of the genetic code, polynucleotides of the present invention may also comprise other nucleic acid sequences encoding this enzyme and derivatives, variants or active fragments thereof.
  • the present invention also relates to variants of these polynucleotides which may be naturally occurring, i.e., present in microorganisms such as Amycolatopsis orientalis and Amycolata autotrophica, or in soil or other sources from which nucleic acids can be isolated, or mutants prepared by well known mutagenesis techniques.
  • Exemplary variants polynucleotides of the present invention are depicted in SEQ ID NO: 36-42.
  • mutants as used herein it is meant to be inclusive of nucleic acid sequences with one or more point mutations, or deletions or additions of nucleic acids as compared to SEQ ID NO: 1 or 3, but which still encode a polypeptide or fragment with similar activity to the polypeptides encoded by SEQ ID NO: 1 or 3.
  • mutations are made which alter the substrate specificity and/or yield of the enzyme.
  • a preferred region of mutation with respect to the epothilone B hydroxylase gene is that region of the nucleic acid sequence coding for the approximately 113 amino acids residues comprising the active site of the enzyme.
  • mutants encoding a polypeptide with at least one amino acid substitution at amino acid position GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU 231, SER294, PHE237, or ILE365 of SEQ ID NO: 1.
  • Exemplary polynucleotide mutants of the present invention are depicted in SEQ ID NO: 30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74.
  • Cloning of the nucleic acid sequence of SEQ ID NO: 1 encoding epothilone B hydroxylase was performed using PCR primers designed by aligning the nucleic acid sequences of six cytochrome P450 genes from bacteria. The following cytochrome P450 genes were aligned:
  • Sequence 1 Locus: STMSUACB; Accession number: M32238; Reference: Omer, C.A., J. Bacteriol. 172: 3335-3345 (1990)
  • Sequence 2 Locus: STMSUBCB; Accession number: M32239; Reference:
  • Primers P450-l + (SEQ ID NO:23) and P450-la + (SEQ ID NO:24) were designed from the I helix
  • Primer P450-2 + (SEQ ID NO:25) was designed from the B-Bulge and L-helix region
  • Primer P450-3 " was designed as the reverse complement to the heme binding protein. Genomic fragments were then amplified via polymerase chain reaction (PCR).
  • the reaction products were separated by gel electrophoresis and fragments of the expected size were excised.
  • the DNA was extracted from the agarose gel slices using the Qiaquick gel extraction procedure (Qiagen, Santa Clarita, California, USA). The fragments were then cloned into the PCRscript vector (Stratagene, La Jolla, California, USA) using the PCRscript Amp cloning kit
  • the cloned PCR products were sequenced using the Big-Dye sequencing kit from Applied Biosystems, (Foster City, California, USA) and were analyzed using the ABD 10 sequencer (Applied Biosystems, Foster City, California, USA).
  • the sequence of the inserts was used to perform a TblastX search, using the protocol of Altschul, S.F, et al., Mol. Biol. 215:403-410 (1990), of the non-redundant protein database.
  • the nucleic acid sequence of the genomic DNA was determined using the Big-Dye sequencing system (Applied Biosystems) and analyzed using an ABD 10 sequencer. This sequence is depicted in SEQ ID NO: 1. An open reading frame coding for a protein of 404 amino acids and a predicted molecular weight of 44.7 kDa was found within the cloned Bglll fragment. The deduced amino acid sequence of this polypeptide is depicted in SEQ ID NO: 2. The amino acid sequence of this polypeptide was found to share 51% identity with the NikF protein of Streptomyces tendae (Bruntner, C. et al, 1999, Mol. Gen. Genet. 262: 102-114) and 48% identity with the Sca-2 protein of S.
  • ebh gene sequence was also used to isolate variant cytochrome P450 genes from other microorganisms.
  • Exemplary variant polynucleotides ebh43491, ebhl4930, ebh5363 , ebh53550, ebh.39444, eM43333 and ebh.35165 of the present invention and the species from which they were isolated are depicted in Table 1 below.
  • the nucleic acid sequences for these variants are depicted in SEQ ID NO:36- 42, respectively.
  • Table 1 Variant polynucleotides
  • Amycolatopsis orientalis ebh 14930 Amycolatopsis orientalis ebh 14930
  • amino acid sequences encoded by the exemplary variants ebh43491, ebh!4930, ebh53630, ebh53550, ebh39444, eb/ ⁇ 43333 and ebh35165 are depicted in SEQ ID NO:43-49, respectively.
  • Table 2 provides a summary of the amino acid substitutions of these exemplary variants.
  • Mutations were also introduced into the coding region of the ebh gene to identify mutants with improved yield, and or rate of biocon version and/or altered substrate specificity.
  • Exemplary mutant nucleic acid sequences of the present invention are depicted in SEQ ID NO: 30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74.
  • the nucleic acid sequence of SEQ ID NO:30 encodes a mutant ebh25-l which exhibits altered substrate specificity.
  • Plasmid pANT849eb/ ⁇ 25-l containing this mutant gene was deposited and accepted by an International Depository Authority under the provisions of the Budapest Treaty. The deposit was made on November , 2002 to the American Type Culture Collection at 10801 University Boulevard in Manassas, Virginia 20110-2209. The ATCC Accession Number is . All restrictions upon public access to this plasmid will be irrevocably removed upon granting of this patent application. The Deposit will be maintained in a public depository for a period of thirty years after the date of deposit or five years after the last request for a sample or for the enforceable life of the patent, whichever is longer. The above-referenced plasmid was viable at the time of the deposit. The deposit will be replaced if viable samples cannot be dispensed by the depository.
  • This S. lividans transformant identified in the screening of mutation 25 was found to produce a product with a different HPLC elution time than epothilone B or epothilone F.
  • a sample of this unknown was analyzed by LC-MS and was found to have a molecular weight of 523 (M.W.), consistent with a single hydroxylation of epothilone B. Plasmid DNA was isolated from the S.
  • SEQ ID NO:32 encodes a mutant eM10-53, which exhibits improved bioconversion yield.
  • This S. lividans transformant identified in the screening of mutation 10 (primers NPB29-mutl0f (SEQ ID NO:54) and
  • NPB29-mutl0r (SEQ ID NO:55)) produced a greater yield of epothilone F.
  • Plasmid DNA was isolated from the S. lividans culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29)(see Example 16). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh 10-53 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, glutamic acid 231 is changed to arginine and phenylalanine 190 is changed to tyrosine.
  • the position 231 was the target of the mutagenesis, the change at residue 190 is an inadvertent change that is an artifact of the mutagenesis procedure.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:32 is depicted in SEQ ID NO:33.
  • SEQ ID NO:34 encodes a mutant ebh24- ⁇ 6, which also exhibits improved bioconversion yield.
  • This S. lividans transformant, ebh24-16 identified in the screening of mutation 24 (primers NPB29-mut24f (SEQ ID NO:56) and NPB29-mut24r (SEQ ID NO:57) also produced a greater yield of epothilone F.
  • Plasmid DNA was isolated from the S. lividans culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh.24- 6 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, phenylalanine 237 is changed to alanine and isoleucine 92 is changed to valine.
  • the position 237 was the target of the mutagenesis, the change at residue 92 is an inadvertent change that is an artifact of the mutagenesis procedure.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:34 is depicted in SEQ ID NO:35.
  • SEQ ID NO:60 The nucleic acid sequence of SEQ ID NO:60 encodes a mutant ebh24-16dS, which also exhibits improved bioconversion yield.
  • Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh24-16dS mutant was found to have one mutation resulting in a change in the amino acid sequence of the protein, arginine 67 is changed to glutamine. This change is an artifact of the mutagenesis procedure.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:60 is SEQ ID NO:61.
  • the nucleic acid sequence of SEQ ID NO:62 encodes a mutant ebh24-16zl 1, which also exhibits improved bioconversion yield.
  • rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29).
  • the expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh24-16c ⁇ l mutant was found to have two additional mutations resulting in changes in the amino acid sequence of the protein, alanine 93 is changed to glycine and isoleucine 365 is changed to threonine.
  • the position 93 is the target of the mutagenesis, the change at 365 is an artifact of the mutagenesis procedure.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:62 is depicted in SEQ ID NO:63.
  • SEQ ID NO: 64 The nucleic acid sequence of SEQ ID NO: 64 encodes a mutant ebh.24-16-16, which also exhibits improved bioconversion yield.
  • Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29- 6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh24- ⁇ 6-16 mutant was found to have one additional mutation resulting in changes in the amino acid sequence of the protein, valine 106 is changed to alanine.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:64 is depicted in SEQ ID NO:65.
  • the nucleic acid sequence of SEQ ID NO:66 encodes a mutant ebh24- 16-74, which also exhibits improved bioconversion yield.
  • This S. rimosus transformant, ebh24- 16-74 identified in the screening of random mutants of ebh24-16 also produced a greater yield of epothilone F.
  • Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29- 6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh24- 16-74 mutant was found to have one additional mutation resulting in changes in the amino acid sequence of the protein, arginine 88 is changed to histidine.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:66 is SEQ ID NO:67.
  • the nucleic acid sequence of SEQ ID NO:68 encodes a mutant eM24-M18, which also exhibits improved bioconversion yield.
  • This S. rimosus transformant, ebhM-lS identified in the screening of random mutants of ebh also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S.
  • rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29).
  • the expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the eb ⁇ M-18 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, glutamic acid 31 is changed to lysine and methionine 176 is changed to valine.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 68 is depicted in SEQ ID NO:69.
  • SEQ DD NO:72 The nucleic acid sequence of SEQ DD NO:72 encodes a mutant eW ⁇ 24-16g8, which also exhibits improved bioconversion yield.
  • This S. rimosus transformant, ebh24-16gS identified in the screening of mutation 50 also produced a greater yield of epothilone F.
  • Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ DD NO:28) and NPB29-7r (SEQ DD NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the ebh24-16g% mutant was found to have two additional mutations resulting in changes in the amino acid sequence of the protein, methionine 176 is changed to alanine and isoleucine 130 is changed to threonine.
  • the position 176 is the target of the mutagenesis, the change at 130 is an artifact of the mutagenesis procedure.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ DD NO:72 is depicted in SEQ DD NO:73.
  • the nucleic acid sequence of SEQ DD NO:74 encodes a mutant eb/z24-16b9, which also exhibits improved bioconversion yield. This S.
  • rimosus transformant ebh24-16 >9 identified in the screening of mutation 50 (primer NPB29mut50 (SEQ DD NO:78) also produced a greater yield of epothilone F.
  • Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ DD NO:28) and NPB29-7r (SEQ DD NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system.
  • the eb/*24-16b9 mutant was found to have two additional mutations resulting in changes in the amino acid sequence of the protein, methionine 176 is changed to serine and alanine 140 is changed to threonine.
  • the position 176 is the target of the mutagenesis
  • the change at 140 is an artifact of the mutagenesis procedure.
  • the amino acid sequence of the mutant polypeptide encoded by SEQ DD NO:74 is depicted in SEQ DD NO:75.
  • polypeptide it is meant to include the amino acid sequence of SEQ DD NO: 2, and fragments or variants, which retain essentially the same biological activity and/or function as this epothilone B hydroxylase.
  • polypeptide it is meant to include the amino acid sequence of SEQ DD NO:4, and fragments and/or variants, which retain essentially the same biological activity and/or function as this ferredoxin.
  • variants as used herein it is meant to include polypeptides with amino acid sequences with conservative amino acid substitutions as compared to SEQ DD NO: 2 or 4 which are demonstrated to exhibit similar biological activity and/or function to SEQ DD NO:2 or 4.
  • conservative amino acid substitutions it is meant to include replacement, one for another, of the aliphatic amino acids such as Ala, Val, Leu and De, the hydroxyl residues Ser and Thr, the acidic residues Asp and Glu, and the amide residues Asn and Gin.
  • aliphatic amino acids such as Ala, Val, Leu and De
  • the hydroxyl residues Ser and Thr the acidic residues Asp and Glu
  • the amide residues Asn and Gin Exemplary variant amino acid sequences of the present invention are depicted in SEQ DD NO:43-49 and the amino acid substitutions of these exemplary variants are described in Table 2, supra.
  • mutants as used herein it is meant to include polypeptides encoded by nucleic acid sequences with one or more point mutations, or deletions or additions of nucleic acids as compared to SEQ DD NO: 1 or 3, but which still have similar activity to the polypeptides encoded by SEQ DD NO: 1 or 3.
  • mutations are made to the nucleic acid that alter the substrate specificity and/or yield from the polypeptide encoded thereby.
  • a preferred region of mutation with respect to the epothilone B hydroxylase gene is that region of the nucleic acid sequence coding for the approximately 113 amino acid residues comprising the active site of the enzyme.
  • Exemplary mutants ebh25-l, ebhl0-53, ebh24-16, eWz24-16d8, e 24-16cl 1, ebh24-16-16, ebh24-16-14, ebh24-16g , ebh24-16b9 and the nucleic acid sequences encoding such mutants of the present invention are depicted in SEQ DD NO:31, 33, 35, 61, 63, 65, 67, 69, 71, 73 and 75, and SEQ DD NO:30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74, respectively.
  • a 3-dimensional model of epothilone B hydroxylase has also been constructed in accordance with general teachings of Greer et al. (Comparative modeling of homologous proteins. Methods In Enzymology 202239-52, 1991), Lesk et al. (Homology Modeling: Inferences from Tables of Aligned Sequences. Curr. Op. Struc Biol. (2) 242-247, 1992), and Cardozo et al. (Homology modeling by the ICM method. Proteins 23, 403-14, 1995) on the basis of the known structure of a homologous protein EryF (PDB Code 1KTN chain A). Homology between these sequences is 34%.
  • An energy plot of the epothilone B hydroxylase model relative to EryF (PDB code 1 JL ⁇ ) was also prepared and is depicted in Figure 5.
  • An averaging window size of 51 residues was used at a given residue position to calculate the average of the energies of the 51 residues in the sequence that lie with the given residue at the central position. As shown in Figure 5, all energies along the sequence lie below zero thus indicating that the modeled structure as set forth in Figure 4 and Appendix 1 is reasonable.
  • the three-dimensional structure represented in the homology model of epothilone B hydroxylase of Figure 4 is defined by a set of structure coordinates as set forth in Appendix 1.
  • the term "structure coordinates" refers to Cartesian coordinates generated from the building of a homology model.
  • a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions.
  • an entirely different set of coordinates could define a similar or identical shape.
  • slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates and/or using different methods in generating the homology model, will have minor effects on the overall shape.
  • Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates.
  • the structure coordinates set forth in Appendix 1 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • the superimposition tool in the program SYBYL allows comparisons to be made between different structures and different conformations of the same structure.
  • the procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure.
  • equivalent atoms are defined as protein backbone atoms (N, C ⁇ , C and O) for all conserved residues between the two structures being compared.
  • rigid fitting operations are considered .
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by SYBYL.
  • any homology model of epothilone B hydroxylase that has a root mean square deviation of conserved residue backbone atoms (N, C ⁇ , C, O) of less than about 4.0 A when superimposed on the corresponding backbone atoms described by structure coordinates listed in Appendix 1 are considered identical. More preferably, the root mean square deviation is less than about 3.0 A. More preferably the root mean square deviation is less than about 2.0 A.
