JP2005514031A - Compositions and methods for hydroxylating epothilone - Google Patents

Abstract

Epothilone B hydroxylase and mutants and variants thereof and an isolated nucleic acid sequence of ferredoxin located downstream of the epothilone B hydroxylase gene and polypeptides encoded by the sequence are provided. Vectors and cells containing the vectors are also provided. Further provided are methods of producing recombinant microorganisms, methods of using these recombinant microorganisms to produce hydroxyalkyl-containing epothilones, and epothilone analogs produced by mutants of epothilone B hydroxylase.

Description

  The present invention relates to epothilone B hydroxylase and mutants and variants thereof and to the isolated nucleic acid sequence of ferredoxin located downstream of the epothilone B hydroxylase gene and the polypeptide encoded thereby. The present invention also provides an epothilone B hydroxylase or mutant or variant thereof and / or capable of hydroxylating a small organic molecule compound having a terminal alkyl group such as epothilone to produce a compound having a terminal hydroxyalkyl group. The present invention relates to a recombinant microorganism that expresses ferredoxin. Also provided are methods of recombinantly producing such microorganisms and methods of using these recombinant microorganisms for the synthesis of compounds having terminal hydroxyalkyl groups. The compositions and methods of the present invention are useful for producing epothilones having various utilities in the pharmaceutical field. Also described are novel epothilone analogs produced using mutants of the epothilone B hydroxylase of the present invention.

を有するエポチロンＡおよびＢは、パクリタキセル（タキソール^Ｒ）と類似の微小管安定化作用を発揮し、それゆえ腫瘍細胞などの迅速に増殖する細胞や他の過剰増殖細胞性疾患に関連する細胞に対して細胞障害作用を発揮することがわかっている（Bollagら、Cancer Res., Vol. 55, No. 11, 2325-2333 (1995)を参照）。 Epothilone is a macrolide compound that has found utility in the field of medicine. For example, the structure:

Epothilone A and B having, compared cells paclitaxel (Taxol ^R) and exert microtubule-stabilizing effects similar, associated with rapidly proliferating cells or other hyperproliferative cellular disease, such as thus tumor cells (See Bollag et al., Cancer Res ., Vol. 55, No. 11, 2325-2333 (1995)).

Epothilones A and B are natural anticancer agents produced by Sorangium cellulosum, first isolated and characterized by Hofle et al . (DE 4138042; WO 93/10121; Angew. Chem. Int. Ed. Engl . Vol. 35, No13 / 14, 1567-1569 (1996); and J. Antibiot ., Vol. 49, No. 6, 560-563 (1996)). Subsequently, the total synthesis of epothilones A and B was performed by Balog et al . , Angew. Chem. Int. Ed. Engl . Vol. 35, No. 23/24, 2801-2803, 1996; Meng et al., J. Am. Chem. , Vol. 119, No. 42, 10073-10092 (1997); Nicolaou et al., J. Am. Chem. Soc ., Vol. 119, No. 34, 7974-7991 (1997); Schinzer et al . , Angew. Chem Int. Ed. Engl . Vol. 36, No. 5, 523-524 (1997); and Yang et al . , Angew. Chem. Int. Ed. Engl ., Vol. 36, No. 1/2, 166-168 , 1997. WO 98/25929 discloses a method for chemical synthesis of epothilone A, epothilone B, epothilone analogs and a library of epothilone analogs. The structure of epothilone C, D, E and F and the production from Solandium cellulosum DSM 6773 is disclosed in WO 98/22461. FIG. 1 shows Epothilone B to Epothilone F in Actinomyces sp. Strain SC15847 (ATCC PT-1043) (which was subsequently identified as Amycolatopsis orientalis) as described in WO 00/39276. An illustration of biotransformation of is shown.

  Cytochrome P450 enzymes are found in prokaryotic and eukaryotic cells and have in common a heme-binding domain that can be distinguished by an absorption maximum at 450 nm when complexed with carbon monoxide. The cytochrome P450 enzyme performs a broad spectrum oxidation reaction on predominantly hydrophobic substrates including aromatic and benzylic rings and alkanes. In prokaryotes, the cytochrome P450 enzyme has been found as a detoxification system and as the first enzymatic process to metabolize substrates such as toluene, benzene and camphor. The cytochrome P450 gene is also known as nikkomycin (Bruntner, C. et al., 1999, Mol. Gen. Genet. 262: 102-114), doxorubicin (Dickens, ML, Strohl, WR, 1996, Streptomyces tendae). J. Bacteriol., 178: 3389-3395) and secondary metabolites such as Sorandium cellulosum epothilone biosynthetic cluster (Julien, B. et al., 2000, Gene, 249: 153-160). Has been issued. With some exceptions, the cytochrome P450 system in prokaryotes consists of three proteins, ferredoxin NADH or NADPH-dependent reductase, iron-sulfur ferredoxin and cytochrome P450 enzymes (Lewis, DF, Hlavica, P., 2000). , Biochim. Biophys. Acta., 1460: 353-374). The electrons are transported from ferredoxin reductase to ferredoxin and finally to the cytochrome P450 enzyme for molecular oxygen cleavage.

  It is an object of the present invention to provide an isolated nucleic acid sequence encoding epothilone B hydroxylase and variants or mutants thereof and an isolated nucleic acid sequence encoding ferredoxin or variants or mutants thereof.

  Another object of the present invention provides an isolated polypeptide comprising the amino acid sequence of epothilone B hydroxylase and variants or mutants thereof and an isolated polypeptide comprising the amino acids of ferredoxin and variants or mutants thereof. That is.

  Another object of the present invention is to provide the structural coordinates of the homology model of epothilone B hydroxylase. The structural coordinates are listed in Appendix Table 1. This model of the invention provides a modulator of the biological function of epothilone B hydroxylase as well as a means to design additional mutants of epothilone B hydroxylase with altered specificity.

  Another object of the present invention is to provide a vector comprising an isolated nucleic acid sequence encoding epothilone B hydroxylase or a variant or mutant thereof and / or ferredoxin or a variant or mutant thereof. In a preferred embodiment, these vectors further comprise a nucleic acid sequence encoding ferredoxin.

  Another object of the present invention is to provide a host cell comprising a vector comprising an isolated nucleic acid sequence encoding epothilone B hydroxylase or a variant or mutant thereof and / or ferredoxin or a variant or mutant thereof. It is.

  Another object of the present invention is to provide a method for producing a recombinant microorganism capable of producing a compound having a terminal alkyl group, particularly a compound having a terminal hydroxyalkyl group by hydroxylating an epothilone.

  Another object of the present invention is to provide a recombinantly produced microorganism capable of producing a compound having a terminal alkyl group, particularly a compound having a terminal hydroxyalkyl group by hydroxylating an epothilone.

  Another object of the present invention is to provide a method for hydroxylating compounds in these recombinant microorganisms. In particular, the present invention provides a process for producing hydroxyalkyl-containing epothilones, which are useful as anticancer agents and as starting materials in the preparation of other epothilone analogs.

で示される化合物、並びに式Ａの化合物を含む組成物および該組成物の製造方法を提供することである。 Another object of the invention is a compound of formula A, designated herein as 24-OH epothilone B or 24-OH EpoB:

And a composition comprising the compound of formula A and a process for preparing the composition.

  The present invention relates to isolated nucleic acid sequences and methods for obtaining polypeptides and compounds having the desired substituent at the terminal carbon atom position. In particular, the present invention provides compositions and methods for producing hydroxyalkyl-containing epothilones, which are useful as anticancer agents and as starting materials in the preparation of other epothilone analogs.

を含む残基をいい、上記式において置換基は以下のとおりである：
Ｑは、

よりなる群から選ばれる；
Ｗは、ＯまたはＮＲ_６；
Ｘは、Ｏ、ＨおよびＯＲ_７よりなる群から選ばれる；
Ｍは、Ｏ、Ｓ、ＮＲ_８、ＣＲ_９Ｒ_１０；
Ｂ_１およびＢ_２は、ＯＲ_１１、ＯＣＯＲ_１２よりなる群から選ばれる；
Ｒ_１−Ｒ_５およびＲ_１２−Ｒ_１７は、Ｈ、アルキル、置換アルキル、アリール、およびへテロシクロよりなる群から選ばれ、Ｒ_１およびＲ_２がアルキルである場合はこれらは一緒になってシクロアルキルを形成することができる；
Ｒ_６は、Ｈ、アルキル、および置換アルキルよりなる群から選ばれる；
Ｒ_７およびＲ_１１は、Ｈ、アルキル、置換アルキル、トリアルキルシリル、アルキルジアリールシリルおよびジアルキルアリールシリルよりなる群から選ばれる；
Ｒ_８は、Ｈ、アルキル、置換アルキル、Ｒ_１３Ｃ＝Ｏ、Ｒ_１４ＯＣ＝ＯおよびＲ_１５ＳＯ_２よりなる群から選ばれる；および
Ｒ_９およびＲ_１０は、Ｈ、ハロゲン、アルキル、置換アルキル、アリール、へテロシクロ、ヒドロキシ、Ｒ_１６Ｃ＝Ｏ、およびＲ_１７ＯＣ＝Ｏよりなる群から選ばれる。 As used herein, “epothilone” refers to a compound containing an epothilone core and a side chain group as defined herein. As used herein, “epothilone core” refers to a core structure (indicating the numbering of the ring system positions used herein):

In the above formula, the substituents are as follows:
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 ₁₀ ;
B ₁ and B ₂ are selected from the group consisting of OR ₁₁ , OCOR ₁₂ ;
R ₁ -R ₅ and R ₁₂ -R ₁₇ are selected from the group consisting of H, alkyl, substituted alkyl, aryl, and heterocyclo, and when R ₁ and R ₂ are alkyl, these together are cyclo Can form alkyl;
R ₆ is selected from the group consisting of H, alkyl, and substituted alkyl;
R ₇ and R ₁₁ are selected from the group consisting of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl and dialkylarylsilyl;
R ₈ is selected from the group consisting of H, alkyl, substituted alkyl, R ₁₃ C═O, R ₁₄ OC═O and R ₁₅ SO ₂ ; and R ₉ and R ₁₀ are H, halogen, alkyl, substituted alkyl , Aryl, heterocyclo, hydroxy, R ₁₆ C═O, and R ₁₇ OC═O.

The “side chain group” refers to the substituent G defined above for epothilone A or B or G ₁ and G ₂ shown below.

G ₁ represents the following formula V:
_{HO-CH 2 - (A 1} ) n - (Q) m - (A 2) o (V)
And
G ₂ represents the following formula VI:
_{CH 3 - (A 1) n} - (Q) m - (A 2) o (VI)
Wherein A ₁ and A ₂ are independently selected from the group consisting of optionally substituted C ₁ -C ₃ alkyl and alkenyl;
Q is an optionally substituted ring containing 1 to 3 rings and at least one carbon-carbon double bond in at least one ring;
n, m and o are integers independently selected from the group consisting of 0 and 1, and at least one of n, m or o is 1.
It is.

  “Terminal carbon atom” or “terminal alkyl group” means the terminal carbon atom of the residue directly bonded to the epothilone core at the 15th position, the terminal alkyl group or the terminal carbon atom of the side chain group bonded to the 15th position, or Refers to a terminal alkyl group. It should be understood that “alkyl group” includes alkyl and substituted alkyl as described herein.

  “Alkyl” refers to an optionally substituted linear or branched saturated hydrocarbon group having 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms. The term “lower alkyl” refers to an optionally substituted alkyl group having 1 to 4 carbon atoms.

“Substituted alkyl” means, for example, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, aralkyl Amino, cycloalkylamino, heterocycloamino, disubstituted amine (the two amino substituents are selected from alkyl, aryl or aralkyl), alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted arano Lucanoylamino, thiol, alkylthio, arylthio, aralkylthio, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aralkylthiono, alkyl Ruhoniru, arylsulfonyl, aralkylsulfonyl, sulfonamido _(e.g., SO 2 _NH 2), substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g., CONH _2), substituted carbamyl (e.g., CONH alkyl, CONH aryl, CONH aralkyl Or when there are two substituents selected from alkyl, aryl or aralkyl on the nitrogen atom), alkoxycarbonyl, aryl, substituted aryl, guanidino and heterocyclo (eg, indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl) , Pyridyl, pyrimidyl, and the like). Where the substituent is further substituted in the above, it will be substituted with halogen, alkyl, alkoxy, aryl or aralkyl.

  According to one aspect of the present invention, there is provided an isolated polynucleotide encoding an epothilone B hydroxylase, an enzyme that can hydroxylate an epothilone having a terminal alkyl group to produce an epothilone having a terminal hydroxyalkyl group.

  According to another aspect of the present invention, an isolated polynucleotide encoding ferredoxin, a gene located downstream of the epothilone B hydroxylase gene, is provided. Ferredoxin is a cytochrome P450 protein.

