Recombinant methods and materials for producing epothilone and epothilone derivatives

Abstract

Recombinant nucleic acids that encode all or a portion of the epothilone polyketide synthase (PKS) are used to express recombinant PKS genes in host cells for the production of epothilones, epothilone derivatives, and polyketides that are useful as cancer chemotherapeutics, fungicides, and immunosuppressants.

Description

FIELD OF THE INVENTION

[0003] The present invention provides recombinant methods and materials for producing epothilone and epothilone derivatives. The invention relates to the fields of agriculture, chemistry, medicinal chemistry, medicine, molecular biology, and pharmacology.

BACKGROUND OF THE INVENTION

[0004] The epothilones were first identified by Gerhard Hofle and colleagues at the National Biotechnology Research Institute as an antifungal activity extracted from the myxobacterium Sorangium cellulosum (see K. Gerth et al., 1996, J. Antibiotics 49: 560-563 and Germany Patent No. DE 41 38 042). The epothilones were later found to have activity in a tubulin polymerization assay (see D. Bollag et al., 1995, Cancer Res. 55:2325-2333) to identify antitumor agents and have since been extensively studied as potential antitumor agents for the treatment of cancer.

[0005] The chemical structure of the epothilones produced by Sorangium cellulosum strain So ce 90 was described in Hofle et al., 1996, Epothilone A and B—novel 16-membered macrolides with cytotoxic activity: isolation, crystal structure, and conformation in solution, Angew. Chem. Int. Ed. Engl. 35(13/14): 1567-1569, incorporated herein by reference. The strain was found to produce two epothilone compounds, designated A (R═H) and B (R═CH3), as shown below, which showed broad cytotoxic activity against eukaryotic cells and noticeable activity and selectivity against breast and colon tumor cell lines. 1

[0006] The desoxy counterparts of epothilones A and B, also known as epothilones C (R═H) and D (R═CH3), are known to be less cytotoxic, and the structures of these epothilones are shown below. 2

[0007] Two other naturally occurring epothilones have been described. These are epothilones E and F, in which the methyl side chain of the thiazole moiety of epothilones A and B has been hydroxylated to yield epothilones E and F, respectively.

[0008] Because of the potential for use of the epothilones as anticancer agents, and because of the low levels of epothilone produced by the native So ce 90 strain, a number of research teams undertook the effort to synthesize the epothilones. This effort has been successful (see Balog et al., 1996, Total synthesis of (−)-epothilone A, Angew. Chem. Int. Ed. Engl. 35(23/24): 2801-2803; Su et al., 1997, Total synthesis of (−)-epothilone B: an extension of the Suzuki coupling method and insights into structure-activity relationships of the epbthilones, Angew. Chem. Int. Ed. Engl. 36(7): 757-759; Meng et al., 1997, Total syntheses of epothilones A and B, JACS 119(42): 10073-10092; and Balog et al., 1998, A novel aldol condensation with 2-methyl-4-pentenal and its application to an improved total synthesis of epothilone B, Angew. Chem. Int. Ed. Engl. 37(19): 2675-2678, each of which is incorporated herein by reference). Despite the success of these efforts, the chemical synthesis of the epothilones is tedious, time-consuming, and expensive. Indeed, the methods have been characterized as impractical for the full-scale pharmaceutical development of an epothilone.

[0009] A number of epothilone derivatives, as well as epothilones A—D, have been studied in vitro and in vivo (see Su et al., 1997, Structure-activity relationships of the epothilones and the first in vivo comparison with paclitaxel, Angew. Chem. Int. Ed. Engl. 36(19): 2093-2096; and Chou et al., August 1998, Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B, Proc. Natl. Acad. Sci. USA 95: 9642-9647, each of which is incorporated herein by reference). Additional epothilone derivatives and methods for synthesizing epothilones and epothilone derivatives are described in PCT patent publication Nos. 99/54330, 99/54319, 99/54318, 99/43653, 99/43320, 99/42602, 99/40047, 99/27890, 99/07692, 99/02514, 99/01124,98/25929, 98/22461, 98/08849, and 97/19086; U.S. Pat. No. 5,969,145; and Germany patent publication No. DE 41 38 042, each of which is incorporated herein by reference.

[0010] There remains a need for economical means to produce not only the naturally occurring epothilones but also the derivatives or precursors thereof, as well as new epothilone derivatives with improved properties. There remains a need for a host cell that produces epothilones or epothilone derivatives that is easier to manipulate and ferment than the natural producer Sorangium cellulosum. The present invention meets these and other needs.

SUMMARY OF THE INVENTION

[0011] In one embodiment, the present invention provides recombinant DNA compounds that encode the proteins required to produce epothilones A, B, C, and D. The present invention also provides recombinant DNA compounds that encode portions of these proteins. The present invention also provides recombinant DNA compounds that encode a hybrid protein, which hybrid protein includes all or a portion of a protein involved in epothilone biosynthesis and all or a portion of a protein involved in the biosynthesis of another polyketide or non-ribosomal-derived peptide. In a preferred embodiment, the recombinant DNA compounds of the invention are recombinant DNA cloning vectors that facilitate manipulation of the coding sequences or recombinant DNA expression vectors that code for the expression of one or more of the proteins of the invention in recombinant host cells.

[0012] In another embodiment, the present invention provides recombinant host cells that produce a desired epothilone or epothilone derivative. In one embodiment, the invention provides host cells that produce one or more of the epothilones or epothilone derivatives at higher levels than produced in the naturally occurring organisms that produce epothilones. In another embodiment, the invention provides host cells that produce mixtures of epothilones that are less complex than the mixtures produced by naturally occurring host cells. In another embodiment, the present invention provides non-Sorangium recombinant host cells that produce an epothilone or epothilone derivative.

[0013] In a preferred embodiment, the host cells of the invention produce less complex mixtures of epothilones than do naturally occurring cells that produce epothilones. Naturally occurring cells that produce epothilones typically produce a mixture of epothilones A, B, C, D, E, and F. The table below summarizes the epothilones produced in different illustrative host cells of the invention.

Cell Type	Epothilones Produced	Epothilones Not Produced
1	A, B, C, D, E, F	—
2	A, C, E	B, D, F
3	B, D, F	A, C, E
4	A, B, C, D	E, F
5	A, C	B, D, E, F
6	C	A, B, D, E, F
7	B, D	A, C, E, F
8	D	A, B, C, E, F

[0014] In addition, cell types may be constructed which produce only the newly discovered epothilones G and H, further discussed below, and one or the other of G and H or both in combination with the downstream epothilones. Thus, it is understood, based on the present invention, that the biosynthetic pathway which relates the naturally occurring epothilones is, respectively, G→C→A→E and H→D→B→F. Appropriate enzymes may also convert members of each pathway to the corresponding member of the other.

[0015] Thus, the recombinant host cells of the invention also include host cells that produce only one desired epothilone or epothilone derivative.

[0016] In another embodiment, the invention provides Sorangium host cells that have been modified genetically to produce epothilones either at levels greater than those observed in naturally occurring host cells or as less complex mixtures of epothilones than produced by naturally occurring host cells, or produce an epothilone derivative that is not produced in nature. In a preferred embodiment, the host cell produces the epothilones at equal to or greater than 20 mg/L.

[0017] In another embodiment, the recombinant host cells of the invention are host cells other than Sorangium cellulosum that have been modified genetically to produce an epothilone or an epothilone derivative. In a preferred embodiment, the host cell produces the epothilones at equal to or greater than 20 mg/L. In a more preferred embodiment, the recombinant host cells are Myxococcus, Pseudomonas, or Streptomyces host cells that produce the epothilones or an epothilone derivative at equal to or greater than 20 mg/L.

[0018] In another embodiment, the present invention provides novel compounds useful in agriculture, veterinary practice, and medicine. In one embodiment, the compounds are useful as fungicides. In another embodiment, the compounds are useful in cancer chemotherapy. In a preferred embodiment, the compound is an epothilone derivative that is at least as potent against tumor cells as epothilone B or D. In another embodiment, the compounds are useful as immunosuppressants. In another embodiment, the compounds are useful in the manufacture of another compound. In a preferred embodiment, the compounds are formulated in a mixture or solution for administration to a human or animal.

[0019] These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.

BRIEF DESCRIPTION OF THE FIGURES

[0020] FIG. 1 shows a restriction site map of the insert Sorangium cellulosum genomic DNA in four overlapping cosmid clones (designated 8A3, 1A2, 4, and 85 and corresponding to pKOS35-70.8A3, pKOS35-70. 1A2, pKOS35-70.4, and pKOS35-79.85, respectively) spanning the epothilone gene cluster. A functional map of the epothilone gene cluster is also shown. The loading domain (Loading, epoA), the non-ribosomal peptide synthase (NRPS, Module 1, epoB) module, and each module (Modules 2 through 9, epoC, epoD, epoE, and epoF) of the remaining eight modules of the epothilone synthase gene are shown, as is the location of the epoK gene that encodes a cytochrome P450-like epoxidation enzyme.

[0021] FIG. 2 shows a number of precursor compounds to N-acylcysteamine thioester derivatives that can be supplied to an epothilone PKS of the invention in which the NRPS-like module 1 or module 2 KS domain has been inactivated to produce a novel epothilone derivative. A general synthetic procedure for making such compounds is also shown.

[0022] FIG. 3 shows restriction site and function maps of plasmids pKOS35-82.1 and pKOS35-82.2.

[0023] FIG. 4 shows restriction site and function maps of plasmids pKOS35-154 and pKOS90-22.

[0024] FIG. 5 shows a schematic of a protocol for introducing the epothilone PKS and modification enzyme genes into the chromosome of a Myxococcus xanthus host cell as described in Example 3.

[0025] FIG. 6 shows restriction site and function maps of plasmids pKOS039-124 and pKOS039-124R.

[0026] FIG. 7 shows a restriction site and function map of plasmid pKOS039-126R.

[0027] FIG. 8 shows a restriction site and function map of plasmid pKOS039-141.

[0028] FIG. 9 shows a restriction site and function map of plasmid pKOS045-12.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides the genes and proteins that synthesize the epothilones in Sorangium cellulosum in recombinant and isolated form. As used herein, the term recombinant refers to a compound or composition produced by human intervention, typically by specific and directed manipulation of a gene or portion thereof. The term isolated refers to a compound or composition in a preparation that is substantially free of contaminating or undesired materials or, with respect to a compound or composition found in nature, substantially free of the materials with which that compound or composition is associated in its natural state. The epothilones (epothilone A, B, C, D, E, and F) and compounds structurally related thereto (epothilone derivatives) are potent cytotoxic agents specific for eukaryotic cells. These compounds have application as anti-fungals, cancer chemotherapeutics, and immunosuppressants. The epothilones are produced at very low levels in the naturally occurring Sorangium cellulosum cells in which they have been identified. Moreover, S. cellulosum is very slow growing, and fermentation of S. cellulosum strains is difficult and time-consuming. One important benefit conferred by the present invention is the ability simply to produce an epothilone or epothilone derivative in a non-S. cellulosum host cell. Another advantage of the present invention is the ability to produce the epothilones at higher levels and in greater amounts in the recombinant host cells provided by the invention than possible in the naturally occurring epothilone producer cells. Yet another advantage is the ability to produce an epothilone derivative in a recombinant host cell.

[0030] The isolation of recombinant DNA encoding the epothilone biosynthetic genes resulted from the probing of a genomic library of Sorangium cellulosum SMP44 DNA. As described more fully in Example 1 below, the library was prepared by partially digesting S. cellulosum genomic DNA with restriction enzyme SauIIIA1 and inserting the DNA fragments generated into BamHI-digested Supercos™ cosmid DNA (Stratagene). Cosmid clones containing epothilone gene sequences were identified by probing with DNA probes specific for sequences from PKS genes and reprobing with secondary probes comprising nucleotide sequences identified with the primary probes.

[0031] Four overlapping cosmid clones were identified by this effort. These four cosmids were deposited with the American Type Culture Collection (ATCC), Manassas, Va., USA, under the terms of the Budapest Treaty, and assigned ATCC accession numbers. The clones (and accession numbers) were designated as cosmids pKOS35-70.1A2 (ATCC 203782), pKOS35-70.4 (ATCC 203781), pKOS35-70.8A3 (ATCC 203783), and pKOS35-79.85 (ATCC 203780). The cosmids contain insert DNA that completely spans the epothilone gene cluster. A restriction site map of these cosmids is shown in FIG. 1. FIG. 1 also provides a function map of the epothilone gene cluster, showing the location of the six epothilone PKS genes and the epoK P450 epoxidase gene.

[0032] The epothilone PKS genes, like other PKS genes, are composed of coding sequences organized to encode a loading domain, a number of modules, and a thioesterase domain. As described more fully below, each of these domains and modules corresponds to a polypeptide with one or more specific functions. Generally, the loading domain is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first module. The building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, methylmalonyl, and ethylmalonyl CoA. Other building blocks include amino acid-like acylthioesters. PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks. Each module is responsible for binding a building block, performing one or more functions on that building block, and transferring the resulting compound to the next module. The next module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete. At that point, an enzymatic thioesterase (TE) activity cleaves the polyketide from the PKS.

[0033] Such modular organization is characteristic of the class of PKS enzymes that synthesize complex polyketides and is well known in the art. Recombinant methods for manipulating modular PKS genes are described in U.S. Pat. Nos. 5,672,491; 5,712,146; 5,830,750; and 5,843,718; and in PCT patent publication Nos. 98/49315 and 97/02358, each of which is incorporated herein by reference. The polyketide known as 6-deoxyerythronolide B (6-dEB) is synthesized by a PKS that is a prototypical modular PKS enzyme. The genes, known as eryAI, eryAII, and eryAIII, that code for the multi-subunit protein known as deoxyerythronolide B synthase or DEBS (each subunit is known as DEBS1, DEBS2, or DEBS3) that synthesizes 6-dEB are described in U.S. Pat. Nos. 5,712,146 and 5,824,513, incorporated herein by reference.

[0034] The loading domain of the DEBS PKS consists of an acyltransferase (AT) and an acyl carrier protein (ACP). The AT of the DEBS loading domain recognizes propionyl CoA (other loading domain ATs can recognize other acyl-CoAs, such as acetyl, malonyl, methylmalonyl, or butyryl CoA) and transfers it as a thioester to the ACP of the loading domain. Concurrently, the AT on each of the six extender modules recognizes a methylmalonyl CoA (other extender module ATs can recognize other CoAs, such as malonyl or alpha-substituted malonyl CoAs, i.e., malonyl, ethylmalonyl, and 2-hydroxymalonyl CoA) and transfers it to the ACP of that module to form a thioester. Once DEBS is primed with acyl- and methylmalonyl-ACPs, the acyl group of the loading domain migrates to form a thioester (trans-esterification) at the KS of the first module; at this stage, module one possesses an acyl-KS adjacent to a methylmalonyl ACP. The acyl group derived from the DEBS loading domain is then covalently attached to the alpha-carbon of the extender group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension). The growing polyketide chain is transferred from the ACP to the KS of the next module of DEBS, and the process continues.

[0035] The polyketide chain, growing by two carbons for each module of DEBS, is sequentially passed as a covalently bound thioester from module to module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Commonly, however, additional enzymatic activities modify the beta keto group of each two carbon unit just after it has been added to the growing polyketide chain but before it is transferred to the next module. Thus, in addition to the minimal module containing KS, AT, and ACP necessary to form the carbon-carbon bond, modules may contain a ketoreductase (KR) that reduces the keto group to an alcohol. Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond. Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function. The DEBS modules include those with only a KR domain, only an inactive KR domain, and with all three KR, DH, and ER domains.

[0036] Once a polyketide chain traverses the final module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs. Here, the polyketide is cleaved from the enzyme and, for most but not all polyketides, cyclized. The polyketide can be modified further by tailoring or modification enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, i.e., oxidation or reduction, on the polyketide core molecule. For example, 6-dEB is hydroxylated, methylated, and glycosylated (glycosidated) to yield the well known antibiotic erythromycin A in the Saccharopolyspora erythraea cells in which it is produced naturally.

[0037] While the above description applies generally to modular PKS enzymes and specifically to DEBS, there are a number of variations that exist in nature. For example, many PKS enzymes comprise loading domains that, unlike the loading domain of DEBS, comprise an “inactive” KS domain that functions as a decarboxylase. This inactive KS is in most instances called KSQ, where the superscript is the single-letter abbreviation for the amino acid (glutamine) that is present instead of the active site cysteine required for ketosynthase activity. The epothilone PKS loading domain contains a KSY domain not present in other PKS enzymes for which amino acid sequence is currently available in which the amino acid tyrosine has replaced the cysteine. The present invention provides recombinant DNA coding sequences for this novel KS domain.

[0038] Another important variation in PKS enzymes relates to the type of building block incorporated. Some polyketides, including epothilone, incorporate an amino acid derived building block. PKS enzymes that make such polyketides require specialized modules for incorporation. Such modules are called non-ribosomal peptide synthetase (NRPS) modules. The epothilone PKS, for example, contains an NRPS module. Another example of a variation relates to additional activities in a module. For example, one module of the epothilone PKS contains a methyltransferase (MT) domain, a heretofore unknown domain of PKS enzymes that make modular polyketides.

[0039] The complete nucleotide sequence of the coding sequence of the open reading frames (ORFs) of the epothilone PKS genes and epothilone tailoring (modification) enzyme genes is provided in Example 1, below. This sequence information together with the information provided below regarding the locations of the open reading frames of the genes within that sequence provides the amino acid sequence of the encoded proteins. Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the invention. The native DNA sequence encoding the epothilone PKS and epothilone modification enzymes of Sorangium cellulosum is shown herein merely to illustrate a preferred embodiment of the invention. The present invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity and, in some instances, even an improvement of a desired activity. The present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences shown merely illustrate preferred embodiments of the invention.

[0040] The present invention provides recombinant genes for the production of epothilones. The invention is exemplified by the cloning, characterization, and manipulation of the epothilone PKS and modification enzymes of Sorangium cellulosum SMP44. The description of the invention and the recombinant vectors deposited in connection with that description enable the identification, cloning, and manipulation of epothilone PKS and modification enzymes from any naturally occurring host cell that produces an epothilone. Such host cells include other S. cellulosum strains, such as So ce 90, other Sorangium species, and non-Sorangium cells. Such identification, cloning, and characterization can be conducted by those of ordinary skill in accordance with the present invention using standard methodology for identifying homologous DNA sequences and for identifying genes that encode a protein of function similar to a known protein. Moreover, the present invention provides recombinant epothilone PKS and modification enzyme genes that are synthesized de novo or are assembled from non-epothilone PKS genes to provide an ordered array of domains and modules in one or more proteins that assemble to form a PKS that produces epothilone or an epothilone derivative.

[0041] The recombinant nucleic acids, proteins, and peptides of the invention are many and diverse. To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following discussion describes various regions of the epothilone PKS and corresponding coding sequences. This discussion begins with a general discussion of the genes that encode the PKS, the location of the various domains and modules in those genes, and the location of the various domains in those modules. Then, a more detailed discussion follows, focusing first on the loading domain, followed by the NRPS module, and then the remaining eight modules of the epothilone PKS.

[0042] There are six epothilone PKS genes. The epoA gene encodes the 149 kDa loading domain (which can also be referred to as a loading module). The epoB gene encodes module 1, the 158 kDa NRPS module. The epoC gene encodes the 193 kDa module 2. The epoD gene encodes a 765 kDa protein that comprises modules 3 through 6, inclusive. The epoE gene encodes a 405 kDa protein that comprises modules 7 and 8. The epoF gene encodes a 257 kDa protein that comprises module 9 and the thioesterase domain. Immediately downstream of the epoF gene is epoK, the P450 epoxidase gene which encodes a 47 kDa protein, followed immediately by the epoL gene, which may encode a 24 kDa dehydratase. The epoL gene is followed by a number of ORFs that include genes believed to encode proteins involved in transport and regulation.

[0043] The sequences of these genes are shown in Example 1 in one contiguous sequence or contig of 71,989 nucleotides (SEQ ID NO: 2). This contig also contains two genes that appear to originate from a transposon and are identified below as ORF A and ORF B. These two genes are believe not to be involved in epothilone biosynthesis but could possibly contain sequences that function as a promoter or enhancer. The contig also contains more than 12 additional ORFs, only 12 of which, designated ORF2 through ORF12 and ORF2 complement, are identified below. As noted, ORF2 actually is two ORFs, because the complement of the strand shown also comprises an ORF. The function of the corresponding gene product, if any, of these ORFs has not yet been established. Table below provides the location of various open reading frames, module-coding sequences, and domain encoding sequences within the contig sequence shown in Example 1. Those of skill in the art will recognize, upon consideration of the sequence shown in Example 1, that the actual start locations of several of the genes could differ from the start locations shown in the table, because of the presence in frame codons for methionine or valine in close proximity to the codon indicated as the start codon. The actual start codon can be confirmed by amino acid sequencing of the proteins expressed from the genes.

Start	Stop	Comment
3	992	transposase gene ORF A, not part of the PKS
989	1501	transposase gene ORF B, not part of the PKS
1998	6263	epoA gene, encodes the loading domain
2031	3548	KSY of the loading domain
3621	4661	AT of the loading domain
4917	5810	ER of the loading domain, potentially involved in
		formation of the thiazole moiety
5856	6155	ACP of the loading domain
6260	10493	epoB gene, encodes module 1, the NRPS module
6620	6649	condensation domain C2 of the NRPS module
6861	6887	heterocyclization signature sequence
6962	6982	condensation domain C4 of the NRPS module
7358	7366	condensation domain C7 (partial) of the NRPS
		module
7898	7921	adenylation domain A1 of the NRPS module
8261	8308	adenylation domain A3 of the NRPS module
8411	8422	adenylation domain A4 of the NRPS module
8861	8905	adenylation domain A6 of the NRPS module
8966	8983	adenylation domain A7 of the NRPS module
9090	9179	adenylation domain A8 of the NRPS module
9183	9992	oxidation region for forming thiazole
10121	10138	Adenylation domain A10 of the NRPS module
10261	10306	Thiolation domain (PCP) of the NRPS module
10639	16137	epoC gene, encodes module 2
10654	12033	KS2, the KS domain of module 2
12250	13287	AT2, the AT domain of module 2
13327	13899	DH2, the DH domain of module 2
14962	15756	KR2, the KR domain of module 2
15763	16008	ACP2, the ACP domain of module 2
16134	37907	epoD gene, encodes modules 3-6
16425	17606	KS3
17817	18857	AT3
19581	20396	KR3
20424	20642	ACP3
20706	22082	KS4
22296	23336	AT4
24069	24647	KR4
24867	25151	ACP4
25203	26576	KS5
26793	27833	AT5
27966	28574	DH5
29433	30287	ER5
30321	30869	KR5
31077	31373	ACP5
31440	32807	KS6
33018	34067	AT6
34107	34676	DH6
35760	36641	ER6
36705	37256	KR6
37470	37769	ACP6
37912	49308	epoE gene, encodes modules 7 and 8
38014	39375	KS7
39589	40626	AT7
41341	41922	KR7
42181	42423	ACP7
42478	43851	KS8
44065	45102	AT8
45262	45810	DH (inactive)
46072	47172	MT8, the methyltransferase domain of module 8
48103	48636	KR8, this domain is inactive
48850	49149	ACP8
49323	56642	epoF gene, encodes module 9 and the TE domain
49416	50774	KS9
50985	52025	AT9
52173	53414	DH (inactive)
54747	55313	KR9
55593	55805	ACP9
55878	56600	TE9, the thioesterase domain
56757	58016	epoK gene, encodes the P450 epoxidase
58194	58733	epoL gene (putative dehydratase)
59405	59974	ORF2 complement, complement of strand shown
59460	60249	ORF2
60271	60738	ORF3, complement of strand shown
61730	62647	ORF4 (putative transporter)
63725	64333	ORF5
64372	65643	ORF6
66237	67472	ORF7 (putative oxidoreductase)
67572	68837	ORF8 (putative oxidoreductase membrane subunit)
68837	69373	ORF9
69993	71174	ORF10 (putative transporter)
71171	71542	ORF11
71557	71989	ORF12

[0044] With this overview of the organization and sequence of the epothilone gene cluster, one can better appreciate the many different recombinant DNA compounds provided by the present invention.

[0045] The epothilone PKS is multiprotein complex composed of the gene products of the epoA, epoB, epoC, epoD, epoE, and epoF genes. To confer the ability to produce epothilones to a host cell, one provides the host cell with the recombinant epoA, epoB, epoC, epoD, epoE, and epoF genes of the present invention, and optionally other genes, capable of expression in that host cell. Those of skill in the art will appreciate that, while the epothilone and other PKS enzymes may be referred to as a single entity herein, these enzymes are typically multisubunit proteins. Thus, one can make a derivative PKS (a PKS that differs from a naturally occurring PKS by deletion or mutation) or hybrid PKS (a PKS that is composed of portions of two different PKS enzymes) by altering one or more genes that encode one or more of the multiple proteins that constitute the PKS.

[0046] The post-PKS modification or tailoring of epothilone includes multiple steps mediated by multiple enzymes. These enzymes are referred to herein as tailoring or modification enzymes. Surprisingly, the products of the domains of the epothilone PKS predicted to be functional by analysis of the genes that encode them are compounds that have not been previously reported. These compounds are referred to herein as epothilones G and H. Epothilones G and H lack the C-12-C-13 π-bond of epothilones C and D and the C-12-C-13 epoxide of epothilones A and B, having instead a hydrogen and hydroxyl group at C-13, a single bond between C-12 and C-13, and a hydrogen and H or methyl group at C-12. These compounds are predicted to result from the epothilone PKS, because the DNA and corresponding amino acid sequence for module 4 of the epothilone PKS does not appear to include a DH domain.

[0047] As described below, however, expression of the epothilone PKS genes epoA, epoB, epoC, epoD, epoE, and epoF in certain heterologous host cells that do not express epoK or epoL leads to the production of epothilones C and D, which lack the C-13 hydroxyl and have a double bond between C-12 and C-13. The dehydration reaction that mediates the formation of this double bond may be due to the action of an as yet unrecognized domain of the epothilone PKS (for example, dehydration could occur in the next module, which possesses an active DH domain and could generate a conjugated diene precursor prior to its dehydrogenation by an ER domain) or an endogenous enzyme in the heterologous host cells (Streptomyces coelicolor) in which it was observed. In the latter event, epothilones G and H may be produced in Sorangium cellulosum or other host cells and, to be converted to epothilones C and D, by the action of a dehydratase, which may be encoded by the epoL gene. In any event, epothilones C and D are converted to epothilones A and B by an epoxidase encoded by the epoK gene. Epothilones A and B are converted to epothilones E and F by a hydroxylase gene, which may be encoded by one of the ORFs identified above or by another gene endogenous to Sorangium cellulosum. Thus, one can produce an epothilone or epothilone derivative modified as desired in a host cell by providing that host cell with one or more of the recombinant modification enzyme genes provided by the invention or by utilizing a host cell that naturally expresses (or does not express) the modification enzyme. Thus, in general, by utilizing the appropriate host and by appropriate inactivation, if desired, of modification enzymes, one may interrupt the progression of G→C→A→E or the corresponding downstream processing of epothilone H at any desired point; by controlling methylation, one or both of the pathways can be selected.

[0048] Thus, the present invention provides a wide variety of recombinant DNA compounds and host cells for expressing the naturally occurring epothilones A, B, C, and D and derivatives thereof. The invention also provides recombinant host cells, particularly Sorangium cellulosum host cells that produce epothilone derivatives modified in a manner similar to epothilones E and F. Moreover, the invention provides host cells that can produce the heretofore unknown epothilones G and H, either by expression of the epothilone PKS genes in host cells that do not express the dehydratase that converts epothilones G and H to C and D or by mutating or altering the PKS to abolish the dehydratase function, if it is present in the epothilone PKS.

[0049] The macrolide compounds that are products of the PKS cluster can thus be modified in various ways. In addition to the modifications described above, the PKS products can be glycosylated, hydroxylated, dehydroxylated, oxidized, methylated and demethylated using appropriate enzymes. Thus, in addition to modifying the product of the PKS cluster by altering the number, functionality, or specificity of the modules contained in the PKS, additional compounds within the scope of the invention can be produced by additional enzyme-catalyzed activity either provided by a host cell in which the polyketide synthases are produced or by modifying these cells to contain additional enzymes or by additional in vitro modification using purified enzymes or crude extracts or, indeed, by chemical modification.

[0050] The present invention also provides a wide variety of recombinant DNA compounds and host cells that make epothilone derivatives. As used herein, the phrase “epothilone derivative” refers to a compound that is produced by a recombinant epothilone PKS in which at least one domain has been either rendered inactive, mutated to alter its catalytic function, or replaced by a domain with a different function or in which a domain has been inserted. In any event, the “epothilone derivative PKS” functions to produce a compound that differs in structure from a naturally occurring epothilone but retains its ring backbone structure and so is called an “epothilone derivative.” To faciliate a better understanding of the recombinant DNA compounds and host cells provided by the invention, a detailed discussion of the loading domain and each of the modules of the epothilone PKS, as well as novel recombinant derivatives thereof, is provided below.

[0051] The loading domain of the epothilone PKS includes an inactive KS domain, KSY, an AT domain specific for malonyl CoA (which is believed to be decarboxylated by the KSY domain to yield an acetyl group), and an ACP domain. The present invention provides recombinant DNA compounds that encode the epothilone loading domain. The loading domain coding sequence is contained within an ˜8.3 kb EcoRI restriction fragment of cosmid pKOS35-70.8A3. The KS domain is referred to as inactive, because the active site region “TAYSSSL”(SEQ ID NO: 20) of the KS domain of the loading domain has a Y residue in place of the cysteine required for ketosynthase activity; this domain does have decarboxylase activity. See Witkowski et al., 7 Sep. 1999, Biochem. 38(36): 11643-11650, incorporated herein by reference.

[0052] The presence of the Y residue in place of a Q residue (which occurs typically in an inactive loading domain KS) may make the KS domain less efficient at decarboxylation. The present invention provides a recombinant epothilone PKS loading domain and corresponding DNA sequences that encode an epothilone PKS loading domain in which the Y residue has been changed to a Q residue by changing the codon therefor in the coding sequence of the loading domain. The present invention also provides recombinant PKS enzymes comprising such loading domains and host cells for producing such enzymes and the polyketides produced thereby. These recombinant loading domains include those in which just the Y residue has been changed, those in which amino acids surrounding and including the Y domain have been changed, and those in which the complete KSY domain has been replaced by a complete KSQ domain. The latter embodiment includes but is not limited to a recombinant epothilone loading domain in which the KSY domain has been replaced by the KSQ domain of the oleandolide PKS or the narbonolide PKS (see the references cited below in connection with the oleandomycin, narbomycin, and picromycin PKS and modification enzymes).

[0053] The epothilone loading domain also contains an AT domain believed to bind malonyl CoA. The sequence “QTAFTQPALFTFEYALAALW . . . GHSIG”(SEQ ID NO: 1) in the AT domain is consistent with malonyl CoA specificity. As noted above, the malonyl CoA is believed to be decarboxylated by the KSY domain to yield acetyl CoA. The present invention provides recombinant epothilone derivative loading domains or their encoding DNA sequences in which the malonyl specific AT domain or its encoding sequence has been changed to another specificity, such as methylmalonyl CoA, ethylmalonyl CoA, and 2-hydroxymalonyl CoA. When expressed with the other proteins of the epothilone PKS, such loading domains lead to the production of epothilones in which the methyl substituent of the thiazole ring of epothilone is replaced with, respectively, ethyl, propyl, and hydroxymethyl. The present invention provides recombinant PKS enzymes comprising such loading domains and host cells for producing such enzymes and the polyketides produced thereby.

[0054] Those of skill in the art will recognize that an AT domain that is specific for 2-hydroxymalonyl CoA will result in a polyketide with a hydroxyl group at the corresponding location in the polyketide produced, and that the hydroxyl group can be methylated to yield a methoxy group by polyketide modification enzymes. See, e.g., the patent applications cited in connection with the FK-520 PKS in the table below. Consequently, reference to a PKS that has a 2-hydroxymalonyl specific AT domain herein similarly refers to polyketides produced by that PKS that have either a hydroxyl or methoxyl group at the corresponding location in the polyketide.

[0055] The loading domain of the epothilone PKS also comprises an ER domain. While, this ER domain may be involved in forming one of the double bonds in the thiazole moiety in epothilone (in the reverse of its normal reaction), or it may be non-functional. In either event, the invention provides recombinant DNA compounds that encode the epothilone PKS loading domain with and without the ER region, as well as hybrid loading domains that contain an ER domain from another PKS (either active or inactive, with or without accompanying KR and DH domains) in place of the ER domain of the epothilone loading domain. The present invention also provides recombinant PKS enzymes comprising such loading domains and host cells for producing such enzymes and the polyketides produced thereby.

[0056] The recombinant nucleic acid compounds of the invention that encode the loading domain of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone loading domain is coexpressed with the proteins of a heterologous PKS. As used herein, reference to a heterologous modular PKS (or to the coding sequence therefor) refers to all or part of a PKS, including each of the multiple proteins constituting the PKS, that synthesizes a polyketide other than an epothilone or epothilone derivative (or to the coding sequences therefor). This coexpression can be in one of two forms. The epothilone loading domain can be coexpressed as a discrete protein with the other proteins of the heterologous PKS or as a fusion protein in which the loading domain is fused to one or more modules of the heterologous PKS. In either event, the hybrid PKS formed, in which the loading domain of the heterologous PKS is replaced by the epothilone loading domain, provides a novel PKS. Examples of a heterologous PKS that can be used to prepare such hybrid PKS enzymes of the invention include but are not limited to DEBS and the picromycin (narbonolide), oleandolide, rapamycin, FK-506, FK-520, rifamycin, and avermectin PKS enzymes and their corresponding coding sequences.

[0057] In another embodiment, a nucleic acid compound comprising a sequence that encodes the epothilone loading domain is coexpressed with the proteins that constitute the remainder of the epothilone PKS (i.e., the epoB, epoC, epoD, epoE, and epoF gene products) or a recombinant epothilone PKS that produces an epothilone derivative due to an alteration or mutation in one or more of the epoB, epoC, epoD, epoE, and epoF genes. As used herein, reference to an epothilone or a PKS that produces an epothilone derivative (or to the coding sequence therefor) refers to all or any one of the proteins that comprise the PKS (or to the coding sequences therefor).

[0058] In another embodiment, the invention provides recombinant nucleic acid compounds that encode a loading domain composed of part of the epothilone loading domain and part of a heterologous PKS. In this embodiment, the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT. This replacement, like the others described herein, is typically mediated by replacing the coding sequences therefor to provide a recombinant DNA compound of the invention; the recombinant DNA is used to prepare the corresponding protein. Such changes (including not only replacements but also deletions and insertions) may be referred to herein either at the DNA or protein level.

[0059] The compounds of the invention also include those in which both the KSY and AT domains of the epothilone loading domain have been replaced but the ACP and/or linker regions of the epothilone loading domain are left intact. Linker regions are those segments of amino acids between domains in the loading domain and modules of a PKS that help form the tertiary structure of the protein and are involved in correct alignment and positioning of the domains of a PKS. These compounds include, for example, a recombinant loading domain coding sequence in which the KSY and AT domain coding sequences of the epothilone PKS have been replaced by the coding sequences for the KSQ and AT domains of, for example, the oleandolide PKS or the narbonolide PKS. There are also PKS enzymes that do not employ a KSQ domain but instead merely utilize an AT domain that binds acetyl CoA, propionyl CoA, or butyryl CoA (the DEBS loading domain) or isobutyryl CoA (the avermectin loading domain). Thus, the compounds of the invention also include, for example, a recombinant loading domain coding sequence in which the KSY and AT domain coding sequences of the epothilone PKS have been replaced by an AT domain of the DEBS or avermectin PKS. The present invention also provides recombinant DNA compounds encoding loading domains in which the ACP domain or any of the linker regions of the epothilone loading domain has been replaced by another ACP or linker region.