  • any homology model of epothilone B hydroxylase that has a root mean square deviation of conserved residue backbone atoms (N, C ⁇ , C, O) of less than about 2.0 A when superimposed on the corresponding backbone atoms described by structure coordinates listed in Appendix 1 are considered identical. More preferably, the root mean square deviation is less than about 1.0 A.
  • structural models wherein backbone atoms have been substituted with other elements which when superimposed on the corresponding backbone atoms have low root mean square deviations are considered to be identical.
  • an homology model where the original backbone carbon, and/or nitrogen and/or oxygen atoms are replaced with other elements having a root mean square deviation of about 4.0 A, more preferably about 3.0 A, even more preferably less than about 2A, when superimposed on the corresponding backbone atoms described by structure coordinates listed in Appendix 1 is considered identical.
  • root mean square deviation means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object.
  • the "root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the epothilone B hydroxylase portion of the complex as defined by the structure coordinates described herein.
  • the present invention as embodied by the homology model enables the structure-based design of additional mutants of epothilone B hydroxylase.
  • residues lying within lOA of the binding site of epothilone B hydroxylase have now been defined.
  • Mutants with mutations at one or more of these positions are expected to exhibit altered biological function and/or specificity and thus comprise another embodiment of preferred mutants of the present invention.
  • Another embodiment of preferred mutants are molecules that have a root mean square deviation from the backbone atoms of said epothilone B hydroxylase of not more than about 4. ⁇ A.
  • the structure coordinates of an epothilone B hydroxylase homology model or portions thereof are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.
  • Another aspect of the present invention relates to machine- readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Appendix 1.
  • the three-dimensional model structure of epothilone B hydroxylase can also be used to identify modulators of biological function and potential substrates of the enzyme. Various methods or combinations thereof can be used to identify such modulators.
  • a test compound can be modeled that fits spatially into a binding site in epothilone B hydroxylase, according to Appendix 1.
  • Structure coordinates of amino acids within 10 A of the binding region of epothilone B hydroxylase defined by amino acids LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, LYS85, PRO86, PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS 172, SER173, SER174, ARG175, MET176, LEU177, SER178, ARG179
  • Identified structural or chemical features can then be employed to design or select compounds as potential epothilone B hydroxylase ligands.
  • structural and chemical features it is meant to include, but is not limited to, covalent bonding, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction.
  • Compounds identified as potential epothilone B hydroxylase ligands can then be synthesized and screened in an assay characterized by binding of a test compound to epothilone B hydroxylase, or in characterizing the ability of epothilone B hydroxylase to modulate a protease target in the presence of a small molecule.
  • Examples of assays useful in screening of potential epothilone B hydroxylase ligands include, but are not limited to, screening in silico, in vitro assays and high throughput assays.
  • other structure-based design methods can be used.
  • Various computational structure-based design methods have been disclosed in the art. For example, a number of computer modeling systems are available in which the sequence of epothilone B hydroxylase and the epothilone B hydroxylase structure (i.e., atomic coordinates of epothilone B hydroxylase as provided in Appendix 1 and/or the atomic coordinates within lOA of the binding region as provided above) can be input.
  • This computer system then generates the structural details of one or more these regions in which a potential epothilone B hydroxylase modulator binds so that complementary structural details of the potential modulators can be determined.
  • Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with epothilone B hydroxylase. In addition, the compound must be able to assume a conformation that allows it to associate with epothilone B hydroxylase.
  • Some modeling systems estimate the potential inhibitory or binding effect of a potential epothilone B hydroxylase substrate or modulator prior to actual synthesis and testing.
  • Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in a binding region of epothilone B hydroxylase. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic force fields such as, MMFF, CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRDD (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).
  • compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor.
  • Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Inc., St. Louis MO).
  • DOCK Zantz et al. 1982
  • Programs such as DOCK can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the active site binding region which may therefore be suitable candidates for synthesis and testing.
  • vectors comprising polynucleotides of the present invention and host cells which are genetically engineered with vectors of the present invention to produce epothilone B hydroxylase or active fragments and variants or mutants of this enzyme and/or ferredoxin or active fragments thereof.
  • any vector suitable to maintain, propagate or express polynucleotides to produce these polypeptides in the host cell may be used for expression in this regard.
  • the vector may be, for example, a plasmid vector, a single- or double- stranded phage vector, or a single- or double- stranded RNA or DNA viral vector.
  • Vectors may be extra-chromosomal or designed for integration into the host chromosome.
  • Such vectors include, but are not limited to, chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses such as baculoviruses, papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids.
  • Useful expression vectors for prokaryotic hosts include, but are not limited to, bacterial plasmids, such as those from E. coli, Bacillus or Streptomyces, including pBluescript, pGEX-2T, pUC vectors, pET vectors, ColEl, pCRl, ⁇ BR322, pMB9, pCW, pBMS200, pBMS2020, PU101, PIJ702, pANT849, pOJ260, pOJ446, pSET152, pKCl 139, pKC1218, pFD666 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, ⁇ GTIO and ⁇ GTl 1, and other phages, e.g., M13 and filamentous single stranded phage DNA.
  • Vectors of the present invention for use in yeast will typically contain an origin of replication suitable for use in yeast and a selectable marker that is functional in yeast.
  • yeast vectors useful in the present invention include, but are not limited to, Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp and YEp series plasmids), Yeast Centromere plasmids (the YCp series plasmids), Yeast Artificial Chromosomes (YACs) which are based on yeast linear plasmids, denoted YLp, pGPD-2, 2 ⁇ plasmids and derivatives thereof, and improved shuttle vectors such as those described in Gietz et al, Gene, 74: 527-34 (1988) (YIplac, YEplac and YCplac).
  • Mammalian vectors useful for recombinant expression may include a viral origin, such as the SV40 origin (for replication in cell lines expressing the large T-antigen, such as COS1 and COS7 cells), the papillomavirus origin, or the EBV origin for long term episomal replication (for use, e.g., in 293-EBNA cells, which constitutively express the EBV EBNA-1 gene product and adenovirus EIA).
  • a viral origin such as the SV40 origin (for replication in cell lines expressing the large T-antigen, such as COS1 and COS7 cells), the papillomavirus origin, or the EBV origin for long term episomal replication (for use, e.g., in 293-EBNA cells, which constitutively express the EBV EBNA-1 gene product and adenovirus EIA).
  • Expression in mammalian cells can be achieved using a variety of plasmids, including, but not limited to, pSV2, pBC12BI, and p91023, pCDNA vectors as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retro viruses).
  • lytic virus vectors e.g., vaccinia virus, adeno virus, and baculovirus
  • episomal virus vectors e.g., bovine papillomavirus
  • retroviral vectors e.g., murine retro viruses.
  • Useful vectors for insect cells include baculo viral vectors and pVL941.
  • expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initiating codon at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • phage promoters such as phage lambda pL promoter, the trc promoter, a hybrid derived from the trp and lac promoters, the bacteriophage T7 promoter, the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, snpA promoter, melC promotor, ermE* promoter or the ⁇ r BAD operon.
  • phage promoters such as phage lambda pL promoter, the trc promoter, a hybrid derived from the trp and lac promoters, the bacteriophage T7 promoter, the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, snpA promoter, melC promotor, ermE* promoter or the ⁇ r BAD operon.
  • Examples of useful promoters for yeast include, but are not limited to, the CYCl promoter, the GALl promoter, the GAL 10 promoter, ADHl promoter, the promoters of the yeast ⁇ -mating system, and the GPD promoter.
  • Examples of promoters routinely used in mammalian expression vectors include, but are not limited to, the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus(RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • RSV Rous Sarcoma Virus
  • Vectors comprising the polynucleotides can be introduced into host cells using any number of well known techniques including infection, transduction, transfection, transvection and transformation.
  • the polynucleotides may be introduced into a host alone or with additional polynucleotides encoding, for example, a selectable marker or ferredoxin reductase.