  As used herein, “polynucleotide” is meant to encompass any form of DNA or RNA encoding these enzymes or active fragments thereof, eg, cDNA or genomic DNA or mRNA, respectively, such polynucleotides It can be obtained by cloning or synthesized by well-known chemical methods. The DNA may be double-stranded or single-stranded. Single stranded DNA may contain either a coding or sense strand or a non-coding or antisense strand. Therefore, the term polynucleotide also exhibits at least 60% or more, preferably at least 80% homology to the sequences disclosed herein and hybridizes to the polynucleotide under stringent conditions. Polynucleotides are also included. As used herein, the term “stringent conditions” means hybridization conditions at 60 ° C. in 2 × SSC buffer. Further preferred is shown in SEQ ID NO: 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. Shown in the nucleic acid sequence or in SEQ ID NO: 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. It can hybridize to the complementary sequence of the nucleic acid sequence at 65 ° C. under 3 × SSC hybridization conditions for 16 hours, and SEQ ID NOs: 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72 or 74 or the nucleic acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 1, 30, 32, 34, 36, 37, 38, 39, 40, 4 , 42, 60, 62, 64, 66, 68, 70, 72 or 74 or the complementary sequence of the nucleic acid sequence shown in SEQ ID NO: 3 for 30 minutes under a wash condition of 0.5 × SSC at 55 ° C. An isolated nucleic acid that can remain intact.

  In one embodiment, the polynucleotide of the present invention comprises the genomic DNA shown in SEQ ID NO: 1 or a homologous sequence thereof or a fragment encoding a polypeptide having the same activity as this epothilone B hydroxylase. Alternatively, the polynucleotide of the present invention may comprise the genomic DNA shown in SEQ ID NO: 3 or a homologous sequence thereof or a fragment encoding a polypeptide having the same activity as this ferredoxin. Because of the degeneracy of the genetic code, the polynucleotides of the present invention also include other nucleic acid sequences that encode this enzyme and its derivatives, variants, or active fragments.

  The invention also relates to these polynucleotides that are naturally occurring, ie, in microorganisms such as Amycolatopsis orientalis and Amicolata autorophica, or in soil or other sources from which nucleic acids can be isolated. Or mutants prepared by well-known mutagenesis methods. Examples of variants of the present invention are shown in SEQ ID NOs: 36-42.

  As used herein, a “mutant” has one or more point mutations or deletions or additions of nucleic acids compared to SEQ ID NO: 1 or SEQ ID NO: 3, but SEQ ID NO: 1 or SEQ ID NO: 3 Is meant to include a nucleic acid sequence encoding a polypeptide or fragment having similar activity as the polypeptide encoded by. In a preferred embodiment, mutants with altered substrate specificity and / or enzyme yield are produced. A preferred region of mutation for the epothilone B hydroxylase gene is a region of nucleic acid sequence encoding about 113 amino acids that includes the active site of the enzyme. Mutant encoding a polypeptide having at least one amino acid substitution at amino acid position GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU231, SER294, PHE237, or ILE365 of SEQ ID NO: 1 Is also preferred. Illustrative polynucleotide mutants of the invention are shown in SEQ ID NOs: 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; literature: Omer, CA, J. Bacteriol. 172: 3335-3345 (1990)
Sequence 2: locus: STMSUBCB; accession number: M32239; literature: Omer, CA, J. Bacteriol. 172: 3335-3345 (1990)
Sequence 3: locus: AB018807 (formerly STMORFA); accession number: AB018807; literature: Ueda, K., J. Antibiot. 48: 638-646 (1995)
Sequence 4: Locus: SSU65940; Accession number: U65940; Literature: Motamedi, H., J. Bacteriol. 178: 5243-5248 (1996)
Sequence 5: locus: STMOLEP; accession number: L37200; literature: Rodriguez, AM, FEMS Microbiol. Lett. 127: 117-120 (1995)
Sequence 6: locus: SERCP450A; accession number: M83110; literature: Andersen, JF and Hutchinson, CR, J. Bacteriol. 174: 725-735 (1992).

Alignment was performed by executing the algorithm of Myers, EW and W. Miller. 1988. CABIOS 4: 1, 11-17, an Align program from Scientific and Educational Software (Durham, North Carolina, USA). Three highly conserved regions were identified in the I-helix (including the oxygen binding domain), in the K-helix, and during spanning of the B-bulge and L-helix containing the conserved heme-binding domain. . Primers were designed for the three conserved regions identified in the alignment. Primers P450-1 ⁺ (SEQ ID NO: 23) and P450-1a ⁺ (SEQ ID NO: 24) are designed from the I helix, and primer P450-2 ⁺ (SEQ ID NO: 25) is designed from the B-bulge and L-helix regions. and, primer P450-3 ^- (SEQ ID NO: 27) was designed as the reverse complement for the heme-binding protein.

  Subsequently, the genomic fragment was amplified by polymerase chain reaction (PCR). After PCR amplification, the reaction products were separated by gel electrophoresis and excised fragments of the expected size. DNA was extracted from agarose gel slices using the Qiaquick gel extraction method (Qiagen, Santa Clarita, California, USA). The fragment was then cloned into a PCRscript vector (Stratagene) using a PCRscript Amp cloning kit (Stratagene, La Jolla, California, USA). Colonies containing the insert were picked in 1-2 ml LB broth containing 100 μg / ml ampicillin at 30-37 ° C. for 16-24 hours at 230-300 rpm. Plasmid isolation was performed using the Mo Bio mini-plasmid prep kit (Mo Bio, Sorano Beach, California, USA). This plasmid DNA was used as a template for PCR and sequencing and for restriction digest analysis.

Cloned PCR products were sequenced using the Big-Dye sequencing kit from Applied Biosystems (Foster City, CA, USA) and analyzed using the ABI310 sequencer (Applied Biosystems, Foster City, CA, USA). A TblastX search was performed using the sequence of the insert, using the protocol of Altschul, SF et al . , Mol. Biol. 215: 403-410 (1990) of the non-redundant protein database. A unique sequence with significant similarity to the known P450 protein was retained. Using this approach, a total of 9 different P450 sequences were identified from SC15847, 7 from the genomic DNA template, and 2 from the cDNA. Two P450 sequences were found together in the DNA and cDNA templates. Of the 50 cDNA clones analyzed, two sequences were dominant, 20 clones each. These two genes were then cloned from genomic DNA.

  The genomic DNA nucleic acid sequence was determined using the Big-Dye sequencing system (Applied Biosystems) and analyzed using the ABI310 sequencer. This sequence is shown in SEQ ID NO: 1. An open reading frame encoding a protein of 404 amino acids and a predicted molecular weight of 44.7 kDa was found in the cloned BglII fragment. The deduced amino acid sequence of this polypeptide is shown in SEQ ID NO: 2. The amino acid sequence of this polypeptide is 51% identical to Streptomyces tendae NikF protein (Bruntner, C. et al., 1999, Mol. Gen. Genet. 262: 102-114) S. carbophilus) Sca-2 protein (Watanabe, I. et al., 1995, Gene 163: 81-85) was found to have 48% identity. Both these enzymes belong to the cytochrome P450 family 105. An invariant cysteine found in the heme binding domain of all cytochrome P450 enzymes is found at residue 356. This epothilone B gene was named ebh. The ATG start codon of the 64 amino acid putative ferredoxin gene is found 9 base pairs downstream of the ebh stop codon. This enzyme is a ferredoxin gene from S. griseoulus (O'Keefe, DP et al., 1991, Biochemistry 30: 447-455) and a ferredoxin gene from S. noursei (Brautaset, T Et al., 2000, Chem. Biol. 7: 395-403) and 50% identity. The nucleic acid sequence encoding this ferredoxin is shown in SEQ ID NO: 3, and the amino acid sequence of this ferredoxin polypeptide is shown in SEQ ID NO: 4.

  The ebh gene sequence was also used to isolate mutant cytochrome P450 genes from other microorganisms. Exemplary mutant polynucleotides of the present invention, ebh43491, ebh14930, ebh53630, ebh53550, ebh39444, ebh43333 and ebh35165, along with the species from which they were isolated, are shown in Table 1 below. The nucleic acid sequences of these variants are shown in SEQ ID NOs: 36-42, respectively.

Table 1 : Mutant polynucleotides

  The amino acid sequences encoded by the exemplary mutants ebh43491, ebh14930, ebh53630, ebh53550, ebh39444, ebh43333 and ebh35165 are shown in SEQ ID NOs: 43-49, respectively. Table 2 shows a summary of amino acid substitutions for these exemplary variants.

Table 2 : Amino acid substitution

  Mutations were also introduced into the coding region of the ebh gene to identify mutants with improved yield, and / or rate of bioconversion, and / or altered substrate specificity. Exemplary mutant nucleic acid sequences of the present invention are shown in SEQ ID NOs: 30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74.

The nucleic acid sequence of SEQ ID NO: 30 encodes mutant ebh25-1, which exhibits altered substrate specificity. Plasmid pANT849ebh25-1 containing this mutated gene has been deposited and is deposited by the International Depositary Authority under the provisions of the Budapest Treaty. Deposit is November 2002 The day went to the American Type Culture Collection (10801, University Boulevard, Manassas, Virginia 20110-2209). The ATCC accession number is It is. All restrictions on public access to this plasmid will be irrevocably removed as soon as this patent application is patented. The deposit will be maintained at a public depository for a period of 30 years after the date of deposit or 5 years after a recent sample request or the validity period of the patent, whichever is longer. The plasmid was alive at the time of deposit. The deposit will be replaced when live samples cannot be dispensed by the depository.

  This S. lividans transformant identified during the screening for mutation 25 (primers NPB29-mut25f (SEQ ID NO: 58) and NPB29-mut25r (SEQ ID NO: 59)) is epothilone B or epothilone It was found to produce a product with a different HPLC elution time than F. This unknown sample was analyzed by LC-MS and found to have a molecular weight of 523 (M.W.), consistent with a single hydroxylation of epothilone B. Plasmid DNA was isolated from a culture of Streptomyces lividans 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 17). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh25-1 mutant was found to have two mutations that resulted in changes in the amino acid sequence of the protein (asparagine 195 changed to serine and serine 294 changed to proline). The position targeted for mutation at codon 238 was found to result in two nucleotide changes, but this did not result in a change in the amino acid sequence of the protein. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 30 is shown in SEQ ID NO: 31.

  The nucleic acid sequence of SEQ ID NO: 32 encodes mutant ebh10-53 (showing improved bioconversion yield). This Streptomyces lividans transformant identified during screening for mutation 10 (primers NPB29-mut10f (SEQ ID NO: 54) and NPB29-mut10r (SEQ ID NO: 55)) resulted in a higher yield of epothilone F. It was. Plasmid DNA was isolated from a culture of Streptomyces lividans 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 ebh10-53 mutant was found to have two mutations that resulted in changes in the amino acid sequence of the protein (glutamic acid 231 changed to arginine and phenylalanine 190 changed to tyrosine). Position 231 was the target for mutagenesis, but the change at residue 190 is a coincidence change and is a mutagenesis artifact. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 32 is shown in SEQ ID NO: 33.

  The nucleic acid sequence of SEQ ID NO: 34 encodes mutant ebh24-16 (which also exhibits improved bioconversion yield). This Streptomyces lividans transformant ebh24-16 identified during the screening of mutation 24 (primers NPB29-mut24f (SEQ ID NO: 56) and NPB29-mut24r (SEQ ID NO: 57)) is also higher of epothilone F. Yielded yield. Plasmid DNA was isolated from a Streptomyces 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 ebh24-16 mutant was found to have two mutations that resulted in changes in the amino acid sequence of the protein (phenylalanine 237 changed to alanine and isoleucine 92 changed to valine). Although position 237 was the target for mutagenesis, the change at residue 92 is a coincidence change and is a mutagenesis artifact. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 34 is shown in SEQ ID NO: 35.

  The nucleic acid sequence of SEQ ID NO: 60 encodes mutant ebh24-16d8 (which also exhibits improved bioconversion yield). This S. rimosus transformant ebh24-16d8 identified during screening of mutation 59 (primer NPB29mut59 (SEQ ID NO: 77)) also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 ebh24-16d8 mutant was found to have a single mutation that resulted in a change in the amino acid sequence of the protein (arginine 67 changed to glutamine). This change is an artifact of mutagenesis. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 60 is shown in SEQ ID NO: 61.

  The nucleic acid sequence of SEQ ID NO: 62 encodes the mutant ebh24-16c11 (which also exhibits improved bioconversion yield). This Streptomyces limos transformant ebh24-16c11 identified during the screening of mutation 59 (primer NPB29mut59 (SEQ ID NO: 77)) also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 ebh24-16c11 mutant was found to have two additional mutations that resulted in a change in the amino acid sequence of the protein (alanine 93 changed to glycine and isoleucine 365 changed to threonine). Position 93 was the target for mutagenesis, but the change at 365 is an artifact of the mutagenesis method. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 62 is shown in SEQ ID NO: 63.

  The nucleic acid sequence of SEQ ID NO: 64 encodes mutant ebh24-16-16 (which also exhibits improved bioconversion yield). This Streptomyces limos transformant ebh24-16-16, which was identified during screening of ebh24-16 random mutants, also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 ebh24-16-16 mutant was found to have one more mutation that resulted in a change in the amino acid sequence of the protein (valine 106 changed to alanine). The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 64 is shown in SEQ ID NO: 65.

  The nucleic acid sequence of SEQ ID NO: 66 encodes mutant ebh24-16-74, which also exhibits improved bioconversion yield. This Streptomyces limos transformant ebh24-16-74, identified during screening of ebh24-16 random mutants, also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 more mutation that resulted in a change in the amino acid sequence of the protein (arginine 88 changed to histidine). The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 66 is shown in SEQ ID NO: 67.