[0060] Any of the above loading domain coding sequences is coexpressed with the other proteins that constitute a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide to provide a PKS of the invention. If the product desired is epothilone or an epothilone derivative, then the loading domain coding sequence is typically expressed as a discrete protein, as is the loading domain in the naturally occurring epothilone PKS. If the product desired is produced by the loading domain of the invention and proteins from one or more non-epothilone PKS enzymes, then the loading domain is expressed either as a discrete protein or as a fusion protein with one or more modules of the heterologous PKS.

[0061] The present invention also provides hybrid PKS enzymes in which the epothilone loading domain has been replaced in its entirety by a loading domain from a heterologous PKS with the remainder of the PKS proteins provided by modified or unmodified epothilone PKS proteins. The present invention also provides recombinant expression vectors and host cells for producing such enzymes and the polyketides produced thereby. In one embodiment, the heterologous loading domain is expressed as a discrete protein in a host cell that expresses the epoB, epoC, epoD, epoE, and epoF gene products. In another embodiment, the heterologous loading domain is expressed as a fusion protein with the epoB gene product in a host cell that expresses the epoC, epoD, epoE, and epoF gene products. In a related embodiment, the present invention provides recombinant epothilone PKS enzymes in which the loading domain has been deleted and replaced by an NRPS module and corresponding recombinant DNA compounds and expression vectors. In this embodiment, the recombinant PKS enzymes thus produce an epothilone derivative that comprises a dipeptide moiety, as in the compound leinamycin. The invention provides such enzymes in which the remainder of the epothilone PKS is identical in function to the native epothilone PKS as well as those in which the remainder is a recombinant PKS that produces an epothilone derivative of the invention.

[0062] The present invention also provides reagents and methods useful in deleting the loading domain coding sequence or any portion thereof from the chromosome of a host cell, such as Sorangium cellulosum, or replacing those sequences or any portion thereof with sequences encoding a recombinant loading domain. Using a recombinant vector that comprises DNA complementary to the DNA including and/or flanking the loading domain coding sequence in the Sorangium chromosome, one can employ the vector and homologous recombination to replace the native loading domain coding sequence with a recombinant loading domain coding sequence or to delete the sequence altogether.

[0063] Moreover, while the above discussion focuses on deleting or replacing the epothilone loading domain coding sequences, those of skill in the art will recognize that the present invention provides recombinant DNA compounds, vectors, and methods useful in deleting or replacing all or any portion of an epothilone PKS gene or an epothilone modification enzyme gene. Such methods and materials are useful for a variety of purposes. One purpose is to construct a host cell that does not make a naturally occurring epothilone or epothilone derivative. For example, a host cell that has been modified to not produce a naturally occurring epothilone may be particularly preferred for making epothilone derivatives or other polyketides free of any naturally occurring epothilone. Another purpose is to replace the deleted gene with a gene that has been altered so as to provide a different product or to produce more of one product than another.

[0064] If the epothilone loading domain coding sequence has been deleted or otherwise rendered non-functional in a Sorangium cellulosum host cell, then the resulting host cell will produce a non-functional epothilone PKS. This PKS could still bind and process extender units, but the thiazole moiety of epothilone would not form, leading to the production of a novel epothilone derivative. Because this derivative would predictably contain a free amino group, it would be produced at most in low quantities. As noted above, however, provision of a heterologous or other recombinant loading domain to the host cell would result in the production of an epothilone derivative with a structure determined by the loading domain provided.

[0065] The loading domain of the epothilone PKS is followed by the first module of the PKS, which is an NRPS module specific for cysteine. This NRPS module is naturally expressed as a discrete protein, the product of the epoB gene. The present invention provides the epoB gene in recombinant form. The recombinant nucleic acid compounds of the invention that encode the NRPS module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a nucleic acid compound comprising a sequence that encodes the epothilone NRPS module is coexpressed with genes encoding one or more proteins of a heterologous PKS. The NRPS module can be expressed as a discrete protein or as a fusion protein with one of the proteins of the heterologous PKS. The resulting PKS, in which at least a module of the heterologous PKS is replaced by the epothilone NRPS module or the NRPS module is in effect added as a module to the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the epothilone NRPS module is coexpressed with the other epothilone PKS proteins or modified versions thereof to provide a recombinant epothilone PKS that produces an epothilone or an epothilone derivative.

[0066] Two hybrid PKS enzymes provided by the invention illustrate this aspect. Both hybrid PKS enzymes are hybrids of DEBS and the epothilone NRPS module. The first hybrid PKS is composed of four proteins: (i) DEBS1; (ii) a fusion protein composed of the KS domain of module 3 of DEBS and all but the KS domain of the loading domain of the epothilone PKS; (iii) the epothilone NRPS module; and (iv) a fusion protein composed of the KS domain of module 2 of the epothilone PKS fused to the AT domain of module 5 of DEBS and the rest of DEBS3. This hybrid PKS produces a novel polyketide with a thiazole moiety incorporated into the macrolactone ring and a molecular weight of 413.53 when expressed in Streptomyces coelicolor. Glycosylated, hydroxylated, and methylated derivatives can be produced by expression of the hybrid PKS in Saccharopolyspora erythraea.

[0067] Diagrammatically, the construct is represented: 3

[0068] The structure of the product is: 4

[0069] The second hybrid PKS illustrating this aspect of the invention is composed of five proteins: (i) DEBS1; (ii) a fusion protein composed of the KS domain of module 3 of DEBS and all but the KS domain of the loading domain of the epothilone PKS; (iii) the epothilone NRPS module; and (iv) a fusion protein composed of the KS domain of module 2 of the epothilone PKS fused to the AT domain of module 4 of DEBS and the rest of DEBS2; and (v) DEBS3. This hybrid PKS produces a novel polyketide with a thiazole moiety incorporated into the macrolactone ring and a molecular weight of 455.61 when expressed in Streptomyces coelicolor. Glycosylated, hydroxylated, and methylated derivatives can be produced by expression of the hybrid PKS in Saccharopolyspora erythraea.

[0070] Diagrammatically, the construct is represented: 5

[0071] The structure of the product is: 6

[0072] In another embodiment, a portion of the NRPS module coding sequence is utilized in conjunction with a heterologous coding sequence. In this embodiment, the invention provides, for example, changing the specificity of the NRPS module of the epothilone PKS from a cysteine to another amino acid. This change is accomplished by constructing a coding sequence in which all or a portion of the epothilone PKS NRPS module coding sequences have been replaced by those coding for an NRPS module of a different specificity. In one illustrative embodiment, the specificity of the epothilone NRPS module is changed from cysteine to serine or threonine. When the thus modified NRPS module is expressed with the other proteins of the epothilone PKS, the recombinant PKS produces an epothilone derivative in which the thiazole moiety of epothilone (or an epothilone derivative) is changed to an oxazole or 5-methyloxazole moiety, respectively. Alternatively, the present invention provides recombinant PKS enzymes composed of the products of the epoA, epoC, epoD, epoE, and epoF genes (or modified versions thereof) without an NRPS module or with an NRPS module from a heterologous PKS. The heterologous NRPS module can be expressed as a discrete protein or as a fusion protein with either the epoA or epoC genes.

[0073] The invention also provides methods and reagents useful in changing the specificity of a heterologous NRPS module from another amino acid to cysteine. This change is accomplished by constructing a coding sequence in which the sequences that determine the specificity of the heterologous NRPS module have been replaced by those that specify cysteine from the epothilone NRPS module coding sequence. The resulting heterologous NRPS module is typically coexpressed in conjunction with the proteins constituting a heterologous PKS that synthesizes a polyketide other than epothilone or an epothilone derivative, although the heterologous NRPS module can also be used to produce epothilone or an epothilone derivative.

[0074] In another embodiment, the invention provides recombinant epothilone PKS enzymes and corresponding recombinant nucleic acid compounds and vectors in which the NRPS module has been inactivated or deleted. Such enzymes, compounds, and vectors are constructed generally in accordance with the teaching for deleting or inactivating the epothilone PKS or modification enzyme genes above. Inactive NRPS module proteins and the coding sequences therefore provided by the invention include those in which the peptidyl carrier protein (PCP) domain has been wholly or partially deleted or otherwise rendered inactive by changing the active site serine (the site for phosphopantetheinylation) to another amino acid, such as alanine, or the adenylation domains have been deleted or otherwise rendered inactive. In one embodiment, both the loading domain and the NRPS have been deleted or rendered inactive. In any event, the resulting epothilone PKS can then function only if provided a substrate that binds to the KS domain of module 2 (or a subsequent module) of the epothilone PKS or a PKS for an epothilone derivative. In a method provided by the invention, the thus modified cells are then fed activated acylthioesters that are bound by preferably the second, but potentially any subsequent, module and processed into novel epothilone derivatives.

[0075] Thus, in one embodiment, the invention provides Sorangium and non-Sorangium host cells that express an epothilone PKS (or a PKS that produces an epothilone derivative) with an inactive NRPS. The host cell is fed activated acylthioesters to produce novel epothilone derivatives of the invention. The host cells expressing, or cell free extracts containing, the PKS can be fed or supplied with N-acylcysteamine thioesters (NACS) of novel precursor molecules to prepare epothilone derivatives. See U.S. patent application Ser. No. 60/117,384, filed 27 Jan. 1999, and PCT patent publication No. US99/03986, both of which are incorporated herein by reference, and Example 6, below.

[0076] The second (first non-NRPS) module of the epothilone PKS includes a KS, an AT specific for methylmalonyl CoA, a DH, a KR, and an ACP. This module is encoded by a sequence within an ˜13.1 kb EcoRI-NsiI restriction fragment of cosmid pKOS35-70.8A3.

[0077] The recombinant nucleic acid compounds of the invention that encode the second module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. The second module of the epothilone PKS is produced as a discrete protein by the epoC gene. The present invention provides the epoC gene in recombinant form. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone second module is coexpressed with the proteins constituting a heterologous PKS either as a discrete protein or as a fusion protein with one or more modules of the heterologous PKS. The resulting PKS, in which a module of the heterologous PKS is either replaced by the second module of the epothilone PKS or the latter is merely added to the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the second module of the epothilone PKS is coexpressed with the other proteins constituting the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative.

[0078] In another embodiment, all or only a portion of the second module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting either the DH or KR or both; replacing the DH or KR or both with a DH or KR or both that specify a different stereochemistry; and/or inserting an ER. Generally, any reference herein to inserting or replacing a PKS KR, DH, and/or ER domain includes the replacement of the associated KR, DH, or ER domains in that module, typically with corresponding domains from the module from which the inserted or replacing domain is obtained. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a gene for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous second module coding sequence can be coexpressed with the other proteins that constitute a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide. Alternatively, one can delete or replace the second module of the epothilone PKS with a module from a heterologous PKS, which can be expressed as a discrete protein or as a fusion protein fused to either the epoB or epoD gene product.

[0079] Illustrative recombinant PKS genes of the invention include those in which the AT domain encoding sequences for the second module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a methylmalonyl specific AT to a malonyl specific AT. Such malonyl specific AT domain encoding nucleic acids can be isolated, for example and without limitation, from the PKS genes encoding the narbonolide PKS, the rapamycin PKS (i.e., modules 2 and 12), and the FK-520 PKS (i.e., modules 3, 7, and 8). When such a hybrid second module is coexpressed with the other proteins constituting the epothilone PKS, the resulting epothilone derivative produced is a 1 6-desmethyl epothilone derivative.

[0080] In addition, the invention provides DNA compounds and vectors encoding recombinant epothilone PKS enzymes and the corresponding recombinant proteins in which the KS domain of the second (or subsequent) module has been inactivated or deleted. In a preferred embodiment, this inactivation is accomplished by changing the codon for the active site cysteine to an alanine codon. As with the corresponding variants described above for the NRPS module, the resulting recombinant epothilone PKS enzymes are unable to produce an epothilone or epothilone derivative unless supplied a precursor that can be bound and extended by the remaining domains and modules of the recombinant PKS enzyme. Illustrative diketides are described in Example 6, below.

[0081] The third module of the epothilone PKS includes a KS, an AT specific for malonyl CoA, a KR, and an ACP. This module is encoded by a sequence within an ˜8 kb BglII-NsiI restriction fragment of cosmid pKOS35-70.8A3.

[0082] The recombinant DNA compounds of the invention that encode the third module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. The third module of the epothilone PKS is expressed in a protein, the product of the epoD gene, which also contains modules 4, 5, and 6. The present invention provides the epoD gene in recombinant form. The present invention also provides recombinant DNA compounds that encode each of the epothilone PKS modules 3, 4, 5, and 6, as discrete coding sequences without coding sequences for the other epothilone modules. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone third module is coexpressed with proteins constituting a heterologous PKS. The third module of the epothilone PKS can be expressed either as a discrete protein or as a fusion protein fused to one or more modules of the heterologous PKS. The resulting PKS, in which a module of the heterologous PKS is either replaced by that for the third module of the epothilone PKS or the latter is merely added to the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the third module of the epothilone PKS is coexpressed with proteins comprising the remainder of the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative, typically as a protein comprising not only the third but also the fourth, fifth, and sixth modules.

[0083] In another embodiment, all or a portion of the third module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; replacing the KR with a KR that specifies a different stereochemistry; and/or inserting a DH or a DH and an ER. As above, the reference to inserting a DH or a DH and an ER includes the replacement of the KR with a DH and KR or an ER, DH, and KR. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous third module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide.

[0084] Illustrative recombinant PKS genes of the invention include those in which the AT domain encoding sequences for the third module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a malonyl specific AT to a methylmalonyl specific AT. Such methylmalonyl specific AT domain encoding nucleic acids can be isolated, for example and without limitation, from the PKS genes encoding DEBS, the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS. When coexpressed with the remaining modules and proteins of the epothilone PKS or an epothilone PKS derivative, the recombinant PKS produces the 14-methyl epothilone derivatives of the invention.

[0085] Those of skill in the art will recognize that the KR domain of the third module of the PKS is responsible for forming the hydroxyl group involved in cyclization of epothilone. Consequently, abolishing the KR domain of the third module or adding a DH or DH and ER domains will interfere with the cyclization, leading either to a linear molecule or to a molecule cyclized at a different location than is epothilone.

[0086] The fourth module of the epothilone PKS includes a KS, an AT that can bind either malonyl CoA or methylmalonyl CoA, a KR, and an ACP. This module is encoded by a sequence within an ˜10 kb NsiI-HindIII restriction fragment of cosmid pKOS35-70.1A2.

[0087] The recombinant DNA compounds of the invention that encode the fourth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone fourth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS. The resulting construct encodes a protein in which a module of the heterologous PKS is either replaced by that for the fourth module of the epothilone PKS or the latter is merely added to the modules of the heterologous PKS. Together with other proteins that constitute the heterologous PKS, this protein provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the fourth module of the epothilone PKS is expressed in a host cell that also expresses the remaining modules and proteins of the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative. For making epothilone or epothilone derivatives, the recombinant fourth module is usually expressed in a protein that also contains the epothilone third, fifth, and sixth modules or modified versions thereof.

[0088] In another embodiment, all or a portion of the fourth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the malonyl CoA and methylmalonyl specific AT with a malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; and/or replacing the KR, including, optionally, to specify a different stereochemistry; and/or inserting a DH or a DH and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a gene for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous fourth module coding sequence is incorporated into a protein subunit of a recombinant PKS that synthesizes epothilone, an epothilone derivative, or another polyketide. If the desired polyketide is an epothilone or epothilone derivative, the recombinant fourth module is typically expressed as a protein that also contains the third, fifth, and sixth modules of the epothilone PKS or modified versions thereof. Alternatively, the invention provides recombinant PKS enzymes for epothilones and epothilone derivatives in which the entire fourth module has been deleted or replaced by a module from a heterologous PKS.

[0089] In a preferred embodiment, the invention provides recombinant DNA compounds comprising the coding sequence for the fourth module of the epothilone PKS modified to encode an AT that binds methylmalonyl CoA and not malonyl CoA. These recombinant molecules are used to express a protein that is a recombinant derivative of the epoD protein that comprises the modified fourth module as well as modules 3, 5, and 6, any one or more of which can optionally be in derivative form, of the epothilone PKS. In another preferred embodiment, the invention provides recombinant DNA compounds comprising the coding sequence for the fourth module of the epothilone PKS modified to encode an AT that binds malonyl CoA and not methylmalonyl CoA. These recombinant molecules are used to express a protein that is a recombinant derivative of the epoD protein that comprises the modified fourth module as well as modules 3, 5, and 6, any one or more of which can optionally be in derivative form, of the epothilone PKS.

[0090] Prior to the present invention, it was known that Sorangium cellulosum produced epothilones A, B, C, D, E, and F and that epothilones A, C, and E had a hydrogen at C-12, while epothilones B, D, and F had a methyl group at this position. Unappreciated prior to the present invention was the order in which these compounds were synthesized in S. cellulosum, and the mechanism by which some of the compounds had a hydrogen at C-12 where others had a methyl group at this position. The present disclosure reveals that epothilones A and B are derived from epothilones C and D by action of the epoK gene product and that the presence of a hydrogen or methyl moiety at C-12 is due to the AT domain of module 4 of the epothilone PKS. This domain can bind either malonyl or methylmalonyl CoA and, consistent with its having greater similarity to malonyl specific AT domains than to methylmalonyl specific AT domains, binds malonyl CoA more often than methylmalonyl CoA.

[0091] Thus, the invention provides recombinant DNA compounds and expression vectors and the corresponding recombinant PKS in which the hybrid fourth module with a methylmalonyl specific AT has been incorporated. The methylmalonyl specific AT coding sequence can originate, for example and without limitation, from coding sequences for the oleandolide PKS, DEBS, the narbonolide PKS, the rapamycin PKS, or any other PKS that comprises a methylmalonyl specific AT domain. In accordance with the invention, the hybrid fourth module expressed from this coding sequence is incorporated into the epothilone PKS (or the PKS for an epothilone derivative), typically as a derivative epoD gene product. The resulting recombinant epothilone PKS produces epothilones with a methyl moiety at C-12, i.e., epothilone H (or an epothilone H derivative) if there is no dehydratase activity to form the C-12-C-13 alkene; epothilone D (or an epothilone D derivative), if the dehydratase activity but not the epoxidase activity is present; epothilone B (or an epothilone B derivative), if both the dehydratase and epoxidase activity but not the hydroxylase activity are present; and epothilone F (or an epothilone F derivative), if all three dehydratase, epoxidase, and hydroxylase activities are present. As indicated parenthetically above, the cell will produce the corresponding epothilone derivative if there have been other changes to the epothilone PKS.

[0092] If the recombinant PKS comprising the hybrid methylmalonyl specific fourth module is expressed in, for example, Sorangium cellulosum, the appropriate modifying enzymes are present (unless they have been rendered inactive in accordance with the methods herein), and epothilones D, B, and/or F are produced. Such production is typically carried out in a recombinant S. cellulosum provided by the present invention in which the native epothilone PKS is unable to function at all or unable to function except in conjunction with the recombinant fourth module provided. In an illustrative example, one can use the methods and reagents of the invention to render inactive the epoD gene in the native host. Then, one can transform that host with a vector comprising the recombinant epoD gene containing the hybrid fourth module coding sequence. The recombinant vector can exist as an extrachromosomal element or as a segment of DNA integrated into the host cell chromosome. In the latter embodiment, the invention provides that one can simply integrate the recombinant methylmalonyl specific module 4 coding sequence into wild-type S. cellulosum by homologous recombination with the native epoD gene to ensure that only the desired epothilone is produced. The invention provides that the S. cellulosum host can either express or not express (by mutation or homologous recombination of the native genes therefor) the dehydratase, epoxidase, and/or oxidase gene products and thus form or not form the corresponding epothilone D, B, and F compounds, as the practitioner elects.

[0093] Sorangium cellulosum modified as described above is only one of the recombinant host cells provided by the invention. In a preferred embodiment, the recombinant methylmalonyl specific epothilone fourth module coding sequences are used in accordance with the methods of invention to produce epothilone D, B, and F (or their corresponding derivatives) in heterologous host cells. Thus, the invention provides reagents and methods for introducing the epothilone or epothilone derivative PKS and epothilone dehydratase, epoxidase, and hydroxylase genes and combinations thereof into heterologous host cells.

[0094] The recombinant methylmalonyl specific epothilone fourth module coding sequences provided by the invention afford important alternative methods for producing desired epothilone compounds in host cells. Thus, the invention provides a hybrid fourth module coding sequence in which, in addition to the replacement of the endogenous AT coding sequence with a coding sequence for an AT specific for methylmalonyl Co A, coding sequences for a DH and KR for, for example and without limitation, module 10 of the rapamycin PKS or modules 1 or 5 of the FK-520 PKS have replaced the endogenous KR coding sequences. When the gene product comprising the hybrid fourth module and epothilone PKS modules 3, 5, and 6 (or derivatives thereof) encoded by this coding sequence is incorporated into a PKS comprising the other epothilone PKS proteins (or derivatives thereof) produced in a host cell, the cell makes either epothilone D or its trans stereoisomer (or derivatives thereof), depending on the stereochemical specificity of the inserted DH and KR domains.

[0095] Similarly, and as noted above, the invention provides recombinant DNA compounds comprising the coding sequence for the fourth module of the epothilone PKS modified to encode an AT that binds malonyl CoA and not methylmalonyl CoA. The invention provides recombinant DNA compounds and vectors and the corresponding recombinant PKS in which this hybrid fourth module has been incorporated into a derivative epoD gene product. When incorporated into the epothilone PKS (or the PKS for an epothilone derivative), the resulting recombinant epothilone PKS produces epothilones C, A, and E, depending, again, on whether epothilone modification enzymes are present. As noted above, depending on the host, whether the fourth module includes a KR and DH domain, and on whether and which of the dehydratase, epoxidase, and oxidase activities are present, the practitioner of the invention can produce one or more of the epothilone G, C, A, and E compounds and derivatives thereof using the compounds, host cells, and methods of the invention.

[0096] The fifth module of the epothilone PKS includes a KS, an AT that binds malonyl CoA, a DH, an ER, a KR, and an ACP. This module is encoded by a sequence within an ˜12.4 kb NsiI-NotI restriction fragment of cosmid pKOS35-70.1A2.

[0097] The recombinant DNA compounds of the invention that encode the fifth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone fifth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, can be incorporated into an expression vector and used to produce the recombinant protein encoded thereby. When the recombinant protein is combined with the other proteins of the heterologous PKS, a novel PKS is produced. In another embodiment, a DNA compound comprising a sequence that encodes the fifth module of the epothilone PKS is inserted into a DNA compound that comprises coding sequences for the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative. In the latter constructs, the epothilone fifth module is typically expressed as a protein comprising the third, fourth, and sixth modules of the epothilone PKS or derivatives thereof.

[0098] In another embodiment, a portion of the fifth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module coding sequence and the hybrid module encoded thereby. In this embodiment, the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER, including, optionally, to specify a different stereochemistry. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting hybrid fifth module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide. Alternatively, the fifth module of the epothilone PKS can be deleted or replaced in its entirety by a module of a heterologous PKS to produce a protein that in combination with the other proteins of the epothilone PKS or derivatives thereof constitutes a PKS that produces an epothilone derivative.

[0099] Illustrative recombinant PKS genes of the invention include recombinant epoD gene derivatives in which the AT domain encoding sequences for the fifth module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a malonyl specific AT to a methylmalonyl specific AT. Such methylmalonyl specific AT domain encoding nucleic acids can be isolated, for example and without limitation, from the PKS genes encoding DEBS, the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS. When such recombinant epoD gene derivatives are coexpressed with the epoA, epoB, epoC, epoE, and epoF genes (or derivatives thereof), the PKS composed thereof produces the 10-methyl epothilones or derivatives thereof. Another recombinant epoD gene derivative provided by the invention includes not only this altered module 5 coding sequence but also module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA. When incorporated into a PKS with the epoA, epoB, epoC, epoE, and epoF genes, the recombinant epoD gene derivative product leads to the production of 10-methyl epothilone B and/or D derivatives.

[0100] Other illustrative recombinant epoD gene derivatives of the invention include those in which the ER, DH, and KR domain encoding sequences for the fifth module of the epothilone PKS have been replaced with those encoding (i) a KR and DH domain; (ii) a KR domain; and (iii) an inactive KR domain. These recombinant epoD gene derivatives of the invention are coexpressed with the epoA, epoB, epoC, epoE, and epoF genes to produce a recombinant PKS that makes the corresponding (i) C-11 alkene, (ii) C-11 hydroxy, and (iii) C-11 keto epothilone derivatives. These recombinant epoD gene derivatives can also be coexpressed with recombinant epo genes containing other alterations or can themselves be further altered to produce a PKS that makes the corresponding C-11 epothilone derivatives. For example, one recombinant epoD gene derivative provided by the invention also includes module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA. When incorporated into a PKS with the epoA, epoB, epoC, epoE, and epoF genes, the recombinant epoD gene derivative product leads to the production of the corresponding C-11 epothilone B and/or D derivatives.

[0101] Functionally similar epoD genes for producing the epothilone C-11 derivatives can also be made by inactivation of one, two, or all three of the ER, DH, and KR domains of the epothilone fifth module. However, the preferred mode for altering such domains in any module is by replacement with the complete set of desired domains taken from another module of the same or a heterologous PKS coding sequence. In this manner, the natural architecture of the PKS is conserved. Also, when present, KR and DH or KR, DH, and ER domains that function together in a native PKS are preferably used in the recombinant PKS. Illustrative replacement domains for the substitutions described above include, for example and without limitation, the inactive KR domain from the rapamycin PKS module 3 to form the ketone, the KR domain from the rapamycin PKS module 5 to form the alcohol, and the KR and DH domains from the rapamycin PKS module 4 to form the alkene. Other such inactive KR, active KR, and active KR and DH domain encoding nucleic acids can be isolated from, for example and without limitation, the PKS genes encoding DEBS, the narbonolide PKS, and the FK-520 PKS. Each of the resulting PKS enzymes produces a polyketide compound that comprises a functional group at the C-11 position that can be further derivatized in vitro by standard chemical methodology to yield semi-synthetic epothilone derivatives of the invention.

[0102] The sixth module of the epothilone PKS includes a KS, an AT that binds methylmalonyl CoA, a DH, an ER, a KR, and an ACP. This module is encoded by a sequence within an ˜14.5 kb HindIII-NsiI restriction fragment of cosmid pKOS35-70.1A2.

[0103] The recombinant DNA compounds of the invention that encode the sixth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone sixth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS. The resulting protein encoded by the construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS when coexpressed with the other proteins comprising the PKS. In another embodiment, a DNA compound comprising a sequence that encodes the sixth module of the epothilone PKS is inserted into a DNA compound that comprises the coding sequence for modules 3, 4, and 5 of the epothilone PKS or a recombinant epothilone PKS that produces an epothilone derivative and coexpressed with the other proteins of the epothilone or epothilone derivative PKS to produce a PKS that makes epothilone or an epothilone derivative in a host cell.

[0104] In another embodiment, a portion of the sixth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER, including, optionally, to specify a different stereochemistry. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous sixth module coding sequence can be utilized in conjunction with a coding sequence for a protein subunit of a PKS that makes epothilone, an epothilone derivative, or another polyketide. If the PKS makes epothilone or an epothilone derivative, the hybrid sixth module is typically expressed as a protein comprising modules 3, 4, and 5 of the epothilone PKS or derivatives thereof. Alternatively, the sixth module of the epothilone PKS can be deleted or replaced in its entirety by a module from a heterologous PKS to produce a PKS for an epothilone derivative.

[0105] Illustrative recombinant PKS genes of the invention include those in which the AT domain encoding sequences for the sixth module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a methylmalonyl specific AT to a malonyl specific AT. Such malonyl specific AT domain encoding nucleic acids can be isolated from, for example and without limitation, the PKS genes encoding the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS. When a recombinant epoD gene of the invention encoding such a hybrid module 6 is coexpressed with the other epothilone PKS genes, the recombinant PKS makes the 8-desmethyl epothilone derivatives. This recombinant epoD gene derivative can also be coexpressed with recombinant epo gene derivatives containing other alterations or can itself be further altered to produce a PKS that makes the corresponding 8-desmethyl epothilone derivatives. For example, one recombinant epoD gene provided by the invention also includes module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA. When incorporated into a PKS with the epoA, epoB, epoC, epoE, and epoF genes, the recombinant epoD gene product leads to the production of the 8-desmethyl derivatives of epothilones B and D.

[0106] Other illustrative recombinant epoD gene derivatives of the invention include those in which the ER, DH, and KR domain encoding sequences for the sixth module of the epothilone PKS have been replaced with those that encode (i) a KR and DH domain; (ii) a KR domain; and (iii) an inactive KR domain. These recombinant epoD gene derivatives of the invention, when coexpressed with the other epothilone PKS genes make the corresponding (i) C-9 alkene, (ii) C-9 hydroxy, and (iii) C-9 keto epothilone derivatives. These recombinant epoD gene derivatives can also be coexpressed with other recombinant epo gene derivatives containing other alterations or can themselves be further altered to produce a PKS that makes the corresponding C-9 epothilone derivatives. For example, one recombinant epoD gene derivative provided by the invention also includes module 4 coding sequences that encode an AT domain that binds only methylmalonyl CoA. When incorporated into a PKS with the epoA, epoB, epoC, epoE, and epoF genes, the recombinant epoD gene product leads to the production of the C-9 derivatives of epothilones B and D.

[0107] Functionally equivalent sixth modules can also be made by inactivation of one, two, or all three of the ER, DH, and KR domains of the epothilone sixth module. The preferred mode for altering such domains in any module is by replacement with the complete set of desired domains taken from another module of the same or a heterologous PKS coding sequence. Illustrative replacement domains for the substitutions described above include but are not limited to the inactive KR domain from the rapamycin PKS module 3 to form the ketone, the KR domain from the rapamycin PKS module 5 to form the alcohol, and the KR and DH domains from the rapamycin PKS module 4 to form the alkene. Other such inactive KR, active KR, and active KR and DH domain encoding nucleic acids can be isolated from for example and without limitation the PKS genes encoding DEBS, the narbonolide PKS, and the FK-520 PKS. Each of the resulting PKSs produces a polyketide compound that comprises a functional group at the C-9 position that can be further derivatized in vitro by standard chemical methodology to yield semi-synthetic epothilone derivatives of the invention.

[0108] The seventh module of the epothilone PKS includes a KS, an AT specific for methylmalonyl CoA, a KR, and an ACP. This module is encoded by a sequence within an ˜8.7 kb BglII restriction fragment from cosmid pKOS35-70.4.

[0109] The recombinant DNA compounds of the invention that encode the seventh module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. The seventh module of the epothilone PKS is contained in the gene product of the epoE gene, which also contains the eighth module. The present invention provides the epoE gene in recombinant form, but also provides DNA compounds that encode the seventh module without coding sequences for the eighth module as well as DNA compounds that encode the eighth module without coding sequences for the seventh module. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone seventh module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the seventh module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS coding sequence that can be expressed in a host cell. Alternatively, the epothilone seventh module can be expressed as a discrete protein. In another embodiment, a DNA compound comprising a sequence that encodes the seventh module of the epothilone PKS is expressed to form a protein that, together with other proteins, constitutes the epothilone PKS or a PKS that produces an epothilone derivative. In these embodiments, the seventh module is typically expressed as a protein comprising the eighth module of the epothilone PKS or a derivative thereof and coexpressed with the epoA, epoB, epoC, epoD, and epoF genes or derivatives thereof to constitute the PKS.

[0110] In another embodiment, a portion or all of the seventh module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; replacing the KR with a KR that specifies a different stereochemistry; and/or inserting a DH or a DH and an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous seventh module coding sequence is utilized, optionally in conjunction with other coding sequences, to express a protein that together with other proteins constitutes a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide. When used to prepare epothilone or an epothilone derivative, the seventh module is typically expressed as a protein comprising the eighth module or derivative thereof and coexpressed with the epoA, epoB, epoC, epoD, and epoF genes or derivatives thereof to constitute the PKS. Alternatively, the coding sequences for the seventh module in the epoE gene can be deleted or replaced by those for a heterologous module to prepare a recombinant epoE gene derivative that, together with the epoA, epoB, epoC, epoD, and epoF genes, can be expressed to make a PKS for an epothilone derivative.

[0111] Illustrative recombinant epoE gene derivatives of the invention include those in which the AT domain encoding sequences for the seventh module of the epothilone PKS have been altered or replaced to change the AT domain encoded thereby from a methylmalonyl specific AT to a malonyl specific AT. Such malonyl specific AT domain encoding nucleic acids can be isolated from for example and without limitation the PKS genes encoding the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS. When coexpressed with the other epothilone PKS genes, epoA, epoB, epoC, epoD, and epoF, or derivatives thereof, a PKS for an epothilone derivative with a C-6 hydrogen, instead of a C-6 methyl, is produced. Thus, if the genes contain no other alterations, the compounds produced are the 6-desmethyl epothilones.

[0112] The eighth module of the epothilone PKS includes a KS, an AT specific for methylmalonyl CoA, inactive KR and DH domains, a methyltransferase (MT) domain, and an ACP. This module is encoded by a sequence within an ˜10 kb NotI restriction fragment of cosmid pKOS35-79.85.

[0113] The recombinant DNA compounds of the invention that encode the eighth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone eighth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the eighth module of the epothilone PKS or the latter is merely added to coding sequences for modules of the heterologous PKS, provides a novel PKS coding sequence that is expressed with the other proteins constituting the PKS to provide a novel PKS. Alternatively, the eighth module can be expressed as a discrete protein that can associate with other PKS proteins to constitute a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the eighth module of the epothilone PKS is coexpressed with the other proteins constituting the epothilone PKS or a PKS that produces an epothilone derivative. In these embodiments, the eighth module is typically expressed as a protein that also comprises the seventh module or a derivative thereof.

[0114] In another embodiment, a portion or all of the eighth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the inactive KR and/or the inactive DH; replacing the inactive KR and/or DH with an active KR and/or DH; and/or inserting an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous eighth module coding sequence is expressed as a protein that is utilized in conjunction with the other proteins that constitute a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide. When used to prepare epothilone or an epothilone derivative, the heterologous or hybrid eighth module is typically expressed as a recombinant epoE gene product that also contains the seventh module. Alternatively, the coding sequences for the eighth module in the epoE gene can be deleted or replaced by those for a heterologous module to prepare a recombinant epoE gene that, together with the epoA, epoB, epoC, epoD, and epoF genes, can be expressed to make a PKS for an epothilone derivative.