  • the polynucleotide for epothilone B hydroxylase and ferredoxin are introduced into the host cell.
  • Host cells for the various expression constructs are well known, and those of skill can routinely select a host cell for expressing the epothilone B hydroxylase and/or ferredoxin in accordance with this aspect of the present invention.
  • Examples of mammalian expression systems useful in the present invention include, but are not limited to, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines, and the COS-7 line of monkey kidney fibroblasts.
  • epothilone B hydroxylase and ferredoxin can be expressed recombinantly in microorganisms.
  • another aspect of the present invention relates to recombinantly produced microorganisms which express epothilone B hydroxylase alone or in conjunction with the ferredoxin and which are capable of hydroxylating a compound , and in particular an epothilone, having a terminal alkyl group to produce ones having a terminal hydroxyalkyl group.
  • the recombinantly produced microorganisms are produced by transforming cells such as bacterial cells with a plasmid comprising a nucleic acid sequence encoding epothilone B hydroxylase.
  • the cells are transformed with a plasmid comprising a nucleic acid encoding epothilone B hydroxylase or mutants or variants thereof as well as the nucleic acid sequence encoding ferredoxin located downstream of the epothilone B hydroxylase gene.
  • the recombinantly produced microorganisms of the present invention are useful in microbial processes or methods for production of compounds, and in particular epothilones, containing a terminal hydroxyalkyl group.
  • the hydroxyalkyl-bearing product can be produced by culturing the recombinantly produced microorganism or enzyme derived therefrom, capable of selectively hydroxylating a terminal carbon or alkyl, in the presence of a suitable substrate in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, under submerged aerobic conditions.
  • Suitable epothilones employed as substrate for the method of the present invention may be any such compound having a terminal carbon or terminal alkyl group capable of undergoing the enzymatic hydroxylation of the present invention.
  • the starting material, or substrate can be isolated from natural sources, such as Sorangium cellulosum, or they can be synthetically formed epothilones.
  • Other substrates having a terminal carbon or terminal alkyl group capable of undergoing an enzymatic hydroxylation can be employed by the methods herein.
  • compactin can be used as a substrate, which upon hydroxylation forms the compound pravastatin. Methods for hydroxylating compactin to pravastatin via an
  • Actinomadura strain are set forth in U.S. Patent 5,942,423 and U.S. Patent 6,274,360.
  • at least one epothilone can be prepared as described in WO 00/39276, U. S. Serial. No. 09/468,854, filed December 21, 1999, the text of which is inco ⁇ orated herein as if set forth at length.
  • A] and A 2 are independently selected from the group of optionally substituted C ⁇ -C 3 alkyl and alkenyl;
  • Q is an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring;
  • n, m, and o are integers selected from the group consisting of zero and 1 , where at least one of m or n or o is 1 ;
  • E is an epothilone core; can be prepared.
  • This method comprises the steps of contacting at least one epothilone of the following formula II CH 3 -(A,V(Q) m -(A 2 )o-E (II) where Ai, Q, A 2 , E, n, m, and o are defined as above; with a recombinantly produced microorganism, or an enzyme derived therefrom, which is capable of selectively catalyzing the hydroxylation of formula II, and effecting said hydroxylation.
  • the starting material is epothilone B.
  • Epothilone B can be obtained from the fermentation of Sorangium cellulosum So ce90, as described in DE 41 38 042 and WO 93/10121. The strain has been deposited at the Deutsche
  • Epothilone B can also be obtained by chemical means, such as those disclosed by Meng, D., et al., J.
  • a typical medium for growth includes necessary carbon sources, nitrogen sources, and trace elements. Inducers may also be added to the medium.
  • inducer includes any compound enhancing formation of the desired enzymatic activity within the recombinantly produced microbial cell.
  • Typical inducers as used herein may include solvents used to dissolve substrates, such as dimethyl sulfoxide, dimethyl formamide, dioxane, ethanol and acetone. Further, some substrates, such as epothilone B, may also be considered to be inducers.
  • Carbon sources may include sugars such as glucose, fructose, galactose, maltose, sucrose, mannitol, sorbital, glycerol starch and the like; organic acids such as sodium acetate, sodium citrate, and the like; and alcohols such as ethanol, propanol and the like.
  • Preferred carbon sources include, but are not limited to, glucose, fructose, sucrose, glycerol and starch.
  • Nitrogen sources may include an N-Z amine A, corn steeped liquor, soybean meal, beef extract, yeast extract, tryptone, peptone, cottonseed meal, peanut meal, amino acids such as sodium glutamate and the like, sodium nitrate, ammonium sulfate and the like.
  • Trace elements may include magnesium, manganese, calcium, cobalt, nickel, iron, sodium and potassium salts. Phosphates may also be added in trace or preferably, greater than trace amounts.
  • the medium employed for the fermentation may include more than one carbon or nitrogen source or other nutrient.
  • the pH of the medium is preferably from about 5 to about 8 and the temperature is from about 14°C to about 37°C, preferably the temperature is 28°C.
  • the duration of the reaction is 1 to 100 hours, preferably 8 to 72 hours.
  • the medium is incubated for a period of time necessary to complete the biotransformation as monitored by high performance liquid chromatography (HPLC). Typically, the period of time needed to complete the transformation is twelve to one hundred hours and preferably about 72 hours after the addition of the substrate.
  • the medium is placed on a rotary shaker (New Brunswick Scientific Innova 5000) operating at 150 to 300 ⁇ m and preferably about 250 ⁇ m with a throw of 2 inches.
  • the hydroxyalkyl-bearing product can be recovered from the fermentation broth by conventional means that are commonly used for the recovery of other known biologically active substances.
  • recovery means include, but are not limited to, isolation and purification by extraction with a conventional solvent, such as ethyl acetate and the like; by pH adjustment; by treatment with a conventional resin, for example, by treatment with an anion or cation exchange resin or a non-ionic adso ⁇ tion resin; by treatment with a conventional adsorbent, for example, by distillation, by crystallization; or by recrystallization, and the like.
  • a conventional solvent such as ethyl acetate and the like
  • pH adjustment such as ethyl acetate and the like
  • treatment with a conventional resin for example, by treatment with an anion or cation exchange resin or a non-ionic adso ⁇ tion resin
  • a conventional adsorbent for example, by distillation, by crystallization; or by recrystallization, and the like.
  • the extract obtained above from the biotransformation reaction mixture can be further isolated and purified by column chromatography and analytical thin layer chromatography.
  • a recombinantly produced microorganism of the present invention to biotransform an epothilone having a terminal alkyl group to an epothilone having a terminal hydroxyalkyl group was demonstrated.
  • a culture comprising a Streptomyces lividans clone containing a plasmid with the ebh gene as described in more detail in Example 11 was incubated with an epothilone B suspension for 3 days at 30°with agitation. A sample of the incubate was extracted with an equal volume of 25% methanol: 75% n-butanol, vortexed and allowed to settle for 5 minutes.
  • Example 12 Two hundred ⁇ l of the organic phase was transferred to an HPLC vial and analyzed by HPLC/MS (Example 12). A product peak of epothilone F eluted at a retention time of 15.9 minutes and had a protonated molecular weight of 524. The epothilone B substrate eluted at 19.0 minutes and had a protonated molecular weight of 508. The peak retention times and molecular weights were confirmed using known standards.
  • Rates of biotransformation of epothilone B by cells expressing ebh were also compared to rates of biotransformation by ebh mutants.
  • Cells expressing ebh comprised a frozen spore preparation of. S. lividans (pANT849-eM).
  • Cells expressing mutants comprises frozen spore preparations of S. lividans (pANT849- eb O-53) and S. lividans (pANT849-eM24-16).
  • a frozen spore preparation of S. lividans TK24 was used as the control. The cells were pre-incubated for several days at 30°C.
  • epothilone B in 100% EtOH was added to each culture to a final concentration of 0.05% weight/volume.