  The nucleic acid sequence of SEQ ID NO: 68 encodes mutant ebh24-M18 (which also exhibits improved bioconversion yield). This Streptomyces limosus transformant ebhM-18, identified during screening of ebh random mutants, also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 ebhM-18 mutant was found to have two mutations that resulted in changes in the amino acid sequence of the protein (glutamate 31 changed to lysine and methionine 176 changed to valine). The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 68 is shown in SEQ ID NO: 69.

  The nucleic acid sequence of SEQ ID NO: 72 encodes mutant ebh24-16g8 (which also exhibits improved bioconversion yield). This Streptomyces limos transformant ebh24-16g8, identified during mutation 50 screening (primer NPB29mut50 (SEQ ID NO: 78)), also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 ebh24-16g8 mutant was found to have two more mutations that resulted in a change in the amino acid sequence of the protein (methionine 176 changed to alanine and isoleucine 130 changed to threonine). Position 176 was the target for mutagenesis, but the change at 130 is a mutagenesis artifact. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 72 is shown in SEQ ID NO: 73.

  The nucleic acid sequence of SEQ ID NO: 74 encodes mutant ebh24-16b9 (which also exhibits improved bioconversion yield). This Streptomyces limos transformant ebh24-16b9, identified during mutation 50 screening (primer NPB29mut50 (SEQ ID NO: 78)), also resulted in higher yields of epothilone F. Plasmid DNA was isolated from a Streptomyces limos 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 ebh24-16b9 mutant was found to have two additional mutations that resulted in changes in the amino acid sequence of the protein (methionine 176 changed to serine and alanine 140 changed to threonine). Position 176 was the target for mutagenesis, but the change at 140 is a mutagenesis artifact. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO: 74 is shown in SEQ ID NO: 75.

Plasmids pANT849ebb-24-16, pANT849ebh-10-53, pANT849ebb-24-16d8, pANT849ebb-24-16c11, pANT849ebb-16-16, pANT849ebb-24-16-74, pANT849ebb-24 of these nine mutant genes A mixture of -16b9, pANT849ebh-M18 and pANT849ebh-24-24g8 has been deposited and is deposited by the International Depositary Authority under the provisions of the Budapest Treaty. Deposit is November 2002 The day went to the American Type Culture Collection (10801, University Boulevard, Manassas, Virginia 20110-2209). The ATCC accession number is It is. All restrictions on public access to this plasmid will be irrevocably removed as soon as this patent application is patented. The deposit will be maintained at a public depository for a period of 30 years after the date of deposit or 5 years after a recent sample request or the validity period of the patent, whichever is longer. The plasmid was alive at the time of deposit. The deposit will be replaced when live samples cannot be dispensed by the depository.

  Therefore, according to another aspect of the present invention, there are provided isolated polypeptides of epothilone B hydroxylase and variants and mutants thereof, and isolated polypeptides of ferredoxin or variants thereof. In one embodiment of the invention, the “polypeptide” comprises the amino acid sequence of SEQ ID NO: 2 and fragments or variants that retain essentially the same biological activity and / or function as this epothilone B hydroxylase. means. In another embodiment of the invention, “polypeptide” is meant to include the amino acid sequence of SEQ ID NO: 4 and fragments or variants that retain essentially the same biological activity and / or function as this ferredoxin.

  As used herein, “variant” has an amino acid sequence having a conservative amino acid substitution compared to SEQ ID NO: 2 or 4, and exhibits the same biological activity and / or function as SEQ ID NO: 2 or 4. It is meant to include a proven polypeptide. A “conservative amino acid substitution” is an amino acid of an aliphatic amino acid (such as Ala, Val, Leu and Ile), hydroxyl residues Ser and Thr, acidic residues Asp and Glu, and amide residues Asn and Gln. Is meant to include replacing with another amino acid. The amino acid sequences of exemplary variants of the invention are shown in SEQ ID NOs: 43-49, and the amino acid substitutions of these exemplary variants are listed in Table 2 above.

  As used herein, a “mutant” is encoded by a nucleic acid sequence having one or more point mutations, deletions or additions of nucleic acid compared to SEQ ID NO: 1 or 3. Or is meant to include polypeptides that still have similar activity as the polypeptide encoded by 3. In a preferred embodiment, the nucleic acid is mutated to alter substrate specificity and / or yield from the polypeptide encoded thereby. A preferred region of mutation for the epothilone B hydroxylase gene is a region of the nucleic acid sequence that encodes about 113 amino acid residues including the active site of the enzyme. Also preferred are mutations having at least one amino acid substitution at amino acid positions GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU231, SER294, PHE237, or ILE365 of SEQ ID NO: 1 It is. Exemplary mutants ebh25-1, ebh10-53, ebh24-16, ebh24-16d8, ebh24-16c11, ebh24-16-16, ebh24-16-74, ebh24-16g8, ebh24-16b9, and Nucleic acid sequences encoding such mutants are shown in SEQ ID NOs: 31, 33, 35, 61, 63, 65, 67, 69, 71, 73 and 75, and SEQ ID NOs: 30, 32, 34, 60, 62, respectively. 64, 66, 68, 70, 72 and 74.

  The three-dimensional model of epothilone B hydroxylase is also based on the known structure of the homologous protein EryF (PDB code 1KIN chain A), 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). Constructed according to general teachings. The homology of these sequences is 34%. FIG. 3 shows an alignment between the sequence of epothilone B hydroxylase (SEQ ID NO: 2) and the sequence of EryF (PDB code 1KIN chain A; SEQ ID NO: 76). A homology model of epothilone B hydroxylase based on sequence alignment with EryF is shown in FIG.

  An energy plot of the epothilone B hydroxylase model compared to EryF (PDB code 1JIN) was also generated and is shown in FIG. Using the average window size of 51 residues at a residue position, the average of the energy of 51 residues in the sequence with that residue in the middle position was calculated. As shown in FIG. 5, all the energy along the array was less than 0, indicating that the model structure shown in FIG. 4 and Appendix 1 is valid.

  The three-dimensional structure shown in the homology model of epothilone B hydroxylase in FIG. 4 is defined by the structure coordinates shown in Appendix Table 1. “Structural coordinates” refers to Cartesian coordinates generated from the construction of a homology model. However, as will be appreciated by those skilled in the art, the set of structural coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates may define a similar or identical shape. In addition, minor changes in individual coordinates have a minor effect on the overall shape, as may result from the generation of similar homology models using different alignment templates and / or different methods when generating homology models. It will not reach. Changes in coordinates also occur due to mathematical manipulation of structural coordinates. For example, the structural coordinates shown in Appendix 1 could be manipulated by fractionalization of structural coordinates, addition or subtraction of integers to a set of structural coordinates, inversion of structural coordinates, or a combination thereof.

  Therefore, various computer analyzes are required to determine whether a molecule or part thereof is sufficiently similar to be considered the same as all or part of the epothilone B hydroxylase. Such an analysis can be performed with current software applications such as SYBYL version 6.7 or INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) Version 2000, as described in the accompanying user specifications.

  For example, the overlay tool of the program SYBYL allows comparisons between different structures and between different conformations of the same structure. The procedure for comparing the structures used in SYBYL is divided into four steps: (1) loading the structures to be compared; (2) defining atomic equivalence in these structures; (3) matching operations. Then (4) analyze the results. Identify each structure by name. One structure is the target (ie, the fixed structure) and the second structure (ie, the structure to be moved) is the source structure. Since the equivalence of atoms within SYBYL is determined by user input, for the purposes of this aspect of the invention, the equivalent atoms are the same for all conserved residues between the two structures compared. We will define it as protein backbone atoms (N, Cα, C and O). In addition, only rigid fitting operations are considered. When using a rigid fitting method, the working structure is translated and rotated to obtain the best fit with the target structure. The fitting operation uses an algorithm that calculates the optimal translation and rotation to be applied to the moving structure so that the root mean square difference of the fit of a specified pair of equivalent atoms is an absolute minimum. This number (given in Angstroms) is reported by SYBYL.

  For purposes of the present invention, the root mean square of conserved residue skeleton atoms (N, Cα, C and O) when superimposed on the corresponding skeleton atoms described by the structural coordinates listed in Appendix Table 1 All homology models of epothilone B hydroxylase with a deviation of less than about 4.0 angstroms are considered identical. More preferably, the root mean square deviation is less than about 3.0 angstroms. More preferably, the root mean square deviation is less than about 2.0 angstroms.

  For purposes of the present invention, the root mean square of conserved residue skeleton atoms (N, Cα, C and O) when superimposed on the corresponding skeleton atoms described by the structural coordinates listed in Appendix Table 1 All homology models of epothilone B hydroxylase with deviations less than about 2.0 angstroms are considered identical. More preferably, the root mean square deviation is less than about 1.0 angstrom.

  In other embodiments of the invention, the structural models that have a low mean square deviation when the skeletal atoms are replaced with other elements and are superimposed on the corresponding skeletal atoms are considered identical. For example, the original skeletal carbon atom and / or nitrogen atom and / or oxygen atom are substituted with other elements and superimposed on the corresponding skeletal atoms described by the structural coordinates listed in Appendix Table 1. Sometimes homology models having a mean square deviation of about 4.0 angstroms, preferably about 3.0 angstroms, and even more preferably less than about 2 angstroms are considered identical.

  “Root mean square deviation” means the square root of the arithmetic mean of the square of the deviation from the mean. It is one way to express a deviation or variation from a trend or subject. For the purposes of the present invention, the “root mean square deviation” defines the variation of the protein backbone from the relevant portion of the backbone of the epothilone B hydroxylase portion of the complex defined by the structural coordinates described herein.

  The present invention allows structure-based design of additional mutants of epothilone B hydroxylase, as embodied by the homology model. For example, using the homology model of the present invention, residues present within 10 angstroms of the binding site of epothilone B hydroxylase have now been defined. As shown in Appendix Table 1, these residues are 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, GNL170, CYS172, SER173AR17E17U17 SER178, ARG179, ARG186, PHE190, LEU193, VAL233 GLY234, LEU235, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, 2829 SER294, ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352EL35G 58, including the ALA359, GLU362, LYS389, ASP391, SER392, THR393, ILE394 and TYR395. Mutants with mutations at one or more of these positions are expected to exhibit altered biological function and / or specificity and thus constitute another aspect of preferred mutants of the invention . Another embodiment of the preferred mutant is a molecule having a mean square deviation from the backbone atom of the epothilone B hydroxylase of about 4.0 angstroms or less.

  The structural coordinates of the epothilone B hydroxylase homology model or part thereof are stored in a machine-readable storage medium. Such data can be used for various purposes such as drug discovery.

  Accordingly, another aspect of the present invention relates to a machine readable data storage medium comprising a data storage material encoded with structural coordinates shown 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 for the enzyme. Various methods or combinations thereof can be used to identify such modulators.

  For example, according to Appendix 1, a test compound that spatially matches the binding site of epothilone B hydroxylase can be modeled. Amino acids LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, RO86P ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174, ARG175, MET176, LEEU177AR17E17 VAL233, GLY234, LEU2 5, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, THR287, ILE288, ILE288 ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GL3532, CY3353, L35 The structural coordinates of amino acids within 10 angstroms of the binding region of epothilone B hydroxylase as defined by U362, LYS389, ASP391, SER392, THR393, ILE394 and TYR395, and the coordinated heme group HEM1, are also desirable for such a modulator. Can be used to identify structural and chemical characteristics of The identified structural or chemical characteristics can then be used to design or select compounds as potential epothilone B hydroxylase ligands. Structural and chemical features are meant to include, but are not limited to, covalent bonds, van der Waals interactions, hydrogen bonding interactions, charge interactions, hydrophobic interactions, and dipolar interactions. It is not a thing. The compounds identified as potential epothilone B hydroxylase ligands are then synthesized and synthesized in an assay characterized by binding of the test compound to epothilone B hydroxylase, or in the presence of small molecules, epothilone B Screening can be done by characterizing the ability of hydroxylase. Examples of assays useful for screening potential epothilone B hydroxylase ligands include, but are not limited to, in silico screening, in vitro assays, and high throughput assays.

  Other structure-based design methods can be used, as will be appreciated by those skilled in the art from this disclosure. Various computer based structure-based design methods have been disclosed in the art. For example, inputting the sequence of epothilone B hydroxylase and the structure of epothilone B hydroxylase (ie, the atomic coordinates of epothilone B hydroxylase shown in Appendix 1 and / or the atomic coordinates within 10 angstroms of the binding region shown above) Many computer modeling systems are available. The computer system then generates one or more structural details of these regions so that the complementary structural details of the potential modulator can be determined. The designs in these modeling systems are generally based on compounds that can physically and structurally bind to epothilone B hydroxylase. Furthermore, the compound must be able to adopt a conformation that allows binding to epothilone B hydroxylase. Some modeling systems evaluate potential inhibitory or binding effects of potential epothilone B hydroxylase substrates or modulators prior to actual synthesis and testing.

  Methods for screening chemicals or fragments for the ability to bind to a given protein target are also well known. These methods often begin by visual inspection of the binding site on a computer screen. The selected fragment or chemical is then positioned in the binding region of epothilone B hydroxylase. Docking is performed using software such as INSIGHTII, QUANTA, and SYBYL, followed by energy minimization and molecular dynamics using standard molecular mechanical force fields such as MMFF, CHARMM, and AMBER. Examples of computer programs that aid in the selection of chemical fragments or chemicals useful in the present invention include GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990) and DOCK (Kuntz et al., 1982). It is not limited to.