[0115] The eighth module of the epothilone PKS also comprises a methylation or methyltransferase (MT) domain with an activity that methylates the epothilone precursor. This function can be deleted to produce a recombinant epoD gene derivative of the invention, which can be expressed with the other epothilone PKS genes or derivatives thereof that makes an epothilone derivative that lacks one or both methyl groups, depending on whether the AT domain of the eighth module has been changed to a malonyl specific AT domain, at the corresponding C-4 position of the epothilone molecule. In another important embodiment, the present invention provides recombinant DNA compounds that encode a polypeptide with this methylation domain and activity and a variety of recombinant PKS coding sequences that encode recombinant PKS enzymes that incorporate this polypeptide. The availability of this MT domain and the coding sequences therefor provides a significant number of new polyketides that differ from known polyketides by the presence of at least an additional methyl group. The MT domain of the invention can in effect be added to any PKS module to direct the methylation at the corresponding location in the polyketide produced by the PKS. As but one illustrative example, the present invention provides the recombinant nucleic acid compounds resulting from inserting the coding sequence for this MT activity into a coding sequence for any one or more of the six modules of the DEBS enzyme to produce a recombinant DEBS that synthesizes a 6-deoxyerythronolide B derivative that comprises one or more additional methyl groups at the C-2, C-4, C-6, C-8, C-10, and/or C-12 positions. In such constructs, the MT domain can be inserted adjacent to the AT or the ACP.

[0116] The ninth module of the epothilone PKS includes a KS, an AT specific for malonyl CoA, a KR, an inactive DH, and an ACP. This module is encoded by a sequence within an ˜14.7 HindIII-Bg1II kb restriction fragment of cosmid pKOS35-79.85.

[0117] The recombinant DNA compounds of the invention that encode the ninth module of the epothilone PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. The ninth module of the epothilone PKS is expressed as a protein, the product of the epoF gene, that also contains the TE domain of the epothilone PKS. The present invention provides the epoF gene in recombinant form, as well as DNA compounds that encode the ninth module without the coding sequences for the TE domain and DNA compounds that encode the TE domain without the coding sequences for the ninth module. In one embodiment, a DNA compound comprising a sequence that encodes the epothilone ninth module is inserted into a DNA compound that comprises the coding sequence for one or more modules of a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the ninth module of the epothilone PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS protein coding sequence that when coexpressed with the other proteins constituting a PKS provides a novel PKS. The ninth module coding sequence can also be expressed as a discrete protein with or without an attached TE domain. In another embodiment, a DNA compound comprising a sequence that encodes the ninth module of the epothilone PKS is expressed as a protein together with other proteins to constitute an epothilone PKS or a PKS that produces an epothilone derivative. In these embodiments, the ninth module is typically expressed as a protein that also contains the TE domain of either the epothilone PKS or a heterologous PKS.

[0118] In another embodiment, a portion or all of the ninth module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, either replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxy malonyl CoA specific AT; deleting the KR; replacing the KR with a KR that specifies a different stereochemistry; and/or inserting a DH or a DH and an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the epothilone PKS, from a coding sequence for a PKS that produces a polyketide other than epothilone, or from chemical synthesis. The resulting heterologous ninth module coding sequence is coexpressed with the other proteins constituting a PKS that synthesizes epothilone, an epothilone derivative, or another polyketide. Alternatively, the present invention provides a PKS for an epothilone or epothilone derivative in which the ninth module has been replaced by a module from a heterologous PKS or has been deleted in its entirety. In the latter embodiment, the TE domain is expressed as a discrete protein or fused to the eighth module.

[0119] The ninth module of the epothilone PKS is followed by a thioesterase domain. This domain is encoded in the ˜14.7 kb HindIII-BglII restriction comprising the ninth module coding sequence. The present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the ninth module of the epothilone PKS is fused to a heterologous thioesterase or one or more modules of a heterologous PKS are fused to the epothilone PKS thioesterase. Thus, for example, a thioesterase domain coding sequence from another PKS can be inserted at the end of the ninth module ACP coding sequence in recombinant DNA compounds of the invention. Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode a protein of the epothilone PKS, a PKS that produces an epothilone derivative, and a PKS that produces a polyketide other than epothilone or an epothilone derivative.

[0120] In one important embodiment, the present invention thus provides a hybrid PKS and the corresponding recombinant DNA compounds that encode the proteins constituting those hybrid PKS enzymes. For purposes of the present invention a hybrid PKS is a recombinant PKS that comprises all or part of one or more modules, loading domain, and thioesterase/cyclase domain of a first PKS and all or part of one or more modules, loading domain, and thioesterase/cyclase domain of a second PKS. In one preferred embodiment, the first PKS is most but not all of the epothilone PKS, and the second PKS is only a portion or all of a non-epothilone PKS. An illustrative example of such a hybrid PKS includes an epothilone PKS in which the natural loading domain has been replaced with a loading domain of another PKS. Another example of such a hybrid PKS is an epothilone PKS in which the AT domain of module four is replaced with an AT domain from a heterologous PKS that binds only methylmalonyl CoA. In another preferred embodiment, the first PKS is most but not all of a non-epothilone PKS, and the second PKS is only a portion or all of the epothilone PKS. An illustrative example of such a hybrid PKS includes an erythromycin PKS in which an AT specific for methylmalonyl CoA is replaced with an AT from the epothilone PKS specific for malonyl CoA. Another example is an erythromycin PKS that includes the MT domain of the epothilone PKS.

[0121] Those of skill in the art will recognize that all or part of either the first or second PKS in a hybrid PKS of the invention need not be isolated from a naturally occurring source. For example, only a small portion of an AT domain determines its specificity. See U.S. patent application Ser. No. 09/346,860 and PCT patent application No. WO US99/15047, each of which is incorporated herein by reference. The state of the art in DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain. For purposes of the present invention, such synthetic DNA compounds are deemed to be a portion of a PKS.

[0122] The following Table lists references describing illustrative PKS genes and corresponding enzymes that can be utilized in the construction of the recombinant PKSs and the corresponding DNA compounds that encode them of the invention. Also presented are various references describing polyketide tailoring and modification enzymes and corresponding genes that can be employed to make the recombinant DNA compounds of the present invention.

[0123] Avermectin

[0124] U.S. Pat. No. 5,252,474 to Merck.

[0125] MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.

[0126] MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

[0127] Ikeda and Omura, 1997, Chem. Res. 97: 2599-2609, Avermectin biosynthesis.

[0128] Candicidin (FR008)

[0129] Hu et al., 1994, Mol. Microbiol. 14: 163-172.

[0130] Erythromycin

[0131] PCT Pub. No. 93/13663 to Abbott.

[0132] U.S. Pat. No. 5,824,513 to Abbott.

[0133] Donadio et al., 1991, Science 252:675-9.

[0134] Cortes et al., 8 Nov. 1990, Nature 348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea.

[0135] Glycosylation Enzymes

[0136] PCT Pat. App. Pub. No. 97/23630 to Abbott.

[0137] FK-506

[0138] Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK-506, Eur. J. Biochem. 256: 528-534.

[0139] Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK-506, Eur. J. Biochem. 244: 74-80.

[0140] Methyltransferase

[0141] U.S. Pat. No. 5,264,355, issued 23 Nov. 1993, Methylating enzyme from Streptomyces MA6858. 31 -O-desmethyl-FK-506 methyltransferase.

[0142] Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK-506 and FK-520, J. Bacteriol. 178: 5243-5248.

[0143] FK-520

[0144] U.S. patent application Ser. No. 09/154,083, filed 16 Sep. 1998.

[0145] U.S. patent application Ser. No. 09/410,551, filed 1 Oct. 1999.

[0146] Nielsen et al., 1991, Biochem. 30:5789-96.

[0147] Lovastatin

[0148] U.S. Pat. No. 5,744,350 to Merck.

[0149] Narbomycin

[0150] U.S. patent application Ser. No. 60/107,093, filed 5 Nov. 1998.

[0151] Nemadectin

[0152] MacNeil et al., 1993, supra.

[0153] Niddamycin

[0154] Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis, J. Bacteriol. 179: 7515-7522.

[0155] Oleandomycin

[0156] Swan et al., 1994, Characterisation of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Mol. Gen. Genet. 242: 358-362.

[0157] U.S. patent application Ser. No. 60/120,254, filed 16 Feb. 1999, Ser. No. 09/______ , filed 28 Oct. 1999, claiming priority thereto by inventors S. Shah, M. Betlach, R. McDaniel, and L. Tang, attorney docket No. 30063-20029.00.

[0158] Olano et al., 1998, Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308.

[0159] Picromycin

[0160] PCT patent application No. WO US99/11814, filed 28 May 1999.

[0161] U.S. patent application Ser. No. 09/320,878, filed 27 May 1999.

[0162] U.S. patent application Ser. No. 09/141,908, filed 28 Aug. 1998.

[0163] Xue et al., 1998, Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae, Chemistry & Biology 5(11): 661-667.

[0164] Xue et al., October 1998, A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity, Proc. Natl. Acad. Sci. USA 95: 12111 12116.

[0165] Platenolide

[0166] EP Pat. App. Pub. No. 791,656 to Lilly.

[0167] Pradimicin

[0168] PCT Pat. Pub. No. WO 98/11230 to Bristol-Myers Squibb.

[0169] Rapamycin

[0170] Schwecke et al., August 1995, The biosynthetic gene cluster for the polyketide rapamycin, Proc. Natl. Acad. Sci. USA 92:7839-7843.

[0171] Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.

[0172] Rifamycin

[0173] PCT Pat. Pub. No. WO 98/07868 to Novartis.

[0174] August et al., 13 Feb. 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rifbiosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry & Biology, 5(2): 69-79.

[0175] Sorangium PKS

[0176] U.S. patent application Ser. No. 09/144,085, filed 31 Aug. 1998.

[0177] Soraphen

[0178] U.S. Pat. No. 5,716,849 to Novartis.

[0179] Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangium cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.

[0180] Spiramycin

[0181] U.S. Pat. No. 5,098,837 to Lilly.

[0182] Activator Gene

[0183] U.S. Pat. No. 5,514,544 to Lilly.

[0184] Tylosin

[0185] U.S. Pat. No. 5,876,991 to Lilly.

[0186] EP Pub. No. 791,655 to Lilly.

[0187] Kuhstoss et al., 1996, Gene 183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.

[0188] Tailoring Enzymes

[0189] Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355. Analysis of five tylosin biosynthetic genes from the tylBA region of the Streptomyces fradiae genome.

[0190] As the above Table illustrates, there are a wide variety of PKS genes that serve as readily available sources of DNA and sequence information for use in constructing the hybrid PKS-encoding DNA compounds of the invention. Methods for constructing hybrid PKS-encoding DNA compounds are described without reference to the epothilone PKS in U.S. Pat. Nos. 5,672,491 and 6,712,146 and U.S. patent application Ser. Nos. 09/073,538, filed 6 May 1998, and 09/141,908, filed 28 Aug. 1998, each of which is incorporated herein by reference. Preferred PKS enzymes and coding sequences for the proteins which constitute them for purposes of isolating heterologous PKS domain coding sequences for constructing hybrid PKS enzymes of the invention are the soraphen PKS and the PKS described as a Sorangium PKS in the above table.

[0191] To summarize the functions of the genes cloned and sequenced in Example 1:

Gene	Protein	Modules	Domains Present
epoA	EpoA	Load	Ksy mAT ER ACP
epoB	EpoB	1	NRPS, condensation, heterocyclization,
			adenylation, thiolation, PCP
epoC	EpoC	2	KS mmAT DH KR ACP
epoD	EpoD	3	KS mAT KR ACP
		4	KS mAT KR ACP
		5	KS mAT DH ER KR ACP
		6	KS mmAT DH ER KR ACP
epoE	EpoE	7	KS mmAT KR ACP
		8	KS mmAT MT DH* KR* ACP
epoF	EpoF	9	KS mAT KR DH* ACP TE
NRPS—non-ribosomal peptide systhetase;
KS—ketosynthase;
mAT—malonyl CoA specifying acyltransferase;
mmAT—methylmalonyl CoA specifying acyltransferase;
DH—dehydratase;
ER—enoylreductase;
KR—ketoreductase;
MT—methyltransferase;
TE—thioesterase;
*inactive domain.

[0192] The hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes. Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene. Illustrative examples of recombinant epothilone derivative PKS genes of the invention, which are identified by listing the specificities of the hybrid modules (the other modules having the same specificity as the epothilone PKS), include:

[0193] (a) module 4 with methylmalonyl specific AT (mm AT) and a KR and module 2 with a malonyl specific AT (m AT) and a KR;

[0194] (b) module 4 with mM AT and a KR and module 3 with mM AT and a KR;

[0195] (c) module 4 with mM AT and a KR and module 5 with mM AT and a ER,DH, and KR;

[0196] (d) module 4 with mM AT and a KR and module 5 with mM AT and a DH and KR;

[0197] (e) module 4 with mM AT and a KR and module 5 with mM AT and a KR;

[0198] (f) module 4 with mM AT and a KR and module 5 with mM AT and an inactive KR;

[0199] (g) module 4 with mM AT and a KR and module 6 with m AT and a ER, DH, and KR;

[0200] (h) module 4 with mM AT and a KR and module 6 with m AT and a DH and KR;

[0201] (i) module 4 with mM AT and a KR and module 6 with m AT and a KR;

[0202] (j) module 4 with mM AT and a KR and module 6 with m AT and an inactive KR;

[0203] (k) module 4 with mM AT and a KR and module 7 with m AT;

[0204] (l) hybrids (c) through (f), except that module 5 has a m AT;

[0205] (m) hybrids (g) through (j) except that module 6 has a mM AT; and

[0206] (n) hybrids (a) through (m) except that module 4 has a m AT.

[0207] The above list is illustrative only and should not be construed as limiting the invention, which includes other recombinant epothilone PKS genes and enzymes with not only two hybrid modules other than those shown but also with three or more hybrid modules.

[0208] Those of skill in the art will appreciate that a hybrid PKS of the invention includes but is not limited to a PKS of any of the following types: (i) an epothilone or epothilone derivative PKS that contains a module in which at least one of the domains is from a heterologous module; (ii) an epothilone or epothilone derivative PKS that contains a module from a heterologous PKS; (iii) an epothilone or epothilone derivative PKS that contains a protein from a heterologous PKS; and (iv) combinations of the foregoing.

[0209] While an important embodiment of the present invention relates to hybrid PKS genes, the present invention also provides recombinant epothilone PKS genes in which there is no second PKS gene sequence present but which differ from the epothilone PKS gene by one or more deletions. The deletions can encompass one or more modules and/or can be limited to a partial deletion within one or more modules. When a deletion encompasses an entire module other than the NRPS module, the resulting epothilone derivative is at least two carbons shorter than the compound produced from the PKS from which the deleted version was derived. The deletion can also encompass the NRPS module and/or the loading domain, as noted above. When a deletion is within a module, the deletion typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains.

[0210] The catalytic properties of the domains and modules of the epothilone PKS and of epothilone modification enzymes can also be altered by random or site specific mutagenesis of the corresponding genes. A wide variety of mutagenizing agents and methods are known in the art and are suitable for this purpose. The technique known as DNA shuffling can also be employed. See, e.g., U.S. Pat. Nos. 5,830,721; 5,811,238; and 5,605,793; and references cited therein, each of which is incorporated herein by reference.

[0211] Recombinant Manipulations

[0212] To construct a hybrid PKS or epothilone derivative PKS gene of the invention, or simply to express unmodified epothilone biosynthetic genes, one can employ a technique, described in PCT Pub. No. 98/27203 and U.S. patent application Ser. Nos. 08/989,332, filed 11 Dec. 1997, and 60/129,731, filed 16 April 1999, each of which is incorporated herein by reference, in which the various genes of the PKS are divided into two or more, often three, segments, and each segment is placed on a separate expression vector. In this manner, the full complement of genes can be assembled and manipulated more readily for heterologous expression, and each of the segments of the gene can be altered, and various altered segments can be combined in a single host cell to provide a recombinant PKS of the invention. This technique makes more efficient the construction of large libraries of recombinant PKS genes, vectors for expressing those genes, and host cells comprising those vectors. In this and other contexts, the genes encoding the desired PKS are not only present on two or more vectors, but also can be ordered or arranged differently than in the native producer organism from which the genes were derived. Various examples of this technique as applied to the epothilone PKS are described in the Examples below. In one embodiment, the epoa, epoB, epoC, and epoD genes are present on a first plasmid, and the epoE and epoF and optionally either the epoK or the epoK and epoL genes are present on a second (or third) plasmid.

[0213] Thus, in one important embodiment, the recombinant nucleic acid compounds of the invention are expression vectors. As used herein, the term “expression vector” refers to any nucleic acid that can be introduced into a host cell or cell-free transcription and translation medium. An expression vector can be maintained stably or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm. An expression vector also comprises a gene that serves to produce RNA that is translated into a polypeptide in the cell or cell extract. Thus, the vector typically includes a promoter to enhance gene expression but alternatively may serve to incorporate the relevant coding sequence under the control of an endogenous promoter. Furthermore, expression vectors may typically contain additional functional elements, such as resistance-conferring genes to act as selectable markers and regulatory genes to enhance promoter activity.

[0214] The various components of an expression vector can vary widely, depending on the intended use of the vector. In particular, the components depend on the host cell(s) in which the vector will be used or is intended to function. Vector components for expression and maintenance of vectors in E. coli are widely known and commercially available, as are vector components for other commonly used organisms, such as yeast cells and Streptomyces cells.

[0215] In one embodiment, the vectors of the invention are used to transform Sorangium host cells to provide the recombinant Sorangium host cells of the invention. U.S. Pat. No. 5,686,295, incorporated herein by reference, describes a method for transforming Sorangium host cells, although other methods may also be employed. Sorangium is a convenient host for expressing epothilone derivatives of the invention in which the recombinant PKS that produces such derivatives is expressed from a recombinant vector in which the epothilone PKS gene promoter is positioned to drive expression of the recombinant coding sequence. The epothilone PKS gene promoter is provided in recombinant form by the present invention and is an important embodiment thereof. The promoter is contained within an ˜500 nucleotide sequence between the end of the transposon sequences and the start site of the open reading frame of the epoA gene. Optionally, one can include sequences from further upstream of this 500 bp region in the promoter. Those of skill in the art will recognize that, if a Sorangium host that produces epothilone is used as the host cell, the recombinant vector need drive expression of only a portion of the PKS containing the altered sequences. Thus, such a vector may comprise only a single altered epothilone PKS gene, with the remainder of the epothilone PKS polypeptides provided by the genes in the host cell chromosomal DNA. If the host cell naturally produces an epothilone, the epothilone derivative will thus be produced in a mixture containing the naturally occurring epothilone(s).

[0216] Those of skill will also recognize that the recombinant DNA compounds of the invention can be used to construct Sorangium host cells in which one or more genes involved in epothilone biosynthesis have been rendered inactive. Thus, the invention provides such Sorangium host cells, which may be preferred host cells for expressing epothilone derivatives of the invention so that complex mixtures of epothilones are avoided. Particularly preferred host cells of this type include those in which one or more of any of the epothilone PKS gene ORFs has been disrupted, and/or those in which any or more of the epothilone modification enzyme genes have been disrupted. Such host cells are typically constructed by a process involving homologous recombination using a vector that contains DNA homologous to the regions flanking the gene segment to be altered and positioned so that the desired homologous double crossover recombination event desired will occur.

[0217] Homologous recombination can thus be used to delete, disrupt, or alter a gene. In a preferred illustrative embodiment, the present invention provides a recombinant epothilone producing Sorangium cellulosum host cell in which the epoK gene has been deleted or disrupted by homologous recombination using a recombinant DNA vector of the invention. This host cell, unable to make the epoK epoxidase gene product is unable to make epothilones A and B and so is a preferred source of epothilones C and D.

[0218] Homologous recombination can also be used to alter the specificity of a PKS module by replacing coding sequences for the module or domain of a module to be altered with those specifying a module or domain of the desired specificity. In another preferred illustrative embodiment, the present invention provides a recombinant epothilone producing Sorangium cellulosum host cell in which the coding sequence for the AT domain of module 4 encoded by the epoD gene has been altered by homologous recombination using a recombinant DNA vector of the invention to encode an AT domain that binds only methylmalonyl CoA. This host cell, unable to make epothilones A, C, and E is a preferred source of epothilones B, D, and F. The invention also provides recombinant Sorangium host cells in which both alterations and deletions of epothilone biosynthetic genes have been made. For example, the invention provides recombinant Sorangium cellulosum host cells in which both of the foregoing alteration and deletion have been made, producing a host cell that makes only epothilone D.

[0219] In similar fashion, those of skill in the art will appreciate the present invention provides a wide variety of recombinant Sorangium cellulosum host cells that make less complex mixtures of the epothilones than do the wild type producing cells as well as those that make one or more epothilone derivatives. Such host cells include those that make only epothilones A, C, and E; those that make only epothilones B, D, and F, those that make only epothilone D; and those that make only epothilone C.

[0220] In another preferred embodiment, the present invention provides expression vectors and recombinant Myxococcus, preferably M. xanthus, host cells containing those expression vectors that express a recombinant epothilone PKS or a PKS for an epothilone derivative. Presently, vectors that replicate extrachromosomally in M. xanthus are not known. There are, however, a number of phage known to integrate into M. xanthus chromosomal DNA, including Mx8, Mx9, Mx81, and Mx82. The integration and attachment function of these phages can be placed on plasmids to create phage-based expression vectors that integrate into the M. xanthus chromosomal DNA. Of these, phage Mx9 and Mx8 are preferred for purposes of the present invention. Plasmid pPLH343, described in Salmi et al., February 1998, Genetic determinants of immunity and integration of temperate Myxococcus xanthus phage Mx8, J. Bact. 180(3): 614-621, is a plasmid that replicates in E. coli and comprises the phage Mx8 genes that encode the attachment and integration functions.

[0221] The promoter of the epothilone PKS gene functions in Myxococcus xanthus host cells. Thus, in one embodiment, the present invention provides a recombinant promoter for use in recombinant host cells derived from the promoter of the Sorangium cellulosum epothilone PKS gene. The promoter can be used to drive expression of one or more epothilone PKS genes or another useful gene product in recombinant host cells. The invention also provides an epothilone PKS expression vector in which one or more of the epothilone PKS or epothilone modification enzyme genes are under the control of their own promoter. Another preferred promoter for use in Myxococcus xanthus host cells for purposes of expressing a recombinant PKS of the invention is the promoter of the pilA gene of M. xanthus. This promoter, as well as two M. xanthus strains that express high levels of gene products from genes controlled by the pilA promoter, a pilA deletion strain and a pilS deletion strain, are described in Wu and Kaiser, December 1997, Regulation of expression of the pilA gene in Myxococcus xanthus, J. Bact. 179(24):7748-7758, incorporated herein by reference. Optionally, the invention provides recombinant Myxococcus host cells comprising both the pilA and pilS deletions. Another preferred promoter is the starvation dependent promoter of the sdcK gene.

[0222] Selectable markers for use in Myxococcus xanthus include kanamycin, tetracycline, chloramphenicol, zeocin, spectinomycin, and streptomycin resistance conferring genes. The recombinant DNA expression vectors of the invention for use in Myxococcus typically include such a selectable marker and may further comprise the promoter derived from an epothilone PKS or epothilone modification enzyme gene.

[0223] The present invention provides preferred expression vectors for use in preparing the recombinant Myxococcus xanthus expression vectors and host cells of the invention. These vectors, designated plasmids pKOS35-82.1 and pKOS35-82.2 (FIG. 3), are able to replicate in E. coli host cells as well as integrate into the chromosomal DNA of M. xanthus . The vectors comprise the Mx8 attachment and integration genes as well as the pilA promoter with restriction enzyme recognition sites placed conveniently downstream. The two vectors differ from one another merely in the orientation of the pilA promoter on the vector and can be readily modified to include the epothilone PKS and modification enzyme genes of the invention. The construction of the vectors is described in Example 2.

[0224] Especially preferred Myxococcus host cells of the invention are those that produce an epothilone or epothilone derivative or mixtures of epothilones or epothilone derivatives at equal to or greater than 20 mg/L, more preferably at equal to or greater than 200 mg/L, and most preferably at equal to or greater than 1 g/L. Especially preferred are M. xanthus host cells that produce at these levels. M. xanthus host cells that can be employed for purposes of the invention include the DZ1 (Campos et al., 1978, J. Mol. Biol. 119: 167-178, incorporated herein by reference), the TA-producing cell line ATCC 31046, DK1219 (Hodgkin and Kaiser, 1979, Mol. Gen. Genet. 171: 177-191, incorporated herein by reference), and the DK1622 cell lines (Kaiser, 1979, Proc. Natl. Acad. Sci. USA 76: 5952-5956, incorporated herein by reference).

[0225] In another preferred embodiment, the present invention provides expression vectors and recombinant Pseudomonas fluorescens host cells that contain those expression vectors and express a recombinant PKS of the invention. A plasmid for use in constructing the P. fluorescens expression vectors and host cells of the invention is plasmid pRSF1010, which replicates in E. coli and P. fluorescens host cells (see Scholz et al., 1989, Gene 75:271-8, incorporated herein by reference). Low copy number replicons and vectors can also be used. As noted above, the invention also provides the promoter of the Sorangium cellulosum epothilone PKS and epothilone modification enzyme genes in recombinant form. The promoter can be used to drive expression of an epothilone PKS gene or other gene in P. fluorescens host cells. Also, the promoter of the soraphen PKS genes can be used in any host cell in which a Sorangium promoter functions. Thus, in one embodiment, the present invention provides an epothilone PKS expression vector for use in P. fluorescens host cells.

[0226] In another preferred embodiment, the expression vectors of the invention are used to construct recombinant Streptomyces host cells that express a recombinant PKS of the invention. Streptomyces host cells useful in accordance with the invention include S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. fradiae, and the like. Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and S. lividans K4-114 and K4-155 host cells, which do not produce actinorhodin, and expression vectors derived from the pRM1 and pRM5 vectors, as described in U.S. Pat. No. 5,830,750 and U.S. patent application Ser. Nos. 08/828,898, filed 31 Mar. 1997, and 09/181,833, filed 28 Oct. 1998. Especially preferred Streptomyces host cells of the invention are those that produce an epothilone or epothilone derivative or mixtures of epothilones or epothilone derivatives at equal to or greater than 20 mg/L, more preferably at equal to or greater than 200 mg/L, and most preferably at equal to or greater than 1 g/L. Especially preferred are S. coelicolor and S. lividans host cells that produce at these levels. Also, species of the closely related genus Saccharopolyspora can be used to produce epothilones, including but not limited to S. erythraea.

[0227] The present invention provides a wide variety of expression vectors for use in Streptomyces. For replicating vectors, the origin of replication can be, for example and without limitation, a low copy number replicon and vectors comprising the same, such as SCP2* (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, U.K., 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51-62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol. Gen. Genet. 219: 341-348, and Bierman et al., 1992, Gene 116: 43-49, each of which is incorporated herein by reference), or a high copy number replicon and vectors comprising the same, such as pIJ101 and pJV1 (see Katz et al., 1983, J. Gen. Microbiol. 129: 2703-2714; Vara et al., 1989, J. Bacteriol. 171: 5782-5781; and Servin-Gonzalez, 1993, Plasmid 30: 131-140, each of which is incorporated herein by reference). High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes. For non-replicating and integrating vectors and generally for any vector, it is useful to include at least an E. coli origin of replication, such as from pUC, p1P, p1I, and pBR. For phage based vectors, the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra). Also, plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211, all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.

[0228] Typically, the expression vector will comprise one or more marker genes by which host cells containing the vector can be identified and/or selected. Useful antibiotic resistance conferring genes for use in Streptomyces host cells include the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes.

[0229] The recombinant PKS gene on the vector will be under the control of a promoter, typically with an attendant ribosome binding site sequence. A preferred promoter is the actI promoter and its attendant activator gene actII-ORF4, which is provided in the pRM1 and pRM5 expression vectors, supra. This promoter is activated in the stationary phase of growth when secondary metabolites are normally synthesized. Other useful Streptomyces promoters include without limitation those from the ernE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage. In addition, the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention. In this system, the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA promoter, and the gene of interest is placed under the control of the T7 promoter. As noted above, one or more activator genes can also be employed to enhance the activity of a promoter. Activator genes in addition to the actII-ORF4 gene discussed above include dnrI, redD, and ptpA genes (see U.S. patent application Ser. No. 09/181,833, supra), which can be employed with their cognate promoters to drive expression of a recombinant gene of the invention.

[0230] The present invention also provides recombinant expression vectors that drive expression of the epothilone PKS and PKS enzymes that produce epothilone or epothilone derivatives in plant cells. Such vectors are constructed in accordance with the teachings in U.S. patent application Ser. No. 09/114,083, filed 10 July 1998, and PCT patent publication No. 99/02669, each of which is incorporated herein by reference. Plants and plant cells expressing epothilone are disease resistant and able to resist fungal infection. For improved production of an epothilone or epothilone derivative in any heterologous host cells, including plant, Myxococcus, Pseudomonas, and Streptomyces host cells, one can also transform the cell to express a heterologous phosphopantetheinyl transferase. See U.S. patent application Ser. No. 08/728,742, filed 11 Oct. 1996, and PCT patent publication No. 97/13845, both of which are incorporated herein by reference.

[0231] In addition to providing recombinant expression vectors that encode the epothilone or an epothilone derivative PKS, the present invention also provides, as discussed above, DNA compounds that encode epothilone modification enzyme genes. As discussed above, these gene products convert epothilones C and D to epothilones A and B, and convert epothilones A and B to epothilones E and F. The present invention also provides recombinant expression vectors and host cells transformed with those vectors that express any one or more of those genes and so produce the corresponding epothilone or epothilone derivative. In one aspect, the present invention provides the epoK gene in recombinant form and host cells that express the gene product thereof, which converts epothilones C and D to epothilones A and B, respectively.

[0232] In another important embodiment, and as noted above, the present invention provides vectors for disrupting the function of any one or more of the epoL, epoK, and any of the ORFs associated with the epothilone PKS gene cluster in Sorangium cells. The invention also provides recombinant Sorangium host cells lacking (or containing inactivated forms of) any one or more of these genes. These cells can be used to produce the corresponding epothilones and epothilone derivatives that result from the absence of any one or more of these genes.

[0233] The invention also provides non-Sorangium host cells that contain a recombinant epothilone PKS or a PKS for an epothilone derivative but do not contain (or contain non-functional forms of) any epothilone modification enzyme genes. These host cells of the invention are expected produce epothilones G and H in the absence of a dehydratase activity capable of forming the C-12-C-13 alkene of epothilones C and D. This dehydration reaction is believed to take place in the absence of the epoL gene product in Streptomyces host cells. The host cells produce epothilones C and D (or the corresponding epothilone C and D derivative) when the dehydratase activity is present and the P450 epoxidase and hydroxylase (that converts epothilones A and B to epothilones E and F, respectively) genes are absent. The host cells also produce epothilones A and B (or the corresponding epothilone A and B derivatives) when the hydroxylase gene only is absent. Preferred for expression in these host cells is the recombinant epothilone PKS enzymes of the invention that contain the hybrid module 4 with an AT specific for methylmalonlyl CoA only, optionally in combination with one or more additional. hybrid modules. Also preferred for expression in these host cells is the recombinant epothilone PKS enzymes of the invention that contain the hybrid module 4 with an AT specific for malonyl CoA only, optionally in combination with one or more additional hybrid modules.

[0234] The recombinant host cells of the invention can also include other genes and corresponding gene products that enhance production of a desired epothilone or epothilone derivative. As but one non-limiting example, the epothilone PKS proteins require phosphopantetheinylation of the ACP domains of the loading domain and modules 2 through 9 as well as of the PCP domain of the NRPS. Phosphopantethein-ylation is mediated by enzymes that are called phosphopantetheinyl transferases (PPTases). To produce functional PKS enzyme in host cells that do not naturally express a PPTase able to act on the desired PKS enzyme or to increase amounts of functional PKS enzyme in host cells in which the PPTase is rate-limiting, one can introduce a heterologous PPTase, including but not limited to Sfp, as described in PCT Pat. Pub. Nos. 97/13845 and 98/27203, and U.S. patent application Ser. Nos. 08/728,742, filed 11 Oct. 1996, and 08/989,332, each of which is incorporated herein by reference.

[0235] The host cells of the invention can be grown and fermented under conditions known in the art for other purposes to produce the compounds of the invention. The compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures. Fermentation conditions for producing the compounds of the invention from Sorangium host cells can be based on the protocols described in PCT patent publication Nos. 93/10121, 97/19086, 98/22461, and 99/42602, each of which is incorporated herein by reference. The novel epothilone analogs of the present invention, as well as the epothilones produced by the host cells of the invention, can be derivatized and formulated as described in PCT patent publication Nos. 93/10121, 97/19086, 98/08849, 98/22461, 98/25929, 99/01124, 99/02514, 99/07692, 99/27890, 99/39694, 99/40047, 99/42602, 99/43653, 99/43320, 99/54319, 99/54319, and 99/54330, and U.S. Pat. No. 5,969,145, each of which is incorporated herein by reference.

[0236] Invention Compounds

[0237] Preferred compounds of the invention include the 14-methyl epothilone derivatives (made by utilization of the hybrid module 3 of the invention that has an AT that binds methylmalonyl CoA instead of malonyl CoA); the 8,9-dehydro epothilone derivatives (made by utilization of the hybrid module 6 of the invention that has a DH and KR instead of an ER, DH, and KR); the 10-methyl epothilone derivatives (made by utilization of the hybrid module 5 of the invention that has an AT that binds methylmalonyl CoA instead of malonyl CoA); the 9-hydroxy epothilone derivatives (made by utilization of the hybrid module 6 of the invention that has a KR instead of an ER, DH, and KR); the 8-desmethyl-14-methyl epothilone derivatives (made by utilization of the hybrid module 3 of the invention that has an AT that binds methylmalonyl CoA instead of malonyl CoA and a hybrid module 6 that binds malonyl CoA instead of methylmalonyl CoA); and the 8-desmethyl-8,9-dehydro epothilone derivatives (made by utilization of the hybrid module 6 of the invention that has a DH and KR instead of an ER, DH, and KR and an AT that specifies malonyl CoA instead of methylmalonyl CoA).

[0238] More generally, preferred epothilone derivative compounds of the invention are those that can be produced by altering the epothilone PKS genes as described herein and optionally by action of epothilone modification enzymes and/or by chemically modifying the resulting epothilones produced when those genes are expressed. Thus, the present invention provides compounds of the formula: 7

[0239] including the glycosylated forms thereof and stereoisomeric forms where the stereochemistry is not shown,

[0240] wherein A is a substituted or unsubstituted straight, branched chain or cyclic alkyl, alkenyl or alkynyl residue optionally containing 1-3 heteroatoms selected from O, S and N; or wherein A comprises a substituted or unsubstituted aromatic residue;

[0241] R2 represents H,H, or H,lower alkyl, or lower alkyl,lower alkyl;

[0242] X5 represents ═O or a derivative thereof, or H,OH or H,NR2 wherein R is H, or alkyl, or acyl or H,OCOR or H,OCONR2 wherein R is H, or alkyl, or is H,H;

[0243] R6 represents H or lower alkyl, and the remaining substituent on the corresponding carbon is H;

[0244] X7 represents OR, NR2, wherein R is H, or alkyl or acyl or is OCOR, or OCONR2 wherein R is H or alkyl or X7 taken together with X9 forms a carbonate or carbamate cycle, and wherein the remaining substituent on the corresponding carbon is H;

[0245] R8 represents H or lower alkyl and the remaining substituent on the carbon is H;

[0246] X9 represents ═O or a derivative thereof, or is H,OR or H,NR2, wherein R is H, or alkyl or acyl or is H,OCOR or H,OCONR2 wherein R is H or alkyl, or represents H,H or wherein X9 together with X7 or with X11 can form a cyclic carbonate or carbamate;

[0247] R10 is H,H or H,lower alkyl, or lower alkyl,lower alkyl;

[0248] X11 is ═O or a derivative thereof, or is H,OR, or H,NR2 wherein R is H, or alkyl or acyl or is H,OCOR or H,OCONR2 wherein R is H or alkyl, or is H,H or wherein X11 in combination with X9 may form a cyclic carbonate or carbamate;

[0249] R12 is H,H, or H,lower alkyl, or lower alkyl,lower alkyl;

[0250] X13 is ═O or a derivative thereof, or H,OR or H,NR2 wherein R is H, alkyl or acyl or is H,OCOR or H,OCONR2 wherein R is H or alkyl;

[0251] R14 is H,H, or H,lower alkyl, or lower alkyl,lower alkyl;

[0252] R16 is H or lower alkyl; and

[0253] wherein optionally H or another substituent may be removed from positions 12 and 13 and/or 8 and 9 to form a double bond, wherein said double bond may optionally be converted to an epoxide.