  • Samples were then taken at 0, 24, 48 and 72 hours with the exception of the S. lividans (pANT849- ebh24-16) culture, in which the epothilone B had been completely converted to epothilone F at 48 hours.
  • the samples were analyzed by HPLC. The results are calculated as a percentage of the epothilone B at time 0 hours.
  • Epothilone B
  • Epothilone F
  • mutant e 25-l exhibits altered substrate specificity and biotransformation of epothilone B by this mutant resulted in a product with a different HPLC elution time than epothilone B or epothilone F.
  • a sample of this unknown was analyzed by LC-MS and was found to have a molecular weight of 523 (M.W.), consistent with a single hydroxylation of epothilone B.
  • the structure of the biotransformation product was determined as 24-hydroxyl-epothilone B, based on MS and NMR data (compared with data of epothilone B):
  • compositions and methods of the present invention are useful in producing known compounds that are microtubule-stabilizing agents as well as new compounds comprising epothilone analogs such as 24-hydroxyl-epothilone B (Formula A) and pharmaceutically acceptable salts thereof expected to be useful as microtubule-stabilizing agents.
  • microtubule stabilizing agents produced using these compositions and methods are useful in the treatment of a variety of cancers and other proliferative diseases including, but not limited to, the following; carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; - hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma;
  • Microtubule stabilizing agents produced using the compositions and methods of the present invention will also inhibit angiogenesis, thereby affecting the growth of tumors and providing treatment of tumors and tumor-related disorders.
  • Such anti- angiogenesis properties of these compounds will also be useful in the treatment of other conditions responsive to anti-angiogenesis agents including, but not limited to, certain forms of blindness related to retinal vascularization, arthritis, especially inflammatory arthritis, multiple sclerosis, restinosis and psoriasis.
  • Microtubule stabilizing agents produced using the compositions and methods of the present invention will induce or inhibit apoptosis, a physiological cell death process critical for normal development and homeostasis. Alterations of apoptotic pathways contribute to the pathogenesis of a variety of human diseases.
  • Compounds of the present invention such as those set forth in formula I and ⁇ and Formula A, as modulators of apoptosis, will be useful in the treatment of a variety of human diseases with aberrations in apoptosis including, but not limited to, cancer and precancerous lesions, immune response related diseases, viral infections, degenerative diseases of the musculoskeletal system and kidney disease.
  • microtubule stabilizing agents produced using the compositions and methods of the present invention may also be used to treat conditions other than cancer or other proliferative diseases.
  • Such conditions include, but are not limited to viral infections such as he ⁇ esvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus; autoimmune diseases such as systemic lupus erythematosus, immune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases and autoimmune diabetes mellitus; neurodegenerative disorders such as Alzheimer's disease, ADDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration; ADDS; myelodysplastic syndromes; aplastic anemia; ischemic injury associated myocardial infarctions; stroke and reperfusion injury; restenosis; arrhythmi
  • R2 Medium was prepared as follows:
  • auxotrophs 50 ⁇ g/ml
  • Cysteine 37 ⁇ g/ml
  • adenine, guanine, thymidine and uracil 7.5 ⁇ g/ml
  • Vitamins 0.5 ⁇ g/ml
  • R2YE medium was prepared in the same fashion as R2 medium. However, 5 ml of Difco yeast extract (10%) was added to each 100 ml flask at time of use.
  • P (protoplast) buffer was prepared as follows:
  • Tris maleic acid buffer prepared from 1 M solution of Tris adjusted to pH 8.0 by adding maleic acid.
  • L (lysis) buffer was prepared by mixing the following sterile solutions: 100 ml Sucrose (10.3%) 10 ml TES buffer (5.73%, adjusted to pH 7.2) 1 ml K 2 SO 4 (2.5%)
  • a solution containing the following components was prepared in 1 liter of dH 2 O: glucose (10 grams), sucrose (103 grams), MgCl 2 »6H 2 O (10.12 grams), BBLTM trypticase soy broth (15 grams) (Becton Dickinson Microbiology Systems, Sparks, Maryland, USA), and BBLTM yeast extract (5 grams) (Becton Dickinson Microbiology Systems).
  • the solution was autoclaved for 30 minutes. Thiostrepton was added to a concentration of 10 ⁇ g/ml for cultures propagated with plasmids.
  • a solution containing 30% (wt/vol) PEG 1000, 10% glycerol, and 6.5 % sucrose was prepared in dH 2 O.
  • the solution was sterilized by vacuum filtration through a 0.22 ⁇ m cellulose acetate filter.
  • Genomic DNA was isolated from an Amycolatopsis orientalis soil isolate strain designation SC15847 (ATCC PT-1043) using a guanidine-detergent lysis method, DNAzol reagent (Invitrogen, Carlsbad, California, USA).
  • the SCI 5847 culture was grown 24 hours at 28°C in F7 medium (glucose 2.2%, yeast extract 1.0%, malt extract 1.0 %, peptone 0.1%, pH 7.0). Twenty ml of culture was harvested by centrifugation and resuspended in 20 ml of DNAzol, mixed by pipetting and centrifuged 10 minutes in the Beckman TJ6 centrifuge. Ten ml of 100% ethanol was added, inverted several times and stored at room temperature 3 minutes.
  • the DNA was spooled on a glass pipette washed in 100% ethanol and allowed to air dry 10 minutes. The pellet was resuspended in 500 ⁇ l of 8mM NaOH and once dissolved it was neutralized with 30 ⁇ l of IM HEPES pH7.2.
  • PCR reactions were prepared in a volume of 50 ⁇ l, containing 200-500 ng of genomic DNA or 1.0 ⁇ l of the cDNA, a forward and reverse primer, and the forward primer being either P450-l + (SEQ DD NO:23) or P450-la + (SEQ DD NO:24) or P450- 2 + (SEQ DD NO:25) and the reverse primer P450-3 " (SEQ DD NO:27)or P450-2 " (SEQ DD NO:26). All primers were added to a final concentration of 1.4- 2.0 ⁇ M.
  • the PCR reaction was prepared with 1 ⁇ l of Taq enzyme (2.5 units) (Stratagene), 5 ⁇ l of Taq buffer and 4 ⁇ l of 2.5 mM of dNTPs with dH 2 O to 50 ⁇ l.
  • the cycling reactions were performed on a Geneamp® PCR system with the following protocol: 95°C for 5 minutes, 5 cycles [95°C 30 seconds, 37°C 15 seconds (30% ramp), 72°C 30 seconds], 35 cycles (94°C 30 seconds, 65°C 15 seconds, 72°C 30 seconds), 72°C 7 minutes.
  • the expected sizes for the reactions are 340 bp for the P450-l + (SEQ DD NO:23) or P450-la + (SEQ DD NO:24) and P450-3 " (SEQ DD NO:27) primer pairs, 240 bp for the P450-l + (SEQ DD NO:23) and P450-2 " (SEQDD NO:26) primer pairs and 130 bp for the P450-2 + (SEQ DD NO:25) and P450-3 " (SEQ DD NO:27) primer pairs.
  • Patent 5,516,679 which had been digested with BamHI and dephosphorylated using the SAP I enzyme (Roche Molecular Biochemicals, Indianapolis, Indiana, catalog#l 758 250). Ligation reactions were performed in a 15 ⁇ l volume with IU of T4 DNA ligase (Invitrogen) for 1 hour at room temperature. One ⁇ l of the ligation was transformed to 100 ⁇ l of chemically competent DH10B cells (Invitrogen) and 100 ⁇ l plated to five LB agar plates with 30 ⁇ g/ml of neomycin, 37°C overnight.
  • T4 DNA ligase Invitrogen
  • the cloned PCR products were sequenced using fluorescent-dye-labeled terminator cycle sequencing, Big-Dye sequencing kit (Applied Biosystems, Foster city, California, USA) and were analyzed using laser-induced fluorescence capillary electrophoresis, ABI Prism 310 sequencer (Applied Biosystems).