  Once preferred chemicals or fragments are selected, the relationship between each other and the epothilone B hydroxylase can be visualized and then assembled into a single potential modulator. Useful programs for assembling individual chemicals include, but are not limited to, CAVEAT (Bartlett et al., 1989) and 3D database systems (Martin, 1992).

  Alternatively, compounds can be designed de novo using blank binding sites or including some of the known inhibitors. This type of design method includes, but is not limited to, LUDI (Bohm, 1992) and LeapFrog (Tripos Inc., St. Louis, MO).

  Programs such as DOCK (Kuntz et al., 1982) are used with atomic coordinates from the homology model to identify potential ligands that bind potentially to the binding region in the active site and are therefore suitable candidates for synthesis and testing. It can be identified from a database or a virtual database.

  In addition, a gene comprising the polynucleotide of the present invention and the vector of the present invention to produce epothilone B hydroxylase or an active fragment thereof and a mutant or mutant of the enzyme and / or ferredoxin or an active fragment thereof Engineered host cells are also provided in the present invention. In general, any vector suitable for maintaining, propagating or expressing a polynucleotide to produce these polypeptides in a host cell can be used for expression in this regard. According to this aspect of the invention, the vector may be, for example, a plasmid vector, a single-stranded or double-stranded phage vector, or a single-stranded or double-stranded RNA or DNA viral vector. The vector may be extrachromosomal or designed for integration into the host chromosome. Such vectors include chromosomal, episomal and viral vectors such as bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal sequences, and viruses (baculovirus, papovavirus, SV40, vaccinia virus, adenovirus, chicken). Vectors derived from spider viruses, pseudorabies viruses and retroviruses) and combinations thereof, including but not limited to plasmid and bacteriophage gene sequences, cosmid and phagemid vectors is not.

  Expression vectors useful for prokaryotic hosts include pBluescript, pGEX-2T, pUC vector, pET vector, ColE1, pCR1, pBR322, pMB9, pCW, pBMS200, pBMS2020, PIJ101, PIJ702, pANT849, pOJ260, pOJ46, pOJ4 Bacterial plasmids such as those from E. coli, Bacillus or Streptomyces, including pKC1218, pFD666, and derivatives thereof, broad host range plasmids such as RP4, phage DNA, eg numerous derivatives of phage lambda, such as NM989, λGT10 And λGT11, and other phages such as M13 and filamentous single-stranded phage DNA. Is not something

  Vectors of the invention for use in yeast will typically contain an origin of replication suitable for use in yeast and a selectable marker functional in yeast. Examples of yeast vectors useful in the present invention include yeast integration plasmids (eg, YIp5) and yeast self-replicating plasmids (YRp and YEp series plasmids), yeast centromere plasmids (YCp series plasmids), yeast artificial chromosomes ( YAC) (based on the yeast linear plasmid designated YLp, pGPD-2, 2μ plasmid and its derivatives), and those described in Gietz et al., Gene, 74: 527-34 (1988) (YIplac, YEplac and Improved shuttle vectors such as, but not limited to, YCplac).

  Mammalian vectors useful for recombinant expression are viral origins of replication, eg, SV40 origin of replication (for replication in cell lines that express large T antigens such as COS1 and COS7 cells), papillomavirus origin of replication, or long-term episomal replication. EBV origin of replication (eg, for use in 293-EBNA cells that constitutively express the EBV EBNA-1 gene product and adenovirus E1A). Expression in mammalian cells includes pSV2, pBC12BI, and p91023, pCDNA vectors, and lytic viral vectors (eg, vaccinia virus, adenovirus, and baculovirus), episomal viral vectors (eg, bovine papilloma virus), and retroviruses This can be done using a variety of plasmids, including but not limited to vectors (eg, murine retroviruses). Vectors useful for insect cells include baculovirus vectors and pVL941.

  Selection of appropriate promoters that direct transcription of mRNA and construction of expression vectors are well known. In general, however, expression constructs will contain sites for initiation and termination of transcription, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct will initially contain a translation initiation codon and a stop codon located approximately at the end of the polypeptide to be translated.

Examples of promoters useful in prokaryotes include phage promoters such as phage lambda pL promoter, trc promoter, hybrids derived from trp and lac promoters, bacteriophage T7 promoter, TAC or TRC systems, major operators of phage lambda And promoter region, fd coat protein control region, snpA promoter, melC promoter, ermE ^* promoter or araBAD operon, but not limited thereto. Examples of useful promoters for yeast include, but are not limited to, CYC1 promoter, GAL1 promoter, GAL10 promoter, ADH1 promoter, yeast α-mating system promoter, and GPD promoter. Absent. Examples of promoters routinely used in mammalian expression vectors include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTR promoter, such as those derived from Rous sarcoma virus (RSV), And a metallothionein promoter, such as, but not limited to, the mouse metallothionein I promoter.

  A vector comprising a polynucleotide can be introduced into a host cell using any of the well-known techniques including infection, transduction, transfection, transvection and transformation. The polynucleotide can be introduced into the host alone or with an additional polynucleotide encoding, for example, a selectable marker or ferredoxin reductase. In a preferred embodiment of the invention, polynucleotides encoding epothilone B hydroxylase and ferredoxin are introduced into the host cell. Host cells for various expression constructs are well known and one of skill in the art can routinely select host cells for expressing epothilone B hydroxylase and / or ferredoxin according to this aspect of the invention. Examples of mammalian expression systems useful in the present invention include, but are not limited to, C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines, and monkey kidney fibroblast COS-7 cell lines. It is not something that can be done.

  Alternatively, as exemplified herein, epothilone B hydroxylase and ferredoxin can be recombinantly expressed in microorganisms.

  Accordingly, another aspect of the present invention is to express epothilone B hydroxylase alone or epothilone B hydroxylase with ferredoxin and to hydroxylate the epothilone to produce a compound having a terminal hydroxyalkyl group, in particular epothilone. The present invention relates to a recombinantly produced microorganism. A microorganism produced by recombination is produced by transforming a cell such as a bacterial cell with a plasmid containing a nucleic acid sequence encoding epothilone B hydroxylase. In a preferred embodiment, cells are transformed with a plasmid comprising a nucleic acid encoding epothilone B hydroxylase or a mutant or variant thereof and a nucleic acid sequence encoding ferredoxin located downstream of the epothilone B hydroxylase gene. Examples of microorganisms that can be transformed with these plasmids to produce the recombinant microorganism of the present invention include Escherichia coli, Bacillus megaterium, Amycolatopsis orientalis, Solandium cellulosum, Rhodococcus erythropolis ( Rhodococcus erythropolis), Streptomyces virginiae, Streptomyces venezuelae, Streptomyces albus, Streptomyces coelis And Streptomyces species such as, but not limited to, Streptomyces griseus.

  The recombinantly produced microorganisms of the present invention are useful in microbial processes or methods for the production of compounds containing terminal hydroxyalkyl groups, particularly epothilones. In general, hydroxyalkyl-containing products selectively hydroxylate terminal carbon atoms or alkyls under aerobic conditions in water in the presence of a suitable substrate in an aqueous nutrient medium containing assimilable carbon and nitrogen sources. Can be produced by culturing a recombinantly produced microorganism or an enzyme from the microorganism.

  A suitable epothilone for use as a substrate in the method of the present invention is any such compound having a terminal carbon atom or terminal alkyl group capable of undergoing the enzymatic hydroxylation of the present invention. The starting material, i.e. the substrate, can be isolated from a natural source such as Sorandium cellulosum, or it can be a synthetically produced epothilone. Other substrates having terminal carbon atoms or terminal alkyl groups and capable of undergoing enzymatic hydroxylation can be used by the methods described herein. For example, compactin can be used as a substrate, which when hydroxylated produces the compound pravastatin. Methods for hydroxylating compactin with the Actinomadura strain to produce pravastatin are described in US Pat. Nos. 5,942,423 and 6,274,360.

  For example, using the recombinant microorganism of the present invention, WO 00/39276, U.S. Patent Application No. 09 / 468,854 (filed on December 21, 1999) (the text of which is incorporated herein by reference) At least one epothilone can be prepared as described in

Formula I below:
_{HO-CH 2 - (A 1} ) n - (Q) m - (A 2) o -E (I)
Wherein A ₁ and A ₂ are each independently selected from the group consisting of optionally substituted C ₁ -C ₃ alkyl and alkenyl;
Q is an optionally substituted ring system comprising 1 to 3 rings and at least one carbon-carbon double bond in at least one ring;
n, m and o are integers selected from the group consisting of 0 and 1, where at least one of m or n or o is 1; and E is an epothilone core). be able to.

The method of the present invention comprises the following formula II:
_{CH 3 - (A 1) n} - (Q) m - (A 2) o -E (II)
(Wherein A ₁ , Q, A ₂ , E, n, m, and o are as defined above) can selectively catalyze the hydroxylation of the compound of formula II. Contacting with a recombinantly produced microorganism or an enzyme from the microorganism, followed by the hydroxylation step.

  In a preferred embodiment, the starting material is epothilone B. Epothilone B can be obtained from fermentation of Solandium Cellulosum Source 90 as described in DE 418042 and WO 93/10121. This strain has been deposited with Deutsche Zamlung von Microorganismen (German Collection of Microorganisms) (DSM) under accession number 6773. The fermentation process is also described in Hofle, G. et al., Angew. Chem. Int. Ed. Engl., Vol. 35, No. 13/14, 1567-1569 (1996). Epothilone B can also be obtained by chemical means such as Meng, D. et al., J. Am. Chem. Soc., Vol. 119, No. 42, 10073-10092 (1996); Nicolaou, K. et al., J. Am Chem. Soc., Vol. 119, No. 34, 7974-7991 (1997) and Schinzer, D. et al., Chem. Eur. J., Vol. 5, No. 9, 2483-2491 (1999). It can be obtained by the following chemical means.

Growth of recombinantly produced microorganisms selected for use in the methods of the present invention can be achieved by one skilled in the art through the use of appropriate nutrient media. Suitable media for the growth of recombinantly produced microorganisms include those that provide nutrients necessary for the growth of microbial cells. For example, T. Nagodawithana and JM Wasileski, Chapter 2: “Media Design for Industrial Fermentations”, Nutritional Requirements of Commercially Important Microorganism , TW Nagodawithana and G. Reed, Estekay Associates, Inc., Milwaukee, Wisconsin, 18-45 ( 1998); TL Miller and BW Churchill, Chapter 10: “Substrates for Large-Scale Fermentations”, Manual of Industrial Microbiology and Biotechnology , edited by AL Demain and NA Solomon, American Society for Microbiology, Washington, D.C., 122- See 136 (1986). A typical medium for growth contains the necessary carbon source, nitrogen source, and trace elements. Inducers can also be added to the medium. As used herein, the term inducer includes any compound that promotes the production of a desired enzymatic activity in a recombinantly produced microbial cell. Typical inducers used herein include the solvents used to dissolve the substrate, such as dimethyl sulfoxide, dimethylformamide, dioxane, ethanol and acetone. In addition, certain substrates such as epothilone B are also considered inducers.

  Examples of the carbon source include sugars such as glucose, galactose, maltose, sucrose, mannitol, sorbitol, glycerin and starch; organic acids such as sodium acetate and sodium citrate; and alcohols such as ethanol and propanol. Preferred carbon sources include, but are not limited to, glucose, fructose, sucrose, glycerin and starch.

  Examples of the nitrogen source include NZ amine A, corn dipping solution, soybean meal, beef extract, yeast extract, tryptone, peptone, cottonseed meal, peanut meal, sodium glutamate and other amino acids, sodium nitrate, ammonium sulfate, and the like.

  Examples of the trace element include magnesium, manganese, calcium, cobalt, nickel, iron, sodium salt, and potassium salt. Phosphate can also be added in trace amounts, or preferably in quantities greater than trace amounts.

  The medium used for fermentation may contain more than one carbon or nitrogen source or other nutrient.

  For the growth and / or hydroxylation of recombinantly produced microorganisms according to the method of the invention, 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 reaction time is 1 to 100 hours, preferably 8 to 72 hours.

  The medium is monitored by high performance liquid chromatography (HPLC) and incubated for the time required to complete biotransformation. Typically, the time required to complete the biotransformation is 12 to 100 hours, preferably about 72 hours, after adding the substrate. The medium is placed on a rotary shaker (New Brunswick Scientific Innova 5000) operating at 150-300 rpm, preferably about 250 rpm, and a stroke of 2 inches.

  Recovery of the hydroxyalkyl-containing product from the fermentation broth can be done by conventional means commonly used for recovery of other known biologically active substances. Examples of such recovery means are by isolation and purification by extraction with common solvents such as ethyl acetate; by pH adjustment; by normal resin treatment, eg anion or cation exchange Resin or non-ionic adsorbent resin treatment; ordinary adsorbent treatment, such as, but not limited to, distillation or crystallization; or recrystallization .

  The extract obtained from the biotransformation reaction mixture above can be further isolated and purified by column chromatography and analytical thin layer chromatography.