[0254] Particularly preferred are compounds of the formulas 8

[0255] wherein the noted substituents are as defined above.

[0256] Especially preferred are compounds of the formulas 9

[0257] wherein both Z are O or one Z is N and the other Z is O, and the remaining substituents are as defined above.

[0258] As used herein, a substituent which “comprises an aromatic moiety” contains at least one aromatic ring, such as phenyl, pyridyl, pyrimidyl, thiophenyl, or thiazolyl. The substituent may also include fused aromatic residues such as naphthyl, indolyl, benzothiazolyl, and the like. The aromatic moiety may also be fused to a nonaromatic ring and/or may be coupled to the remainder of the compound in which it is a substituent through a nonaromatic, for example, alkylene residue. The aromatic moiety may be substituted or unsubstituted as may the remainder of the substituent.

[0259] Preferred embodiments of A include the “R” groups shown in FIG. 2.

[0260] As used herein, the term alkyl refers to a C1-C8 saturated, straight or branched chain hydrocarbon radical derived from a hydrocarbon moiety by removal of a single hydrogen atom. Alkenyl and alkynyl refer to the corresponding unsaturated forms. Examples of alkyl include but are not limited to methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, i-hexyl, n-heptyl, n-octyl. Lower alkyl (or alkenyl or alkynyl) refers to a 1-4C radical. Methyl is preferred. Acyl refers to alkylCO, alkenylCO or alkynylCO.

[0261] The terms halo and halogen as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine. The term haloalkyl as used herein denotes an alkyl group to which one, two, or three halogen atoms are attached to any one carbon and includes without limitation chloromethyl, bromoethyl, trifluoromethyl, and the like.

[0262] The term heteroaryl as used herein refers to a cyclic aromatic radical having from five to ten ring atoms of which one ring atom is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon, the radical being joined to the rest of the molecule via any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

[0263] The term heterocyle includes but is not limited to pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuiryl.

[0264] The term “substituted” as used herein refers to a group substituted by independent replacement of any of the hydrogen atoms thereon with, for example, Cl, Br, F, I, OH, CN, alkyl, alkoxy, alkoxy substituted with aryl, haloalkyl, alkylthio, amino, alkylamino, dialkylamino, mercapto, nitro, carboxaldehyde, carboxy, alkoxycarbonyl, or carboxamide. Any one substituent may be an aryl, heteroaryl, or heterocycloalkyl group.

[0265] It will apparent that the nature of the substituents at positions 2, 4, 6, 8, 10, 12, 14 and 16 in formula (1) is determined at least initially by the specificity of the AT catalytic domain of modules 9, 8, 7, 6, 5, 4, 3 and 2, respectively. Because AT domains that accept malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA (and in general, lower alkyl malonyl CoA), as well as hydroxymalonyl CoA, are available, one of the substituents at these positions may be H, and the other may be H, lower alkyl, especially methyl and ethyl, or OH. Further reaction at these positions, e.g., a methyl transferase reaction such as that catalyzed by module 8 of the epothilone PKS, may be used to replace H at these positions as well. Further, an H,OH embodiment may be oxidized to ═O or, with the adjacent ring C, be dehydrated to form a π-bond. Both OH and ═O are readily derivatized as further described below.

[0266] Thus, a wide variety of embodiments of R2, R6, R8, R10, R12, R14 and R16 is synthetically available. The restrictions set forth with regard to embodiments of these substituents set forth in the definitions with respect to Formula (1) above reflect the information described in the SAR description in Example 8 below.

[0267] Similarly, β-carbonyl modifications (or absence of modification) can readily be controlled by modifying the epothilone PKS gene cluster to include the appropriate sequences in the corresponding positions of the epothilone gene cluster which will or will not contain active KR, DH and/or ER domains. Thus, the embodiments of X5, X7, X9, X11 and X13 synthetically available are numerous, including the formation of π-bonds with the adjacent ring positions.

[0268] Positions occupied by OH are readily converted to ethers or esters by means well known in the art; protection of OH at positions not to be derivatized may be required. Further, a hydroxyl may be converted to a leaving group, such as a tosylate, and replaced by an amino or halo substituent. A wide variety of “hydroxyl derivatives” such as those discussed above is known in the art.

[0269] Similarly, ring positions which contain oxo groups may be converted to “carbonyl derivatives” such as oximes, ketals, and the like. Initial reaction products with the oxo moieties may be further reacted to obtain more complex derivatives. As described in Example 8, such derivatives may ultimately result in a cyclic substituent linking two ring positions.

[0270] The enzymes useful in modification of the polyketide initially synthesized, such as transmethylases, dehydratases, oxidases, glycosylation enzymes and the like, can be supplied endogenously by a host cell when the polyketide is synthesized intracellularly, by modifying a host to contain the recombinant materials for the production of these modifying enzymes, or can be supplied in a cell-free system, either in purified forms or as relatively crude extracts. Thus, for example, the epoxidation of the π-bond at position 12-13 may be effected using the protein product of the epoK gene directly in vitro.

[0271] The nature of A is most conveniently controlled by employing an epothilone PKS which comprises an inactivated module 1 NRPS (using a module 2 substrate) or a KS2 knockout (using a module 3 substrate) as described in Example 6, hereinbelow. Limited variation can be obtained by altering the AT catalytic specificity of the loading module; further variation is accomplished by replacing the NRPS of module 1 with an NRPS of different specificity or with a conventional PKS module. However, at present, variants are more readily prepared by feeding the synthetic module 2 substrate precursors and module 3 substrate precursors to the appropriately altered epothilone PKS as described in Example 6.

[0272] Pharmaceutical Compositions

[0273] The compounds can be readily formulated to provide the pharmaceutical compositions of the invention. The pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use.

[0274] The carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used. For example, the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat. No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference.

[0275] Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference. Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference. The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.

[0276] For the treatment of conditions and diseases caused by infection, immune system disorder (or to suppress immune function), or cancer, a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intrathecal, intramuscular, and intrasternal injection or infusion techniques.

[0277] Dosage levels of the compounds of the present invention are of the order from about 0.01 mg to about 100 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 50 mg per kilogram of body weight per day. The dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient). In addition, the compounds of the present invention may be administered on an intermittent basis, i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.

[0278] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain from 0.5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition. Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient. For external administration, the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from about 0.005% to 0.8% by weight.

[0279] It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.

[0280] A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the present invention and shall not be construed as being a limitation on the scope of the invention or claims.

EXAMPLE 1

DNA Sequencing of Cosmid Clones and Subclones Thereof

[0281] The epothilone producing strain, Sorangium cellulosum SMP44, was grown on a cellulose-containing medium, see Bollag et al., 1995, Cancer Research 55: 2325-2333, incorporated herein by reference, and epothilone production was confirmed by LC/MS analysis of the culture supernatant. Total DNA was prepared from this strain using the procedure described by Jaoua et al., 1992, Plasmid 28: 157-165, incorporated herein by reference. To prepare a cosmid library, S. cellulosum genomic DNA was partially digested with Sau3AI and ligated with BaniHI-digested pSupercos (Stratagene). The DNA was packaged in lambda phage as recommended by the manufacturer and the mixture then used to infect E. coli XL1-Blue MR cells. This procedure yielded approximately 3,000 isolated colonies on LB-ampicillin plates. Because the size of the S. cellulosum genome is estimated to be circa 107 nucleotides, the DNA inserts present among 3000 colonies would correspond to circa 10 S. cellulosum genomes.

[0282] To screen the library, two segments of KS domains were used to design oligonucleotide primers for a PCR with Sorangium cellulosum genomic DNA as template. The fragment generated was then used as a probe to screen the library. This approach was chosen, because it was found, from the examination of over a dozen PKS genes, that KS domains are the most highly conserved (at the amino acid level) of all the PKS domains examined. Therefore, it was expected that the probes produced would detect not only the epothilone PKS genes but also other PKS gene clusters represented in the library. The two degenerate oligonucleotides synthesized using conserved regions within the ketosynthase (KS) domains compiled from the DEBS and soraphen PKS gene sequences were (standard nomenclature for degenerate positions is used): CTSGTSKCSSTBCACCTSGCSTGC (SEQ ID NO: 21) and TGAYRTGSGCGTTSGTSCCGSWGA (SEQ ID NO: 22). A single band of ˜750 bp, corresponding to the predicted size, was seen in an agarose gel after PCR employing the oligos as primers and S. cellulosum SMP44 genomic DNA as template. The fragment was removed from the gel and cloned in the HincII site of pUC118 (which is a derivative of pUC18 with an insert sequence for making single stranded DNA). After transformation of E. coli, plasmid DNA from ten independent clones was isolated and sequenced. The analysis revealed nine unique sequences that each corresponded to a common segment of KS domains in PKS genes. Of the nine, three were identical to a polyketide synthase gene cluster previously isolated from this organism and determined not to belong to the epothilone gene cluster from the analysis of the modules. The remaining six KS fragments were excised from the vector, pooled, end-labeled with 32P and used as probe in hybridizations with the colonies containing the cosmid library under high stringency conditions.

[0283] The screen identified 15 cosmids that hybridized to the pooled KS probes. DNA was prepared from each cosmid, digested with NotI, separated on an agarose gel, and transferred to a nitrocellulose membrane for Southern hybridization using the pooled KS fragments as probe. The results revealed that two of the cosmids did not contain KS-hybridizing inserts, leaving 13 cosmids to analyze further. The blot was stripped of the label and re-probed , under less stringent conditions, with labeled DNA containing the sequence corresponding to the enoylreductase domain from module four of the DEBS gene cluster. Because it was anticipated that the epothilone PKS gene cluster would encode two consecutive modules that contain an ER domain, and because not all PKS gene clusters have ER domain containing modules, hybridization with the ER probe was predicted to identify cosmids containing insert DNA from the epothilone PKS gene cluster. Two cosmids were found to hybridize strongly to the ER probe, one hybridized moderately, and a final cosmid hybridized weakly. Analysis of the restriction pattern of the NotI fragments indicated that the two cosmids that hybridized strongly with the ER probe overlapped one another. The nucleotide sequence was also obtained from the ends of each of the 13 cosmids using the T7 and T3 primer binding sites. All contained sequences that showed homology to PKS genes. In FIG. 1, the T7 site is to the left side of cosmid 8A3 and to the right side of cosmids 1A2 and 4. The PKS gene sequence is to the left of cosmid 1A2, because the sequences generated from the left of cosmid 8A3 are non-PKS sequences. Sequence from one of the cosmids that hybridized strongly to the ER probe showed homology to NRPs and, in particular, to the adenylation domain of an NRPS. Because it was anticipated that the thiazole moiety of epothilone might be derived from the formation of an amide bond between an acetate and cysteine molecule (with a subsequent cyclization step), the presence of an NRPS domain in a cosmid that also contained ER domain(s) supported the prediction that this cosmid might contain all or part of the epothilone PKS gene cluster.

[0284] Preliminary restriction analysis of the 12 remaining cosmids suggested that three might overlap with the cosmid of interest. To verify this, oligonucleotides were synthesized for each end of the four cosmids (determined from the end sequencing described above) and used as primer sets in PCRs with each of the four cosmid DNAs. Overlap would be indicated by the appearance of a band from a non-cognate primer-template reaction. The results of this experiment verified that two of the cosmids overlapped with the cosmid containing the NRPS. Restriction mapping of the three cosmids revealed that the cosmids did, in fact, overlap. Furthermore, because PKS sequences extended to the end of the insert in the last overlapping fragment, based on the assumption that the NRPS would map to the 5′-end of the cluster, the results also indicated that the 3′ end of the gene cluster had not been isolated among the clones identified.

[0285] To isolate the remaining segment of the epothilone biosynthesis genes, a PCR fragment was generated from the cosmid containing the most 3′-terminal region of the putative gene cluster. This fragment was used as a probe to screen a newly prepared cosmid library of Sorangium cellulosum genomic DNA of again approximately 3000 colonies. Several hybridizing clones were identified; DNA was made from six of them. Analysis of NotI-digested fragments indicated that all contained overlapping regions. The cosmid containing the largest insert DNA that also had the shortest overlap with the cosmid used to make the probe was selected for further analysis.

[0286] Restriction maps were created for the four cosmids, as shown in FIG. 1. Sequence obtained from one of the ends of cosmid pKOS35-70.8A3 showed no homology to PKS sequences or any associated modifying enzymes. Similarly, sequence from one end of cosmid pKOS35-79.85 also did not contain sequences corresponding to a PKS region. These findings supported the observation that the epothilone cluster was contained within the ˜70 kb region encompassed by the four cosmid inserts.

[0287] (A) The right (T3) side of cosmid 8A3 was sequenced, and the complement of the sequence generated is shown below, followed by the encoded amino acid sequence, which is from a ketosynthase domain.

[0288] T3 end Reverse and complement from the original sequence

TCCTGGGTCTGCACGGCCCGACGCTGGCCATGGATACGGCGTGCTCGTCCTCCCTGGTC (SEQ ID NO: 23)
GCGCTGCACCTCGCCTGCCAGAGCCTGCGACTGGGCGAGTGCGATCAAGCGCTGGTTG
GCGGGGTCAACGTGCTGCTCGCGCCGGAGACCTTCGTGCTGCTCTCACGGATGCGCGCG
CTTTCGCCCGACGGGCGGTGCAAGACGTTCTCGGCCGACGCGGACGGCTACGCGCGGG
GCGAGGGGTGCGCCGTGGTGGTGCTCAAGCGGCTGCGCAATGCGCAGCGCGCTCGGCG.

[0289] Protein translation

(SEQ ID NO: 24)
Leu Gly Leu His Gly Pro Thr Leu Ala Met Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Leu His Leu
Ala Cys Gln Ser Leu Arg Leu Gly Glu Cys Asp Gln Ala Leu Val Gly Gly Val Asn Val Leu Leu Ala
Pro Glu Thr Phe Val Leu Leu Ser Arg Met Arg Ala Leu Ser Pro Asp Gly Arg Cys Lys Thr Phe Ser
Ala Asp Ala Asp Gly Tyr Ala Arg Gly Glu Gly Cys Ala Val Val Val Leu Lys Arg Leu Arg Asn Ala
Gln Arg Ala Arg.

[0290] (B) The left (T3) side of cosmid 1A2 was sequenced, and the sequence generated is shown below, followed by the encoded amino acid sequence, which is from a ketosynthase domain.

[0291] T3 end

CATAGATCGTAAGCTGTGCTAGTGATCTGCCTTACGTTACGTCTTCCGCACCTCGAGCG (SEQ ID NO: 25)
AATTCTCTCGGATAACTTTCAAGTTTTCTGAGGGGGCTTGGTCTCTGGTTCCTCAGGAAG
CCTGATCGGGACGAGCTAATTCCCATCCATTTTTTTGAGACTCTGCTCAAAGGGATTAG
ACCGAGTGAGACAGTTCTTTTGCAGTGAGCGAAGAACCTGGGGCTCGACCGGAGGACG
ATCGACGTCCGCGAGCGGGTCAGCCGCTGAGGATGTGCCCGTCGTGGCGGATCGTCCC
ATCGAGCGCGCAGCCGAAGATCCGATTGCGATCGTCGGAGCGGGCTGCCGTCTGCCCG
GTGGCGTGATCGATCTGAGCGGGTTCTGGACGCTCCTCGAGGGCTCGCGCGACACCGTC
GGGCAAGTCCCCGCCGAACGCTGG

[0292] Protein translation

(SEQ ID NO: 26)
His Arg Ser STP Ala Val Leu Val Ile Cys Leu Thr Leu Arg Leu Pro His Leu Glu Arg Ile Leu
Ser Asp Asn Phe Gln Val Phe STP
(SEQ ID NO: 27)
Gly Gly Leu Val Ser Gly Ser Ser Gly Ser Leu Ile Gly Thr Ser STP
(SEQ ID NO: 28)
Phe Pro Ser Ile Phe Leu Arg Leu Cys Ser Lys Gly Leu Asp Arg Val Arg Gln Phe Phe Cys Ser Glu
Arg Arg Thr Trp Gly Ser Thr Gly Gly Arg Ser Thr Ser Ala Ser Gly Ser Ala Ala Glu Asp Val Pro
Val Val Ala Asp Arg Pro Ile Glu Arg Ala Ala Glu Asp Pro Ile Ala Ile Val Gly Ala Gly Cys Arg Leu
Pro Gly Gly Val Ile Asp Leu Ser Gly Phe Trp Thr Leu Leu Glu Gly Ser Arg Asp Thr Val Gly Gln
Val Pro Ala Glu Arg Trp.

[0293] (C) The right (T7) side of cosmid 1A2 was sequenced, and the complement of the sequence generated is shown below, followed by the encoded amino acid sequence, which is from an acyltransferase-ketoreductase domain junction.

[0294] T7 end Reverse and complement from the original sequence

GACGCGCGGACTTTCCTGCACGGTGCTTCATGCGTCGGCTGACGCCTCCACCGTCGCCG (SEQ ID NO: 29)
AGCAGGTATCCGAAGCTGCCAGTCGCCGAAACGACTGGCAGGGAGTCCTCTACCTGTG
GGGCCTCGACGCCGTCGTCGATGCTGGGGCATCGGCCGACGAAGTCAGCGAGGCTACC
CGCCGTGCCACCGCACCCGTCCTTGGGCTGGTTCGATTCCTGAGCGCTGCGCCCCATCC
TCCTCGCTTCTGGGTGGTGACCCGCGGGGCATGCACGGTGGGCGGCGAGCCAGAGGTC
TCTCTTTGCCAAGCGGCGTTGTGGGGCCTCGCGCGCGTCGTGGCGCTGGAGCATCCCGC
TGCCTGTGGGTGGCC.

[0295] Protein translation

(SEQ ID NO: 30
Thr Arg Gly Leu Ser Cys Thr Val Leu His Ala Ser Ala Asp Ala Ser Thr Val Ala Glu Gln Val Ser
Glu Ala Ala Ser Arg Arg Asn Asp Trp Gln Gly Val Leu Tyr Leu Trp Gly Leu Asp Ala Val Val Asp
Ala Gly Ala Ser Ala Asp Glu Val Ser Glu Ala Thr Arg Arg Ala Thr Ala Pro Val Leu Gly Leu Val
Arg Phe Leu Ser Ala Ala Pro His Pro Pro Arg Phe Trp Val Val Thr Arg Gly Ala Cys Thr Val Gly
Gly Glu Pro Glu Val Ser Leu Cys Gln Ala Ala Leu Trp Gly Leu Ala Arg Val Val Ala Leu Glu His
Pro Ala Ala Cys Gly Trp.

[0296] (D) The left (T3) side of cosmid 4 was sequenced, and the sequence generated is shown below, followed by the encoded amino acid sequence, which is from an adenylation domain of the non-ribosomal peptide synthase portion of the epothilone PKS.

[0297] T3 end.

CGTCCAGCCTGCGCGATCCGGCGCATTGGGCGCGTTGATCGAACGAGAGAAGGTGACG (SEQ ID NO: 31)
GTGTGGAACTCGGTGCCGGCGCTGATGCGGATGCTCGTCGAGCATTCCGAGGGTCGCCC
CGATTCGCTCGCTAGGTCTCCTGCGGCTTTCGCTGCTGAGCGGCGACTGGATCCCGGTG
GGCCTGCCTGGCGAGCTCCAGGCCATCAGGCCCGGCGTGTCGGTGATCAGCCTGGGCG
GGGCCACCGAAGCGTCGATCTGGTCCATCGGGTACCCCGTGAGGAACGTCGATCCATC
GTGGGCGAGCATCCCCTACGGCCGTCCGCTGCGCAACCAGACGTTCCACGTGCTCGATG
AGGCGCTCGAACCGCGCCCGGTCTGGGTTCCGGGGCAACTCTACATTGGCGGGGTCGG
ACTGGCACTGGGCTACTGGCGCGATGAAGAGAAGACGCGCACAGCT.

[0298] Protein translation

(SEQ ID NO: 32)
Val Gln Pro Ala Arg Ser Gly Ala Leu Gly Ala Leu Ile Glu Arg Glu Lys Val Thr Val Trp Asn Ser
Val Pro Ala Leu Met Arg Met Leu Val Glu His Ser Glu Gly Arg Pro Asp Ser Leu Ala Arg Ser Leu
Arg Leu Ser Leu Leu Ser Gly Asp Trp Ile Pro Val Gly Leu Pro Gly Glu Leu Gln Ala Ile Arg Pro
Gly Val Ser Val Ile Ser Leu Gly Gly Ala Thr Glu Ala Ser Ile Trp Ser Ile Gly Tyr Pro Val Arg Asn
Val Asp Pro Ser Trp Ala Ser Ile Pro Tyr Gly Arg Pro Leu Arg Asn Gln Thr Phe His Val Leu Asp
Glu Ala Leu Glu Pro Arg Pro Val Trp Val Pro Gly Gln Leu Tyr Ile Gly Gly Val Gly Leu Ala Leu
Gly Tyr Trp Arg Asp Glu Glu Lys Thr Arg Thr Ala.

[0299] (E) The right (T7) side of cosmid 4 was sequenced, and the complement of the sequence generated is shown below, followed by the encoded amino acid sequence, which is from a dehydratase domain.

[0300] T7 end Reverse and complement from the original sequence

GGCCCGGTCGGGCCTCATTCCAGGTATCGAGCCGTGAGGAGGCAGGTAGAAGCTGGGT (SEQ ID NO: 33)
TCGGCACGCCACGGGGCACGTGTGTAGCGACCAGAGCTCAGCAGTGGGAGCGTTGAAG
GAAGCTCCGTGGGAGATTCAACAGCGATGTCCGAGCGTCCTGTCGTCGGAGGCGCTCT
ATCCGCTGCTCAACGAGCACGCCCTCGACTATGGCCCCTGCTTCCAGGGTGTGGAGCAG
GTGTGGCTCGGCACGGGGGAGGTGCTCGGCCGGGTACGCTTGCCAGAAGACATGGCAT
CCTCAAGTGGCGCCTATCGGATTCATCCCGCCTTGTTGGATGCAGTTTTCATAGTGCTGA
CCGCGCTGCTCGACCACGCCGGAATCCATCGT.

[0301] Protein translation

(SEQ ID NO: 34)
Pro Gly Arg Ala Ser Phe Gln Val Ser Ser Arg Glu Glu Ala Gly Arg Ser Trp Val Arg His Ala Thr
Gly His Val Cys Ser Asp Gln Ser Ser Ala Val Gly Ala Leu Lys Glu Ala Pro Trp Glu Ile Gln Gln
Arg Cys Pro Ser Val Leu Ser Ser Glu Ala Leu Tyr Pro Leu Leu Asn Glu His Ala Leu Asp Tyr Gly
Pro Cys Phe Gln Gly Val Glu Gln Val Trp Leu Gly Thr Gly Glu Val Leu Gly Arg Val Arg Leu Pro
Glu Asp Met Ala Ser Ser Ser Gly Ala Tyr Arg Ile His Pro Ala Leu Leu Asp Ala Val Phe Ile Val Leu
Thr Ala Leu Leu Asp His Ala Gly Ile His Arg.

[0302] To sequence the inserts in the cosmids, each of the NotI restriction fragments from the four cosmids was cloned into the NotI site of the commercially available pBluescript plasmid. Initial sequencing was performed on the ends of each of the clones. Analysis of the sequences allowed the prediction, before having the complete sequence, that there would be 10 modules in this PKS gene cluster, a loading domain plus 9 modules.

[0303] Sequence was obtained for the complete PKS as follows. Each of the 13 non-overlapping NotI fragments was isolated and subjected to partial HinPI digestion. Fragments of ˜2 to 4 kb in length were removed from an agarose gel and cloned in the AccI site of pUC118. Sufficient clones from each library of the NotI fragments were sequenced to provide at least 4-fold coverage of each. To sequence across each of the NotI sites, a set of oligos, one 5′ and the other 3′ to each NotI site, was made and used as primers in PCR amplification of a fragment that contained each NotI site. Each fragment produced in this manner was cloned and sequenced.

[0304] The nucleotide sequence was determined for a linear segment corresponding to ˜72 kb. Analysis revealed a PKS gene cluster with a loading domain and nine modules. Downstream of the PKS sequence is an ORF, designated epoK, that shows strong homology to cytochrome P450 oxidase genes and encodes the epothilone epoxidase. The nucleotide sequence of 15 kb downstream of epoK has also been determined: a number of additional ORFs have been identified but an ORF that shows homology to any known dehydratase has not been identified. The epoL gene may encode a dehydratase activity, but this activity may instead be resident within the epothilone PKS or encoded by another gene.

[0305] The PKS genes are organized in 6 open reading frames. At the polypeptide level, the loading domain and modules 1, 2, and 9 appear on individual polypeptides; their corresponding genes are designated epoA, epoB, epoC and epoF respectively. Modules 3, 4, 5, and 6 are contained on a single polypeptide whose gene is designated epoD, and modules 7 and 8 are on another polypeptide whose gene is designated epoE. It is clear from the spacing between ORFs that epoC, epoD, epoE and epoF constitute an operon. The epoA, epoB, and epoK gene may be also part of the large operon, but there are spaces of approximately 100 bp between epoB and epoC and 115 bp between epoF and epoK which could contain a promoter. The present invention provides the intergenic sequences in recombinant form. At least one, but potentially more than one, promoter is used to express all of the epothilone genes. The epothilone PKS gene cluster is shown schematically below. 10

[0306] A detailed examination of the modules shows an organization and composition that is consistent with one able to be used for the biosynthesis of epothilone. The description that follows is at the polypeptide level. The sequence of the AT domain in the loading module and in modules 3, 4, 5, and 9 shows similarity to the consensus sequence for malonyl loading domains, consistent with the presence of an H side chain at C-14, C-12 (epothilones A and C), C-10, and C-2, respectively, as well as the loading region. The AT domains in modules 2, 6, 7, and 8 resemble the consensus sequence for methylmalonyl specifying AT domains, again consistent with the presence of methyl side chains at C-16, C-8, C-6, and C-4 respectively.

[0307] The loading module contains a KS domain in which the cysteine residue usually present at the active site is instead a tyrosine. This domain is designated as KSy and serves as a decarboxylase, which is part of its normal function, but cannot function as a condensing enzyme. Thus, the loading domain is expected to load malonyl CoA, move it to the ACP, and decarboxylate it to yield the acetyl residue required for condensation with cysteine.

[0308] Module 1 is the non-ribosomal peptide synthetase that activates cysteine and catalyzes the condensation with acetate on the loading module. The sequence contains segments highly similar to ATP-binding and ATPase domains, required for activation of amino acids, a phosphopantotheinylation site, and an elongation domain. In database searches, module 1 shows very high similarity to a number of previously identified peptide synthetases.

[0309] Module 2 determines the structure of epothilone at C-15—C-17. The presence of the DH domain in module 2 yields the C-16-17 dehydro moiety in the molecule. The domains in module 3 are consistent with the structure of epothilone at C-14 and C-15; the OH that comes from the action of the KR is employed in the lactonization of the molecule.

[0310] Module 4 controls the structure at C-12 and C-13 where a double bond is found in epothilones C and D, consistent with the presence of a DH domain. Although the sequence of the AT domain appears to resemble those that specify malonate loading, it can also load methylmalonate, thereby accounting in part for the mixture of epothilones found in the fermentation broths of the naturally producing organisms.

[0311] A significant departure from the expected array of functions was found in module 4. This module was expected to contain a DH domain, thereby directing the synthesis of epothilones C and D as the products of the PKS. Rigorous analysis revealed that the space between the AT and KR domains of module 4 was not large enough to accommodate a functional DH domain. Thus, the extent of reduction at module 4 does not proceed beyond the ketoreduction of the beta-keto formed after the condensation directed by module 4. Because the C-12,13 unsaturation has been demonstrated (epothilones C and D), there must be an additional dehydratase function that introduces the double bond, and this function is believed to be in the PKS itself or resident in an ORF in the epothilone biosynthetic gene cluster.

[0312] Thus, the action of the dehydratase could occur either during the synthesis of the polyketide or after cyclization has taken place. In the former case, the compounds produced at the end of acyl chain growth would be epothilones C and D. If the C-12,13 dehydration were a post-polyketide event, the completed acyl chain would have a hydroxyl group at C-13, as shown below. The names epothilones G and H have been assigned to the 13-hydroxy compounds produced in the absence of or prior to the action of the dehydratase. 11

Epothilones G (R═H) and H (R═CH3)

[0313] Modules 5 and 6 each have the full set of reduction domains (KR, DH and ER) to yield the methylene functions at C-11 and C-9. Modules 7 and 9 have KR domains to yield the hydroxyls at C-7 and C-3, and module 8 does not have a fimctional KR domain, consistent with the presence of the keto group at C-5. Module 8 also contains a methyltransferase (MT) domain that results in the presence of the geminal dimethyl function at C-4. Module 9 has a thioesterase domain that terminates polyketide synthesis and catalyzes ring closure. The genes, proteins, modules, and domains of the epothilone PKS are summarized in the Table hereinabove.

[0314] Inspection of the sequence has revealed translational coupling between epoA and epoB (loading domain and module 1) and between epoC and epoD. Very small gaps are seen between epoD and epoE and epoE and epoF but gaps exceeding 100 bp are found between epoB and epoC and epoF and epoK. These intergenic regions may contain promoters. Sequencing efforts have not revealed the presence of regulatory genes, and it is possible that epothilone synthesis is not regulated by operon specific regulation in Sorangium cellulosum.

[0315] The sequence of the epothilone PKS and flanking regions has been compiled into a single contig, as shown below (SEQ ID NO: 2).