  • Example 6 Extraction of Total RNA Total RNA was isolated from the SC15847 culture using a modification of the
  • Fifty-five milligrams of epothilone B was dissolved in 1 ml of 100% ethanol and added to the culture. A second ml of ethanol was used to rinse the residual epothilone B from the tube and added to the culture. The culture was incubated at 16°C, 230 ⁇ m for 30 hours. Thirty ml of the culture was transferred to a 50 ml tube, 150 mg of lysozyme was added to the culture and the culture was incubated 5 minutes at room temperature. Ten ml of the culture was placed in a 50 ml Falcon tube and centrifuged 5 minutes, 4°C in a TJ6 centrifuge. Two ml of chloroform was added and the tube was mixed vigorously for 15 seconds.
  • the tube was incubated 2 minutes at room temperature and centrifuged 10 minutes, top speed in the TJ6 centrifuge. The aqueous layer was transferred to a fresh tube and 2.5 ml of isopropanol was added to precipitate the RNA. The tube was incubated 10 minutes at room temperature and centrifuged 10 minutes, 4°C. The supernatant was removed, the pellet was rinsed with 70% ethanol and dried briefly under vacuum. The pellet was resuspended in 150 ⁇ l of RNase-free dH 2 O. Fifty ⁇ l of 7.5M LiCI was added to the RNA and incubated at -20°C for 30 minutes. The RNA was pelleted by centrifugation 10 minutes, 4°C in a microcentrifuge. The pellet was rinsed with 200 ⁇ l of 70% ethanol, dried briefly under vacuum and resuspended in 150 ⁇ l of RNase free dH 2 O.
  • RNA was treated with DNasel (Ambion, Austin, Texas, USA). Twenty- five ⁇ l of total RNA (5.3 ⁇ g/ ⁇ l), 2.5 ⁇ l of DNasel buffer, 1.0 ⁇ l of DNase I added and incubated at 37°C for 25 minutes. Five ⁇ l of DNase I inactivation buffer added, incubated 2 minutes, centrifuged 1 minute, the supernatant was transferred to a fresh tube.
  • Example 7 cDNA Synthesis cDNA was synthesized from the total RNA using the Superscript II enzyme (Invitrogen). The reaction was prepared with 1 ⁇ l of total RNA (5.3 ⁇ g/ ⁇ l), 9 ⁇ l of dH 0, 1 ⁇ l of dNTP mix (10 mM), and 1 ⁇ l of random hexamers. The reaction was incubated at 65°C for 5 minutes then placed on ice. The following components were then added: 4 ⁇ l of 1 st strand buffer, 1 ⁇ l of RNase Inhibitor, 2.0 ⁇ l of 0.1 M DTT, and 1 ⁇ l of Superscript II enzyme. The reaction was incubated at room temperature 10 minutes, 42°C for 50 minutes and 70°C for 15 minutes. One ⁇ l of RNaseH was added and incubated 20 minutes at 37°C, 15 minutes at 70°C and stored at 4°C.
  • Superscript II enzyme Invitrogen
  • Plasmid pCRscript-29 contains a 340bp PCR fragment amplified from SC15847 genomic DNA using primers P450 1 + (SEQ ID NO:23) and P450 3 " (SEQ DD NO:27). Two ⁇ l of the plasmid prep was used as a template, with a total of 25 cycles.
  • the amplified product was gel purified using the Qiaquick gel extraction system (Qiagen). The extracted DNA was ethanol precipitated and resuspended in 5 ⁇ l of TE, the yield was estimated to be 500 ng.
  • the membrane was prehybridized 1 hour at 42°C in 20 ml of Dig Easy Hyb buffer (Roche Molecular Biochemicals). The probe was denatured 10 minutes at 65°C and then placed on ice. Five ml of probe in Dig-Easy Hyb at an approximate concentration on 20 ng/ml was incubated with the membrane at 42°C overnight. The membrane was washed 2 times in 2X SCC with 0.1% SDS at room temperature, then 2 times in 0.5X SSC with 0.1% SDS at 65°C.
  • the membrane was equilibrated in Genius buffer 1 (10 mM maleic acid, 15 mM NaCl; pH 7.5; 0.3% v/v Tween 20) (Roche Molecular Biochemicals, Indianapolis, Indiana) for 2 minutes, then incubated with 2% blocking solution (2% Blocking reagent in Genius Buffer l)(Roche Molecular Biochemicals Indianapolis, Indiana) for 1 hour at room temperature. The membrane was incubated with a 1:20,000 dilution of anti-dig antibody in 50 ml of blocking solution for 30 minutes. The membrane was washed 2 times, 15 minutes each in 50 ml of Genius buffer 1.
  • the membrane was equilibrated for two minutes in Genius Buffer 3 (lOmM Tris-HCl, lOmM NaCl; pH 9.5).
  • Genius Buffer 3 lOmM Tris-HCl, lOmM NaCl; pH 9.5.
  • One ml of a 1:100 dilution of CSPD (disodium 3-(4-methoxyspiro ⁇ l,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1 3 ' 7 ]decan ⁇ -4-yl)phenyl phosphate) (Roche Molecular Biochemicals) in Genius buffer 3 was added to the membrane and incubated 5 minutes at room temperature, then placed at 37°C for 15 minutes.
  • the membrane was exposed to Biomax ML film (Kodak, Rochester, New York, USA) for 1 hour.
  • Example 10 E. coli Transformation Competent cells were purchased from Invitrogen. E. coli strain DH10B was used as a host for genomic cloning. The chemically competent cells were thawed on ice and 100 ⁇ l aliquoted to a 17 x 100-mm polypropylene tube on ice. One ⁇ l of the ligation mixture was added to the cells and incubated on ice for 30 minutes. The cells were incubated at 42°C for 45 seconds, then placed on ice 1-2 minutes. 0.9 ml pf SOC. medium(Invitrogen) was added and the cells were incubated one hour at 30- 37°C at 200-240 ⁇ m. Cells were plated on a selective medium (Luria agar with neomycin or ampicillin at a concentration of 30 ⁇ g /ml or 100 ⁇ g /ml respectively).
  • a selective medium Lia agar with neomycin or ampicillin at a concentration of 30 ⁇ g /m
  • Plasmid pWB 19N849 was constructed by digesting plasmid pWB 19N with
  • Plasmids constructed from the ligation of pWB19N and pANT849 were determined by electrophoretic mobility on 0.7 % agarose.
  • the plasmid pWB19N849 was digested with HindUI and BglH to excise a 5.3 kb fragment equivalent to plasmid pANT849 digested with Bglll and Hindm. This 5.3 kb fragment was purified on an agarose gel and extracted using the Qiaquick gel extraction system.
  • a 1.469 kb DNA fragment containing the epothilone B hydroxylase gene and the downstream ferredoxin gene was amplified using PCR.
  • the 50 ⁇ l PCR reaction was composed of 5 ⁇ l of Taq buffer, 2.5 ⁇ l glycerol, 1 ⁇ l of 20 ng/ ⁇ l NPB29-1 plasmid, 0.4 ⁇ l of 25 mM dNTPs, 1.0 ⁇ l each of primers NPB29-6F (SEQ DD NO:28) and NPB29-7R (SEQ DD NO:29) (5 pmole/ ⁇ l), 38.1 ⁇ l of dH 2 O and 0.5 ⁇ l of Taq enzyme (Stratagene).
  • the reactions were performed on a Perkin Elmer 9700, 95°C for 5 minutes, then 30 cycles (96°C for 30 seconds, 60°C 30 seconds, 72°C for 2 minutes), and 72°C for 7 minutes.
  • the PCR product was purified using a Qiagen minielute column with the PCR cleanup procedure.