  The ability of the recombinantly produced microorganism of the present invention to biotransform an epothilone having a terminal alkyl group into an epothilone having a terminal hydroxyalkyl group has been demonstrated. In these experiments, the culture solution containing the Streptomyces lividans clone containing the plasmid having the ebh gene as described in more detail in Example 11 was stirred for 3 days at 30 ° C. with stirring with the epothilone B suspension. Incubated. Samples for incubation were extracted with an equal volume of 25% methanol: 75% n-butanol, vortexed and then allowed to settle for 5 minutes. 200 μl of the organic phase was transferred to an HPLC vial and analyzed by HPLC / MS (Example 12). The epothilone F product peak 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. Peak retention time and molecular weight were confirmed using known standards.

  The rate of biotransformation of epothilone B by cells expressing ebh was compared to the rate of biotransformation by ebh mutants. Cells expressing ebh contain a frozen spore preparation of Streptomyces lividans (pANT849-ebh). Cells expressing the mutant include frozen spore preparations of Streptomyces lividans (pANT849-ebh10-53) and Streptomyces lividans (pANT849-ebh24-16). A frozen spore preparation of Streptomyces lividans TK24 was used as a control. Cells were preincubated at 30 ° C. for several days. After this preincubation, epothilone B in 100% EtOH was added to each culture at a final concentration of 0.05% w / v. The samples were then removed at 0, 24, 48 and 72 hours, except for the culture of Streptomyces lividans (pANT849-ebh24-16), where epothilone B was completely converted to epothilone F in 48 hours. It was collected at the time point. Samples were analyzed by HPLC. Results were calculated as percent of epothilone B at 0 hours.

Epothilone B:

Epothilone F:

  The ability of cells expressing ebh to biotransform compactin to pravastatin was also examined. In these experiments, frozen spore preparations of Streptomyces lividans (pANT849) or Streptomyces lividans (pANT849-ebh) were grown at 30 ° C. for several days. After preincubation, an aliquot of each cell culture was transferred to a polypropylene culture tube, compactin was added to each culture tube, and the culture tube was then incubated at 30 ° C., 250 rpm for 24 hours. An aliquot of the culture broth was then extracted and the compactin and pravastatin values compared to the control Streptomyces lividans (pANT849) culture were measured by HPLC.

Compactin and pravastatin as a percentage of the starting material compactin concentration:

As discussed above, mutant ebh25-1 (SEQ ID NO: 30) exhibits altered substrate specificity and the biotransformation of epothilone B by this mutant is a different HPLC elution time than epothilone B or epothilone F. The result is a product with This unknown sample was analyzed by LC-MS and found to have a molecular weight of 523 (M.W.), consistent with a single hydroxylation of epothilone B. The structure of this biotransformation product was determined to be 24-hydroxyl-epothilone B based on MS and NMR data (compared to the epothilone B data):

＊１.８〜２.１ｐｐｍのピークは溶媒抑制（solvent suppression）のために観察されなかった。 Molecular _formula: C ₂₇ H 41 _NO 7 S
Molecular weight: 523
Mass spectrum: ES + (m / z): 524 ([M + H] ⁺ ), 506
LC / MS / MS: + ESI (m / z): 524, 506, 476, 436, 320
HRMS: As [M + H] ⁺ ; Calculated value: 524.2682; Found: 524.2701
HPLC (Rt): 7.3 minutes (with analytical HPLC system)
LC / NMR: Observe chemical shift
Varian AS-600 (Proton: 599.624 MHz)
Solvent D ₂ O / CD ₃ CN (δ 1.94): ˜4 / 6

* A peak between 1.8 and 2.1 ppm was not observed due to solvent suppression.

＊ＳＳＰ：溶媒抑制のために観察されず。 Proton chemical shifts were assigned as follows:

* SSP: Not observed due to solvent suppression.

Thus, the compositions and methods of the present invention are known compounds that are microtubule stabilizers and epothilone analogs that are expected to be useful as microtubule stabilizers (such as 24-hydroxyl-epothilone B (Formula A)). ) And pharmacologically acceptable salts thereof. Microtubule stabilizers produced using these compositions and methods are useful for the treatment of various cancers and other proliferative diseases, including but not limited to the following:
Carcinomas including those of the bladder, breast, rectum, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma;
Hematopoietic tumors of the lymphatic system including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma and Burkitt lymphoma;
Hematopoietic tumors of the myeloid lineage including acute and chronic myeloid leukemias and promyelocytic leukemias;
Tumors derived from mesenchyme including fibrosarcoma and rhabdomyosarcoma;
Other tumors including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioblastoma;
Tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioblastoma, and schwannoma;
Mesenchymal-derived tumors including fibrosarcoma, rhabdomyosarcoma and osteosarcoma;
Other tumors, including melanoma, xeroderma pigmentosum, keratinous squamous cell tumor, seminoma, thyroid follicular cancer and teratocarcinoma.

  Microtubule stabilizers produced using the compositions and methods of the present invention will also inhibit angiogenesis, thereby affecting tumor growth and resulting in the treatment of tumors and tumor-related diseases. Such anti-angiogenic properties of these compounds also include certain types of blindness, arthritis, especially inflammatory arthritis, multiple sclerosis, restenosis and psoriasis associated with retinal neovascularization. Not) and may be useful in the treatment of other conditions where anti-angiogenic agents are applied.

  Microtubule stabilizers produced using the compositions and methods of the present invention will induce or inhibit apoptosis, a process of physiological cell death important for normal development and homeostasis. Changes in the apoptotic pathway contribute to the pathogenesis of various human diseases. The compounds of the present invention, such as the compounds represented by Formula I, Formula II and Formula A, can be used as modulators of apoptosis to treat cancer, precancerous lesions, immune response related diseases, viral infections, musculoskeletal degenerative diseases and kidney diseases Including, but not limited to, will be useful in the treatment of various diseases in humans with abnormalities in apoptosis.

  Without intending to be limited to any mechanism or morphology, microtubule stabilizers produced using the compositions and methods of the present invention also treat conditions other than cancer and other proliferative diseases Can also be used. Such conditions include viral infections such as herpes virus, pox virus, Epstein Barr virus, Sindbis virus and adenovirus; systemic lupus erythematosus, glomerulonephritis immune system, rheumatoid arthritis, psoriasis, inflammatory bowel disease and Autoimmune diseases such as autoimmune diabetes mellitus; neurodegenerative diseases such as Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration; AIDS; bone marrow Dysplasia syndrome; aplastic anemia; ischemic disorder-related myocardial infarction; stroke and reperfusion injury; restenosis; arrhythmia; atherosclerosis; toxin-induced or alcohol-induced liver disease; chronic anemia and aplastic anemia Blood diseases; musculoskeletal degenerative diseases such as osteoporosis and arthritis; Emissions sensitive rhinosinusitis; cystic fibrosis; multiple sclerosis; kidney disease; including but and cancer pain is not limited thereto.

The following non-limiting examples are provided to further illustrate the present invention.
Example
Example 1: Reagent
R2 medium was prepared as follows.
A solution containing sucrose (103 g), K ₂ SO ₄ (0.25 g), MgCl ₂ .6H ₂ O (10.12 g), glucose (10 g), Difco casamino acid (0.1 g) and distilled water (800 ml) Was prepared. Then 80 ml of this solution was poured into a 200 ml screw cap bottle containing 2.2 g Difco Bacto agar. The bottle was capped and autoclaved. In use, the medium was redissolved and the following autoclave solutions were added in the order listed:
1 ml of KH ₂ PO ₄ (0.5%)
8 ml of CaCl ₂ · 2H ₂ O (3.68%)
1.5 ml L-proline (20%)
10 ml of TES buffer (adjusted to 5.73%, pH 7.2)
ZnCl ₂ (40 mg), FeCl ₃ .6H ₂ O (200 mg), CuCl ₂ .2H ₂ O (10 mg), MnCl ₂ .4H ₂ O (10 mg), Na ₂ B ₄ O ₇ · 10H ₂ O (10 mg) and 0.2 ml trace element solution containing (NH ₄ ) ₆ Mo ₇ O ₂₄ · H ₂ O 0.5 ml NaOH (1N) (no sterilization required)
Growth factors required for 0.5 ml auxotrophs (histidine (50 μg / ml); cysteine (37 μg / ml); adenine, guanine, thymidine and uracil (7.5 μg / ml); and vitamins (0.5 μg / ml) ))

R2YE medium was prepared in the same manner as R2 medium. However, 5 ml of Difco yeast extract (10%) was added to each 100 ml flask at the time of use.

P (protoplast) buffer was prepared as follows.
A base solution consisting of the following was prepared:
Sucrose (103g)
K ₂ SO ₄ (0.25 g)
MgCl ₂ · 6H ₂ O (2.02 g)
Trace element solution described for R2 medium (2 ml)
Distilled water (up to 800ml)

The 80 ml aliquot of the base solution was then dispensed and autoclaved. Prior to use, the following were added to each flask in the order listed:
1 ml of KH ₂ PO ₄ (0.5%)
10 ml of CaCl ₂ · 2H ₂ O (3.68%)
TES buffer (adjusted to 5.75%, pH 7.2)

A T (transformation) buffer was prepared by mixing the following sterile solutions:
25 ml sucrose (10.3%)
75 ml distilled water 1 ml trace element solution described for R2 medium 1 ml K ₂ SO ₄ (2.5%)

The following is then added to 9.3 ml of this solution:
0.2 ml of CaCl ₂ (5M)
0.5 ml of trismaleic acid buffer prepared from a 1 M solution of Tris and adjusted to pH 8.0 with maleic acid.
In use, 3 parts by weight of the above solution is added to 1 part by weight of PEG 1000 previously autoclaved.

L (lysis) buffer was prepared by mixing the following sterile solutions:
100 ml sucrose (10.3%)
10 ml of TES buffer (adjusted to 5.73%, pH 7.2)
1 ml of K ₂ SO ₄ (2.5%)
1 ml trace element solution described for R2 medium 1 ml KH ₂ PO ₄ (0.5%)
0.1 ml MgCl ₂ .6H ₂ O (2.5M)
1 ml of CaCl ₂ (0.25M)

A solution containing the following components in 1 liter of dH ₂ O was prepared: glucose (10 g), sucrose (103 g), MgCl ₂ .6H ₂ O (10.12 g), BBL ^™ trypticase soy broth (15 g) ) (Becton Dickinson Microbiology Systems, Sparks, MD, USA), and BBL ^™ yeast extract (5 g) (Becton Dickinson Microbiology Systems). This solution was autoclaved for 30 minutes. To grow the culture with the plasmid, thiostrepton was added at a concentration of 10 μg / ml.

A solution containing electroporation buffer 30% (w / v) PEG 1000, 10% glycerin, and 6.5% sucrose was prepared in dH ₂ O. This solution was sterilized by vacuum filtration through a 0.22 μm cellulose acetate membrane.

Example 2: Extraction of chromosomal DNA from strain SC15847 Using a guanidine-surfactant lysis method, DNAzol reagent (Invitrogen, Carlsbad, CA, USA), representation of a soil isolate of Amycolatopsis orientalis SC15847 (ATCC) Genomic DNA was isolated from PT-1043). The SC15847 culture was grown at 28 ° C. for 24 hours in F7 medium (glucose 2.2%, yeast extract 1.0%, malt extract 1.0%, peptone 0.1%, pH 7.0). 20 ml culture was collected by centrifugation, resuspended in 20 ml DNAzol, mixed by pipetting and then centrifuged in a Beckman TJ6 centrifuge tube for 10 minutes. 10 ml of 100% ethanol was added, inverted several times and stored at room temperature for 3 minutes. The DNA was wrapped around a glass pipette, washed with 100% ethanol and air dried for 10 minutes. The pellet was resuspended in 500 μl 8 mM NaOH, and once dissolved, neutralized with 30 μl 1M HEPES (pH 7.2).

Example 3: PCR reaction 200-500 ng genomic DNA or 0.1 μl cDNA, forward primer and reverse primer [Forward primer is P450-1 ⁺ (SEQ ID NO: 23) or P450-1a ⁺ (SEQ ID NO: 24 ) or P450-2 ⁺ (SEQ ID NO: 25) is any one of, the reverse primer P450-3 ^- (SEQ ID NO: 26) was either ^- (SEQ ID NO: 27) or P450-2 A PCR reaction was prepared in a volume of 50 μl. All primers were added at a final concentration of 1.4-2.0 μM. The PCR reaction solution was prepared by using 1 μl Taq enzyme (2.5 units) (Stratagene), 5 μl Taq buffer and 4 μl 2.5 mM dNTP, and adding dH ₂ O to 50 μl. Cycle reaction was carried out in the following protocol in Geneamp ^R PCR System: 5 min at 95 ° C., 30 seconds at 5 cycles [95 ° C., 15 sec at 37 ° C. (30% ramp (ramp)), 72 30 ° C.], 35 cycles (94 ° C. for 30 seconds, 65 ° C. for 15 seconds, 72 ° C. for 30 seconds), 72 ° C. for 7 minutes. The expected size from the reaction, P450-1 ⁺ (SEQ ID NO: 23) or P450-1a ⁺ (SEQ ID NO: 24) P450-3 ^- 340bp for the primer pair (SEQ ID NO: 27), P450-1 ⁺ ( SEQ ID NO: 23) and P450-2 ^- (for primer pairs SEQ ID NO: 26) is 240 bp, and P450-2 ⁺ (SEQ ID NO: 25) P450-3 ^- (for primer pairs SEQ ID NO: 27) is a 130bp is there.