1	TCGTGCGCGG	GCACGTCGAG	GCGTTTGCCG	ACTTCGGCGG	CGTCCCGCGC	GTGCTGCTCT
61	ACGACAACCT	CAAGAACGCC	GTCGTCGAGC	GCCACGGCGA	CGCGATCCGG	TTCCACCCCA
121	CGCTGCTGGC	TCTGTCGGCG	GATTACCGCT	TCGAGCCGCG	CCCCGTCGCC	GTCGCCCGCG
181	GCAACGAGAA	GGGCCGCGTC	GAGCGCGCCA	TCCGCTACGT	CCGCGAGGGC	TTCTTCGAGG
241	CCCGGGCCTA	CGCCGACCTC	GGAGACCTCA	ACCGCCAAGC	GACCGAGTGG	ACCAGCTCCG
301	CGGCGCTCGA	TCGCTCCTGG	GTCGAGGACC	GCGCCCGCAC	CGTGCGTCAG	GCCTTCGACG
361	ACGAGCGCAG	CGTGCTGCTG	CGACACCCTG	ACACACCGTT	TCCGGACCAC	GAGCGCGTCG
421	AGGTCGAGGT	CGGAAAGACC	CCCTACGCGC	GCTTCGATCT	CAACGACTAC	TCGGTCCCCC
481	ACGACCGGAC	GCGCCGCACG	CTGGTCGTCC	TCGCCGACCT	CAGTCAGGTA	CGCATCGCCG
541	ACGGCAACCA	GATCGTCGCG	ACCCACGTCC	GTTCGTGGGA	CCGCGGCCAG	CAGATCGAGC
601	AGCCCGAGCA	CCTCCAGCGC	CTGGTCGACG	AGAAGCGCCG	CGCCCGCGAG	CACCGCGGCC
661	TTGATCGCCT	CGCGCGCGCC	GCCCGCAGCA	GCCAGGCATT	CCTGCGCATC	GTCGCCGAGC
721	GCGGCGATAA	CGTCGGCAGC	GCGATCGCCC	GGCTTCTGCA	ACTGCTCGAC	GCCGTGGGCG
781	CCGCCGAGCT	CGAAGAGGCC	CTGGTCGAGG	TGCTTGAGCG	CGACACCATC	CACATCGGTG
841	CCGTCCGCCA	GGTGATCGAC	CGCCGCCGCT	CCGAGCGCCA	CCTGCCGCCT	CCAGTCTCAA
901	TCCCCGTCAC	CCGCGGCGAG	CACGCCGCCC	TCGTCGTCAC	GCCGCATTCC	CTCACCACCT
961	ACGACGCCCT	GAAGAAGGAC	CCGACGCCAT	GACCGACCTG	ACGCCCACCG	AGACCAAAGA
1021	CCGGCTCAAG	AGCCTCGGCC	TCTTCGGCCT	GCTCGCCTGC	TGGGAGCAGC	TCGCCGACAA
1081	GCCCTGGCTT	CGCGAGGTGC	TCGCCATCGA	GGAGCGCGAG	CGCCACAAGC	GCAGCCTCGA
1141	ACGCCGCCTG	AAGAACTCCC	GCGTCGCCGC	CTTCAAGCCC	ATGACCGACT	TCGACTCGTC
1201	CTGGCCCAAG	AAGATCGACC	GCGAGGCCGT	CGACGACCTC	TACGATAGCC	GCTACGCGGA
1261	CCTGCTCTTC	GAGGTCGTCA	CCCGTCGCTA	CGACGCGCAG	AAGCCGCTCT	TGCTCAGCAC
1321	GAACAAGGCA	TTCGCCGACT	GGGGCCAGGT	CTTCCCGCAC	GCCGCGTGCG	TCGTCACGCT
1381	CGTCGACCGG	CTCGTGCACC	GCGCCGAGGT	GATCGAGATC	GAGGCCGAGA	GCTACCGGCT
1441	GAAGGAAGCC	AAGGAGCTCA	ACGCCACCCG	CACCAAGCAG	CGCCGCACCA	AGAAGCACTG
1501	AGCGGCATTT	TCACCGGTGA	ACTTCACCGA	AATCCCGCGT	GTTGCCGAGA	TCATCTACAG
1561	GCGGATCGAG	ACCGTGCTCA	CGGCGTGGAC	GACATGGCGC	GGAAACGTCG	TCGTAACTGC
1621	CCAGCAATGT	CATGGGAATG	GCCCCTTGAG	GGGCTGGCCG	GGGTCGACGA	TATCGCGCGA
1681	TCTCCCCGTC	AATTCCCGAG	CGTAAAAGAA	AAATTTGTCA	TAGATCGTAA	GCTGTGCTAG
1741	TGATCTGCCT	TACGTTACGT	CTTCCGCACC	TCGAGCGAAT	TCTCTCGGAT	AACTTTCAAG
1801	TTTTCTGAGG	GGGCTTGGTC	TCTGGTTCCT	CAGGAAGCCT	GATCGGGACG	AGCTAATTCC
1861	CATCCATTTT	TTTGAGACTC	TGCTCAAAGG	GATTAGACCG	AGTGAGACAG	TTCTTTTGCA
1921	GTGAGCGAAG	AACCTGGGGC	TCGACCGGAG	GACGATCGAC	GTCCGCGAGC	GGGTCAGCCG
1981	CTGAGGATGT	GCCCGTCGTG	GCGGATCGTC	CCATCGAGCG	CGCAGCCGAA	GATCCGATTG
2041	CGATCGTCGG	AGCGGGCTGC	CGTCTGCCCG	GTGGCGTGAT	CGATCTGAGC	GGGTTCTGGA
2101	CGCTCCTCGA	GGGCTCGCGC	GACACCGTCG	GGCAAGTCCC	CGCCGAACGC	TGGGATGCAG
2161	CAGCGTGGTT	TGATCCCGAC	CTCGATGCCC	CGGGGAAGAC	GCCCGTTACG	CGCGCATCTT
2221	TCCTGAGCGA	CGTAGCCTGC	TTCGACGCCT	CCTTCTTCGG	CATCTCGCCT	CGCGAAGCGC
2281	TGCGGATGGA	CCCTGCACAT	CGACTCTTGC	TGGAGGTGTG	CTGGGAGGCG	CTGGAGAACG
2341	CCGCGATCGC	TCCATCGGCG	CTCGTCGGTA	CGGAAACGGG	AGTGTTCATC	GGGATCGGCC
2401	CGTCCGAATA	TGAGGCCGCG	CTGCCGCGAG	CGACGGCGTC	CGCAGAGATC	GACGCTCATG
2461	GCGGGCTGGG	GACGATGCCC	AGCGTCGGAG	CGGGCCGAAT	CTCGTATGTC	CTCGGGCTGC
2521	GAGGGCCGTG	TGTCGCGGTG	GATACGGCCT	ATTCGTCCTC	GCTCGTGGCC	GTTCATCTGG
2581	CCTGTCAGAG	CTTGCGCTCC	GGGGAATGCT	CCACGGCCCT	GGCTGGTGGG	GTATCGCTGA
2641	TGTTGTCGCC	GAGCACCCTC	GTGTGGCTCT	CGAAGACCCG	CGCGCTGGCC	ACGGACGGTC
2701	GCTGCAAGGC	GTTTTCGGCG	GAGGCCGATG	GGTTCGGACG	AGGCGAAGGG	TGCGCCGTCG
2761	TGGTCCTCAA	GCGGCTCAGT	GGAGCCCGCG	CGGACGGCGA	CCGGATATTG	GCGGTGATTC
2821	GAGGATCCGC	GATCAATCAC	GACGGAGCGA	GCAGCGGTCT	GACCGTGCCG	AACGGGAGCT
2881	CCCAAGAAAT	CGTGCTGAAA	CGGGCCCTGG	CGGACGCAGG	CTGCGCCGCG	TCTTCGGTGG
2941	GTTATGTCGA	GGCACACGGC	ACGGGCACGA	CGCTTGGTGA	CCCCATCGAA	ATCCAAGCTC
3001	TGAATGCGGT	ATACGGCCTC	GGGCGAGACG	TCGCCACGCC	GCTGCTGATC	GGGTCGGTGA
3061	AGACCAACCT	TGGCCATCCT	GAGTATGCGT	CGGGGATCAC	TGGGCTGCTG	AAGGTCGTCT
3121	TGTCCCTTCA	GCACGGGCAG	ATTCCTGCGC	ACCTCCACGC	GCAGGCGCTG	AACCCCCGGA
3181	TCTCATGGGG	TGATCTTCGG	CTGACCGTCA	CGCGCGCCCG	GACACCGTGG	CCGGACTGGA
3241	ATACGCCGCG	ACGGGCGGGG	GTGAGCTCGT	TCGGCATGAG	CGGGACCAAC	GCGCACGTGG
3301	TGCTGGAAGA	GGCGCCGGCG	GCGACGTGCA	CACCGCCGGC	GCCGGAGCGG	CCGGCAGAGC
3361	TGCTGGTGCT	GTCGGCAAGG	ACCGCGGCAG	CCTTGGATGC	ACACGCGGCG	CGGCTGCGCG
3421	ACCATCTGGA	GACCTACCCT	TCGCAGTGTC	TGGGCGATGT	GGCGTTCAGT	CTGGCGACGA
3481	CGCGCAGCGC	GATGGAGCAC	CGGCTCGCGG	TGGCGGCGAC	GTCGAGCGAG	GGGCTGCGGG
3541	CAGCCCTGGA	CGCTGCGGCG	CAGGGACAGA	CGCCGCCCGG	TGTGGTGCGC	GGTATCGCCG
3601	ATTCCTCACG	CGGCAAGCTC	GCCTTTCTCT	TCACCGGACA	GGGGGCGCAG	ACGCTGGGCA
3661	TGGGCCGTGG	GCTGTATGAT	GTATGGCCCG	CGTTCCGCGA	GGCGTTCGAC	CTGTGCGTGA
3721	GGCTGTTCAA	CCAGGAGCTC	GACCGGCCGC	TCCGCGAGGT	GATGTGGGCC	GAACCGGCCA
3781	GCGTCGACGC	CGCGCTGCTC	GACCAGACAG	CCTTTACCCA	GCCGGCGCTG	TTCACCTTCG
3841	AGTATGCGCT	CGCCGCGCTG	TGGCGGTCGT	GGGGCGTAGA	GCCGGAGTTG	GTCGCTGGCC
3901	ATAGCATCGG	TGAGCTGGTG	GCTGCCTGCG	TGGCGGGCGT	GTTCTCGCTT	GAGGACGCGG
3961	TGTTCCTGGT	GGCTGCGCGC	GGGCGCCTGA	TGCAGGCGCT	GCCGGCCGGC	GGGGCGATGG
4021	TGTCGATCGC	GGCGCCGGAG	GCCGATGTGG	CTGCTGCGGT	GGCGCCGCAC	GCAGCGTCGG
4081	TGTCGATCGC	CGCGGTCAAC	GGTCCGGACC	AGGTGGTCAT	CGCGGGCGCC	GGGCAACCCG
4141	TGCATGCGAT	CGCGGCGGCG	ATGGCCGCGC	GCGGGGCGCG	AACCAAGGCG	CTCCACGTCT
4201	CGCATGCGTT	CCACTCACCG	CTCATGGCCC	CGATGCTGGA	GGCGTTCGGG	CGTGTGGCCG
4261	AGTCGGTGAG	CTACCGGCGG	CCGTCGATCG	TCCTGGTCAG	CAATCTGAGC	GGGAAGGCTG
4321	GCACAGACGA	GGTGAGCTCG	CCGGGCTATT	GGGTGCGCCA	CGCGCGAGAG	GTGGTGCGCT
4381	TCGCGGATGG	AGTGAAGGCG	CTGCACGCGG	CCGGTGCGGG	CACCTTCGTC	GAGGTCGGTC
4441	CGAAATCGAC	GCTGCTCGGC	CTGGTGCCTG	CCTGCCTGCC	GGACGCCCGG	CCGGCGCTGC
4501	TCGCATCGTC	GCGCGCTGGG	CGTGACGAGC	CAGCGACCGT	GCTCGAGGCG	CTCGGCGGGC
4561	TCTGGGCCGT	CGGTGGCCTG	GTCTCCTGGG	CCGGCCTCTT	CCCCTCAGGG	GGGCGGCGGG
4621	TGCCGCTGCC	CACGTACCCT	TGGCAGCGCG	AGCGCTACTG	GATCGACACG	AAAGCCGACG
4681	ACGCGGCGCG	TGGCGACCGC	CGTGCTCCGG	GAGCGGGTCA	CGACGAGGTC	GAGAAGGGGG
4741	GCGCGGTGCG	CGGCGGCGAC	CGGCGCAGCG	CTCGGCTCGA	CCATCCGCCG	CCCGAGAGCG
4801	GACGCCGGGA	GAAGGTCGAG	GCCGCCGGCG	ACCGTCCGTT	CCGGCTCGAG	ATCGATGAGC
4861	CAGGCGTGCT	CGATCGCCTG	GTGCTTCGGG	TCACGGAGCG	GCGCGCCCCT	GGTCTTGGCG
4921	AGGTCGAGAT	CGCCGTCGAC	GCGGCGGGGC	TCAGCTTCAA	TGATGTCCAG	CTCGCGCTGG
4981	GCATGGTGCC	CGACGACCTG	CCGGGAAAGC	CCAACCCTCC	GCTGCTGCTC	GGAGGCGAGT
5041	GCGCCGGGCG	CATCGTCGCC	GTGGGCGAGG	GCGTGAACGG	CCTTGTGGTG	GGCCAACCGG
5101	TCATCGCCCT	TTCGGCGGGA	GCGTTTGCTA	CCCACGTCAC	CACGTCGGCT	GCGCTGGTGC
5161	TGCCTCGGCC	TCAGGCGCTC	TCGGCGACCG	AGGCGGCCGC	CATGCCCGTC	GCGTACCTGA
5221	CGGCATGGTA	CGCGCTCGAC	GGAATAGCCC	GCCTTCAGCC	GGGGGAGCGG	GTGCTGATCC
5281	ACGCGGCGAC	CGGCGGGGTC	GGTCTCGCCG	CGGTGCAGTG	GGCGCAGCAC	GTGGGAGCCG
5341	AGGTCCATGC	GACGGCCGGC	ACGCCCGAGA	AGCGCGCCTA	CCTGGAGTCG	CTGGGCGTGC
5401	GGTATGTGAG	CGATTCCCGC	TCGGACCGGT	TCGTCGCCGA	CGTGCGCGCG	TGGACGGGCG
5461	GCGAGGGAGT	AGACGTCGTG	CTCAACTCGC	TTTCGGGCGA	GCTGATCGAC	AAGAGTTTCA
5521	ATCTCCTGCG	ATCGCACGGC	CGGTTTGTGG	AGCTCGGCAA	GCGCGACTGT	TACGCGGATA
5581	ACCAGCTCGG	GCTGCGGCCG	TTCCTGCGCA	ATCTCTCCTT	CTCGCTGGTG	GATCTCCGGG
5641	GGATGATGCT	CGAGCGGCCG	GCGCGGGTCC	GTGCGCTCTT	CGAGGAGCTC	CTCGGCCTGA
5701	TCGCGGCAGG	CGTGTTCACC	CCTCCCCCCA	TCGCGACGCT	CCCGATCGCT	CGTGTCGCCG
5761	ATGCGTTCCG	GAGCATGGCG	CAGGCGCAGC	ATCTTGGGAA	GCTCGTACTC	ACGCTGGGTG
5821	ACCCGGAGGT	CCAGATCCGT	ATTCCGACCC	ACGCAGGCGC	CGGCCCGTCC	ACCGGGGATC
5881	GGGATCTGCT	CGACAGGCTC	GCGTCAGCTG	CGCCGGCCGC	GCGCGCGGCG	GCGCTGGAGG
5941	CGTTCCTCCG	TACGCAGGTC	TCGCAGGTGC	TGCGCACGCC	CGAAATCAAG	GTCGGCGCGG
6001	AGGCGCTGTT	CACCCGCCTC	GGCATGGACT	CGCTCATGGC	CGTGGAGCTG	CGCAATCGTA
6061	TCGAGGCGAG	CCTCAAGCTG	AAGCTGTCGA	CGACGTTCCT	GTCCACGTCC	CCCAATATCG
6121	CCTTGTTGAC	CCAAAACCTG	TTGGATGCTC	TCGCCACAGC	TCTCTCCTTG	GAGCGGGTGG
6181	CGGCGGAGAA	CCTACGGGCA	GGCGTGCAAA	GCGACTTCGT	CTCATCGGGC	GCAGATCAAG
6241	ACTGGGAAAT	CATTGCCCTA	TGACGATCAA	TCAGCTTCTG	AACGAGCTCG	AGCACCAGGG
6301	TGTCAAGCTG	GCGGCCGATG	GGGAGCGCCT	CCAGATACAG	GCCCCCAAGA	ACGCCCTGAA
6361	CCCGAACCTG	CTCGCTCGAA	TCTCCGAGCA	CAAAAGCACG	ATCCTGACGA	TGCTCCGTCA
6421	GAGACTCCCC	GCAGAGTCCA	TCGTGCCCGC	CCCAGCCGAG	CGGCACGTTC	CGTTTCCTCT
6481	CACAGACATC	CAAGGATCCT	ACTGGCTGGG	TCGGACAGGA	GCGTTTACGG	TCCCCAGCGG
6541	GATCCACGCC	TATCGCGAAT	ACGACTGTAC	GGATCTCGAC	GTGGCGAGGC	TGAGCCGCGC
6601	CTTTCGGAAA	GTCGTCGCGC	GGCACGACAT	GCTTCGGGCC	CACACGCTGC	CCGACATGAT
6661	GCAGGTGATC	GAGCCTAAAG	TCGACGCCGA	CATCGAGATC	ATCGATCTGC	GCGGGCTCGA
6721	CCGGAGCACA	CGGGAAGCGA	GGCTCGTATC	GTTGCGAGAT	GCGATGTCGC	ACCGCATCTA
6781	TGACACCGAG	CGCCCTCCGC	TCTATCACGT	CGTCGCCGTT	CGGCTGGACG	AGCAGCAAAC
6841	CCGTCTCGTG	CTCAGTATCG	ATCTCATTAA	CGTTGACCTA	GGCAGCCTGT	CCATCATCTT
6901	CAAGGATTGG	CTCAGCTTCT	ACGAAGATCC	CGAGACCTCT	CTCCCTGTCC	TGGAGCTCTC
6961	GTACCGCGAC	TATGTGCTCG	CGCTGGAGTC	TCGCAAGAAG	TCTGAGGCGC	ATCAACGATC
7021	GATGGATTAC	TGGAAGCGGC	GCGTCGCCGA	GCTCCCACCT	CCGCCGATGC	TTCCGATGAA
7081	GGCCGATCCA	TCTACCCTGA	GGGAGATCCG	CTTCCGGCAC	ACGGAGCAAT	GGCTGCCGTC
7141	GGACTCCTGG	AGTCGATTGA	AGCAGCGTGT	CGGGGAGCGC	GGGCTGACCC	CGACGGGCGT
7201	CATTCTGGCT	GCATTTTCCG	AGGTGATCGG	GCGCTGGAGC	GCGAGCCCCC	GGTTTACGCT
7261	CAACATAACG	CTCTTCAACC	GGCTCCCCGT	CCATCCGCGC	GTGAACGATA	TCACCGGGGA
7321	CTTCACGTCG	ATGGTCCTCC	TGGACATCGA	CACCACTCGC	GACAAGAGCT	TCGAACAGCG
7381	CGCTAAGCGT	ATTCAAGAGC	AGCTGTGGGA	AGCGATGGAT	CACTGCGACG	TAAGCGGTAT
7441	CGAGGTCCAG	CGAGAGGCCG	CCCGGGTCCT	GGGGATCCAA	CGAGGCGCAT	TGTTCCCCGT
7501	GGTGCTCACG	AGCGCGCTCA	ACCAGCAAGT	CGTTGGTGTC	ACCTCGCTGC	AGAGGCTCGG
7561	CACTCCGGTG	TACACCAGCA	CGCAGACTCC	TCAGCTGCTG	CTGGATCATC	AGCTCTACGA
7621	GCACGATGGG	GACCTCGTCC	TCGCGTGGGA	CATCGTCGAC	GGAGTGTTCC	CGCCCGACCT
7681	TCTGGACGAC	ATGCTCGAAG	CGTACGTCGC	TTTTCTCCGG	CGGCTCACTG	AGGAACCATG
7741	GAGTGAACAG	ATGCGCTGTT	CGCTTCCGCC	TGCCCAGCTA	GAAGCGCGGG	CGAGCGCAAA
7801	CGAGACCAAC	TCGCTGCTGA	GCGAGCATAC	GCTGCACGGC	CTGTTCGCGG	CGCGGGTCGA
7861	GCAGCTGCCT	ATGCAGCTCG	CCGTGGTGTC	GGCGCGCAAG	ACGCTCACGT	ACGAAGAGCT
7921	TTCGCGCCGT	TCGCGGCGAC	TTGGCGCGCG	GCTGCGCGAG	CAGGGGGCAC	GCCCGAACAC
7981	ATTGGTCGCG	GTGGTGATGG	AGAAAGGCTG	GGAGCAGGTT	GTCGCGGTTC	TCGCGGTGCT
8041	CGAGTCAGGC	GCGGCCTACG	TGCCGATCGA	TGCCGACCTA	CCGGCGGAGC	GTATCCACTA
8101	CCTCCTCGAT	CATGGTGAGG	TAAAGCTCGT	GCTGACGCAG	CCATGGCTGG	ATGGCAAACT
8161	GTCATGGCCG	CCGGGGATCC	AGCGGCTGCT	CGTGAGCGAT	GCCGGCGTCG	AAGGCGACGG
8221	CGACCAGCTT	CCGATGATGC	CCATTCAGAC	ACCTTCGGAT	CTCGCGTATG	TCATCTACAC
8281	CTCGGGATCC	ACAGGGTTGC	CCAAGGGGGT	GATGATCGAT	CATCGGGGTG	CCGTCAACAC
8341	CATCCTGGAC	ATCAACGAGC	GCTTCGAAAT	AGGGCCCGGA	GACAGAGTGC	TGGCGCTCTC
8401	CTCGCTGAGC	TTCGATCTCT	CGGTCTACGA	TGTGTTCGGG	ATCCTGGCGG	CGGGCGGTAC
8461	GATCGTGGTG	CCGGACGCGT	CCAAGCTGCG	CGATCCGGCG	CATTGGGCAG	CGTTGATCGA
8521	ACGAGAGAAG	GTGACGGTGT	GGAACTCGGT	GCCGGCGCTG	ATGCGGATGC	TCGTCGAGCA
8581	TTCCGAGGGT	CGCCCCGATT	CGCTCGCTAG	GTCTCTGCGG	CTTTCGCTGC	TGAGCGGCGA
8641	CTGGATCCCG	GTGGGCCTGC	CTGGCGAGCT	CCAGGCCATC	AGGCCCGGCG	TGTCGGTGAT
8701	CAGCCTGGGC	GGGGCCACCG	AAGCGTCGAT	CTGGTCCATC	GGGTACCCCG	TGAGGAACGT
8761	CGATCCATCG	TGGGCGAGCA	TCCCCTACGG	CCGTCCGCTG	CGCAACCAGA	CGTTCCACGT
8821	GCTCGATGAG	GCGCTCGAAC	CGCGCCCGGT	CTGGGTTCCG	GGGCAACTCT	ACATTGGCGG
8881	GGTCGGACTG	GCACTGGGCT	ACTGGCGCGA	TGAAGAGAAG	ACGCGCAACA	GCTTCCTCGT
8941	GCACCCCGAG	ACCGGGGAGC	GCCTCTACAA	GACCGGCGAT	CTGGGCCGCT	ACCTGCCCGA
9001	TGGAAACATC	GAGTTCATGG	GGCGGGAGGA	CAACCAAATC	AAGCTTCGCG	GATACCGCGT
9061	TGAGCTCGGG	GAAATCGAGG	AAACGCTCAA	GTCGCATCCG	AACGTACGCG	ACGCGGTGAT
9121	TGTGCCCGTC	GGGAACGACG	CGGCGAACAA	GCTCCTTCTA	GCCTATGTGG	TCCCGGAAGG
9181	CACACGGAGA	CGCGCTGCCG	AGCAGGACGC	GAGCCTCAAG	ACCGAGCGGG	TCGACGCGAG
9241	AGCACACGCC	GCCAAAGCGG	ACGGATTGAG	CGACGGCGAG	AGGGTGCAGT	TCAAGCTCGC
9301	TCGACACGGA	CTCCGGAGGG	ATCTGGACGG	AAAGCCCGTC	GTCGATCTGA	CCGGGCTGGT
9361	TCCGCGGGAG	GCGGGGCTGG	ACGTCTACGC	GCGTCGCCGT	AGCGTCCGAA	CGTTCCTCGA
9421	GGCCCCGATT	CCATTTGTTG	AATTCGGCCG	ATTCCTGAGC	TGCCTGAGCA	GCGTGGAGCC
9481	CGACGGCGCG	GCCCTTCCCA	AATTCCGTTA	TCCATCGGCT	GGCAGCACGT	ACCCGGTGCA
9541	AACCTACGCG	TACGCCAAAT	CCGGCCGCAT	CGAGGGCGTG	GACGAGGGCT	TCTATTATTA
9601	CCACCCGTTC	GAGCACCGTT	TGCTGAAGGT	CTCCGATCAC	GGGATCGAGC	GCGGAGCGCA
9661	CGTTCCGCAA	AACTTCGACG	TGTTCGATGA	AGCGGCGTTC	GGCCTCCTGT	TCGTGGGCAG
9721	GATCGATGCC	ATCGAGTCGC	TGTATGGATC	GTTGTCACGA	GAATTCTGCC	TGCTGGAGGC
9781	CGGATATATG	GCGCAGCTCC	TGATGGAGCA	GGCGCCTTCC	TGCAACATCG	GCGTCTGTCC
9841	GGTGGGTCAA	TTCGATTTTG	AACAGGTTCG	GCCGGTTCTC	GACCTGCGGC	ATTCGGACGT
9901	TTACGTGCAC	GGCATGCTGG	GCGGGCGGGT	AGACCCGCGG	CAGTTCCAGG	TCTGTACGCT
9961	CGGTCAGGAT	TCCTCACCGA	GGCGCGCCAC	GACGCGCGGC	GCCCCTCCCG	GCCGCGATCA
10021	GCACTTCGCC	GATATCCTTC	GCGACTTCTT	GAGGACCAAA	CTACCCGAGT	ACATGGTGCC
10081	TACAGTCTTC	GTGGAGCTCG	ATGCGTTGCC	GCTGACGTCC	AACGGCAAGG	TCGATCGTAA
10141	GGCCCTGCGC	GAGCGGAAGG	ATACCTCGTC	GCCGCGGCAT	TCGGGGCACA	CGGCGCCACG
10201	GGACGCCTTG	GAGGAGATCC	TCGTTGCGGT	CGTACGGGAG	GTGCTCGGGC	TGGAGGTGGT
10261	TGGGCTCCAG	CAGAGCTTCG	TCGATCTTGG	TGCGACATCG	ATTCACATCG	TTCGCATGAG
10321	GAGTCTGTTG	CAGAAGAGGC	TGGATAGGGA	GATCGCCATC	ACCGAGTTGT	TCCAGTACCC
10381	GAACCTCGGC	TCGCTGGCGT	CCGGTTTGCG	CCGAGACTCG	AAAGATCTAG	AGCAGCGGCC
10441	GAACATGCAG	GACCGAGTGG	AGGCTCGGCG	CAAGGGCAGG	AGACGTAGCT	AAGAGCGCCG
10501	AACAAAACCA	GGCCGAGCGG	GCCAATGAAC	CGCAAGCCCG	CCTGCGTCAC	CCTGGGACTC
10561	ATCTGATCTG	ATCGCGGGTA	CGCGTCGCGG	GTGTGCGCGT	TGAGCCGTGT	TGCTCGAACG
10621	CTGAGGAACG	GTGAGCTCAT	GGAAGAACAA	GAGTCCTCCG	CTATCGCAGT	CATCGGCATG
10681	TCGGGCCGTT	TTCCGGGGGC	GCGGGATCTG	GACGAATTCT	GGAGGAACCT	TCGAGACGGC
10741	ACGGAGGCCG	TGCAGCGCTT	CTCCGAGCAG	GAGCTCGCGG	CGTCCGGAGT	CGACCCAGCG
10801	CTGGTGCTGG	ACCCGAACTA	CGTCCGGGCG	GGCAGCGTGC	TGGAAGATGT	CGACCGGTTC
10861	GACGCTGCTT	TCTTCGGCAT	CAGCCCGCGC	GAGGCAGAGC	TCATGGATCC	GCAGCACCGC
10921	ATCTTCATGG	AATGCGCCTG	GGAGGCGCTG	GAGAACGCCG	GATACGACCC	GACAGCCTAC
10981	GAGGGCTCTA	TCGGCGTGTA	CGCCGGCGCC	AACATGAGCT	CGTACTTGAC	GTCGAACCTC
11041	CACGAGCACC	CAGCGATGAT	GCGGTGGCCC	GGCTGGTTTC	AGACGTTGAT	CGGCAACGAC
11101	AAGGATTACC	TCGCGACCCA	CGTCTCCTAC	AGGCTGAATC	TGAGAGGGCC	GAGCATCTCC
11161	GTTCAAACTG	CCTGCTCTAC	CTCGCTCGTG	GCGGTTCACT	TGGCGTGCAT	GAGCCTCCTG
11221	GACCGCGAGT	GCGACATGGC	GCTGGCCGGC	GGGATTACCG	TCCGGATCCC	CCATCGAGCC
11281	GGCTATGTAT	ATGCTGAGGG	GGGCATCTTC	TCTCCCGACG	GCCATTGCCG	GGCCTTCGAC
11341	GCCAAGGCGA	ACGGCACGAT	CATGGGCAAC	GGCTGCGGGG	TTGTCCTCCT	GAAGCCGCTG
11401	GACCGGGCGC	TCTCCGATGG	TGATCCCGTC	CGCGCGGTCA	TCCTTGGGTC	TGCCACAAAC
11461	AACGACGGAG	CGAGGAAGAT	CGGGTTCACT	GCGCCCAGTG	AGGTGGGCCA	GGCGCAAGCG
11521	ATCATGGAGG	CGCTGGCGCT	GGCAGGGGTC	GAGGCCCGGT	CCATCCAATA	CATCGAGACC
11581	CACGGGACCG	GCACGCTGCT	CGGAGACGCC	ATCGAGACGG	CGGCGTTGCG	GCGGGTGTTC
11641	GATCGCGACG	CTTCGACCCG	GAGGTCTTGC	GCGATCGGCT	CCGTGAAGAC	CGGCATCGGA
11701	CACCTCGAAT	CGGCGGCTGG	CATCGCCGGT	TTGATCAAGA	CGGTCTTGGC	GCTGGAGCAC
11761	CGGCAGCTGC	CGCCCAGCCT	GAACTTCGAG	TCTCCTAACC	CATCGATCGA	TTTCGCGAGC
11821	AGCCCGTTCT	ACGTCAATAC	CTCTCTTAAG	GATTGGAATA	CCGGCTCGAC	TCCGCGGCGG
11881	GCCGGCGTCA	GCTCGTTCGG	GATCGGCGGC	ACCAACGCCC	ATGTCGTGCT	GGAGGAAGCA
11941	CCCGCGGCGA	AGCTTCCAGC	CGCGGCGCCG	GCGCGCTCTG	CCGAGCTCTT	CGTCGTCTCG
12001	GCCAAGAGCG	CAGCGGCGCT	GGATGCCGCG	GCGGCACGGC	TACGAGATCA	TCTGCAGGCG
12061	CACCAGGGGC	TTTCGTTGGG	CGACGTCGCC	TTCAGCCTGG	CGACGACGCG	CAGTCCCATG
12121	GAGCACCGGC	TCGCGATGGC	GGCACCGTCG	CGCGAGGCGT	TGCGAGAGGG	GCTCGACGCA
12181	GCGGCGCGAG	GCCAGACCCC	GCCGGGCGCC	GTGCGTGGCC	GCTGCTCCCC	AGGCAACGTG
12241	CCGAAGGTGG	TCTTCGTCTT	TCCCGGCCAG	GGCTCTCAGT	GGGTCGGTAT	GGGCCGTCAG
12301	CTCCTGGCTG	AGGAACCCGT	CTTCCACGCG	GCGCTTTCGG	CGTGCGACCG	GGCCATCCAG
12361	GCCGAAGCTG	GTTGGTCGCT	GCTCGCCGAG	CTCGCCGCCG	ACGAAGGGTC	GTCCCAGATC
12421	GAGCGCATCG	ACGTGGTGCA	GCCGGTGCTG	TTCGCGCTCG	CGGTGGCATT	TGCGGCGCTG
12481	TGGCGGTCGT	GGGGTGTCGG	GCCCGACGTC	GTGATCGGCC	ACAGCATGGG	CGAGGTAGCC
12541	GCCGCGCATG	TGGCCGGGGC	GCTGTCGCTC	GAGGATGCGG	TGGCGATCAT	CTGCCGGCGC
12601	AGCCGGCTGC	TCCGGCGCAT	CAGCGGTCAG	GGCGAGATGG	CGGTGACCGA	GCTGTCGCTG
12661	GCCGAGGCCG	AGGCAGCGCT	CCGAGGCTAC	GAGGATCGGG	TGAGCGTGGC	CGTGAGCAAC
12721	AGCCCGCGCT	CGACGGTGCT	CTCGGGCGAG	CCGGCAGCGA	TCGGCGAGGT	GCTGTCGTCC
12781	CTGAACGCGA	AGGGGGTGTT	CTGCCGTCGG	GTGAAGGTGG	ATGTCGCCAG	CCACAGCCCG
12841	CAGGTCGACC	CGCTGCGCGA	GGACCTCTTG	GCAGCGCTGG	GCGGGCTCCG	GCCGCGTGCG
12901	GCTGCGGTGC	CGATGCGCTC	GACGGTGACG	GGCGCCATGG	TAGCGGGCCC	GGAGCTCGGA
12961	GCGAATTACT	GGATGAACAA	TCTCAGGCAG	CCTGTGCGCT	TCGCCGAGGT	AGTCCAGGCG
13021	CAGCTCCAAG	GCGGCCACGG	TCTGTTCGTG	GAGATGAGCC	CGCATCCGAT	CCTAACGACT
13081	TCGGTCGAGG	AGATGCGGCG	CGCGGCCCAG	CGGGCGGGCG	CAGCGGTGGG	CTCGCTGCGG
13141	CGAGGGCAGG	ACGAGCGCCC	GGCGATGCTG	GAGGCGCTGG	GCGCGCTGTG	GGCGCAGGGC
13201	TACCCTGTAC	CCTGGGGGCG	GCTGTTTCCC	GCGGGGGGGC	GGCGGGTACC	GCTGCCGACC
13261	TATCCCTGGC	AGCGCGAGCG	GTACTGGATC	GAAGCGCCGG	CCAAGAGCGC	CGCGGGCGAT
13321	CGCCGCGGCG	TGCGTGCGGG	CGGTCACCCG	CTCCTCGGTG	AAATGCAGAC	CCTATCAACC
13381	CAGACGAGCA	CGCGGCTGTG	GGAGACGACG	CTGGATCTCA	AGCGGCTGCC	GTGGCTCGGC
13441	GACCACCGGG	TGCAGGGAGC	GGTCGTGTTT	CCGGGCGCGG	CGTACCTGGA	GATGGCGATT
13501	TCGTCGGGGG	CCGAGGCTTT	GGGCGATGGC	CCATTGCAGA	TAACCGACGT	GGTGCTCGCC
13561	GAGGCGCTGG	CCTTCGCGGG	CGACGCGGCG	GTGTTGGTCC	AGGTGGTGAC	GACGGAGCAG
13621	CCGTCGGGAC	GGCTGCAGTT	CCAGATCGCG	AGCCGGGCGC	CGGGCGCTGG	CCACGCGTCC
13681	TTCCGGGTCC	ACGCTCGCGG	CGCGTTGCTC	CGAGTGGAGC	GCACCGAGGT	CCCGGCTGGG
13741	CTTACGCTTT	CCGCCGTGCG	CGCACGGCTC	CAGGCCAGCA	TGCCCGCCGC	GGCCACCTAC
13801	GCGGAGCTGA	CCGAGATGGG	GCTGCAGTAC	GGCCCTGCCT	TCCAGGGGAT	TGCTGAGCTA
13861	TGGCGCGGTG	AGGGCGAGGC	GCTGGGACGG	GTACGCCTGC	CCGACGCGGC	CGGCTCGGCA