  • the purified product was digested with Bglll and HindDI and purified on a 0.7 % agarose gel. A 1.469 kb band was excised from the gel and eluted using a Qiagen minielute column.
  • Five ⁇ l of this PCR product was ligated with 2 ⁇ l of the Bglll, Hindlll digested pANT849 vector in a 10 ⁇ l ligation reaction. The reaction was incubated at room temperature for 24 hours and then transformed to S. lividans TK24 protoplasts.
  • YEME media Twenty ml of YEME media was inoculated with a frozen spore suspension of S. lividans TK24 and grown 48 hours in a 125 ml bi -indent flask. Protoplasts were prepared as described in Practical Streptomyces Genetics. The ligation reaction was mixed with protoplasts, then 500 ⁇ l of transformation buffer was added, followed immediately by 5 ml of P buffer. The transformation reactions were spun down 7 minutes at 2,750 ⁇ m, resuspended in 100 ⁇ l of P buffer and plated to one R2YE plate. The plate was incubated at 28°C for 20 hours then overlaid with 5 ml of LB 0.7% agar with 250 ⁇ g/ml thiostrepton. After 7 days colonies were picked to an R2YE grid plate with 50 ⁇ g/ml of thiostrepton. The colonies were grown an additional 5 days at 28°C, then stored at 4°C.
  • This recombinant microorganism has been deposited with the ATCC and designated PTA-4022.
  • S. rimosus strain R6 593 was cultivated in 20 ml of CRM medium at 30 °C on a rotary shaker (250 ⁇ m). The cells were harvested at 24 hrs by centrifugation for 5 minutes, 5,000 ⁇ m, 4 °C, and resuspended in 20 ml of 10% sucrose, 4 °C, and centrifuged for 5 minutes, 5,000 ⁇ m, 4 °C. The pellet was resuspended in 10 ml of 15% glycerol, 4 °C and centrifuged for 5 minutes, 5,000 ⁇ m, 4 °C.
  • the pellet was resuspended in 2 ml of 15% glycerol, 4 °C with 100 ⁇ g/ml lysozyme and incubated at 37 °C for 30 minutes, centrifuged for 5 minutes, 5,000 ⁇ m, 4 °C and resuspended in 2 ml of 15% glycerol, 4 °C.
  • the 15% glycerol wash was repeated once and the pellet was resuspended in 1 to 2 ml of Electroporation Buffer.
  • the cells were stored at -80°C in 50 - 200 ⁇ l aliquots.
  • the ligations were prepared as described for the S. lividans transformation. After the incubation of the ligation reaction, the volume was brought to 100 ⁇ l with dH 2 O, NaCl was added to 0.3M, and the reaction extracted with an equal volume of 24: 1 : 1 phenolxhoroform isoamyl alcohol. Twenty ⁇ g of glycogen was added and the ligated DNA was precipitated with 2 volumes of 100% ethanol at -20 °C for 30 minutes. The DNA was pelleted 10 minutes in a microcentrifuge, washed once with 70% ethanol, dried 5 minutes in a speed-vac concentrator and resuspended in 5 ⁇ l of dH 2 O.
  • the cells were diluted with 0.75 to 1.0 ml of CRM (0-4 °C), transferred to 15 ml culture tubes and incubated with agitation 3hrs at 30 °C.
  • the cells were plated on trypticase soy broth agar plates with 10-30 ⁇ g/ml of thiostrepton.
  • Example 13 High Performance liquid chromatography The liquid chromatography separation was performed using a Waters 2690
  • the gradient mobile phase programming was used with a flow rate of 1.0 ml/minute.
  • Eluent A was water/acetonitrile (20:1) + 10 mM ammonium acetate.
  • Eluent B was acetonitrile/water (20:1).
  • the mobile phase was a linear gradient from 12% B to 28 % B over 6 minutes and held isocratic at 28% B over 4 minutes. This was followed by a 28% B to 100% B linear gradient over 20 minutes and a linear gradient to 12% B over two minutes with a 3 minute hold at 12% B.
  • the column effluent was introduced directly into the electrospray ion source of a ZMD mass spectrometer (Micromass, Manchester, UK).
  • the instrument was calibrated using Test Juice reference standard (Waters Co ⁇ , Milford, MA, USA) and was delivered at a flow of 10 ⁇ l/minute from a syringe pump (Harvard Apparatus, Holliston, MA, USA).
  • the mass spectrometer was operated at a low mass resolution of 13.2 and a high mass resolution of 11.2. Spectra were acquired from using a scan range of m/z 100 to 600 at an acquisition rate of 10 spectra /second.
  • the ionization technique employed was positive electrospray (ES).
  • the sprayer voltage was kept at 2900 V and the cone voltage of the ion source was kept at a potential of 17 V.
  • Example 15 Use of the ebh gene sequence (SEQ ID NO:l) to isolate cytochrome P450 genes from other microorganisms
  • Genomic DNA was isolated from a set of cultures (ATCC43491, ATCC 14930, ATCC53630, ATCC53550, ATCC39444, ATCC43333, ATCC35165) using the DNAzol reagent.
  • the DNA was used as a template for PCR reactions using primers designed to the sequence of the ebh gene.
  • Three sets of primers were used for amplification; NPB29-6f (SEQ DD NO:28) and NPB29-7r (SEQ DD NO:29), NPB29- 16f (SEQ DD NO:50) and NPB29-17r (SEQ DD NO:51), and NPB29-19f (SEQ DD NO:52) and NPB29-20r (SEQ DD NO:53).
  • PCR reactions were prepared in a volume of 20 ⁇ l, containing 200-500 ng of genomic DNA and a forward and reverse primer. All primers were added to a final concentration of 1.4- 2.0 ⁇ M. The PCR reaction was prepared with 0.2 ⁇ l of
  • AdvantageTM 2 Taq enzyme (BD Biosciences Clontech, Palo Alto, California, USA), 2 ⁇ l of AdvantageTM 2 Taq buffer and 0.2 ⁇ l of 2.5 mM of dNTPs with dH 2 O to 20 ⁇ l.
  • the cycling reactions were performed on a Geneamp ® 9700 PCR system or a Mastercycler ® gradient (Eppendorf, Westbury, New York, USA) with the following protocol: 95°C for 5 minutes, 35 cycles (96°C 20 seconds, 54-69°C 30 seconds, 72°C 2 minutes), 72°C for 7 minutes.
  • the expected size of the PCR products is approximately 1469 bp for the NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29) primer pair, 1034 bp for the NPB29-16f (SEQ DD NO:50) and NPB29-17r (SEQ DD NO:51) primer pair and 1318 bp for the NPB29-19f (SEQ DD NO:52) and NPB29-20r (SEQ ID NO:53) primer pair.
  • the PCR reactions were analyzed on 0.7% agarose gels. PCR products of the expected size were excised from the gel and purified using the Qiagen gel extraction method. The purified products were sequenced using the Big-Dye sequencing kit and analyzed using an ABD 10 sequencer.
  • a 1.469 kb DNA fragment containing the epothilone B hydroxylase gene and the downstream ferredoxin gene was amplified using PCR.
  • the 50 ⁇ l PCR reaction was composed of 5 ⁇ l of Taq buffer, 2.5 ⁇ l glycerol, 1 ⁇ l of 20 ng/ ⁇ l NPB29-1 plasmid, 0.4 ⁇ l of 25 mM dNTPs, 1.0 ⁇ l each of primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ DD NO:29) (5 pmole/ ⁇ l), 38.1 ⁇ l of dH 2 O and 0.5 ⁇ l of Taq enzyme (Stratagene).
  • the reactions were performed on a Geneamp ® 9700 PCR system, with the following conditions; 95°C for 5 minutes, then 30 cycles (96°C for 30 seconds, 60°C 30 seconds, 72°C for 2 minutes), and 72°C for 7 minutes.