Example 4 Cloning of Epothilone B Hydroxylase Gene and Ferredoxin Gene 20 μg of SC15847 genomic DNA was digested with BglII restriction enzyme at 37 ° C. for 6 hours. DNA was concentrated using a 30k nanosep column (Gelman Sciences, Ann Arbor, Michigan, USA) to remove enzyme and buffer. The reaction solution was concentrated to 40 μl and washed with 200 μl of TE. The digested products were then separated on a 0.7% agarose gel and genomic DNA in the 12-15 kb range was excised from the gel and purified using the Qiagen gel extraction method. The genomic DNA was then digested with BamHI and dephosphorylated using the SAPI enzyme (Roche Molecular Biochemicals, Indianapolis, Indiana, catalog # 1758 250), plasmid pWB19N (US Pat. No. 5,516,679) Ligated to. The ligation reaction was performed using 1 U of T4 DNA ligase (Invitrogen) at a room temperature of 15 μl for 1 hour. 1 μl of ligation fluid was transformed into 100 μl of chemically competent DH10B cells (Invitrogen) and 100 μl was plated overnight at 37 ° C. on 5 LB agar plates containing 30 μg / ml neomycin.

  Five nylon membrane circles (Roche Molecular Biochemicals, Indianapolis, Indiana) were numbered and the direction was marked. The membrane was placed on the plate for 2 minutes and then allowed to dry for 5 minutes. The membrane was then placed on a Fatman filter disk for 5 minutes with 10% SDS, 5 minutes with 0.5 M NaOH with 1.5 M NaCl, 5 minutes with 1.5 M NaCl with 1.0 M Tris (pH 8.0), and Saturated with 2 × SSC for 15 minutes. Filters were hybridized as previously described for Southern hybridization. Hybridized colonies were picked into 2 ml TB containing 30 μg / ml neomycin and grown overnight at 37 ° C. Plasmid DNA was isolated using the miniprep column method (Mo Bio). This plasmid was designated as NPB29-1.

Example 5: DNA sequencing and analysis Cloned PCR products were subjected to fluorescence-dye-labeled terminator cycle sequencing, Big-Dye sequencing kit (Applied Biosystems, Foster City, California, USA). And sequenced and analyzed using laser-induced fluorescence capillary electrophoresis, ABI Prism 310 sequencer (Applied Biosystems).

Example 6 Extraction of Total RNA Total RNA was isolated from SC15847 culture using a modified version of the Chomczynski and Sacchi method using Trizol reagent (Invitrogen), a single phase solution of phenol and guanidine isothiocyanate. 5 ml of SC15847 frozen stock culture was thawed and used to inoculate 100 ml of F7 medium in a 500 ml Erlenmeyer flask. The culture was grown in a shaker incubator at 230 rpm for 20 hours at 30 ° C. until the optical density at 600 nm (OD ₆₀₀ ) was 9.0. The culture was placed in a 16 ° C. shaker incubator at 230 rpm for 20 minutes. 25 mg of epothilone B was dissolved in 1 ml of 100% ethanol and added to the culture solution. Residual epothilone B was rinsed from the tube with a second ml of ethanol and added to the culture. The culture was incubated at 16 ° C. and 230 rpm for 30 minutes. 30 ml of the culture solution was transferred to a 50 ml tube, 150 mg of lysozyme was added to the culture solution, and the culture solution was incubated at room temperature for 5 minutes. 10 ml of the culture solution was put into a 50 ml falcon tube and centrifuged at 4 ° C. for 5 minutes in a TJ6 centrifuge tube. 2 ml of chloroform was added and the tube was mixed vigorously for 15 seconds. Tubes were incubated for 2 minutes at room temperature and centrifuged for 10 minutes at the maximum speed of a TJ6 centrifuge tube. The aqueous layer was transferred to a new tube and 2.5 ml of isopropanol was added to precipitate the RNA. The tube was incubated at room temperature for 10 minutes and centrifuged at 4 ° C. for 10 minutes. The supernatant was removed and the pellet was rinsed with 70% ethanol and allowed to dry for a while under vacuum. The pellet was resuspended in 150 μl RNase-free dH ₂ O. 50 μl of 7.5 M LiCl was added to this RNA and incubated at −20 ° C. for 30 minutes. RNA was pelleted by centrifuging at 4 ° C. for 10 minutes in a centrifuge tube. The pellet was rinsed with 200 μl 70% ethanol, dried for a while under vacuum and resuspended in 150 μl RNase free dH ₂ O.

  RNA was treated with DNase I (Ambion, Austin, Texas, USA). 25 μl total RNA (5.3 μg / μl), 2.5 μl DNase I buffer, 1.0 μl DNase I were added and incubated at 37 ° C. for 25 minutes. 5 μl of DNase I inactivation buffer was added, incubated for 2 minutes, centrifuged for 1 minute, and the supernatant was transferred to a new tube.

Example 7: cDNA synthesis
CDNA was synthesized from total RNA using Superscript II enzyme (Invitrogen). Reactions were prepared using 1 μl total RNA (5.3 μg / μl), 9 μl dH ₂ O, 1 μl dNTP mixture (10 mM), and 1 μl random hexamer. The reaction was incubated at 65 ° C. for 5 minutes and then placed on ice. The following components were then added: 4 μl first strand buffer, 1 μl RNase inhibitor, 2.0 μl 0.1 M DTT, and 1 μl Superscript II enzyme. The reaction was incubated at room temperature for 10 minutes, 42 ° C. for 50 minutes, then 70 ° C. for 15 minutes. 1 μl of RNase H was added and incubated at 37 ° C. for 20 minutes, 70 ° C. for 15 minutes and stored at 4 ° C.

Example 8: Insertion of plasmid pCRscript-29 was amplified using the PCR conditions used for amplification of P450 specific products from DNA-labeled genomic DNA and cDNA. Plasmid pCRscript-29 is the primer P450-1 ⁺ (SEQ ID NO: 23) and P450-3 ^- contains PCR fragment of 340bp was amplified from SC15847 genomic DNA (SEQ ID NO: 27). A total of 25 cycles were performed using 2 μl of plasmid prep as template. The amplified product was gel purified using a Qiaquick gel extraction system (Qiagen). The extracted DNA was ethanol precipitated and resuspended in 5 μl TE and the yield was estimated to be 500 ng. This fragment was labeled with digoxigenin using a chem link labeling reagent (Roche Molecular Biochemicals, Indianapolis, Indiana, catalog # 1 836 463). 5 μl of PCR product was mixed with 0.5 μl of Dig-chem link and dH ₂ O was added to 20 μl. The reaction was incubated at 85 ° C. for 30 minutes and 5 μl of stop solution was added. The probe concentration was estimated to be 20 ng / μl.

Example 9: Southern DNA Hybridization 10 μl of genomic DNA (0.5 μg / ml) was digested with BamHI, BglII, EcoRI, HindIII or NotI and separated at 12 volts for 16 hours. The gel was depurinated with 0.25 N HCl for 10 min and 90 N with 0.4 N NaOH 5 "Hg using a vacuum blotter (Bio-Rad Laboratories, Inc., Hercules, California, USA, catalog # 165-5000). Transfer to nylon membrane (Roche Molecular Biochemicals) under vacuum for 1 minute, rinse membrane with 1M ammonium acetate and UV crosslink with Stratalinker UV Crosslinker (Stratagene) Rinse membrane with 2x SSC and store at room temperature.

The membrane was prehybridized in 20 ml Dig Easy Hyb buffer (Roche Molecular Biochemicals) for 1 hour at 42 ° C. The probe was denatured at 65 ° C. for 10 minutes and then placed on ice. A 5 ml probe with an approximate concentration in Dig Easy Hyb of 20 ng / ml was incubated with the membrane at 42 ° C. overnight. The membrane was washed twice with 2 × SCC containing 0.1% SDS at room temperature and then twice with 0.5 × SSC containing 0.1% SDS at 65 ° C. The membrane was equilibrated with 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, followed by a 2% blocking solution ( Incubated with 2% blocking reagent in Genius buffer 1) (Roche Molecular Biochemicals, Indianapolis, IN) for 1 hour at room temperature. The membrane was incubated for 30 minutes with a 1: 20,000 dilution of anti-dig antibody in 50 ml blocking solution. The membrane was washed twice in 50 ml Genius buffer 1 for 15 minutes each. The membrane was equilibrated with Genius buffer 3 (10 mM Tris-HCl, 10 mM NaCl; pH 9.5) for 2 minutes. CSPD (3- (4-methoxyspiro {1,2-dioxyethane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decane} -4 in Genius Buffer 3 -Yl) Disodium phenylphosphate) (Roche Molecular Biochemicals) 1: 100 dilution (1 ml) was added to the membrane and incubated at room temperature for 5 minutes, 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: Transformation of E. coli Competent cells were purchased from Invitrogen. E. coli strain DH10B was used as the host for genomic cloning. Chemically competent cells were thawed on ice and 100 μl was aliquoted into 17 × 100 mm polypropylene tubes on ice. 1 μl of ligation mixture was added to the cells and incubated on ice for 30 minutes. The cells were incubated at 42 ° C. for 45 seconds and then placed on ice for 1-2 minutes. 0.9 ml of SOC medium (Invitrogen) was added and the cells were incubated at 30-37 ° C., 200-240 rpm for 1 hour. Cells were placed on selective medium (Luria agar containing neomycin or ampicillin at a concentration of 30 μg / ml or 100 μg / ml, respectively).

Example 11: Transformation of Streptomyces lividans TK24 Plasmid pWB19N is digested with HindIII and treated with SAPI, and plasmid pANT849 (Keiser et al., 2000, Practical Streptomyces Genetics, John Innes) is digested with HindIII to produce plasmid pWB19N849. It was constructed. These two linearized fragments were ligated with 1 U T4 DNA ligase for 1 hour at room temperature. 1 μl ligation solution was used to transform XL-1 Blue electrocompetent cells (Stratagene). The collected cells were plated overnight at 37 ° C. in LB neomycin (30 μg / ml). Colonies were picked into 2 ml LB containing 30 μg / ml neomycin and incubated overnight at 30 ° C. MoBio plasmid minipreps were performed on all cultures. The plasmid constructed by ligation of pWB19N and pANT849 was determined by electrophoretic mobility on 0.7% agarose. Plasmid pWB19N849 was digested with HindIII and BglII, and a 5.3 kb fragment equivalent to plasmid pANT849 digested with BglII and HindIII was excised. This 5.3 kb fragment was purified on an agarose gel and extracted using a 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. 50 μl PCR reaction was 5 μl Taq buffer, 2.5 μl glycerin, 1 μl 20 ng / μl NPB29-1 plasmid, 0.4 μl 25 mM dNTP, 1.0 μl each primer NPB29-6F (SEQ ID NO: 28) And NPB29-7R (SEQ ID NO: 29) (5 pmol / μl), 38.1 μl dH ₂ O and 0.5 μl Taq enzyme (Stratagene). The reaction was run on a Perkin Elmer 9700 at 95 ° C for 5 minutes, then 30 cycles (96 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 2 minutes), and 72 ° C for 7 minutes. The PCR product was purified using a Qiagen minielut column by the PCR cleanup method. The purified product was digested with BglII and HindIII and purified on a 0.7% agarose gel. A 1.469 kb band was excised from the gel and eluted using a Qiagen minielut column. 5 μl of this PCR product was ligated with pANT849 vector digested with BglII and HindIII in 10 μl of ligation reaction. The reaction was incubated at room temperature for 24 hours and then transformed into Streptomyces lividans TK24 protoplasts.

  20 ml of YEME medium was inoculated with a frozen spore suspension of Streptomyces lividans TK24 and grown for 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 μl of transformation buffer was added and then 5 ml of P buffer was added immediately. The transformation reaction was spun down at 2,750 rpm for 7 minutes, resuspended in 100 μl P buffer and plated on one R2YE plate. The plate was incubated at 28 ° C. for 20 hours and then overlaid with 5 ml of 0.7% LB agar containing 250 μg / ml thiostrepton. Seven days later, colonies were picked onto R2YE grid plates containing 50 μg / ml thiostrepton. Colonies were grown for an additional 5 days at 28 ° C and stored at 4 ° C.

  This recombinant microorganism has been deposited with ATCC and is labeled PTA-4022.

Example 12: Transformation of Streptomyces limos
Streptomyces limos was transformed using the procedure of Pigac and Schrempf, Appl. Environ. Microb., Vol. 61, No. 1, 352-356 (1995). Streptomyces limosus strain R6 593 was cultured in a 20 ml CRM medium at 30 ° C. on a rotary shaker (250 rpm). Cells were harvested by centrifugation at 5,000 rpm, 4 ° C for 5 minutes at 24 hours, resuspended in 20 ml of 10% sucrose at 4 ° C, and centrifuged at 5,000 rpm, 4 ° C for 5 minutes. I went to. The pellet was resuspended in 10 ml 15% glycerin at 4 ° C. and centrifuged at 5,000 rpm, 4 ° C. for 5 minutes. The pellet was resuspended in 2 ml 15% glycerin with 100 μg / ml lysozyme at 4 ° C., incubated at 37 ° C. for 30 minutes, centrifuged at 5,000 rpm, 4 ° C. for 5 minutes, And resuspended. The 15% glycerin wash was repeated once and the pellet was resuspended in 1-2 ml of electroporation buffer. Cells were stored as aliquots of 50-200 μl at −80 ° C.