13921	GCGGAGTATC	GGTTGCATCC	TGCGCTGCTG	GACGCGTGCT	TCCAGGTCGT	CGGCAGCCTC
13981	TTCGCCGGCG	GTGGCGAGGC	GACGCCGTGG	GTGCCCGTGG	AAGTGGGCTC	GCTGCGGCTC
14041	TTGCAGCGGC	CTTCGGGGGA	GCTGTGGTGC	CATGCGCGCG	TCGTGAACCA	CGGGCGCCAA
14101	ACCCCCGATC	GGCAGGGCGC	CGACTTTTGG	GTGGTCGACA	GCTCGGGTGC	AGTGGTCGCC
14161	GAAGTCAGCG	GGCTCGTGGC	GCAGCGGCTT	CCGGGAGGGG	TGCGCCGGCG	CGAAGAAGAC
14221	GATTGGTTCC	TGGAGCTCGA	GTGGGAACCC	GCAGCGGTCG	GCACAGCCAA	GGTCAACGCG
14281	GGCCGGTGGC	TGCTCCTCGG	CGGCGGCGGT	GGGCTCGGCG	CCGCGTTGCG	CTCGATGCTG
14341	GAGGCCGGCG	GCCATGCCGT	CGTCCATGCG	GCAGAGAGCA	ACACGAGCGC	TGCCGGCGTA
14401	CGCGCGCTCC	TGGCAAAGGC	CTTTGACGGC	CAGGCTCCGA	CGGCGGTGGT	GCACCTCGGC
14461	AGCCTCGATG	GGGGTGGCGA	GCTCGACCCA	GGGCTCGGGG	CGCAAGGCGC	ATTGGACGCG
14521	CCCCGGAGCG	CCGACGTCAG	TCCCGATGCC	CTCGATCCGG	CGCTGGTACG	TGGCTGTGAC
14581	AGCGTGCTCT	GGACCGTGCA	GGCCCTGGCC	GGCATGGGCT	TTCGAGACGC	CCCGCGATTG
14641	TGGCTTCTGA	CCCGCGGCGC	ACAGGCCGTC	GGCGCCGGCG	ACGTCTCCGT	GACACAGGCA
14701	CCGCTGCTGG	GGCTGGGCCG	CGTCATCGCC	ATGGAGCACG	CGGATCTGCG	CTGCGCTCGG
14761	GTCGACCTCG	ATCCGACCCG	GCCCGATGGG	GAGCTCGGTG	CCCTGCTGGC	CGAGCTGCTG
14821	GCCGACGACG	CCGAAGCGGA	AGTCGCGTTG	CGCGGTGGCG	AGCGATGCGT	CGCTCGGATC
14881	GTCCGCCGGC	AGCCCGAGAC	CCGGCCCCGG	GGGAGGATCG	AGAGCTGCGT	TCCGACCGAC
14941	GTCACCATCC	GCGCGGACAG	CACCTACCTT	GTGACCGGCG	GTCTGGGTGG	GCTCGGTCTG
15001	AGCGTGGCCG	GATGGCTGGC	CGAGCGCGGC	GCTGGTCACC	TGGTGCTGGT	GGGCCGCTCC
15061	GGCGCGGCGA	GCGTGGAGCA	ACGGGCAGCC	GTCGCGGCGC	TCGAGGCCCG	CGGCGCGCGC
15121	GTCACCGTGG	CGAAGGCAGA	TGTCGCCGAT	CGGGCGCAGC	TCGAGCGGAT	CCTCCGCGAG
15181	GTTACCACGT	CGGGGATGCC	GCTGCGGGGC	GTCGTCCATG	CGGCCGGCAT	CTTGGACGAC
15241	GGGCTGCTGA	TGCAGCAGAC	TCCCGCGCGG	TTTCGTAAGG	TGATGGCGCC	CAAGGTCCAG
15301	GGGGCCTTGC	ACCTGCACGC	GTTGACGCGC	GAAGCGCCGC	TTTCCTTCTT	CGTGCTGTAC
15361	GCTTCGGGAG	TAGGGCTCTT	GGGCTCGCCG	GGCCAGGGCA	ACTACGCCGC	GGCCAACACG
15421	TTCCTCGACG	CTCTGGCGCA	CCACCGGAGG	GCGCAGGGGC	TGCCAGCGTT	GAGCGTCGAC
15481	TGGGGCCTGT	TCGCGGAGGT	GGGCATGGCG	GCCGCGCAGG	AAGATCGCGG	CGCGCGGCTG
15541	GTCTCCCGCG	GAATGCGGAG	CCTCACCCCC	GACGAGGGGC	TGTCCGCTCT	GGCACGGCTG
15601	CTCGAAAGCG	GCCGCGTGCA	GGTGGGGGTG	ATGCCGGTGA	ACCCGCGGCT	GTGGGTGGAG
15661	CTCTACCCCG	CGGCGGCGTC	TTCGCGAATG	TTGTCGCGCC	TGGTGACGGC	GCATCGCGCG
15721	AGCGCCGGCG	GGCCAGCCGG	GGACGGGGAC	CTGCTCCGCC	GCCTCGCTGC	TGCCGAGCCG
15781	AGCGCGCGGA	GCGGGCTCCT	GGAGCCGCTC	CTCCGCGCGC	AGATCTCGCA	GGTGCTGCGC
15841	CTCCCCGAGG	GCAAGATCGA	GGTGGACGCC	CCGCTCACGA	GCCTGGGCAT	GAACTCGCTG
15901	ATGGGGCTCG	AGCTGCGCAA	CCGCATCGAG	GCCATGCTGG	GCATCACCGT	ACCGGCAACG
15961	CTGTTGTGGA	CCTATCCCAC	GGTGGCGGCG	CTGAGCGGGC	ATCTGGCGCG	GGAGGCATGC
16021	GAAGCCGCTC	CTGTGGAGTC	ACCGCACACC	ACCGCCGATT	CTGCTGTCGA	GATCGAGGAG
16081	ATGTCGCAGG	ACGATCTGAC	GCAGTTGATC	GCAGCAAAAT	TCAAGGCGCT	TACATGACTA
16141	CTCGCGGTCC	TACGGCACAG	CAGAATCCGC	TGAAACAAGC	GGCCATCATC	ATTCAGCGGC
16201	TGGAGGAGCG	GCTCGCTGGG	CTCGCACAGG	CGGAGCTGGA	ACGGACCGAG	CCGATCGCCA
16261	TCGTCGGTAT	CGGCTGCCGC	TTCCCTGGCG	GTGCGGACGC	TCCGGAAGCG	TTTTGGGAGC
16321	TGCTCGACGC	GGAGCGCGAC	GCGGTCCAGC	CGCTCGACAG	GCGCTGGGCG	CTGGTAGGTG
16381	TCGCTCCCGT	CGAGGCCGTG	CCGCACTGGG	CGGGGCTGCT	CACCGAGCCG	ATAGATTGCT
16441	TCGATGCTGC	GTTCTTCGGC	ATCTCGCCTC	GGGAGGCGCG	ATCGCTCGAC	CCGCAGCATC
16501	GTCTGTTGCT	GGAGGTCGCT	TGGGAGGGGC	TCGAGGACGC	CGGTATCCCG	CCCCGGTCCA
16561	TCGACGGGAG	CCGCACCGGT	GTGTTCGTCG	GCGCTTTCAC	GGCGGACTAC	GCGCGCACGG
16621	TCGCTCGGTT	GCCGCGCGAG	GAGCGAGACG	CGTACAGCGC	CACCGGCAAC	ATGCTCAGCA
16681	TCGCCGCCGG	ACGGCTGTCG	TACACGCTGG	GGCTGCAGGG	ACCTTGCCTG	ACCGTCGACA
16741	CGGCGTGCTC	GTCATCGCTG	GTGGCGATTC	ACCTCGCCTG	CCGCAGCCTG	CGCGCAGGAG
16801	AGAGCGATCT	CGCGTTGGCG	GGAGGGGTCA	GCACGCTCCT	CTCCCCCGAC	ATGATGGAAG
16861	CCGCGGCGCG	CACGCAAGCG	CTGTCGCCCG	ATGGTCGTTG	CCGGACCTTC	GATGCTTCGG
16921	CCAACGGGTT	CGTCCGTGGC	GAGGGCTGTG	GCCTGGTCGT	CCTCAAACGG	CTCTCCGACG
16981	CGCAACGGGA	TGGCGACCGC	ATCTGGGCGC	TGATCCGGGG	CTCGGCCATC	AACCATGATG
17041	GCCGGTCGAC	CGGGTTGACC	GCGCCCAACG	TGCTGGCTCA	GGAGACGGTC	TTGCGCGAGG
17101	CGCTGCGGAG	CGCCCACGTC	GAAGCTGGGG	CCGTCGATTA	CGTCGAGACC	CACGGAACAG
17161	GGACCTCGCT	GGGCGATCCC	ATCGAGGTCG	AGGCGCTGCG	GGCGACGGTG	GGGCCGGCGC
17221	GCTCCGACGG	CACACGCTGC	GTGCTGGGCG	CGGTGAAGAC	CAACATCGGC	CATCTCGAGG
17281	CCGCGGCAGG	CGTAGCGGGC	CTGATCAAGG	CAGCGCTTTC	GCTGACGCAC	GAGCGCATCC
17341	CGAGAAACCT	CAACTTCCGC	ACGCTCAATC	CGCGGATCCG	GCTCGAGGGC	AGCGCGCTCG
17401	CGTTGGCGAC	CGAGCCGGTG	CCGTGGCCGC	GCACGGACCG	TCCGCGCTTC	GCGGGGGTGA
17461	GCTCGTTCGG	GATGAGCGGA	ACGAACGCGC	ATGTGGTGCT	GGAAGAGGCG	CCGGCGGTGG
17521	AGCTGTGGCC	TGCCGCGCCG	GAGCGCTCGG	CGGAGCTTTT	GGTGCTGTCG	GGCAAGAGCG
17581	AGGGGGCGCT	CGACGCGCAG	GCGGCGCGGC	TGCGCGAGCA	CCTGGACATG	CACCCGGAGC
17641	TCGGGCTCGG	GGACGTGGCG	TTCAGCCTGG	CGACGACGCG	CAGCGCGATG	ACCCACCGGC
17701	TCGCGGTGGC	GGTGACGTCG	CGCGAGGGGC	TGCTGGCGGC	GCTTTCGGCC	GTGGCGCAGG
17761	GGCAGACGCC	GGCGGGGGCG	GCGCGCTGCA	TCGCGAGCTC	CTCGCGCGGC	AAGCTGGCGT
17821	TGCTGTTCAC	CGGACAGGGC	GCGCAGACGC	CGGGCATGGG	CCGGGGGCTC	TGCGCGGCGT
17881	GGCCAGCGTT	CCGGGAGGCG	TTCGACCGGT	GCGTGACGCT	GTTCGACCGG	GAGCTGGACC
17941	GCCCGCTGCG	CGAGGTGATG	TGGGCGGAGG	CGGGGAGCGC	CGAGTCGTTG	TTGCTGGACC
18001	AGACGGCGTT	CACCCAGCCC	GCGCTCTTCG	CGGTGGAGTA	CGCGCTGACG	GCGCTGTGGC
18061	GGTCGTGGGG	CGTAGAGCCG	GAGCTCCTGG	TTGGGCATAG	CATCGGGGAG	CTGGTGGCGG
18121	CGTGCGTGGC	GGGGGTGTTC	TCGCTGGAAG	ATGGGGTGAG	GCTCGTGGCG	GCGCGCGGGC
18181	GGCTGATGCA	GGGGCTCTCG	GCGGGCGGCG	CGATGGTGTC	GCTCGGAGCG	CCGGAGGCGG
18241	AGGTGGCCGC	GGCGGTGGCG	CCGCACGCGG	CGTGGGTGTC	GATCGCGGCG	GTCAATGGGC
18301	CGGAGCAGGT	GGTGATCGCG	GGCGTGGAGC	AAGCGGTGCA	GGCGATCGCG	GCGGGGTTCG
18361	CGGCGCGCGG	CGTGCGCACC	AAGCGGCTGC	ATGTCTCGCA	CGCGTTCCAC	TCGCCGCTGA
18421	TGGAACCGAT	GCTGGAGGAG	TTCGGGCGGG	TGGCGGCGTC	GGTGACGTAC	CGGCGGCCAA
18481	GCGTTTCGCT	GGTGAGCAAC	CTGAGCGGGA	AGGTGGTCAC	GGACGAGCTG	AGCGCGCCGG
18541	GCTACTGGGT	GCGGCACGTG	CGGGAGGCGG	TGCGCTTCGC	GGACGGGGTG	AAGGCGCTGC
18601	ACGAAGCCGG	CGCGGGCACG	TTCCTCGAAG	TGGGCCCGAA	GCCGACGCTG	CTCGGCCTGT
18661	TGCCAGCTTG	CCTGCCGGAG	GCGGAGCCGA	CGTTGCTGGC	GTCGTTGCGC	GCCGGGCGCG
18721	AGGAGGCTGC	GGGGGTGCTC	GAGGCGCTGG	GCAGGCTGTG	GGCCGCTGGC	GGCTCGGTCA
18781	GCTGGCCGGG	CGTCTTCCCC	ACGGCTGGGC	GGCGGGTGCC	GCTGCCGACC	TATCCGTGGC
18841	AGCGGCAGCG	GTACTGGATC	GAGGCGCCGG	CCGAAGGGCT	CGGAGCCACG	GCCGCCGATG
18901	CGCTGGCGCA	GTGGTTCTAC	CGGGTGGACT	GGCCCGAGAT	GCCTCGCTCA	TCCGTGGATT
18961	CGCGGCGAGC	CCGGTCCGGC	GGGTGGCTGG	TGCTGGCCGA	CCGGGGTGGA	GTCGGGGAGG
19021	CGGCCGCGGC	GGCGCTTTCG	TCGCAGGGAT	GTTCGTGCGC	CGTGCTCCAT	GCGCCCGCCG
19081	AGGCCTCCGC	GGTCGCCGAG	CAGGTGACCC	AGGCCCTCGG	TGGCCGCAAC	GACTGGCAGG
19141	GGGTGCTGTA	CCTGTGGGGT	CTGGACGCCG	TCGTGGAGGC	GGGGGCATCG	GCCGAAGAGG
19201	TCGGCAAAGT	CACCCATCTT	GCCACGGCGC	CGGTGCTCGC	GCTGATTCAG	GCGGTGGGCA
19261	CGGGGCCGCG	CTCACCCCGG	CTCTGGATCG	TGACCCGAGG	GGCCTGCACG	GTGGGCGGCG
19321	AGCCTGACGC	TGCCCCCTGT	CAGGCGGCGC	TGTGGGGTAT	GGGCCGGGTC	GCGGCGCTGG
19381	AGCATCCCGG	CTCCTGGGGC	GGGCTCGTGG	ACCTGGATCC	GGAGGAGAGC	CCGACGGAGG
19441	TCGAGGCCCT	GGTGGCCGAG	CTGCTTTCGC	CGGACGCCGA	GGATCAGCTG	GCATTCCGCC
19501	AGGGGCGCCG	GCGCGCAGCG	CGGCTCGTGG	CCGCCCCACC	GGAGGGAAAC	GCAGCGCCGG
19561	TGTCGCTGTC	TGCGGAGGGG	AGTTACTTGG	TGACGGGTGG	GCTGGGCGCC	CTTGGCCTCC
19621	TCGTTGCGCG	GTGGTTGGTG	GAGCGCGGGG	CGGGGCACCT	TGTGCTGATC	AGCCGGCACG
19681	GATTGCCCGA	CCGCGAGGAA	TGGGGCCGAG	ATCAGCCGCC	AGAGGTGCGC	GCGCGCATTG
19741	CGGCGATCGA	GGCGCTGGAG	GCGCAGGGCG	CGCGGGTCAC	CGTGGCGGCG	GTCGACGTGG
19801	CCGATGCCGA	AGGCATGGCG	GCGCTCTTGG	CGGCCGTCGA	GCCGCCGCTG	CGGGGGGTCG
19861	TGCACGCCGC	GGGTCTGCTC	GACGACGGGC	TGCTGGCCCA	CCAGGACGCC	GGTCGGCTCG
19921	CCCGGGTGTT	GCGCCCCAAG	GTGGAGGGGG	CATGGGTGCT	GCACACCCTT	ACCCGCGAGC
19981	AGCCGCTGGA	CCTCTTCGTA	CTGTTTTCCT	CGGCGTCGGG	CGTCTTCGGC	TCGATCGGCC
20041	AGGGCAGCTA	CGCGGCAGGC	AATGCCTTTT	TGGACGCGCT	GGCGGACCTC	CGTCGAACGC
20101	AGGGGCTCGC	CGCCCTGAGC	ATCGCCTGGG	GCCTGTGGGC	GGAGGGGGGG	ATGGGCTCGC
20161	AGGCGCAGCG	CCGGGAACAT	GAGGCATCGG	GAATCTGGGC	GATGCCGACG	AGTCGTGCCC
20221	TGGCGGCGAT	GGAATGGCTG	CTCGGTACGC	GCGCGACGCA	GCGCGTGGTC	ATCCAGATGG
20281	ATTGGGCCCA	TGCGGGAGCG	GCTCCGCGCG	ACGCGAGCCG	AGGCCGCTTC	TGGGATCGGC
20341	TGGTAACTGT	CACGAAAGCG	GCCTCCTCCT	CGGCCGTGCC	AGCTGTAGAG	CGCTGGCGCA
20401	ACGCGTCTGT	TGTGGAGACC	CGCTCGGCGC	TCTACGAGCT	TGTGCGCGGC	GTGGTCGCCG
20461	GGGTGATGGG	CTTTACCGAC	CAAGGCACGC	TCGACGTGCG	ACGAGGCTTC	GCCGAGCAGG
20521	GCCTCGACTC	CCTGATGGCT	GTGGAGATCC	GCAAACGGCT	TCAGGGTGAG	CTGGGTATGC
20581	CGCTGTCGGC	GACGCTGGCG	TTCGACCATC	CGACCGTGGA	GCGGCTGGTG	GAATACTTGC
20641	TGAGCCAGGC	GCTGGAGCTG	CAGGACCGCA	CCGACGTGCG	AAGCGTTCGG	TTGCCGGCGA
20701	CAGAGGACCC	GATCGCCATC	GTGGGTGCCG	CCTGCCGCTT	CCCGGGCGGG	GTCGAGGACC
20761	TGGAGTCCTA	CTGGCAGCTG	TTGACCGAGG	GCGTGGTGGT	CAGCACCGAG	GTGCCGGCCG
20821	ACCGGTGGAA	TGGGGCAGAC	GGGCGCGGCC	CCGGCTCGGG	AGAGGCTCCG	AGACAGACCT
20881	ACGTGCCCAG	GGGTGGCTTT	CTGCGCGAGG	TGGAGACGTT	CGATGCGGCG	TTCTTCCACA
20941	TCTCGCCTCG	GGAGGCGATG	AGCCTGGACC	CGCAACAGCG	GCTGCTGCTG	GAAGTGAGCT
21001	GGGAGGCGAT	CGAGCGCGCG	GGCCAGGACC	CGTCGGCGCT	GCGCGAGAGC	CCCACGGGCG
21061	TGTTCGTGGG	CGCGGGCCCC	AACGAATATG	CCGAGCGGGT	GCAGGACCTC	GCCGATGAGG
21121	CGGCGGGGCT	CTACAGCGGC	ACCGGCAACA	TGCTCAGCGT	TGCGGCGGGA	CGGCTGTCAT
21181	TTTTCCTGGG	CCTGCACGGG	CCGACCCTGG	CTGTGGATAC	GGCGTGCTCC	TCGTCGCTCG
21241	TGGCGCTGCA	CCTCGGCTGC	CAGAGCTTGC	GACGGGGCGA	GTGCGACCAA	GCCCTGGTTG
21301	GCGGGGTCAA	CATGCTGCTC	TCGCCGAAGA	CCTTCGCGCT	GCTCTCACGG	ATGCACGCGC
21361	TTTCGCCCGG	CGGGCGGTGC	AAGACGTTCT	CGGCCGACGC	GGACGGCTAC	GCGCGGGCCG
21421	AGGGCTGCGC	CGTGGTGGTG	CTCAAGCGGC	TCTCCGACGC	GCAGCGCGAC	CGCGACCCCA
21481	TCCTGGCGGT	GATCCGGGGT	ACGGCGATCA	ATCATGATGG	CCCGAGCAGC	GGGCTGACAG
21541	TGCCCAGCGG	CCCTGCCCAG	GAGGCGCTGT	TACGCCAGGC	GCTGGCGCAC	GCAGGGGTGG
21601	TTCCGGCCGA	CGTCGATTTC	GTGGAATGCC	ACGGGACCGG	GACGGCGCTG	GGCGACCCGA
21661	TCGAGGTGCG	GGCGCTGAGC	GACGTGTACG	GGCAAGCCCG	CCCTGCGGAC	CGACCGCTGA
21721	TCCTGGGAGC	CGCCAAGGCC	AACCTTGGGC	ACATGGAGCC	CGCGGCGGGC	CTGGCCGGCT
21781	TGCTCAAGGC	GGTGCTCGCG	CTGGGGCAAG	AGCAAATACC	AGCCCAGCCG	GAGCTGGGCG
21841	AGCTCAACCC	GCTCTTGCCG	TGGGAGGCGC	TGCCGGTGGC	GGTGGCCCGC	GCAGCGGTGC
21901	CGTGGCCGCG	CACGGACCGT	CCGCGCTTCG	CGGGGGTGAG	CTCGTTCGGG	ATGAGCGGAA
21961	CGAACGCGCA	TGTGGTGCTG	GAAGAGGCGC	CGGCGGTGGA	GCTGTGGCCT	GCCGCGCCGG
22021	AGCGCTCGGC	GGAGCTTTTG	GTGCTGTCGG	GCAAGAGCGA	GGGGGCGCTC	GACGCGCAGG
22081	CGGCGCGGCT	GCGCGAGCAC	CTGGACATGC	ACCCGGAGCT	CGGGCTCGGG	GACGTGGCGT
22141	TCAGCCTGGC	GACGACGCGC	AGCGCGATGA	ACCACCGGCT	CGCGGTGGCG	GTGACGTCGC
22201	GCGAGGGGCT	GCTGGCGGCG	CTTTCGGCCG	TGGCGCAGGG	GCAGACGCCG	CCGGGGGCGG
22261	CGCGCTGCAT	CGCGAGCTCG	TCGCGCGGCA	AGCTGGCGTT	CCTGTTCACC	GGACAGGGCG
22321	CGCAGACGCC	GGGCATGGGC	CGGGGGCTTT	GCGCGGCGTG	GCCAGCGTTC	CGAGAGGCGT
22381	TCGACCGGTG	CGTGGCGCTG	TTCGACCGGG	AGCTGGACCG	CCCGCTGTGC	GAGGTGATGT
22441	GGGCGGAGCC	GGGGAGCGCC	GAGTCGTTGT	TGCTCGACCA	GACGGCGTTC	ACCCAGCCCG
22501	CGCTCTTCAC	GGTGGAGTAC	GCGCTGACGG	CGCTGTGGCG	GTCGTGGGGC	GTAGAGCCGG
22561	AGCTGGTGGC	TGGGCATAGC	GCCGGGGAGC	TGGTGGCGGC	GTGCGTGGCG	GGGGTGTTCT
22621	CGCTGGAAGA	TGGGGTGAGG	CTCGTGGCGG	CGCGCGGGCG	GCTGATGCAG	GGGCTCTCGG
22681	CGGGCGGCGC	GATGGTGTCG	CTCGGAGCGC	CGGAGGCGGA	GGTGGCCGCG	GCGGTGGCGC
22741	CGCACGCGGC	GTGGGTGTCG	ATCGCGGCGG	TCAATGGGCC	GGAGCAGGTG	GTGATCGCGG
22801	GCGTGGAGCA	AGCGGTGCAG	GCGATCGCGG	CGGGGTTCGC	GGCGCGCGGC	GTGCGCACCA
22861	AGCGGCTGCA	TGTCTCGCAC	GCATCCCACT	CGCCGCTGAT	GGAACCGATG	CTGGAGGAGT
22921	TCGGGCGGGT	GGCGGCGTCG	GTGACGTACC	GGCGGCCAAG	CGTTTCGCTG	GTGAGCAACC
22981	TGAGCGGGAA	GGTGGTCACG	GACGAGCTGA	GCGCGCCGGG	CTACTGGGTG	CGGCACGTGC
23041	GGGAGGCGGT	GCGCTTCGCG	GACGGGGTGA	AGGCGCTGCA	CGAAGCCGGC	GCGGGGACGT
23101	TCCTCGAAGT	GGGCCCGAAG	CCGACGCTGC	TCGGCCTGTT	GCCAGCTTGC	CTGCCGGAGG
23161	CGGAGCCGAC	GCTGCTGGCG	TCGTTGCGCG	CCGGGCGCGA	GGAGGCTGCG	GGGGTGCTCG
23221	AGGCGCTGGG	CAGGCTGTGG	GCCGCCGGCG	GCTCGGTCAG	CTGGCCGGGC	GTCTTCCCCA
23281	CGGCTGGGCG	GCGGGTGCCG	CTGCCGACCT	ATCCGTGGCA	GCGGCAGCGG	TACTGGCCCG
23341	ACATCGAGCC	TGACAGCCGT	CGCCACGCAG	CCGCGGATCC	GACCCAAGGC	TGGTTCTATC
23401	GCGTGGACTG	GCCGGAGATA	CCTCGCAGCC	TCCAGAAATC	AGAGGAGGCG	AGCCGCGGGA
23461	GCTGGCTGGT	ATTGGCGGAT	AAGGGTGGAG	TCGGCGAGGC	GGTCGCTGCA	GCGCTGTCGA
23521	CACGTGGACT	TCCATGCGTC	GTGCTCCATG	CGCCGGCAGA	GACATCCGCG	ACCGCCGAGC
23581	TGGTGACCGA	GGCTGCCGGC	GGTCGAAGCG	ATTGGCAGGT	AGTGCTCTAC	CTGTGGGGTC
23641	TGGACGCCGT	CGTCGGCGCG	GAGGCGTCGA	TCGATGAGAT	CGGCGACGCG	ACCCGTCGTG
23701	CTACCGCGCC	GGTGCTCGGC	TTGGCTCGGT	TTCTGAGCAC	CGTGTCTTGT	TCGCCCCGAC
23761	TCTGGGTCGT	GACCCGGGGG	GCATGCATCG	TTGGCGACGA	GCCTGCGATC	GCCCCTTGTC
23821	AGGCGGCGTT	ATGGGGCATG	GGCCGGGTGG	CGGCGCTCGA	GCATCCCGGG	GCCTGGGGCG
23881	GGCTCGTGGA	CCTGGATCCC	CGAGCGAGCC	CGCCCCAAGC	CAGCCCGATC	GACGGCGAGA
23941	TGCTCGTCAC	CGAGCTATTG	TCGCAGGAGA	CCGAGGACCA	GCTCGCCTTC	CGCCATGGGC
24001	GCCGGCACGC	GGCACGGCTG	GTGGCCGCCC	CGCCACGGGG	GGAAGCGGCA	CCGGCGTCGC
24061	TGTCTGCGGA	GGCGAGCTAC	CTGGTGACGG	GAGGCCTCGG	TGGGCTGGGC	CTGATCGTGG
24121	CCCAGTGGCT	GGTGGAGCTG	GGAGCGCGGC	ACTTGGTGCT	GACCAGCCGG	CGCGGGTTGC
24181	CCGACCGGCA	GGCGTGGCGC	GAGCAGCAGC	CGCCTGAGAT	CCGCGCGCGG	ATCGCAGCGG
24241	TCGAGGCGCT	GGAGGCGCGG	GGTGCACGGG	TGACCGTGGC	AGCGGTGGAC	GTGGCCGACG
24301	TCGAACCGAT	GACAGCGCTG	GTTTCGTCGG	TCGAGCCCCC	GCTGCGAGGG	GTGGTGCACG
24361	CCGCTGGCGT	CAGCGTCATG	CGTCCACTGG	CGGAGACGGA	CGAGACCCTG	CTCGAGTCGG
24421	TGCTCCGTCC	CAAGGTGGCC	GGGAGCTGGC	TGCTGCACCG	GCTGCTGCAC	GGCCGGCCTC
24481	TCGACCTGTT	CGTGCTGTTC	TCGTCGGGCG	CAGCGGTGTG	GGGTAGCCAT	AGCCAGGGTG
24541	CGTACGCGGC	GGCCAACGCT	TTCCTCGACG	GGCTCGCGCA	TCTTCGGCGT	TCGCAATCGC
24601	TGCCTGCGTT	GAGCGTCGCG	TGGGGTCTGT	GGGCCGAGGG	AGGCATGGCG	GACGCGGAGG
24661	CTCATGCACG	TCTGAGCGAC	ATCGGGGTTC	TGCCCATGTC	GACGTCGGCA	GCGTTGTCGG
24721	CGCTCCAGCG	CCTGGTGGAG	ACCGGCGCGG	CTCAGCGCAC	GGTGACCCGG	ATGGACTGGG
24781	CGCGCTTCGC	GCCGGTGTAC	ACCGCTCGAG	GGCGTCGCAA	CCTGCTTTCG	GCGCTGGTCG
24841	CAGGGCGCGA	CATCATCGCG	CCTTCCCCTC	CGGCGGCAGC	AACCCGGAAC	TGGCGTGGCC
24901	TGTCCGTTGC	GGAAGCCCGC	ATGGCTCTGC	ACGAGGTCGT	CCATGGGGCC	GTCGCTCGGG
24961	TGCTGGGCTT	CCTCGACCCG	AGCGCGCTCG	ATCCTGGGAT	GGGGTTCAAT	GAGCAGGGCC
25021	TCGACTCGTT	GATGGCGGTG	GAGATCCGCA	ACCTCCTTCA	GGCTGAGCTG	GACGTGCGGC
25081	TTTCGACGAC	GCTGGCCTTT	GATCATCCGA	CGGTACAGCG	GCTGGTGGAG	CATCTGCTCG
25141	TCGATGTACT	GAAGCTGGAG	GATCGCAGCG	ACACCCAGCA	TGTTCGGTCG	TTGGCGTCAG
25201	ACGAGCCCAT	CGCCATCGTG	GGAGCCGCCT	GCCGCTTCCC	GGGCGGGGTG	GAGGACCTGG
25261	AGTCCTACTG	GCAGCTGTTG	GCCGAGGGCG	TGGTGGTCAG	CGCCGAGGTG	CCGGCCGACC
25321	GGTGGGATGC	GGCGGACTGG	TACGACCCTG	ATCCGGAGAT	CCCAGGCCGG	ACTTACGTGA
25381	CCAAAGGCGC	CTTCCTGCGC	GATTTGCAGA	GATTGGATGC	GACCTTCTTC	CGCATCTCGC
25441	CTCGCGAGGC	GATGAGCCTC	GACCCGCAGC	AGCGGTTGCT	CCTGGAGGTA	AGCTGGGAGG
25501	CGCTCGAGAG	CGCGGGTATC	GCTCCGGATA	CGCTGCGAGA	TAGCCCCACC	GGGGTGTTCG
25561	TGGGTGCGGG	GCCCAATGAG	TACTACACGC	AGCGGCTGCG	AGGCTTCACC	GACGGAGCGG
25621	CAGGGCTGTA	CGGCGGCACC	GGGAACATGC	TCAGCGTTGC	GGCTGGACGG	CTGTCGTTTT
25681	TCCTGGGTCT	GCACGGCCCG	ACGCTGGCCA	TGGATACGGC	GTGCTCGTCC	TCCCTGGTCG
25741	CGCTGCACCT	CGCCTGCCAG	AGCCTGCGAC	TGGGCGAGTG	CGATCAAGCG	CTGGTTGGCG
25801	GGGTCAACGT	GCTGCTCGCG	CCGGAGACCT	TCGTGCTGCT	CTCACGGATG	CGCGCGCTTT
25861	CGCCCGACGG	GCGGTGCAAG	ACGTTCTCGG	CCGACGCGGA	CGGCTACGCG	CGGGGCGAGG
25921	GGTGCGCCGT	GGTGGTGCTC	AAGCGGCTGC	GCGATGCGCA	GCGCGCCGGC	GACTCCATCC
25981	TGGCGCTGAT	CCGGGGAAGC	GCGGTGAACC	ACGACGGCCC	GAGCAGCGGG	CTGACCGTGC
26041	CCAACGGACC	CGCCCAGCAA	GCATTGCTGC	GCCAGGCGCT	TTCGCAAGCA	GGCGTGTCTC
26101	CGGTCGACGT	TGATTTTGTG	GAGTGTCACG	GGACAGGGAC	GGCGCTGGGC	GACCCGATCG
26161	AGGTGCAGGC	GCTGAGCGAG	GTGTATGGTC	CAGGGCGCTC	CGAGGATCGA	CCGCTGGTGC
26221	TGGGGGCCGT	CAAGGCCAAC	GTCGCGCATC	TGGAGGCGGC	ATCCGGCTTG	GCCAGCCTGC
26281	TCAAGGCCGT	GCTTGCGCTG	CGGCACGAGC	AGATCCCGGC	CCAGCCGGAG	CTGGGGGAGC
26341	TCAACCCGCA	CTTGCCGTGG	AACACGCTGC	CGGTGGCGGT	GCCACGTAAG	GCGGTGCCGT
26401	GGGGGCGCGG	CGCACGGCCG	CGTCGGGCCG	GCGTGAGCGC	GTTCGGGTTG	AGCGGAACCA
26461	ACGTGCATGT	CGTGCTGGAG	GAGGCACCGG	AGGTGGAGCT	GGTGCCCGCG	GCGCCGGCGC
26521	GACCGGTGGA	GCTGGTTGTG	CTATCGGCCA	AGAGCGCGGC	GGCGCTGGAC	GCCGCGGCGG
26581	AACGGCTCTC	GGCGCACCTG	TCCGCGCACC	CGGAGCTGAG	CCTCGGCGAC	GTGGCGTTCA
26641	GCCTGGCGAC	GACGCGCAGC	CCGATGGAGC	ACCGGCTCGC	CATCGCGACG	ACCTCGCGCG
26701	AGGCCCTGCG	AGGCGCGCTG	GACGCCGCGG	CGCAGCGGCA	GACGCCGCAG	GGCGCGGTGC
26761	GCGGCAAGGC	CGTGTCCTCA	CGCGGTAAGT	TGGCTTTCCT	GTTCACCGGA	CAGGGCGCGC
26821	AAATGCCGGG	CATGGGCCGT	GGGCTGTACG	AGGCGTGGCC	AGCGTTCCGG	GAGGCGTTCG
26881	ACCGGTGCGT	GGCGCTCTTC	GATCGGGAGC	TCGACCAGCC	TCTGCGCGAG	GTGATGTGGG
26941	CTGCGCCGGG	CCTCGCTCAG	GCGGCGCGGC	TCGATCAGAC	CGCGTACGCG	CAGCCGGCTC
27001	TCTTTGCGCT	GGAGTACGCG	CTGGCTGCCC	TGTGGCGTTC	GTGGGGCGTG	GAGCCGCACG
27061	TACTCCTCGG	TCATAGCATC	GGCGAGCTGG	TCGCCGCCTG	CGTGGCGGGC	GTGTTCTCGC
27121	TCGAAGACGC	GGTGAGGTTG	GTGGCCGCGC	GCGGGCGGCT	GATGCAGGCG	CTGCCCGCCG
27181	GCGGTGCCAT	GGTCGCCATC	GCAGCGTCCG	AGGCCGAGGT	GGCCGCCTCC	GTGGCACCCC
27241	ACGCCGCCAC	GGTGTCGATC	GCCGCGGTCA	ACGGTCCTGA	CGCCGTCGTG	ATCGCTGGCG
27301	CCGAGGTACA	GGTGCTCGCC	CTCGGCGCGA	CGTTCGCGGC	GCGTGGGATA	CGCACGAAGA
27361	GGCTCGCCGT	CTCCCATGCG	TTCCACTCGC	CGCTCATGGA	TCCGATGCTG	GAAGACTTCC
27421	AGCGGGTCGC	TGCGACGATC	GCGTACCGCG	CGCCAGACCG	CCCGGTGGTG	TCGAATGTCA
27481	CCGGCCACGT	CGCAGGCCCC	GAGATCGCCA	CGCCCGAGTA	TTGGGTCCGG	CATGTGCGAA
27541	GCGCCGTGCG	CTTCGGCGAT	GGGGCAAAGG	CGTTGCATGC	CGCGGGTGCC	GCCACGTTCG
27601	TCGAGATTGG	CCCGAAGCCG	GTCCTGCTCG	GGCTATTGCC	AGCGTGCCTC	GGGGAAGCGG