  • the PCR product was purified using a Qiagen Qiaquick column with the PCR cleanup procedure.
  • the purified product was digested with Bglll and Hindi ⁇ and purified on a 0.7 % agarose gel.
  • a 1.469 kb band was excised from the gel and eluted using a Qiagen Qiaquick gel extraction procedure.
  • the fragments were then cloned into the pPCRscript Amp vector using the PCRscript Amp cloning kit.
  • Colonies containing inserts were picked to 1-2 ml of LB (Luria Broth) with 100 ⁇ g/ml ampicillin, 30- 37°C, 16-24 hours, 230-300 ⁇ m. Plasmid isolation was performed using the Mo Bio miniplasmid prep kit. The sequence of the insert was confirmed by cycle sequencing with the Big-Dye sequencing kit. This plasmid was named pPCRscript-eb/i.
  • Example 17 Mutagenesis of the ebh gene for improved yield or altered specificity
  • the Quikchange® XL Site-Directed Mutagenesis Kit and the Quikchange® Multi Site-Directed Mutagenesis kit, both from Stratagene were used to introduce mutations in the coding region of the ebh gene. Both of these methods employ DNA primers 35-45 bases in length containing the desired mutation (SEQ DD NO:54-59 and 77), a methylated circular plasmid template and PfuTurbo® DNA Polymerase (U.S. Patent Nos 5,545,552 and 5,866,395 and 5,948,663) to generate copies of the plasmid template inco ⁇ orating the mutation carried on the mutagenic primers.
  • XL 1 -Blue® electrocompetent or XLIO-Gold® ultracompetent cells (Stratagene). Cells were plated to a density of greater than 100 colonies per plate on LA (Luria Agar) 100 ⁇ g/ml ampicillin plates, and incubated 24-48 hrs at 30-37°C. The entire plate was resuspended in 5 ml of LB containing 100 ⁇ g/ml ampicillin. Plasmid was isolated directly from the resuspended cells by centrifuging the cells and then purifying the plasmid using the Mo Bio miniprep procedure.
  • This plasmid was then used as a template for PCR with primers NPB29-6f (SEQ ID NO:28)and NPB29-7r (SEQ DD NO:29) to amplify a mutated expression cassette. Digestion of the 1.469 kb PCR product with the restriction enzymes Bglll and Hindlll was used to prepare this fragment for ligation to vector pANT849 also digested with Bglll and HindM. Alternatively, the resuspended cells were used to inoculate 20- 50 ml of LB containing 100 ⁇ g/ml ampicillin and grown 18-24 hrs at 30-37°C.
  • Qiagen midi-preps were performed on the cultures to isolate plasmid DNA containing the desired mutation. Digestion with the restriction enzymes Bglll and Hindll was used to excise the mutated expression cassette for ligation to BglH and Hindm digested plasmid pANT849. Screening of mutants was performed in S. lividans or S. rimosus as described.
  • the PCR reaction was prepared with 10 ⁇ l of Not I digested pCRscript-eM (O.lng/ ⁇ l), 10 ⁇ l of polymerase buffer, 1.0 ⁇ l of IM ⁇ -mercaptoethanol, 10.0 ⁇ l of DMSO, 1.0 ⁇ l of NPB29-6f (SEQ ID NO:28) primer (100 pmole/ ⁇ l), 1.0 ⁇ l of NPB29-7r (SEQ ID NO:29) primer (100 pmole/ ⁇ l), 10 ⁇ l of 5 mM MnCl 2 , 10.0 ⁇ l 10 mM dGTP, 10.0 ⁇ l 2 mM dATP, 10 mM dTTP, 10.0 ⁇ l 10 mM dCTP, and 2.0 ⁇ l Taq polymerase.
  • dH 2 O was added to 100 ⁇ l. Reactions were also prepared as described above but without MnCl .
  • the cycling reactions were performed a GeneAmp® PCR system with the following protocol: 95°C for 1 minute, 25-30 cycles (94 °C for 1 minute, 55 °C for 30 seconds, 72 °C for 4 minutes), 72 °C for 7 minutes.
  • the PCR reactions were separated on an agarose gel using a Qiagen spin column.
  • the fragments were then digested with Bglll and Hindm and purified using a Qiagen spin column.
  • the purified fragments were then ligated to BglH and Hindm digested pANT849 plasmids. Screening of mutants was performed in S. lividans and S. rimosus.
  • Example 18 Comparison of epothilone B transformation in cells expressing ebh and mutants thereof
  • Epothilone F
  • the bioconversion of epothilone B to epothilone F was performed in S. rimosus host cells transformed with expression plasmids containing the ebh gene and its variants or mutants.
  • One-hundred ⁇ l of a frozen S. rimosus transformant culture was inoculated to 20 ml CRM media with 10 ⁇ g/ml thiostrepton and cultivated 16-24 hr, 30°C, 230- 300 ⁇ m.
  • Epothilone B in 100% ethanol was added to each culture to a final concentration of 0.05% weight/volume. The reaction was typically incubated 20- 40hrs at 30 °C, 230-300 ⁇ m.
  • the concentration of epothilones B and F was determined by HPLC analysis.
  • Epothilone F yield e -M18 55% ebh24-16dS 75% ebh24-16z ⁇ ⁇ 75% ebh24 ⁇ 6 ⁇ 6 75% eM24-16-74 75% eb/ ⁇ 24-16b9 80% eM24-16g8 85%
  • Example 20 High performance liquid chromatography method for compactin and pravastatin detection The liquid chromatography separation was performed using a Hewlett
  • Packard 1090 Series Separation system (Agilent Technologies, Palo Alto, California, USA) and a column, 50x46 mm, filled with Spherisorb ODS2, particle size 5 ⁇ m (Keystone Scientific, Inc, Bellefonte, Pennsylvania, USA).
  • the gradient mobile phase programming was used with a flow rate of 2.0 ml/minute.
  • Eluent A was water, 10 mM ammonium acetate and 0.05% Phosphoric Acid.
  • Eluent B was acetonitrile. The mobile phase was a linear gradient from 20% B to 90 % B over 4 minutes.
  • Example 21 Structure determination of the biotransformation product of mutant eb lS ⁇ Analytical HPLC was performed using a Hewlett Packard 1100 Series Liquid
  • YMC Packed ODS-AQ column (4.6 mm i.d. x 15 cm 1).
  • the column was eluted at 1 ml/minute flow rate with a gradient system of D 2 O (solvent A) and acetonitrile-d 3 (solvent B): 30% B, 1 minute; 30% to 80% B linear gradient, 11 minutes.
  • the eluent passed a UV detection cell (monitored at 254 nm) before flowing through a F19/H1 NMR probe (60 ⁇ l active volume) in Varian AS -600 NMR spectrometer.
  • the biotransformation product was eluted at around 7.5 minutes and the flow was stopped manually to allow the eluent to remain in the NMR probe for NMR data acquisition.

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Abstract

La présente invention concerne des séquences d'acide nucléiques isolées et des polypeptides codés par ces dernières qui produisent de l'épothilone B hydroxylase ainsi que des mutants et des variants de cette dernière et une ferrédoxine située en aval du gène d'épothilone B hydroxylase. Cette invention concerne également des vecteurs et des cellules contenant ces vecteurs; des méthodes de production de micro-organismes de recombinaison, des méthodes d'utilisation de ces micro-organismes de recombinaison pour produire des épothilones porteuses d'un groupe hydroxyalkyle et un analogue d'épothilone produit par un mutant de l'épothilone B hydroxylase.
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JP2005514031A (ja) 2005-05-19
AU2002357872A1 (en) 2003-07-24
WO2003057830A2 (fr) 2003-07-17
WO2003057830A3 (fr) 2004-08-05
TW200301697A (en) 2003-07-16

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