Ligation fluid was prepared as described for Streptomyces lividans transformation. After incubating the ligation reaction, the volume was made up to 100 μl with dH ₂ O, NaCl was added to 0.3 M, and the reaction was extracted with an equal volume of 24: 1: 1 phenol: chloroform: isoamyl alcohol. 20 μg 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 in a microcentrifuge tube for 10 minutes, washed once with 70% ethanol, dried for 5 minutes on a speed-vac concentrator, and resuspended in 5 μl dH ₂ O.

One frozen aliquot of cells was thawed at room temperature and divided into 50 μl / tube for each DNA sample for electroporation. Cells were stored on ice until use. 1-2 μl of DNA in dH ₂ O was added and mixed. The cell and DNA mixture was transferred to a 2 mm gap electro cuvette (Bio-Rad Laboratories, Richmond, California, USA) that had been previously chilled on ice. Cells were electroporated using Gene Pulser ^™ (Bio-Rad Laboratories) at 2 kV (10 kV / cm), 25 μF, 400 ohm settings. The cells were diluted with 0.75-1.0 ml CRM (0-4 ° C.), transferred to a 15 ml culture tube and incubated for 3 hours at 30 ° C. with agitation. Cells were plated on trypticase soy broth agar plates containing 10-30 μg / ml thiostrepton.

Example 13: High performance liquid chromatography
Using a Waters 2690 Separation Module system (Waters Corp., Milford, Mass., USA) and a 4.6 × 150 mm column (Waters Corp., Milford, Mass., USA) packed with SymmetryShield RP ₈ , particle size 3.5 μm. Liquid chromatography separation. Gradient mobile phase programming was used at a flow rate of 1.0 ml / min. Eluent A was water / acetonitrile (20: 1) +10 mM ammonium acetate. The eluent B was acetonitrile / water (20: 1). The mobile phase was linearly ramped from 12% B to 28% B over 6 minutes and isocratic at 28% B for 4 minutes. This was followed by a linear gradient from 28% B to 100% B over 20 minutes and 12% B over 2 minutes and held at 12% B for 3 minutes.

Example 14: Mass spectrophotometric column eluate was introduced directly into the electrospray ion source of a ZMD mass spectrometer (Micromass, Manchester, UK). The instrument is calibrated using the Test Juice reference standard (Waters Corp., Milford, Massachusetts, USA) and 10 μl / min from a syringe pump (Harvard Apparatus, Holliston, Massachusetts, USA). Delivered at a flow rate. The mass spectrometer was operated with a low mass analysis of 13.2 and a high mass analysis of 11.2. Spectra were captured using a scan range of m / z 100-600 with a capture rate of 10 spectra / second. The ionization method used was positive ion electrospray (ES). The sprayer voltage was held at 2900V and the cone voltage of the ion source was held at a potential of 17V.

Example 15: Use of ebh gene sequence (SEQ ID NO: 1) for isolating cytochrome P450 gene from other microorganisms A set of culture solutions (ATCC 43491, ATCC 14930, ATCC 53630, ATCC 53550, ATCC 39444, genomic DNA using DNAzol reagent ) Isolated from ATCC 43333, ATCC 35165). This DNA was used as a template in a PCR reaction using primers designed for the sequence of the ebh gene. Three sets of primers were used for amplification: NPB29-6f (SEQ ID NO: 28) and NPB29-7r (SEQ ID NO: 29), NPB29-16f (SEQ ID NO: 50) and NPB29-17r (SEQ ID NO: 51), and NPB29-19f (SEQ ID NO: 52) and NPB29-20r (SEQ ID NO: 53).

A PCR reaction solution containing 200-500 ng of genomic DNA and forward and reverse primers was prepared in a volume of 20 μl. All primers were added at a final concentration of 1.4-2.0 μM. The PCR reaction used was 0.2 μl Advantage ^™ 2 Taq enzyme (BD Biosciences Clontech, Palo Alto, Calif., USA), 2 μl Advantage ^™ 2 Taq buffer and 0.2 μl 2.5 mM dNTP, 20 μl dH ₂ O. To prepare. Cycle reaction, Geneamp ^R 9700PCR system or Mastercycler ^R gradient (Eppendorf, Westbury, NY, USA) at was carried out according to the following protocol: 5 min at 95 ° C., 35 cycles (96 ° C. at 20 seconds, 54 to 69 ° C for 30 seconds, 72 ° C for 2 minutes), 72 ° C for 7 minutes. The expected size of the PCR product is approximately 1469 bp for the NPB29-6f (SEQ ID NO: 28) and NPB29-7r (SEQ ID NO: 29) primer pair, NPB29-16f (SEQ ID NO: 50) and NPB29-17r (SEQ ID NO: 29). The primer pair with No. 51) is 1034 bp, and the primer pair with NPB29-19f (SEQ ID No. 52) and NPB29-20r (SEQ ID No. 53) is 1318 bp. The PCR reaction was analyzed on a 0.7% agarose gel. The expected size PCR product was excised from the gel and purified using the Qiagen gel extraction method. The purified product was sequenced using the Big-Dye sequencing kit and analyzed using the ABI310 sequencer.

Example 16: Construction of plasmid pPCRscript-ebh A 1.469 kb DNA fragment containing the epothilone B hydroxylase gene and the downstream ferredoxin gene was amplified using PCR. 50 μl PCR reaction was 5 μl Taq buffer, 2.5 μl glycerin, 1 μl 20 ng / μl NPB29-1 plasmid, 0.4 μl 25 mM dNTP, 1.0 μl each primer NPB29-6f (SEQ ID NO: 28) And NPB29-7r (SEQ ID NO: 29) (5 pmol / μl), 38.1 μl dH ₂ O and 0.5 μl Taq enzyme (Stratagene). The reaction was carried out under the following conditions in the Geneamp ^R 9700PCR system: 95 ° C. for 5 minutes, then 30 cycles (96 for 30 seconds ° C., 30 sec at 60 ° C., 2 minutes at 72 ° C.), and 72 ° C. for 7 min. The PCR product was purified using a Qiagen Qiaquick column with the PCR cleanup method. The purified product was digested with BglII and HindIII and purified on a 0.7% agarose gel. A 1.469 kb band was excised from the gel and eluted using the Qiagen Qiaquick gel extraction method. These fragments were then cloned into the pPCRscript Amp vector using the PCRscript Amp cloning kit. Colonies containing the insert were picked in 1-2 ml LB (Luria Broth) containing 100 μg / ml ampicillin for 16-24 hours at 37 ° C. and 230-300 rpm. Plasmid isolation was performed using the Mo Bio mini plasmid prep kit. The sequence of the insert was confirmed by cycle sequencing using the Big-Dye sequencing kit. This plasmid was called pPCRscript-ebh.

Example 17: Mutagenesis of the ebh gene for improved yield or altered specificity
Using Quikchange ^R XL Site-Directed Mutagenesis Kit and Quikchange ^R Multi Site-Directed Mutagenesis Kit (both from Stratagene), mutations were introduced into the coding region of ebh gene. Both methods are 35 to 45 bases in length of a DNA primer (SEQ ID NO 54 to 59 and 77) containing the desired mutation, methylated cyclic plasmid template and PfuTurbo ^R DNA polymerase (U.S. Patent No. 5,545, 552 and 5,866,395 and 5,948,663) were used to generate a copy of the plasmid template with the mutation introduced by the mutagenic primer. Subsequent digestion of the reaction with the restriction endonuclease enzyme DpnI selectively digests the methylated plasmid template, but leaves the unmethylated mutant plasmid intact. Except for the DpnI digestion step (incubation time was increased from 1 to 3 hours), all procedures followed the manufacturer's instructions. The pPCRscript-ebh vector was used as a mutagenesis template.

1-2 μl of the reaction was transformed into either XL1-Blue ^R electrocompetent cells or XL10-Gold ^R ultracompetent cells (Stratagene). Cells were plated on LA (Luria Agar) 100 μg / ml ampicillin plates at a density of over 100 colonies per plate and incubated at 30-37 ° C. for 24-48 hours. The entire plate was resuspended in 5 ml LB containing 100 μg / ml ampicillin. The plasmid was isolated directly from the resuspended cells by centrifuging the cells and then purifying the plasmid using the Mo Bio miniprep method. The mutated expression cassette was then amplified using this plasmid as a template for PCR using primers NPB29-6f (SEQ ID NO: 28) and NPB29-7r (SEQ ID NO: 29). Digestion of the 1.469 kb PCR product with restriction enzymes BglII and HindIII was used to prepare a fragment for ligation into the vector pANT849, also digested with BglII and HindIII. Alternatively, resuspended cells were used to inoculate 20-50 ml LB containing 100 μg / ml ampicillin and grown at 30-37 ° C. for 18-24 hours. Qiagen miniprep was performed on the culture to isolate plasmid DNA containing the desired mutation. Digestion with restriction enzymes BglII and HindIII was used to excise the mutated expression cassette for ligation into BglII and HindIII digested plasmid pANT849. Mutants were screened with Streptomyces lividans or Streptomyces limos as described.

Alternatively, a random mutation library of the ebh gene was generated using the method of Leung et al. (Technique -A Journal of Methods in Cell and Molecular Biology , Vol. 1, No. 1, 11-15 (1989)). . Manganese and / or reduced dATP concentrations are used to control the frequency of Taq polymerase mutagenesis. Plasmid pCRscript-ebh was digested with NotI to linearize the plasmid. The polymerase buffer was 0.166 M (NH ₄ ) ₂ SO ₄ , 0.67 M Tris-HCl (pH 8.8), 61 mM MgCl ₂ , 67 μM EDTA (pH 8.0), 1.7 mg / ml bovine serum albumin. Prepared. The PCR reaction was mixed with 10 μl NotI digested pCRscript-ebh (0.1 ng / μl), 10 μl polymerase buffer, 1.0 μl 1M β-mercaptoethanol, 10.0 μl DMSO, 1.0 μl NPB29-6f (SEQ ID NO: 28) Primer (100 pmol / μl), 1.0 μl NPB29-7r (SEQ ID NO: 29) Primer (100 pmol / μl), 10 μl 5 mM MnCl ₂ , 10.0 μl 10 mM dGTP, 10.0 μl Prepared using 2 mM dATP, 10 mM dTTP, 10.0 μl 10 mM dCTP, and 2.0 μl Taq polymerase. dH ₂ O was added to make 100 μl. The reaction solution was prepared in the same manner as above, but no MnCl ₂ was used. Was performed in the following protocol cycling reaction in GeneAmp ^R PCR System: 1 minute at 95 ° C., 25 to 30 cycles (1 minute at 94 ° C., 30 seconds at 55 ° C., 4 min at 72 ° C.), 7 at 72 ° C. For minutes. The PCR reaction was separated on an agarose gel using a Qiagen spin column. The resulting fragment was then digested with BglII and HindIII and purified using a Qiagen spin column. The purified fragment was then ligated into the pANT849 plasmid digested with BglII and HindIII. Mutant screening was performed at Streptomyces lividans and Streptomyces limos.

List of characterized mutants

Example 18: Comparison of Epothilone B Transformation in Cells Expressing ebh and its Mutants In these experiments, 20 ml of YEME medium in 125 ml both well flasks were added to Streptomyces lividans TK24, Streptomyces lividans (pANT849). -Ebh), 200 μl of frozen spore preparation of Streptomyces lividans (pANT849-ebh10-53) or Streptomyces lividans (pANT849-ebh24-16) was inoculated and incubated at 230 rpm, 30 ° C. for 48 hours. Thiostrepton 10 μg / ml was added to the medium inoculated with Streptomyces lividans (pANT849-ebh), Streptomyces lividans (pANT849-ebh10-53) and Streptomyces lividans (pANT849-ebh24-16). 4 ml of the culture was transferred to 20 ml of R5 medium in a 125 ml Erlenmeyer flask and incubated at 230 rpm and 30 ° C. for 18 hours. Epothilone B in 100% EtOH was added to each culture at a final concentration of 0.05% w / v. Except for the culture of Streptomyces lividans (pANT849-ebh24-16), where epothilone B was completely converted to epothilone F in 48 hours, samples were collected at 0, 24, 48 and 72 hours. Collected at Samples were analyzed by HPLC. Results were calculated as percent of epothilone B at 0 hours.

Epothilone B:

Epothilone F:

  Alternatively, bioconversion of epothilone B to epothilone F was performed in Streptomyces limos host cells transformed with an expression plasmid containing the ebh gene and variants or mutants thereof. 100 μl of frozen Streptomyces limosus transformant culture was inoculated into 20 ml of CRM medium containing 10 μg / ml thiostrepton and cultured at 30 ° C. and 230-300 rpm for 16-24 hours. Epothilone B in 100% ethanol was added to each culture at a final concentration of 0.05% w / v. The reaction was typically incubated at 30 ° C. and 230-300 rpm for 20-40 hours. The concentrations of epothilone B and epothilone F were determined by HPLC analysis.