27661	ACGCGGTCCT	CGTGCCGTCG	CTACGCGCGG	ACCGCTCGGA	ATGCGAGGTG	GTCCTCGCGG
27721	CGCTCGGGAC	TTGGTATGCC	TGGGGGGGTG	CGCTCGACTG	GAAGGGCGTG	TTCCCCGATG
27781	GCGCGCGCCG	CGTGGCTCTG	CCCATGTATC	CATGGCAGCG	TGAGCGCCAT	TGGATGGACC
27841	TCACCCCGCG	AAGCGCCGCG	CCTGCAGGGA	TCGCAGGTCG	CTGGCCGCTG	GCTGGTGTCG
27901	GGCTCTGCAT	GCCCGGCGCT	GTGTTGCACC	ACGTGCTCTC	GATCGGACCA	CGCCATCAGC
27961	CCTTCCTCGG	TGATCACCTC	GTGTTTGGCA	AGGTGGTGGT	GCCCGGCGCC	TTTCATGTCG
28021	CGGTGATCCT	CAGCATCGCC	GCCGAGCGCT	GGCCCGAGCG	GGCGATCGAG	CTGACAGGCG
28081	TGGAGTTCCT	GAAGGCGATC	GCGATGGAGC	CCGACCAGGA	GGTCGAGCTC	CACGCCGTGC
28141	TCACCCCCGA	AGCCGCCGGG	GATGGCTACC	TGTTCGAGCT	GGCGACCCTG	GCGGCGCCGG
28201	AGACCGAACG	CCGATGGACG	ACCCACGCCC	GCGGTCGGGT	GCAGCCGACA	GACGGCGCGC
28261	CCGGCGCGTT	GCCGCGCCTC	GAGGTGCTGG	AGGACCGCGC	GATCCAGCCC	CTCGACTTCG
28321	CCGGATTCCT	CGACAGGTTA	TCGGCGGTGC	GGATCGGCTG	GGGTCCGCTT	TGGCGATGGC
28381	TGCAGGACGG	GCGCGTCGGC	GACGAGGCCT	CGCTTGCCAC	CCTCGTGCCG	ACCTATCCGA
28441	ACGCCCACGA	CGTGGCGCCC	TTGCACCCGA	TCCTGCTGGA	CAACGGCTTT	GCGGTGAGCC
28501	TGCTGGCAAC	CCGGAGCGAG	CCGGAGGACG	ACGGGACGCC	CCCGCTGCCG	TTCGCCGTGG
28561	AACGGGTGCG	GTGGTGGCGG	GCGCCGGTTG	GAAGGGTGCG	GTGTGGCGGC	GTGCCGCGGT
28621	CGCAGGCATT	CGGTGTCTCG	AGCTTCGTGC	TGGTCGACGA	AACTGGCGAG	GTGGTCGCTG
28681	AGGTGGAGGG	ATTTGTTTGC	CGCCGGGCGC	CGCGAGAGGT	GTTCCTGCGG	CAGGAGTCGG
28741	GCGCGTCGAC	TGCAGCCTTG	TACCGCCTCG	ACTGGCCCGA	AGCCCCCTTG	CCCGATGCGC
28801	CTGCGGAACG	GATGGAGGAG	AGCTGGGTCG	TGGTGGCAGC	ACCTGGCTCG	GAGATGGCCG
28861	CGGCGCTCGC	AACACGGCTC	AACCGCTGCG	TACTCGCCGA	ACCCAAAGGC	CTCGAGGCGG
28921	CCCTCGCGGG	GGTGTCTCCC	GCAGGTGTGA	TCTGCCTCTG	GGAACCTGGA	GCCCACGAGG
28981	AAGCTCCGGC	GGCGGCGCAG	CGTGTGGCGA	CCGAGGGCCT	TTCGGTGGTG	CAGGCGCTCA
29041	GGGATCGCGC	GGTGCGCCTG	TGGTGGGTGA	CCACGGGCGC	CGTGGCTGTC	GAGGCCGGTG
29101	AGCGGGTGCA	GGTCGCCACA	GCGCCGGTAT	GGGGCCTGGG	CCGGACAGTG	ATGCAGGAGC
29161	GCCCGGAGCT	CAGCTGCACT	CTGGTGGATT	TGGAGCCGGA	GGTCGATGCC	GCGCGTTCAG
29221	CTGACGTTCT	GCTGCGGGAG	CTCGGTCGCG	CTGACGACGA	GACCCAGGTG	GTTTTCCGTT
29281	CCGGAGAGCG	CCGCGTAGCG	CGGCTGGTCA	AAGCGACAAC	CCCCGAAGGG	CTCTTGGTCC
29341	CTGACGCAGA	ATCCTATCGA	CTGGAGGCTG	GGCAGAAGGG	CACATTGGAC	CAGCTCCGCC
29401	TCGCGCCGGC	ACAGCGCCGG	GCACCCGGCC	CGGGCGAGGT	CGAGATCAAG	GTAACCGCCT
29461	CGGGGCTCAA	CTTCCGGACC	GTCCTCGCTG	TGCTGGGAAT	GTATCCGGGC	GACGCTGGGC
29521	CGATGGGCGG	AGATTGTGCC	GGTATCGTCA	CGGCGGTGGG	CCAGGGGGTG	CACCACCTCT
29581	CGGTCGGCGA	TGCTGTCATG	ACGCTGGGGA	CGTTGCATCG	ATTCGTCACG	GTCGACGCGC
29641	GGCTGGTGGT	CCGGCAGCCT	GCAGGGCTGA	CTCCCGCGCA	GGCAGCTACG	GTGCCGGTTG
29701	CGTTCCTGAC	GGCCTGGCTC	GCTCTGCACG	ACCTGGGGAA	TCTGCGGCGC	GGCGAGCGGG
29761	TGCTGATCCA	TGCTGCGGCC	GGCGGCGTGG	GCATGGCCGC	GGTGCAAATC	GCCCGATGGA
29821	TAGGGGCCGA	GGTGTTCGCC	ACGGCGAGCC	CGTCCAAGTG	GGCAGCGGTT	CAGGCCATGG
29881	GCGTGCCGCG	CACGCACATC	GCCAGCTCGC	GGACGCTGGA	GTTTGCTGAG	ACGTTCCGGC
29941	AGGTCACCGG	CGGCCGGGGC	GTGGACGTGG	TGCTCAACGC	GCTGGCCGGC	GAGTTCGTGG
30001	ACGCGAGCCT	GTCCCTGCTG	ACGACGGGCG	GGCGGTTCCT	CGAGATGGGC	AAGACCGACA
30061	TACGGGATCG	AGCCGCGGTC	GCGGCGGCGC	ATCCCGGTGT	TCGCTATCGG	GTATTCGACA
30121	TCCTGGAGCT	CGCTCCGGAT	CGAACTCGAG	AGATCCTCGA	GCGCGTGGTC	GAGGGCTTTG
30181	CTGCGGGACA	TCTGCGCGCA	TTGCCGGTGC	ATGCGTTCGC	GATCACCAAG	GCCGAGGCAG
30241	CGTTTCGGTT	CATGGCGCAA	GCGCGGCATC	AGGGCAAGGT	CGTGCTGCTG	CCGGCGCCCT
30301	CCGCAGCGCC	CTTGGCGCCG	ACGGGCACCG	TACTGCTGAC	CGGTGGGCTG	GGAGCGTTGG
30361	GGCTCCACGT	GGCCCGCTGG	CTCGCCCAGC	AGGGCGCGCC	GCACATGGTG	CTCACAGGTC
30421	GGCGGGGCCT	GGATACGCCG	GGCGCTGCCA	AAGCCGTCGC	GGAGATCGAA	GCGCTCGGCG
30481	CTCGGGTGAC	GATCGCGGCG	TCGGATGTCG	CCGATCGGAA	CGCGCTGGAG	GCTGTGCTCC
30541	AGGCCATTCC	GGCGGAGTGG	CCGTTACAGG	GCGTGATCCA	TGCAGCCGGA	GCGCTCGATG
30601	ATGGTGTGCT	TGATGAGCAG	ACCACCGACC	GCTTCTCGCG	GGTGCTGGCA	CCGAAGGTGA
30661	CTGGCGCCTG	GAATCTGCAT	GAGCTCACGG	CGGGCAACGA	TCTCGCTTTC	TTCGTGCTGT
30721	TCTCCTCCAT	GTCGGGGCTC	TTGGGCTCGG	CCGGGCAGTC	CAACTATGCG	GCGGCCAACA
30781	CCTTCCTCGA	CGCGCTGGCC	GCGCATCGGC	GGGCCGAAGG	CCTGGCGGCG	CAGAGCCTCG
30841	CGTGGGGCCC	ATGGTCGGAC	GGAGGCATGG	CAGCGGGGCT	CAGCGCGGCG	CTGCAGGCGC
30901	GGCTCGCTCG	GCATGGGATG	GGAGCGCTGT	CGCCCGCTCA	GGGCACCGCG	CTGCTCGGGC
30961	AGGCGCTGGC	TCGGCCGGAA	ACGCAGCTCG	GGGCGATGTC	GCTCGACGTG	CGTGCGGCAA
31021	GCCAAGCTTC	GGGAGCGGCA	GTGCCGCCTG	TGTGGCGCGC	GCTGGTGCGC	GCGGAGGCGC
31081	GCCATGCGGC	GGCTGGGGCG	CAGGGGGCAT	TGGCCGCGCG	CCTTGGGGCG	CTGCCCGAGG
31141	CGCGTCGCGC	CGACGAGGTG	CGCAAGGTCG	TGCAGGCCGA	GATCGCGCGC	GTGCTTTCAT
31201	GGGGCGCCGC	GAGCGCCGTG	CCCGTCGATC	GGCCGCTGTC	GGACTTGGGC	CTCGACTCGC
31261	TCACGGCGGT	GGAGCTGCGC	AACGTGCTCG	GCCAGCGGGT	GGGTGCGACG	CTGCCGGCGA
31321	CGCTGGCATT	CGATCACCCG	ACGGTCGACG	CGCTCACGCG	CTGGCTGCTC	GATAAGGTCC
31381	TGGCCGTGGC	CGAGCCGAGC	GTATCGCCCG	CAAAGTCGTC	GCCGCAGGTC	GCCCTCGACG
31441	AGCCCATTGC	GGTGATCGGC	ATCGGCTGCC	GTTTCCCAGG	CGGCGTGACC	GATCCGGAGT
31501	CGTTTTGGCG	GCTGCTCGAA	GAGGGCAGCG	ATGCCGTCGT	CGAGGTGCCG	CATGAGCGAT
31561	GGGACATCGA	CGCGTTCTAT	GATCCGGATC	CGGATGTGCG	CGGCAAGATG	ACGACACGCT
31621	TTGGCGGCTT	CCTGTCCGAT	ATCGACCGGT	TCGAGCCGGC	CTTCTTCGGC	ATCTCGCCGC
31681	GCGAAGCGAC	GACCATGGAT	CCGCAGCAGC	GGCTGCTCCT	GGAGACGAGC	TGGGAGGCGT
31741	TCGAGCGCGC	CGGGATTTTG	CCCGAGCGGC	TGATGGGCAG	CGATACCGGC	GTGTTCGTGG
31801	GGCTCTTCTA	CCAGGAGTAC	GCTGCGCTCG	CCGGCGGCAT	CGAGGCGTTC	GATGGCTATC
31861	TAGGCACCGG	CACCACGGCC	AGCGTCGCCT	CGGGCAGGAT	CTCTTATGTG	CTCGGGCTAA
31921	AGGGGCCGAG	CCTGACGGTG	GACACCGCGT	GCTCCTCGTC	GCTGGTCGCG	GTGCACCTGG
31981	CCTGCCAGGC	GCTGCGGCGG	GGCGAGTGTT	CGGTGGCGCT	GGCCGGCGGC	GTGGCGCTGA
32041	TGCTCACGCC	GGCGACGTTC	GTGGAGTTCA	GCCGGCTGCG	AGGCCTGGCT	CCCGACGGAC
32101	GGTGCAAGAG	CTTCTCGGCC	GCAGCCGACG	GCGTGGGGTG	GAGCGAAGGC	TGCGCCATGC
32161	TCCTGCTCAA	ACCGCTTCGC	GATGCTCAGC	GCGATGGGGA	TCCGATCCTG	GCGGTGATCC
32221	GCGGCACCGC	GGTGAACCAG	GATGGGCGCA	GCAACGGGCT	GACGGCGCCC	AACGGGTCGT
32281	CGCAGCAAGA	GGTGATCCGT	CGGGCCCTGG	AGCAGGCGGG	GCTGGCTCCG	GCGGACGTCA
32341	GCTACGTCGA	GTGCCACGGC	ACCGGCACGA	CGTTGGGCGA	CCCCATCGAA	GTGCAGGCCC
32401	TGGGCGCCGT	GCTGGCACAG	GGGCGACCCT	CGGACCGGCC	GCTCGTGATC	GGGTCGGTGA
32461	AGTCCAATAT	CGGACATACG	CAGGCTGCGG	CGGGCGTGGC	CGGTGTCATC	AAGGTGGCGC
32521	TGGCGCTCGA	GCGCGGGCTT	ATCCCGAGGA	GCCTGCATTT	CGACGCGCCC	AATCCGCACA
32581	TTCCGTGGTC	GGAGCTCGCC	GTGCAGGTGG	CCGCCAAACC	CGTCGAATGG	ACGAGAAACG
32641	GCGCGCCGCG	ACGAGCCGGG	GTGAGCTCGT	TTGGCGTCAG	CGGGACCAAC	GCGCACGTGG
32701	TGCTGGAGGA	GGCGCCAGCG	GCGGCGTTCG	CGCCCGCGGC	GGCGCGTTCA	GCGGAGCTTT
32761	TCGTGCTGTC	GGCGAAGAGC	GCCGCGGCGC	TGGACGCGCA	GGCGGCGCGG	CTTTCGGCGC
32821	ATGTCGTTGC	GCACCCGGAG	CTCGGCCTCG	GCGACCTGGC	GTTCAGCCTG	GCGACGACCC
32881	GCAGCCCGAT	GACGTACCGG	CTCGCGGTGG	CGGCGACCTC	GCGCGAGGCG	CTGTCTGCGG
32941	CGCTCGACAC	AGCGGCGCAG	GGGCAGGCGC	CGCCCGCAGC	GGCTCGCGGC	CACGCTTCCA
33001	CAGGCAGCGC	CCCAAAGGTG	GTTTTCGTCT	TTCCTGGCCA	GGGCTCCCAG	TGGCTGGGCA
33061	TGGGCCAAAA	GCTCCTCTCG	GAGGAGCCCG	TCTTCCGCGA	CGCGCTCTCG	GCGTGTGACC
33121	GAGCGATTCA	GGCCGAAGCC	GGCTGGTCGC	TGCTCGCCGA	GCTCGCGGCC	GATGAGACCA
33181	CCTCGCAGCT	CGGCCGCATC	GACGTGGTGC	AGCCGGCGCT	GTTCGCGATC	GAGGTCGCGC
33241	TGTCGGCGCT	GTGGCGGTCG	TGGGGCGTCG	AGCCGGATGC	AGTGGTAGGC	CACAGCATGG
33301	GCGAAGTGGC	GGCCGCGCAC	GTCGCCGGCG	CCCTGTCGCT	CGAGGATGCT	GTAGCGATCA
33361	TCTGCCGGCG	CAGCCTGCTG	CTGCGGCGGA	TCAGCGGCCA	AGGCGAGATG	GCGGTCGTCG
33421	AGCTCTCCCT	GGCCGAGGCC	GAGGCAGCGC	TCCTGGGCTA	CGAAGATCGG	CTCAGCGTGG
33481	CGGTGAGCAA	CAGCCCGCGA	TCGACGGTGC	TGGCGGGCGA	GCCGGCAGCG	CTCGCAGAGG
33541	TGCTGGCGAT	CCTTGCGGCA	AAGGGGGTGT	TCTGCCGTCG	AGTCAAGGTG	GACGTCGCCA
33601	GCCACAGCCC	ACAGATCGAC	CCGCTGCGCG	ACGAGCTATT	GGCAGCATTG	GGCGAGCTCG
33661	AGCCGCGACA	AGCGACCGTG	TCGATGCGCT	CGACGGTGAC	GAGCACGATC	GTGGCGGGCC
33721	CGGAGCTCGT	GGCGAGCTAC	TGGGCGGACA	ACGTTCGACA	GCCGGTGCGC	TTCGCCGAAG
33781	CGGTGCAATC	GTTGATGGAA	GGCGGTCATG	GGCTGTTCGT	GGAGATGAGC	CCGCATCCGA
33841	TCCTGACGAC	GTCGGTCGAG	GAGATCCGAC	GGGCGACGAA	GCGGGAGGGA	GTCGCGGTGG
33901	GCTCGTTGCG	GCGTGGACAG	GACGAGCGCC	TGTCCATGTT	GGAGGCGCTG	GGAGCGCTCT
33961	GGGTACACGG	CCAGGCGGTG	GGCTGGGAGC	GGCTGTTCTC	CGCGGGCGGC	GCGGGCCTCC
34021	GTCGCGTGCC	GCTGCCGACC	TATCCCTGGC	AGCGCGAGCG	GTACTGGGTC	GAAGCGCCGA
34081	CCGGCGGCGC	GGCGAGCGGC	AGCCGCTTTG	CTCATGCGGG	CAGTCACCCG	CTCCTGGGTG
34141	AAATGCAGAC	CCTGTCGACC	CAGAGGAGCA	CGCGCGTGTG	GGAGACGACG	CTGGATCTCA
34201	AACGGCTGCC	GTGGCTCGGC	GATCACCGGG	TGCAGGGGGC	GGTCGTGTTC	CCGGGCGCGG
34261	CGTACCTGGA	GATGGCGCTT	TCGTCTGGGG	CCGAGGCCTT	GGGTGACGGT	CCGCTCCAGG
34321	TCAGCGATGT	GGTGCTCGCC	GAGGCGCTGG	CCTTCGCGGA	TGATACGCCG	GTGGCGGTGC
34381	AGGTCATGGC	GACCGAGGAG	CGACCAGGCC	GCCTGCAATT	CCACGTTGCG	AGCCGGGTGC
34441	CGGGCCACGG	CCGTGCTGCC	TTTCGAAGCC	ATGCCCGCGG	GGTGCTGCGC	CAGACCGAGC
34501	GCGCCGAGGT	CCCGGCGAGG	CTGGATCTGG	CCGCGCTTCG	TGCCCGGCTT	CAGGCCAGCG
34561	CACCCGCTGC	GGCTACCTAT	GCGGCGCTGG	CCGAGATGGG	GCTCGAGTAC	GGCCCAGCGT
34621	TCCAGGGGCT	TGTCGAGCTG	TGGCGGGGGG	AGGGCGAGGC	GCTGGGACGT	GTGCGGCTCC
34681	CCGAGGCCGC	CGGCTCCCCA	GCCGCGTGCC	GGCTCCACCC	CGCGCTCTTG	GATGCGTGCT
34741	TCCACGTGAG	CAGCGCCTTC	GCTGACCGCG	GCGAGGCGAC	GCCATGGGTA	CCCGTCGAAA
34801	TCGGCTCGCT	GCGGTGGTTC	CAGCGGCCGT	CGGGGGAGCT	GTGGTGTCAT	GCGCGGAGCG
34861	TGAGCCACGG	AAAGCCAACA	CCCGATCGGC	GGAGTACCGA	CTTTTGGGTG	GTCGACAGCA
34921	CGGGCGCGAT	CGTCGCCGAG	ATCTCCGGGC	TCGTGGCGCA	GCGGCTCGCG	GGAGGTGTAC
34981	GCCGGCGCGA	AGAAGACGAC	TGGTTCATGG	AGCCGGCTTG	GGAACCGACC	GCGGTCCCCG
35041	GATCCGAGGT	CACGGCGGGC	CGGTGGCTGC	TCATCGGCTC	GGGCGGCGGG	CTCGGCGCTG
35101	CGCTCTACTC	GGCGCTGACG	GAAGCTGGCC	ATTCCGTCGT	CCACGCGACA	GGGCACGGCA
35161	CGAGCGCCGC	CGGGTTGCAG	GCACTCCTGA	CGGCGTCCTT	CGACGGCCAG	GCCCCGACGT
35221	CGGTGGTGCA	CCTCGGCAGC	CTCGATGAGC	GTGGCGTGCT	CGACGCGGAT	GCCCCCTTCG
35281	ACGCCGATGC	CCTCGAGGAG	TCGCTGGTGC	GCGGCTGCGA	CAGCGTGCTC	TGGACCGTGC
35341	AGGCCGTGGC	CGGGGCGGGC	TTCCGAGATC	CTCCGCGGTT	GTGGCTCGTG	ACACGCGGCG
35401	CTCAGGCCAT	CGGCGCCGGC	GACGTCTCCG	TGGCGCAAGC	GCCGCTCCTG	GGGCTGGGCC
35461	GCGTTATCGC	CTTGGAGCAC	GCCGAGCTGC	GCTGCGCTCG	GATCGACCTC	GATCCAGCGC
35521	GGCGCGACGG	AGAGGTCGAT	GAGCTGCTTG	CCGAGCTGTT	GGCCGACGAC	GCCGAGGAGG
35581	AAGTCGCGTT	TCGCGGCGGT	GAGCGGCGCG	TGGCCCGGCT	CGTCCGAAGG	CTGCCCGAGA
35641	CCGACTGCCG	AGAGAAAATC	GAGCCCGCGG	AAGGCCGGCC	GTTCCGGCTG	GAGATCGATG
35701	GGTCCGGCGT	GCTCGACGAC	CTGGTGCTCC	GAGCCACGGA	GCGGCGCCCT	CCTGGCCCGG
35761	GCGAGGTCGA	GATCGCCGTC	GAGGCGGCGG	GGCTCAACTT	TCTCGACGTG	ATGAGGGCCA
35821	TGGGGATCTA	CCCTGGGCCC	GGGGACGGTC	CGGTTGCGCT	GGGCGCCGAG	TGCTCCGGCC
35881	GAATTGTCGC	GATGGGCGAA	GGTGTCGAGA	GCCTTCGTAT	CGGCCAGGAC	GTCGTGGCCG
35941	TCGCGCCCTT	CAGTTTCGGC	ACCCACGTCA	CCATCGACGC	CCGGATGGTC	GCACCTCGCC
36001	CCGCGGCGCT	GACGGCCGCG	CAGGCAGCCG	CGCTGCCCGT	CGCATTCATG	ACGGCCTGGT
36061	ACGGTCTCGT	CCATCTGGGG	AGGCTCCGGG	CCGGCGAGCG	CGTGCTCATC	CACTCGGCGA
36121	CGGGGGGCAC	CGGGCTCGCT	GCTGTGCAGA	TCGCCCGCCA	CCTCGGCGCG	GAGATATTTG
36181	CGACCGCTGG	TACGCCGGAG	AAGCGGGCGT	GGCTGCGCGA	GCAGGGGATC	GCGCACGTGA
36241	TGGACTCGCG	GTCGCTGGAC	TTCGCCGAGC	AAGTGCTGGC	CGCGACGAAG	GGCGAGGGGG
36301	TCGACGTCGT	GTTGAACTCG	CTGTCTGGCG	CCGCGATCGA	CGCGAGCCTT	GCGACCCTCG
36361	TGCCGGACGG	CCGCTTCATC	GAGCTCGGCA	AGACGGACAT	CTATGCAGAT	CGCTCGCTGG
36421	GGCTCGCTCA	CTTTAGGAAG	AGCCTGTCCT	ACAGCGCCGT	CGATCTTGCG	GGTTTGGCCG
36481	TGCGTCGGCC	CGAGCGCGTC	GCAGCGCTGC	TGGCGGAGGT	GGTGGACCTG	CTCGCACGGG
36541	GAGCGCTGCA	GCCGCTTCCG	GTAGAGATCT	TCCCCCTCTC	GCGGGCCGCG	GACGCGTTCC
36601	GGAAAATGGC	GCAAGCGCAG	CATCTCGGGA	AGCTCGTGCT	CGCGCTGGAG	GACCCGGACG
36661	TGCGGATCCG	CGTTCCGGGC	GAATCCGGCG	TCGCCATCCG	CGCGGACGGC	ACCTACCTCG
36721	TGACCGGCGG	TCTGGGTGGG	CTCGGTCTGA	GCGTGGCTGG	ATGGCTGGCC	GAGCAGGGGG
36781	CTGGGCATCT	GGTGCTGGTG	GGCCGCTCCG	GTGCGGTGAG	CGCGGAGCAG	CAGACGGCTG
36841	TCGCCGCGCT	CGAGGCGCAC	GGCGCGCGTG	TCACGGTAGC	GAGGCCAGAC	GTCGCCGATC
36901	GGGCGCAGAT	CGAGCGGATC	CTCCGCGAGG	TTACCGCGTC	GGGGATGCCG	CTCCGCGGCG
36961	TCGTTCATGC	GGCCGGTATC	CTGGACGACG	GGCTGCTGAT	GCAGCAAACC	CCCGCGCGGT
37021	TCCGCGCGGT	CATGGCGCCC	AAGGTCCGAG	GGGCCTTGCA	CCTGCATGCG	TTGACACGCG
37081	AAGCGCCGCT	CTCCTTCTTC	GTGCTGTACG	CTTCGGGAGC	AGGGCTCTTG	GGCTCGCCGG
37141	GCCAGGGCAA	CTACGCCGCG	GCCAACACGT	TCCTCGACGC	TCTGGCACAC	CACCGGAGGG
37201	CGCAGGGGCT	GCCAGCATTG	AGCATCGACT	GGGGCCTGTT	CGCGGACGTG	GGTTTGGCCG
37261	CCGGGCAGCA	AAATCGCGGC	GCACGGCTGG	TCACCCGCGG	GACGCGGAGC	CTCACCCCCG
37321	ACGAAGGGCT	GTGGGCGCTC	GAGCGTCTGC	TCGACGGCGA	TCGCACCCAG	GCCGGGGTCA
37381	TGCCGTTCGA	CGTGCGGCAG	TGGGTGGAGT	TCTACCCGGC	GGCGGCATCT	TCGCGGAGGT
37441	TGTCGCGGCT	GGTGACGGCA	CGGCGCGTGG	CTTCCGGTCG	GCTCGCCGGG	GATCGGGACC
37501	TGCTCGAACG	GCTCGCCACC	GCCGAGGCGG	GCGCGCGGGC	AGGAATGCTG	CAGGAGGTCG
37561	TGCGCGCGCA	GGTCTCGCAG	GTGCTGCGCC	TCCCCGAAGG	CAAGCTCGAC	GTGGATGCGC
37621	CGCTCACGAG	CCTGGGAATG	GACTCGCTGA	TGGGGCTAGA	GCTGCGCAAC	CGCATCGAGG
37681	CCGTGCTCGG	CATCACCATG	CCGGCGACCC	TGCTGTGGAC	CTACCCCACG	GTGGCAGCGC
37741	TGAGTGCGCA	TCTGGCTTCT	CATGTCGTCT	CTACGGGGGA	TGGGGAATCC	GCGCGCCCGC
37801	CGGATACAGG	GAACGTGGCT	CCAATGACCC	ACGAAGTCGC	TTCGCTCGAC	GAAGACGGGT
37861	TGTTCGCGTT	GATTGATGAG	TCACTCGCGC	GTGCGGGAAA	GAGGTGATTG	CGTGACAGAC
37921	CGAGAAGGCC	AGCTCCTGGA	GCGCTTGCGT	GAGGTTACTC	TGGCCCTTCG	CAAGACGCTG
37981	AACGAGCGCG	ATACCCTGGA	GCTCGAGAAG	ACCGAGCCGA	TCGCCATCGT	GGGGATCGGC
38041	TGCCGCTTCC	CCGGCGGAGC	GGGCACTCCG	GAGGCGTTCT	GGGAGCTGCT	CGACGACGGG
38101	CGCGACGCGA	TCCGGCCGCT	CGAGGAGCGC	TGGGCGCTCG	TAGGTGTCGA	CCCAGGCGAC
38161	GACGTACCGC	GCTGGGCGGG	GCTGCTCACC	GAAGCCATCG	ACGGCTTCGA	CGCCGCGTTC
38221	TTCGGTATCG	CCCCCCGGGA	GGCACGGTCG	CTCGACCCGC	AGCATCGCTT	GCTGCTGGAG
38281	GTCGCCTGGG	AGGGGTTCGA	AGACGCCGGC	ATCCCGCCTA	GGTCCCTCGT	CGGGAGCCGC
38341	ACCGGCGTGT	TCGTCGGCGT	CTGCGCCACG	GAGTATCTCC	ACGCCGCCGT	CGCGCACCAG
38401	CCGCGCGAAG	AGCGGGACGC	GTACAGCACC	ACCGGCAACA	TGCTCAGCAT	CGCCGCCGGA
38461	CGGCTATCGT	ACACGCTGGG	GCTGCAGGGA	CCTTGCCTGA	CCGTCGACAC	GGCGTGCTCG
38521	TCATCGCTGG	TGGCCATTCA	CCTCGCCTGC	CGCAGCCTGC	GCGCTCGAGA	GAGCGATCTC
38581	GCGCTGGCGG	GAGGGGTCAA	CATGCTTCTC	TCCCCCGACA	CGATGCGAGC	TCTGGCGCGC
38641	ACCCAGGCGC	TGTCGCCCAA	TGGCCGTTGC	CAGACCTTCG	ACGCGTCGGC	CAACGGGTTC
38701	GTCCGTGGGG	AGGGCTGCGG	TCTGATCGTG	CTCAAGCGAT	TGAGCGACGC	GCGGCGGGAT
38761	GGGGACCGGA	TCTGGGCGCT	GATCCGAGGA	TCGGCCATCA	ATCAGGACGG	CCGGTCGACG
38821	GGGTTGACGG	CGCCCAACGT	GCTCGCCCAG	GGGGCGCTCT	TGCGCGAGGC	GCTGCGGAAC
38881	GCCGGCGTCG	AGGCCGAGGC	CATCGGTTAC	ATCGAGACCC	ACGGGGCGGC	GACCTCGCTG
38941	GGCGACCCCA	TCGAGATCGA	AGCGCTGCGC	ACCGTGGTGG	GGCCGGCGCG	AGCCGACGGA
39001	GCGCGCTGCG	TGCTGGGCGC	GGTGAAGACC	AACCTCGGCC	ACCTGGAGGG	CGCTGCCGGC
39061	GTGGCGGGCC	TGATCAAGGC	TACACTTTCG	CTACATCACG	AGCGCATCCC	GAGGAACCTC
39121	AACTTTCGTA	CGCTCAATCC	GCGGATCCGG	ATCGAGGGGA	CCGCGCTCGC	GTTGGCGACC
39181	GAACCGGTGC	CCTGGCCGCG	GACGGGCCGG	ACGCGCTTCG	CGGGAGTGAG	CTCGTTCGGG
39241	ATGAGCGGGA	CCAACGCGCA	TGTGGTGTTG	GAGGAGGCGC	CGGCGGTGGA	GCCTGAGGCC
39301	GCGGCCCCCG	AGCGCGCTGC	GGAGCTGTTC	GTCCTGTCGG	CGAAGAGCGT	GGCGGCGCTG
39361	GATGCGCAGG	CAGCCCGGCT	GCGGGACCAC	CTGGAGAAGC	ATGTCGAGCT	TGGCCTCGGC
39421	GATGTGGCGT	TCAGCCTGGC	GACGACGCGC	AGCGCGATGG	AGCACCGGCT	GGCGGTGGCC
39481	GCGAGCTCGC	GCGAGGCGCT	GCGAGGGGCG	CTTTCGGCCG	CAGCGCAGGG	GCATACGCCG
39541	CCGGGAGCCG	TGCGTGGGCG	GGCCTCCGGC	GGCAGCGCGC	CGAAGGTGGT	CTTCGTGTTT
39601	CCCGGCCAGG	GCTCGCAGTG	GGTGGGCATG	GGCCGAAAGC	TCATGGCCGA	AGAGCCGGTC
39661	TTCCGGGCGG	CGCTGGAGGG	TTGCGACCGG	GCCATCGAGG	CGGAAGCGGG	CTGGTCGCTG
39721	CTCGGGGAGC	TCTCCGCCGA	CGAGGCCGCC	TCGCAGCTCG	GGCGCATCGA	CGTGGTTCAG
39781	CCGGTGCTCT	TCGCCATGGA	AGTAGCGCTT	TCTGCGCTGT	GGCGGTCGTG	GGGAGTGGAG
39841	CCGGAAGCGG	TGGTGGGCCA	CAGCATGGGC	GAGGTGGCGG	CGGCGCACGT	GGCCGGCGCG
39901	CTGTCGCTCG	AGGACGCGGT	GGCGATCATC	TGCCGGCGCA	GCCGGCTGCT	GCGGCGGATC
39961	AGCGGTCAGG	GCGAGATGGC	GCTGGTCGAG	CTGTCGCTGG	AGGAGGCCGA	GGCGGCGCTG
40021	CGTGGCCATG	AGGGTCGGCT	GAGCGTGGCG	GTGAGCAACA	GCCCGCGCTC	GACCGTGCTC
40081	GCAGGCGAGC	CGGCGGCGCT	CTCGGAGGTG	CTGGCGGCGC	TGACGGCCAA	GGGGGTGTTC
40141	TGGCGGCAGG	TGAAGGTGGA	CGTCGCCAGC	CATAGCCCGC	AGGTCGACCC	GCTGCGCGAA
40201	GAGCTGATCG	CGGCGCTGGG	GGCGATCCGG	CCGCGAGCGG	CTGCGGTGCC	GATGCGCTCG
40261	ACGGTGACGG	GCGGGGTGAT	CGCGGGTCCG	GAGCTCGGTG	CGAGCTACTG	GGCGGACAAT
40321	CTTCGGCAGC	CGGTGCGCTT	CGCTGCGGCG	GCGCAAGCGC	TGCTGGAAGG	TGGCCCCACG
40381	CTGTTCATCG	AGATGAGCCC	GCACCCGATC	CTGGTGCCGC	CCCTGGACGA	GATCCAGACG
40441	GCGGTCGAGC	AAGGGGGCGC	TGCGGTGGGC	TCGCTGCGGC	GAGGGCAGGA	CGAGCGCGCG
40501	ACGCTGCTGG	AGGCGCTGGG	GACGCTGTGG	GCGTCCGGCT	ATCCGGTGAG	CTGGGCTCGG
40561	CTGTTCCCCG	CGGGCGGCAG	GCGGGTTCCG	CTGCCGACCT	ATCCCTGGCA	GCACGAGCGG
40621	TGCTGGATCG	AGGTCGAGCC	TGACGCCCGC	CGCCTCGCCG	CAGCCGACCC	CACCAAGGAC
40681	TGGTTCTACC	GGACGGACTG	GCCCGAGGTG	CCCCGCGCCG	CCCCGAAATC	GGAGACAGCT
40741	CATGGGAGCT	GGCTGCTGTT	GGCCGACAGG	GGTGGGGTCG	GCGAGGCGGT	CGCTGCAGCG
40801	CTGTCGACGC	GCGGACTTTC	CTGCACCGTG	CTTCATGCGT	CGGCTGACGC	CTCCACCGTC
40861	GCCGAGCAGG	TATCCGAAGC	TGCCAGTCGC	CGAAACGACT	GGCAGGGAGT	CCTCTACCTG
40921	TGGGGCCTCG	ACGCCGTCGT	CGATGCTGGG	GCATCGGCCG	ACGAAGTCAG	CGAGGCTACC
40981	CGCCGTGCCA	CCGCACCCGT	CCTTGGGCTG	GTTCGATTCC	TGAGCGCTGC	GCCCCATCCT
41041	CCTCGCTTCT	GGGTGGTGAC	CCGCGGGGCA	TGCACGGTGG	GCGGCGAGCC	AGAGGTCTCT
41101	CTTTGCCAAG	CGGCGTTGTG	GGGCCTCGCG	CGCGTCGTGG	CGCTGGAGCA	TCCCGCTGCC
41161	TGGGGTGGCC	TCGTGGACCT	GGATCCTCAG	AAGAGCCCGA	CGGAGATCGA	GCCCCTGGTG
41221	GCCGAGCTGC	TTTCGCCGGA	CGCCGAGGAT	CAACTGGCGT	TCCGCAGCGG	TCGCCGGCAC
41281	GCAGCACGCC	TTGTAGCCGC	CCCGCCGGAG	GGCGACGTCG	CACCGATATC	GCTGTCCGCG
41341	GAGGGAAGCT	ACCTGGTGAC	GGGTGGGCTG	GGTGGCCTTG	GTCTGCTCGT	GGCTCGGTGG