Evaluation of mutants in Streptomyces limos

Example 19: Biotransformation of compactin to pravastatin 20 ml R2YE medium containing 10 μg / ml thiostrepton in a 125 ml flask was added to Streptomyces lividans (pANT849), Streptomyces lividans (pANT849-ebh). Frozen spore preparation (200 μl) was inoculated and incubated at 230 rpm, 28 ° C. for 72 hours. 4 ml of the culture was inoculated into 20 ml of R2YE medium and grown at 230 rpm, 28 ° C. for 24 hours. 1 ml of the culture solution was transferred to a 15 ml polypropylene tube, 10 μl of compactin (40 mg / ml) was added to each culture solution, and incubated at 28 ° C. and 250 rpm for 24 hours. 500 μl of broth broth was transferred to a new 15 ml polypropylene culture tube. 500 μl of 50 mM sodium hydroxide was added and vortexed. 3 ml of methanol was added and vortexed, and the tube was centrifuged at 3000 rpm for 10 minutes in a TJ-6 tabletop centrifuge tube. The organic phase was analyzed by HPLC. The compactin and pravastatin values were evaluated compared to the control Streptomyces lividans (pANT849) culture.

Compactin and pravastatin as a percentage of the starting material compactin concentration:

Example 20: High performance liquid chromatography method for detection of compactin and pravastatin
Liquid chromatography using a Hewlett Packard 1090 Series Separation system (Agilent Technologies, Palo Alto, Calif., USA) and a 50 × 46 mm column (Keystone Scientific, Inc., Belfonte, Pennsylvania, USA) packed with Spherisorb ODS2, 5 μm particle size. Separation was performed. Gradient mobile phase programming was used at a flow rate of 2.0 ml / min. Eluent A was water, 10 mM ammonium acetate and 0.05% phosphoric acid. The eluent B was acetonitrile. The mobile phase was linearly gradient from 20% B to 90% B over 4 minutes.

Example 21: Structure determination of biotransformation product of mutant ebh25-1
Analytical HPLC was performed using a Hewlett Packard 1100 Series Liquid chromatograph with a YMC packed ODS-AQ column, 4.6 mm ID x 15 cm length. A gradient system of water (solvent A) and acetonitrile (solvent B) was used: a linear gradient from 20% to 90% B for 10 minutes; a linear gradient from 90% to 20% for 2 minutes. The flow rate was 1 ml / min and UV detection was performed at 254 nm.

Preparative HPLC was performed using the following equipment and conditions:
Pump: Varian ProStar Solvent Delivery Module (Varian Inc., Palo Alto, California, USA). Detector: Gynkotek UVD340S
Column: YMC ODS-A column (30 mm inner diameter × 100 mm length, 5 μ particle size)
Elution flow rate: 30 ml / min Elution gradient: (solvent A: water, solvent B: acetonitrile), 20% B, 2 minutes; linear gradient from 20% to 60% B, 18 minutes; 60% B, 2 minutes Linear gradient from 60% to 90% B, 1 minute; 90% B, 3 minutes; linear gradient from 90% to 20% B, 2 minutes detection: UV, 210 nm

LC / NMR was performed as follows: 40 μl of sample was injected onto a YMC packed ODS-AQ column (4.6 mm inner diameter × 15 cm length). The column was eluted with a gradient system of D ₂ O (solvent A) and acetonitrile-d ₃ (solvent B) at a flow rate of 1 ml / min: 30% B, 1 minute; linear from 30% to 80% B Gradient, 11 minutes. The eluate was passed through a UV detection cell (monitored at 254 nm) before flowing through a F19 / H1 NMR probe (60 μl active volume) in a Varian AS-600 NMR analyzer. Since the biotransformation product eluted at approximately 7.5 minutes, the elution flow was manually stopped so that the eluate remained in the NMR probe for NMR data capture.

Isolation and analysis were performed as follows. Butanol / methanol extract (about 10 ml) was evaporated to dryness under a stream of nitrogen. 1 ml of methanol was added to the residue (38 mg) and the insoluble material was removed by centrifugation (13000 rpm, 2 minutes). 0.1 ml of the supernatant was used for LC / NMR studies and the remaining 0.9 ml was subjected to preparative HPLC (0.2-0.4 ml / injection). Two major peaks were observed and collected: Peak A eluted at 14-15 minutes, while Peak B eluted at 16.5-17.5 minutes. Analytical HPLC analysis showed that peak B was the parent compound epothilone B (Rt 8.5 min) and peak A was a biotransformation product (Rt 7.3 min). Peak A fractions were pooled and MS analysis data was obtained on these pooled fractions. Pooled fractions were evaporated to a small volume and then lyophilized to give 3 mg of a white solid. NMR and HPLC analysis of this white solid (dissolved in methanol) revealed that the biotransformation product was partially decomposed during the drying process.

Appendix Table 1

FIG. 1 is shown in WO 00/39276 (US patent application Ser. No. 09 / 468,854 filed Dec. 21, 1999) to Epothilone F of Epothilone B by Amycolatopsis orientalis strain SC15847 (PTA1043). A schematic diagram of biotransformation is shown.

FIG. 2 shows the nucleic acid sequence alignment of SEQ ID NOs: 5-22 used to design PCR primers for cloning of nucleic acid sequences encoding epothilone B hydroxylase.

FIG. 3 shows a sequence alignment between epothilone B hydroxylase (SEQ ID NO: 2) and EryF (PDB code 1 JIN chain A; SEQ ID NO: 76). The asterisk indicates the same sequence, and the colon (:) indicates a similar residue.

FIG. 4 shows a homology model of epothilone B hydroxylase based on the sequence alignment with EryF shown in FIG.

FIG. 5 shows an energy plot of the epothilone B hydroxylase model (shown in dashed line) compared to EryF (PDB code 1 JIN chain A; shown in solid line). An average window size of 51 residues was used (ie, the energy at a residue position was calculated as the average of the energy of 51 residues in the sequence centered on the residue).

Claims (41)

  An isolated nucleic acid sequence encoding epothilone B hydroxylase or a mutant or variant thereof.   2. The isolated nucleic acid sequence of claim 1 comprising SEQ ID NO: 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 72 or 74. .   2. The isolated nucleic acid sequence of claim 1 comprising SEQ ID NO: 1.   The isolated nucleic acid sequence of claim 1, which encodes a mutant having at least one amino acid substitution in the active site of the epothilone B hydroxylase enzyme.   The invention encodes a mutant having at least one amino acid substitution at amino acids GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU231, SER294, PHE237, or ILE365 of SEQ ID NO: 2. 2. The isolated nucleic acid sequence according to 1.   Amino acids LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, L84 , PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174, ARG175, MET176, ER1767, ER1767 , LEU193, VAL233, GLY234 LEU235, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, THR287, THR287, THR287 ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352, CY35, LU35, CY3353, LU35 59, GLU362, LYS389, ASP391, SER392, THR393, ILE394, or encoding a mutant having at least one amino acid substitution in TYR395, isolated nucleic acid sequence of claim 1.   2. The isolated nucleic acid sequence of claim 1, which encodes a mutant comprising SEQ ID NO: 43, 44, 45, 46, 47, 48 or 49.   A polypeptide encoded by the isolated nucleic acid sequence of claim 1.   An isolated nucleic acid molecule capable of hybridizing to the nucleic acid sequence according to claim 2 or a complementary sequence of the nucleic acid sequence under hybridization conditions of 3 x SSC at 65 ° C for 16 hours, comprising: Alternatively, an isolated nucleic acid molecule which can remain hybridized to a complementary sequence of the nucleic acid sequence under washing conditions of 0.5 × SSC at 55 ° C. for 30 minutes.   An isolated polypeptide comprising SEQ ID NO: 2.   An isolated mutant polypeptide of the epothilone B hydroxylase of SEQ ID NO: 2 comprising an amino acid sequence having at least one amino acid substitution in the active site of the epothilone B hydroxylase enzyme of SEQ ID NO: 2.   Comprising the amino acid sequence having at least one amino acid substitution in amino acid GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU231, SER294, PHE237, or ILE365 of SEQ ID NO: 2. An isolated mutant polypeptide of epothilone B hydroxylase.   Amino acids LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, L84 , PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174, ARG175, MET176, ER1767, ER1767 , LEU193, VAL233, GLY234 LEU235, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, THR287, THR287, THR287 ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352, CY35, LU35, CY3353, LU35 59, GLU362, LYS389, ASP391, SER392, THR393, ILE394 or TYR395 comprising an amino acid sequence having at least one amino acid substitution in, the isolated mutant polypeptide of epothilone B hydroxylase of SEQ ID NO: 2,.   An isolated mutant polypeptide of an epothilone B hydroxylase comprising SEQ ID NO: 31, 33, 35, 61, 63, 65, 67, 69, 71, 73 or 75.   An isolated variant polypeptide of an epothilone B hydroxylase comprising SEQ ID NO: 43, 44, 45, 46, 47, 48 or 49.   An isolated nucleic acid sequence encoding ferredoxin.   The isolated nucleic acid sequence of claim 16 comprising SEQ ID NO: 3.   A polypeptide encoded by the isolated nucleic acid sequence of claim 16.   An isolated nucleic acid molecule capable of hybridizing to the nucleic acid sequence shown in SEQ ID NO: 3 or a complementary sequence of the nucleic acid sequence shown in SEQ ID NO: 3 under hybridization conditions of 3 × SSC at 65 ° C. for 16 hours, The nucleic acid sequence shown in SEQ ID NO: 3 or a complementary sequence of the nucleic acid sequence shown in SEQ ID NO: 3 can remain hybridized under washing conditions of 0.5 × SSC at 55 ° C. for 30 minutes. Isolated nucleic acid molecule.   A vector comprising the isolated nucleic acid sequence of claim 1.   21. The vector of claim 20, further comprising an isolated nucleic acid sequence encoding ferredoxin.   A host cell comprising the vector of claim 20.   A host cell comprising the vector of claim 21.   A method for producing a recombinant microorganism that hydroxylates an epothilone having a terminal alkyl group to produce an epothilone having a terminal hydroxyalkyl group, the method comprising transfecting the microorganism with the vector of claim 20 or 21 .   A recombinant microorganism produced by hydroxylating an epothilone having a terminal alkyl group to produce an epothilone having a terminal hydroxyalkyl group.   26. The nucleic acid sequence of SEQ ID NO: 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 72 or 74 is expressed according to claim 25. Microorganisms produced by recombination. Formula I below:
_{HO-CH 2 - (A 1} ) n - (Q) m - (A 2) o -E (I)
Wherein A ₁ and A ₂ are each independently selected from the group consisting of optionally substituted C ₁ -C ₃ alkyl and alkenyl;
Q is an optionally substituted ring system comprising 1 to 3 rings and at least one carbon-carbon double bond in at least one ring;
n, m and o are integers selected from the group consisting of 0 and 1, wherein at least one of m or n or o is 1; and E is an epothilone core) A method for producing epothilone comprising the following formula II:
_{CH 3 - (A 1) n} - (Q) m - (A 2) o -E (II)
(Wherein A ₁ , Q, A ₂ , E, n, m, and o are as defined above) can selectively catalyze the hydroxylation of the compound of formula II. Contacting with a recombinantly produced microorganism or an enzyme from the microorganism, followed by the hydroxylation. Formula A:

A method of producing an epothilone analog represented by: comprising biotransforming epothilone B to an epothilone analog represented by formula A by incubating with a mutant epothilone B hydroxylase enzyme comprising SEQ ID NO: 31. Formula A:

Or a pharmacologically acceptable salt thereof.   A homology model of epothilone B hydroxylase, wherein the mean square deviation of conserved residue backbone atoms is less than about 4.0 Angstroms when superimposed on the corresponding backbone atoms described by the structural coordinates listed in Appendix Table 1 .   A method for producing a mutant having altered biological properties, function, yield of desired product, reaction rate, substrate specificity, or activity compared to epothilone B hydroxylase, the sequence to be mutated A method comprising identifying the amino acid in number 2 and then mutating the identified amino acid to produce a mutant protein.   A homology model of an epothilone B hydroxylase with a mean square deviation of conserved residue backbone atoms of less than about 4.0 angstroms when superimposed on the corresponding backbone atoms described by the structural coordinates listed in Appendix Table 1. 32. The method of claim 31, wherein the method is used to identify an amino acid in SEQ ID NO: 2 to be mutated.   The identified amino acids are LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GL80, GL80, ILESP, SEQ ID NO: 2. , LYS85, PRO86, PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174, ARG175R, T17, 17G , ARG186, PHE190, LEU193, VAL233, LY234, LEU235, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, E28 SER294, ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352EL35GL35E 8, ALA359, GLU362, LYS389, ASP391, SER392, THR393, ILE394, or TYR395, The method of claim 31.   32. The method of claim 31, wherein the identified amino acid is GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU231, SER294, PHE237, or ILE365 of SEQ ID NO: 2.   32. The method of claim 31, wherein the yield of the desired product of the mutant protein is improved compared to the yield of the desired product obtained using epothilone B hydroxylase.   36. The method of claim 35, wherein the desired product is epothilone F.   32. The method of claim 31, wherein the mutant has an improved reaction rate compared to a reaction rate obtained using epothilone B hydroxylase.   32. The method of claim 31, wherein the mutant exhibits altered substrate specificity compared to the substrate specificity of epothilone B hydroxylase.   40. The method of claim 38, wherein amino acid SER294 is mutated.   32. The method of claim 31, wherein the mutant exhibits biological activity or function essentially similar to epothilone B hydroxylase.   A machine-readable data storage medium comprising a data storage material encoded with the structural coordinates shown in Appendix 1.

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