41401	CTGGTGGAGC	GGGGAGCTCG	ACATCTGGTG	CTCACCAGCC	GGCACGGGCT	GCCAGAGCGA
41461	CAGGCGTCGG	GCGGAGAGCA	GCCGCCGGAG	GCCCGCGCGC	GCATCGCAGC	GGTCGAGGGG
41521	CTGGAAGCGC	AGGGCGCGCG	GGTGACCGTG	GCAGCGGTGG	ATGTCGCCGA	GGCCGATCCC
41581	ATGACGGCGC	TGCTGGCCGC	CATCGAGCCC	CCGTTGCGCG	GGGTGGTGCA	CGCCGCCGGC
41641	GTCTTCCCCG	TGCGTCCCCT	GGCGGAGACG	GACGAGGCCC	TGCTGGAGTC	GGTGCTCCGT
41701	CCCAAGGTGG	CCGGGAGCTG	GCTGCTGCAC	CGGCTGCTGC	GCGACCGGCC	TCTCGACCTG
41761	TTCGTGCTGT	TCTCGTCGGG	CGCGGCGGTG	TGGGGTGGCA	AAGGCCAAGG	CGCATACGCC
41821	GCGGCCAATG	CGTTCCTCGA	CGGGCTCGCG	CACCATCGCC	GCGCGCACTC	CCTGCCGGCG
41881	TTGAGCCTCG	CCTGGGGCCT	ATGGGCCGAG	GGAGGCGTGG	TTGATGCAAA	GGCTCATGCA
41941	CGTCTGAGCG	ACATCGGAGT	CCTGCCCATG	GCCACGGGGC	CGGCCTTGTC	GGCGCTGGAG
42001	CGCCTGGTGA	ACACCAGCGC	TGTCCAGCGT	TCGGTCACAC	GGATGGACTG	GGCGCGCTTC
42061	GCGCCGGTCT	ATGCCGCGCG	AGGGCGGCGC	AACTTGCTTT	CGGCTCTGGT	CGCGGAGGAC
42121	GAGCGCACTG	CGTCTCCCCC	GGTGCCGACG	GCAAACCGGA	TCTGGCGCGG	CCTGTCCGTT
42181	GCGGAGAGCC	GCTCAGCCCT	CTACGAGCTC	GTTCGCGGCA	TCGTCGCCCG	GGTGCTGGGC
42241	TTCTCCGACC	CGGGCGCGCT	CGACGTCGGC	CGAGGCTTCG	CCGAGCAGGG	GCTCGACTCC
42301	CTGATGGCTC	TGGAGATCCG	TAACCGCCTT	CAGCGCGAGC	TGGGCGAACG	GCTGTCGGCG
42361	ACTCTGGCCT	TCGACCACCC	GACGGTGGAG	CGGCTGGTGG	CGCATCTCCT	CACCGACGTG
42421	CTGAAGCTGG	AGGACCGGAG	CGACACCCGG	CACATCCGGT	CGGTGGCGGC	GGATGACGAC
42481	ATCGCCATCG	TCGGTGCCGC	CTGCCGGTTC	CCGGGCGGGG	ATGAGGGCCT	GGAGACATAC
42541	TGGCGGCATC	TGGCCGAGGG	CATGGTGGTC	AGCACCGAGG	TGCCAGCCGA	CCGGTGGCGC
42601	GCGGCGGACT	GGTACGACCC	CGATCCGGAG	GTTCCGGGCC	GGACCTATGT	GGCCAAGGGG
42661	GCCTTCCTCC	GCGATGTGCG	CAGCTTGGAT	GCGGCGTTCT	TCTCCATCTC	CCCTCGTGAG
42721	GCGATGAGCC	TGGACCCGCA	ACAGCGGCTG	TTGCTGGAGG	TGAGCTGGGA	GGCGATCGAG
42781	CGCGCTGGCC	AGGACCCGAT	GGCGCTGCGC	GAGAGCGCCA	CGGGCGTGTT	CGTGGGCATG
42841	ATCGGGAGCG	AGCACGCCGA	GCGGGTGCAG	GGCCTCGACG	ACGACGCGGC	GTTGCTGTAC
42901	GGCACCACCG	GCAACCTGCT	CAGCGTCGCC	GCTGGACGGC	TGTCGTTCTT	CCTGGGTCTG
42961	CACGGCCCGA	CGATGACGGT	GGACACCGCG	TGCTCGTCGT	CGCTGGTGGC	GTTGCACCTC
43021	GCCTGCCAGA	GCCTGCGATT	GGGCGAGTGC	GACCAGGCAC	TGGCCGGCGG	GTCCAGCGTG
43081	CTTTTGTCGC	CGCGGTCATT	CGTCGCGGCA	TCGCGCATGC	GTTTGCTTTC	GCCAGATGGG
43141	CGGTGCAAGA	CGTTCTCGGC	CGCTGCAGAC	GGCTTTGCGC	GGGCCGAGGG	CTGCGCCGTG
43201	GTGGTGCTCA	AGCGGCTCCG	TGACGCGCAG	CGCGACCGCG	ACCCCATCCT	GGCGGTGGTC
43261	CGGAGCACGG	CGATCAACCA	CGATGGCCCG	AGCAGCGGGC	TCACGGTGCC	CAGCGGTCCT
43321	GCCCAGCAGG	CGTTGCTAGG	CCAGGCGCTG	GCGCAAGCGG	GCGTGGCACC	GGCCGAGGTC
43381	GATTTCGTGG	AGTGCCACGG	GACGGGGACA	GCGCTGGGTG	ACCCGATCGA	GGTGCAGGCG
43441	CTGGGCGCGG	TGTATGGCCG	GGGCCGCCCC	GCGGAGCGGC	CGCTCTGGCT	GGGCGCTGTC
43501	AAGGCCAACC	TCGGCCACCT	GGAGGCCGCG	GCGGGCTTGG	CCGGCGTGCT	CAAGGTGCTC
43561	TTGGCGCTGG	AGCACGAGCA	GATTCCGGCT	CAACCGGAGC	TCGACGAGCT	CAACCCGCAC
43621	ATCCCGTGGG	CAGAGCTGCC	AGTGGCCGTT	GTCCGCGCGG	CGGTCCCCTG	GCCGCGCGGC
43681	GCGCGCCCGC	GTCGTGCAGG	CGTGAGCGCT	TTCGGCCTGA	GCGGGACCAA	CGCGCATGTG
43741	GTGTTGGAGG	AGGCGCCGGC	GGTGGAGCCT	GAGGCCGCGG	CCCCCGAGCG	CGCTGCGGAG
43801	CTGTTCGTCC	TGTCGGCGAA	GAGCGTGGCG	GCGCTGGATG	CGCAGGCAGC	CCGGCTGCGG
43861	GATCATCTGG	AGAAGCATGT	CGAGCTTGGC	CTCGGCGATG	TGGCGTTCAG	CCTGGCGACG
43921	ACGCGCAGCG	CGATGGAGCA	CCGGCTGGCG	GTGGCCGCGA	GCTCGCGCGA	GGCGCTGCGA
43981	GGGGCGCTTT	CGGCCGCAGC	GCAGGGGCAT	ACGCCGCCGG	GAGCCGTGCG	TGGGCGGGCC
44041	TCCGGCGGCA	GCGCGCCGAA	GGTGGTCTTC	GTGTTTCCCG	GCCAGGGCTC	GCAGTGGGTG
44101	GGCATGGGCC	GAAAGCTCAT	GGCCGAAGAG	CCGGTCTTCC	GGGCGGCGCT	GGAGGGTTGC
44161	GACCGGGCCA	TCGAGGCGGA	AGCGGGCTGG	TCGCTGCTCG	GGGAGCTCTC	CGCCGACGAG
44221	GCCGCCTCGC	AGCTCGGGCG	CATCGACGTG	GTTCAGCCGG	TGCTCTTCGC	CGTGGAAGTA
44281	GCGCTTTCAG	CGCTGTGGCG	GTCGTGGGGA	GTGGAGCCGG	AAGCGGTGGT	GGGCCACAGC
44341	ATGGGCGAGG	TTGCGGCGGC	GCACGTGGCC	GGCGCGCTGT	CGCTCGAGGA	TGCGGTGGCG
44401	ATCATCTGCC	GGCGCAGCCG	GCTGCTGCGG	CGGATCAGCG	GTCAGGGCGA	GATGGCGCTG
44461	GTCGAGCTGT	CGCTGGAGGA	GGCCGAGGCG	GCGCTGCGTG	GCCATGAGGG	TCGGCTGAGC
44521	GTGGCGGTGA	GCAACAGCCC	GCGCTCGACC	GTGCTCGCAG	GCGAGCCGGC	GGCGCTCTCG
44581	GAGGTGCTGG	CGGCGCTGAC	GGCCAAGGGG	GTGTTCTGGC	GGCAGGTGAA	GGTGGACGTC
44641	GCCAGCCATA	GCCCGCAGGT	CGACCCGCTG	CGCGAAGAGC	TGGTCGCGGC	GCTGGGAGCG
44701	ATCCGGCCGC	GAGCGGCTGC	GGTGCCGATG	CGCTCGACGG	TGACGGGCGG	GGTGATTGCG
44761	GGTCCGGAGC	TCGGTGCGAG	CTACTGGGCG	GACAATCTTC	GGCAGCCGGT	GCGCTTCGCT
44821	GCGGCGGCGC	AAGCGCTGCT	GGAAGGTGGC	CCCACGCTGT	TCATCGAGAT	GAGCCCGCAC
44881	CCGATCCTGG	TGCCGCCTCT	GGACGAGATC	CAGACGGCGG	TCGAGCAAGG	GGGCGCTGCG
44941	GTGGGCTCGC	TGCGGCGAGG	GCAGGACGAG	CGCGCGACGC	TGCTGGAGGC	GCTGGGGACG
45001	CTGTGGGCGT	CCGGCTATCC	GGTGAGCTGG	GCTCGGCTGT	TCCCCGCGGG	CGGCAGGCGG
45061	GTTCCGCTGC	CGACCTATCC	CTGGCAGCAC	GAGCGGTACT	GGATCGAGGA	CAGCGTGCAT
45121	GGGTCGAAGC	CCTCGCTGCG	GCTTCGGCAG	CTTCATAACG	GCGCCACGGA	CCATCCGCTG
45181	CTCGGGGCTC	CATTGCTCGT	CTCGGCGCGA	CCCGGAGCTC	ACTTGTGGGA	GCAAGCGCTG
45241	AGCGACGAGA	GGCTATCCTA	TCTTTCGGAA	CATAGGGTCC	ATGGCGAAGC	CGTGTTGCCC
45301	AGCGCGGCGT	ATGTAGAGAT	GGCGCTCGCC	GCCGGCGTAG	ATCTCTATGG	CGCGGCGACG
45361	CTGGTGCTGG	AGCAGCTGGC	GCTCGAGCGA	GCCCTCGCCG	TGCCTTCCGA	AGGCGGACGC
45421	ATCGTGCAAG	TGGCCCTCAG	CGAAGAAGGG	CCCGGTCGGG	CCTCATTCCA	GGTATCGAGC
45481	CGTGAGGAGG	CAGGTAGAAG	CTGGGTTCGG	CACGCCACGG	GGCACGTGTG	TAGCGACCAG
45541	AGCTCAGCAG	TGGGAGCGTT	GAAGGAAGCT	CCGTGGGAGA	TTCAACAGCG	ATGTCCGAGC
45601	GTCCTGTCGT	CGGAGGCGCT	CTATCCGCTG	CTCAACGAGC	ACGCCCTCGA	CTATGGCCCC
45661	TGCTTCCAGG	GTGTGGAGCA	GGTGTGGCTC	GGCACGGGGG	AGGTGCTCGG	CCGGGTACGC
45721	TTGCCAGAAG	ACATGGCATC	CTCAAGTGGC	GCCTATCGGA	TTCATCCCGC	CTTGTTGGAT
45781	GCATGTTTTC	AAGTGCTGAC	CGCGCTGCTC	ACCACGCCGG	AATCCATCGA	GATTCGGAGG
45841	CGGCTGACGG	ATCTCCACGA	ACCGGATCTC	CCGCGGTCCA	GGGCTCCGGT	GAATCAAGCG
45901	GTGAGTGACA	CCTGGCTGTG	GGACGCCGCG	CTGGACGGTG	GACGGCGCCA	GAGCGCGAGC
45961	GTGCCCGTCG	ACCTGGTGCT	CGGCAGCTTC	CACGCGAAGT	GGGAGGTCAT	GGATCGCCTC
46021	GCGCAGACGT	ACATCATCCG	CACTCTCCGC	ACATGGAACG	TCTTCTGCGC	TGCTGGAGAG
46081	CGTCACACGA	TAGACGAGTT	GCTCGTCAGG	CTCCAAATCT	CTGCTGTCTA	CAGGAAGGTC
46141	ATCAAGCGAT	GGATGGATCA	CCTTGTCGCG	ATCGGCGTCC	TTGTAGGGGA	CGGAGAGCAT
46201	CTTGTGAGCT	CTCAGCCGCT	GCCGGAGCAT	GATTGGGCGG	CGGTGCTCGA	GGAGGCCGCG
46261	ACGGTGTTCG	CCGACCTCCC	AGTCCTACTT	GAGTGGTGCA	AGTTTGCCGG	GGAACGGCTC
46321	GCGGACGTGT	TGACCGGGAA	GACGCTGGCG	CTCGAGATCC	TCTTCCCTGG	CGGCTCGTTC
46381	GATATGGCGG	AGCGAATCTA	TCAAGATTCG	CCCATCGCCC	GTTACTCGAA	CGGCATCGTG
46441	CGCGGTGTCG	TCGAGTCGGC	GGCGCGGGTG	GTAGCACCGT	CGGGAACGTT	CAGCATCTTG
46501	GAGATCGGAG	CAGGGACGGG	CGCGACCACC	GCCGCCGTCC	TCCCGGTGTT	GCTGCCTGAC
46561	CGGACAGAAT	ACCATTTCAC	CGATGTTTCT	CCGCTCTTCC	TTGCTCGTGC	GGAGCAAAGA
46621	TTTCGAGATC	ATCCATTCCT	GAAGTATGGT	ATTCTGGATA	TCGACCAGGA	GCCAGCTGGC
46681	CAGGGATACG	CACATCAGAA	GTTCGACGTC	ATCGTCGCGG	CCAACGTCAT	CCATGCGACC
46741	CGCGATATAA	GAGCCACGGC	GAAGCGTCTC	CTGTCGTTGC	TCGCGCCCGG	AGGCCTTCTG
46801	GTGCTGGTCG	AGGGCACAGG	GCATCCGATC	TGGTTCGATA	TCACCACGGG	ATTGATCGAG
46861	GGGTGGCAGA	AGTACGAAGA	TGATCTTCGT	ACCGACCATC	CGCTCCTGCC	TGCTCGGACC
46921	TGGTGTGACG	TCCTGCGCCG	GGTAGGCTTT	GCGGATGCCG	TGAGTCTGCC	AGGCGACGGA
46981	TCTCCGGCGG	GGATCCTCGG	ACAGCACGTG	ATCCTCTCGC	GCGCTCCGGG	CATAGCAGGA
47041	GCCGCTTGTG	ACAGCTCCGG	TGAGTCGGCG	ACCGAATCGC	CGGCCGCGCG	TGCAGTACGG
47101	CAGGAATGGG	CCGATGGCTC	CGCTGACGGC	GTCCATCGGA	TGGCGTTGGA	GAGAATGTAC
47161	TTCCACCGCC	GGCCGGGCCG	GCAGGTTTGG	GTCCACGGTC	GATTGCGTAC	CGGTGGAGGC
47221	GCGTTCACGA	AGGCGCTCAC	TGGAGATCTG	CTCCTGTTCG	AAGAGACCGG	GCAGGTCGTG
47281	GCAGAGGTTC	AGGGGCTCCG	CCTGCCGCAG	CTCGAGGCTT	CTGCTTTCGC	GCCGCGGGAC
47341	CCGCGGGAAG	AGTGGTTGTA	CGCGTTGGAA	TGGCAGCGCA	AAGACCCTAT	ACCAGAGGCT
47401	CCGGCAGCCG	CGTCTTCTTC	CACCGCGGGG	GCTTGGCTCG	TGCTGATGGA	CCAGGGCGGG
47461	ACAGGCGCTG	CGCTCGTATC	GCTGCTGGAA	GGGCGAGGCG	AGGCGTGCGT	GCGCGTCGTC
47521	GCGGGTACGG	CATACGCCTG	CCTCGCGCCG	GGGCTGTATC	AAGTCGATCC	GGCGCAGCCA
47581	GATGGCTTTC	ATACCCTGCT	CCGCGATGCA	TTCGGCGAGG	ACCGGATGTG	CCGCGCGGTA
47641	GTGCATATGT	GGAGCCTTGA	TGCGAAGGCA	GCAGGGGAGA	GGACGACAGC	GGAGTCGCTT
47701	CAGGCCGATC	AACTCCTGGG	GAGCCTGAGC	GCGCTTTCTC	TGGTGCAGGC	GCTGGTGCGC
47761	CGGAGGTGGC	GCAACATGCC	GCGACTTTGG	CTCTTGACCC	GCGCCGTGCA	TGCGGTGGGC
47821	GCGGAGGACG	CAGCGGCCTC	GGTGGCGCAG	GCGCCGGTGT	GGGGCCTCGG	TCGGACGCTC
47881	GCGCTCGAGC	ATCCAGAGCT	GCGGTGCACG	CTCGTGGACG	TGAACCCGGC	GCCGTCTCCA
47941	GAGGACGCAG	CTGCACTCGC	GGTGGAGCTC	GGGGCGAGCG	ACAGAGAGGA	CCAGATCGCA
48001	TTGCGCTCGA	ATGGCCGCTA	CGTGGCGCGC	CTCGTGCGGA	GCTCCTTTTC	CGGCAAGCCT
48061	GCTACGGATT	GCGGCATCCG	GGCGGACGGC	AGTTATGTGA	TCACCGATGG	CATGGGGAGA
48121	GTGGGGCTCT	CGGTCGCGCA	ATGGATGGTG	ATGCAGGGGG	CCCGCCATGT	GGTGCTCGTG
48181	GATCGCGGCG	GCGCTTCCGA	CGCCTCCCGG	GATGCCCTCC	GGTCCATGGC	CGAGGCTGGC
48241	GCAGAGGTGC	AGATCGTGGA	GGCCGACGTG	GCTCGGCGCG	TCGATGTCGC	TCGGCTTCTC
48301	TCGAAGATCG	AACCGTCGAT	GCCGCCGCTT	CGGGGGATCG	TGTACGTGGA	CGGGACCTTC
48361	CAGGGCGACT	CCTCGATGCT	GGAGCTGGAT	GCCCATCGCT	TCAAGGAGTG	GATGTATCCC
48421	AAGGTGCTCG	GAGCGTGGAA	CCTGCACGCG	CTGACCAGGG	ATAGATCGCT	GGACTTCTTC
48481	GTCCTGTACT	CCTCGGGCAC	CTCGCTTCTG	GGCTTGCCCG	GACAGGGGAG	CCGCGCCGCC
48541	GGTGACGCCT	TCTTGGACGC	CATCGCGCAT	CACCGGTGTA	GGCTGGGCCT	CACAGCGATG
48601	AGCATCAACT	GGGGATTGCT	CTCCGAAGCA	TCATCGCCGG	CGACCCCGAA	CGACGGCGGC
48661	GCACGGCTCC	AATACCGGGG	GATGGAAGGT	CTCACGCTGG	AGCAGGGAGC	GGAGGCGCTC
48721	GGGCGCTTGC	TCGCACAACC	CAGGGCGCAG	GTAGGGGTAA	TGCGGCTGAA	TCTGCGCCAG
48781	TGGCTGGAGT	TCTATCCCAA	CGCGGCCCGA	CTGGCGCTGT	GGGCGGAGTT	GCTGAAGGAG
48841	CGTGACCGCA	CCGACCGGAG	CGCGTCGAAC	GCATCGAACC	TGCGCGAGGC	GCTGCAGAGC
48901	GCCAGGCCCG	AAGATCGTCA	GTTGGTTCTG	GAGAAGCACT	TGAGCGAGCT	GTTGGGGCGG
48961	GGGCTGCGCC	TTCCGCCGGA	GAGGATCGAG	CGGCACGTGC	CGTTCAGCAA	TCTCGGCATG
49021	GACTCGTTGA	TAGGCCTGGA	GCTCCGCAAC	CGCATCGAGG	CCGCGCTCGG	CATCACCGTG
49081	CCGGCGACCC	TGCTATGGAC	TTACCCTACC	GTAGCAGCTC	TGAGCGGGAA	CCTGCTAGAT
49141	ATTCTGTTCC	CGAATGCCGG	CGCGACTCAC	GCTCCGGCCA	CCGAGCGGGA	GAAGAGCTTC
49201	GAGAACGATG	CCGCAGATCT	CGAGGCTCTG	CGGGGTATGA	CGGACGAGCA	GAAGGACGCG
49261	TTGCTCGCCG	AAAAGCTGGC	GCAGCTCGCG	CAGATCGTTG	GTGAGTAAGG	GACTGAGGGA
49321	GTATGGCGAC	CACGAATGCC	GGGAAGCTTG	AGCATGCCCT	TCTGCTCATG	GACAAGCTTG
49381	CGAAAAAGAA	CGCGTCTTTG	GAGCAAGAGC	GGACCGAGCC	GATCGCCATC	ATAGGTATTG
49441	GCTGCCGCTT	CCCCGGCGGA	GCGGACACTC	CGGAGGCATT	CTGGGAGCTG	CTCGACTCGG
49501	GCCGAGACGC	GGTCCAGCCG	CTCGACCGGC	GCTGGGCGCT	GGTCGGCGTC	CATCCCAGCG
49561	AGGAGGTGCC	GCGCTGGGCC	GGACTGCTCA	CCGAGGCGGT	GGACGGCTTC	GACGCCGCGT
49621	TCTTTGGCAC	CTCGCCTCGG	GAGGCGCGGT	CGCTCGATCC	TCAGCAACGC	CTGCTGCTGG
49681	AGGTCACCTG	GGAAGGGCTC	GAGGACGCCG	GCATCGCACC	CCAGTCCCTC	GACGGCAGCC
49741	GCACCGGGGT	ATTCCTGGGC	GCATGCAGCA	GCGACTACTC	GCATACCGTT	GCGCAACAGC
49801	GGCGCGAGGA	GCAGGACGCG	TACGACATCA	CCGGCAATAC	GCTCAGCGTC	GCCGCCGGAC
49861	GGTTGTCTTA	TACGCTAGGG	CTGCAGGGAC	CCTGCCTGAC	CGTCGACACG	GCCTGCTCGT
49921	CGTCGCTCGT	GGCCATCCAC	CTTGCCTGCC	GCAGCCTGCG	CGCTCGCGAG	AGCGATCTCG
49981	CGCTGGCGGG	GGGCGTCAAC	ATGCTCCTTT	CGTCCAAGAC	GATGATAATG	CTGGGGCGCA
50041	TCCAGGCGCT	GTCGCCCGAT	GGCCACTGCC	GGACATTCGA	CGCCTCGGCC	AACGGGTTCG
50101	TCCGTGGGGA	GGGCTGCGGT	ATGGTCGTGC	TCAAACGGCT	CTCCGACGCC	CAGCGACATG
50161	GCGATCGGAT	CTGGGCTCTG	ATCCGGGGTT	CGGCCATGAA	TCAGGATGGC	CGGTCGACAG
50221	GGTTGATGGC	ACCCAATGTG	CTCGCTCAGG	AGGCGCTCTT	ACGCCAGGCG	CTGCAGAGCG
50281	CTCGCGTCGA	CGCCGGGGCC	ATCGATTATG	TCGAGACCCA	CGGAACGGGG	ACCTCGCTCG
50341	GCGACCCGAT	CGAGGTCGAT	GCGCTGCGTG	CCGTGATGGG	GCCGGCGCGG	GCCGATGGGA
50401	GCCGCTGCGT	GCTGGGCGCA	GTGAAGACCA	ACCTCGGCCA	CCTGGAGGGC	GCTGCAGGCG
50461	TGGCGGGTTT	GATCAAGGCG	GCGCTGGCTC	TGCACCACGA	ATCGATCCCG	CGAAACCTCC
50521	ATTTTCACAC	GCTCAATCCG	CGGATCCGGA	TCGAGGGGAC	CGCGCTCGCG	CTGGCGACGG
50581	AGCCGGTGCC	GTGGCCGCGG	GCGGGCCGAC	CGCGCTTCGC	GGGGGTGAGC	GCGTTCGGCC
50641	TCAGCGGCAC	CAACGTCCAT	GTCGTGCTGG	AGGAGGCGCC	GGCCACGGTG	CTCGCACCGG
50701	CGACGCCGGG	GCGCTCAGCA	GAGCTTTTGG	TGCTGTCGGC	GAAGAGCACC	GCCGCGCTGG
50761	ACGCACAGGC	GGCGCGGCTC	TCAGCGCACA	TCGCCGCGTA	CCCGGAGCAG	GGCCTCGGAG
50821	ACGTCGCGTT	CAGCCTGGTA	GCGACGCGGA	GCCCGATGGA	GCACCGGCTC	GCGGTGGCGG
50881	CGACCTCGCG	CGAGGCGCTG	CGAAGCGCGC	TGGAAGCTGC	GGCGCAGGGG	CAGACCCCGG
50941	CAGGCGCGGC	GCGCGGCAGG	GCCGCTTCCT	CGCCCGGCAA	GCTCGCCTTC	CTGTTCGCCG
51001	GGCAGGGCGC	GCAGGTGCCG	GGCATGGGCC	GTGGGTTGTG	GGAGGCGTGG	CCGGCGTTCC
51061	GCGAGACCTT	CGACCGGTGC	GTCACGCTCT	TCGACCGGGA	GCTCCATCAG	CCGCTCTGCG
51121	AGGTGATGTG	GGCCGAGCCG	GGCAGCAGCA	GGTCGTCGTT	GCTGGACCAG	ACGGCATTCA
51181	CCCAGCCGGC	GCTCTTTGCG	CTGGAGTACG	CGCTGGCCGC	GCTCTTCCGG	TCGTGGGGCG
51241	TGGAGCCGGA	GCTCATCGCT	GGCCATAGCC	TCGGCGAGCT	GGTGGCCGCC	TGCGTGGCGG
51301	GTGTGTTCTC	CCTCGAGGAC	GCCGTGCGCT	TGGTGGTCGC	GCGCGGCCGG	TTGATGCAGG
51361	CGCTGCCGGC	CGGCGGTGCG	ATGGTATCGA	TCGCCGCGCC	GGAGGCCGAC	GTGGCTGCCG
51421	CGGTGGCGCC	GCACGCAGCG	TCGGTGTCGA	TCGCGGCAGT	CAATGGGCCG	GAGCAGGTGG
51481	TGATCGCGGG	CGCCGAGAAA	TTCGTGCAGC	AGATCGCGGC	GGCGTTCGCG	GCGCGGGGGG
51541	CGCGAACCAA	ACCGCTGCAT	GTTTCGCACG	CGTTCCACTC	GCCGCTCATG	GATCCGATGC
51601	TGGAGGCGTT	CCGGCGGGTG	ACCGAGTCGG	TGACGTATCG	GCGGCCTTCG	ATGGCGCTGG
51661	TGAGCAACCT	GAGCGGGAAG	CCCTGCACGG	ATGAGGTGTG	CGCGCCGGGT	TACTGGGTGC
51721	GTCACGCGCG	AGAGGCGGTG	CGCTTCGCGG	ACGGCGTGAA	GGCGCTGCAC	GCGGCCGGTG
51781	CGGGCATCTT	CGTCGAGGTG	GGCCCGAAGC	CGGCGCTGCT	CGGCCTTTTG	CCGGCCTGCC
51841	TGCCGGATGC	CAGGCCGGTG	CTGCTCCCAG	CGTCGCGCGC	CGGGCGTGAC	GAGGCTGCGA
51901	GCGCGCTGGA	GGCGCTGGGT	GGGTTCTGGG	TCGTCGGTGG	ATCGGTCACC	TGGTCGGGTG
51961	TCTTCCCTTC	GGGCGGACGG	CGGGTACCGC	TGCCAACCTA	TCCCTGGCAG	CGCGAGCGTT
52021	ACTGGATCGA	AGCGCCGGTC	GATGGTGAGG	CGGACGGCAT	CGGCCGTGCT	CAGGCGGGGG
52081	ACCACCCCCT	TCTGGGTGAA	GCCTTTTCCG	TGTCGACCCA	TGCCGGTCTG	CGCCTGTGGG
52141	AGACGACGCT	GGACCGAAAG	CGGCTGCCGT	GGCTCGGCGA	GCACCGGGCG	CAGGGGGAGG
52201	TCGTGTTTCC	TGGCGCCGGG	TACCTGGAGA	TGGCGCTGTC	GTCGGGGGCC	GAGATCTTGG
52261	GCGATGGACC	GATCCAGGTC	ACGGATGTGG	TGCTCATCGA	GACGCTGACC	TTCGCGGGCG
52321	ATACGGCGGT	ACCGGTCCAG	GTGGTGACGA	CCGAGGAGCG	ACCGGGACGG	CTGCGGTTCC
52381	AGGTAGCGAG	TCGGGAGCCG	GGGGCACGTC	GCGCGTCCTT	CCGGATCCAC	GCCCGCGGCG
52441	TGCTGCGCCG	GGTCGGGCGC	GCCGAGACCC	CGGCGAGGTT	GAACCTCGCC	GCCCTGCGCG
52501	CCCGGCTTCA	TGCCGCCGTG	CCCGCTGCGG	CTATCTATGG	GGCGCTCGCC	GAGATGGGGC
52561	TTCAATACGG	CCCGGCGTTG	CGGGGGCTCG	CCGAGCTGTG	GCGGGGTGAG	GGCGAGGCGC
52621	TGGGCAGAGT	GAGACTGCCT	GAGTCCGCCG	GCTCCGCGAC	AGCCTACCAG	CTGCATCCGG
52681	TGCTGCTGGA	CGCGTGCGTC	CAAATGATTG	TTGGCGCGTT	CGCCGATCGC	GATGAGGCGA
52741	CGCCGTGGGC	GCCGGTGGAG	GTGGGCTCGG	TGCGGCTGTT	CCAGCGGTCT	CCTGGGGAGC
52801	TATGGTGCCA	TGCGCGCGTC	GTGAGCGATG	GTCAACAGGC	CCCCAGCCGG	TGGAGCGCCG
52861	ACTTTGAGTT	GATGGACGGT	ACGGGCGCGG	TGGTCGCCGA	GATCTCCCGG	CTGGTGGTGG
52921	AGCGGCTTGC	GAGCGGTGTA	CGCCGGCGCG	ACGCAGACGA	CTGGTTCCTG	GAGCTGGATT
52981	GGGAGCCCGC	GGCGCTCGAG	GGGCCCAAGA	TCACAGCCGG	CCGGTGGCTG	CTGCTCGGCG
53041	AGGGTGGTGG	GCTCGGGCGC	TCGTTGTGCT	CAGCGCTGAA	GGCCGCCGGC	CATGTCGTCG
53101	TCCACGCCGC	GGGGGACGAC	ACGAGCGCTG	CAGGAATGCG	CGCGCTCCTG	GCCAACGCGT
53161	TCGACGGCCA	GGCCCCGACG	GCCGTGGTGC	ACCTCAGCAG	CCTCGACGGG	GGCGGCCAGC
53221	TCGACCCGGG	GCTCGGGGCG	CAGGGCGCGC	TCGACGCGCC	CCGGAGCCCA	GATGTCGATG
53281	CCGATGCCCT	CGAGTCGGCG	CTGATGCGTG	GTTGCGACAG	CGTGCTCTCC	CTGGTGCAAG
53341	CGCTGGTCGG	CATGGACCTC	CGAAATGCGC	CGCGGCTGTG	GCTTTTGACC	CGCGGGGCTC
53401	AGGCGGCCGC	CGCCGGCGAT	GTCTCCGTGG	TGCAAGCGCC	GCTGTTGGGG	CTGGGCCGCA
53461	CCATCGCCTT	GGAGCACGCC	GAGCTGCGCT	GTATCAGCGT	CGACCTCGAT	CCAGCCCAGC
53521	CTGAAGGGGA	AGCCGATGCT	TTGCTGGCCG	AGCTACTTGC	AGATGATGCC	GAGGAGGAGG
53581	TCGCGCTGCG	CGGTGGCGAG	CGGTTTGTTG	CGCGGCTCGT	CCACCGGCTG	CCCGAGGCTC
53641	AACGCCGGGA	GAAGATCGCG	CCCGCCGGTG	ACAGGCCGTT	CCGGCTAGAG	ATCGATGAAC
53701	CCGGCGTGCT	GGACCAACTG	GTGCTCCGGG	CCACGGGGCG	GCGCGCTCCT	GGTCCGGGCG
53761	AGGTCGAGAT	CGCCGTCGAA	GCGGCGGGGC	TCGACTCCAT	CGACATCCAG	CTGGCGGTGG
53821	GCGTTGCTCC	CAATGACCTG	CCTGGAGGAG	AAATCGAGCC	GTCGGTGCTC	GGAAGCGAGT
53881	GCGCCGGGCG	CATCGTCGCT	GTGGGCGAGG	GCGTGAACGG	CCTTGTGGTG	GGCCAGCCGG
53941	TGATCGCCCT	TGCGGCGGGA	GTATTTGCTA	CCCATGTCAC	CACGTCGGCC	ACGCTGGTGT
54001	TGCCTCGGCC	TCTGGGGCTC	TCGGCGACCG	AGGCGGCCGC	GATGCCCCTC	GCGTATTTGA
54061	CGGCCTGGTA	CGCCCTCGAC	AAGGTCGCCC	ACCTGCAGGC	GGGGGAGCGG	GTGCTGATCC
54121	GTGCGGAGGC	CGGTGGTATC	GGTCTTTGCG	CGGTGCGATG	GGCGCAGCGC	GTGGGCGCCG
54181	AGGTGTATGC	GACCGCCGAC	ACGCCCGAGA	AACGTGCCTA	CCTGGAGTCG	CTGGGCGTGC
54241	GGTACGTGAG	CGATTCCCGC	TCGGGCCGGT	TCGCCGCAGA	CGTGCATGCA	TGGACGGACG
54301	GCGAGGGTGT	GGACGTCGTG	CTCGACTCGC	TTTCGGGCGA	GCACATCGAC	AAGAGCCTCA
54361	TGGTCCTGCG	CGCCTGTGGC	CGCCTTGTGA	AGCTGGGCAG	GCGCGACGAC	TGCGCCGACA
54421	CGCAGCCTGG	GCTGCCGCCG	CTCCTACGGA	ATTTTTCCTT	CTCGCAGGTG	GACTTGCGGG
54481	GAATGATGCT	CGATCAACCG	GCGAGGATCC	GTGCGCTCCT	CGACGAGCTG	TTCGGGTTGG
54541	TCGCAGCCGG	TGCCATCAGC	CCACTGGGGT	CGGGGTTGCG	CGTTGGCGGA	TCCCTCACGC
54601	CACCGCCGGT	CGAGACCTTC	CCGATCTCTC	GCGCAGCCGA	GGCATTCCGG	AGGATGGCGC
54661	AAGGACAGCA	TCTCGGGAAG	CTCGTGCTCA	CGCTGGACGA	CCCGGAGGTG	CGGATCCGCG
54721	CTCCGGCCGA	ATCCAGCGTC	GCCGTCCGCG	CGGACGGCAC	CTACCTTGTG	ACCGGCGGTC
54781	TGGGTGGGCT	CGGTCTGCGC	GTGGCCGGAT	GGCTGGCCGA	GCGGGGCGCG	GGGCAACTGG
54841	TGCTGGTGGG	CCGCTCCGGT	GCGGCGAGCG	CAGAGCAGCG	AGCCGCCGTG	GCGGCGCTAG
54901	AGGCCCACGG	CGCGCGCGTC	ACGGTGGCGA	AAGCGGATGT	CGCCGATCGG	TCACAGATCG
54961	AGCGGGTCCT	CCGCGAGGTT	ACCGCGTCGG	GGATGCCGCT	GCGGGGTGTC	GTGCATGCGG
55021	CAGGTCTTGT	GGATGACGGG	CTGCTGATGC	AGCAGACTCC	GGCGCGGCTC	CGCACGGTGA
55081	TGGGACCTAA	GGTCCAGGGA	GCCTTGCACT	TGCACACGCT	GACACGCGAA	GCGCCTCTTT
55141	CCTTCTTCGT	GCTGTACGCT	TCTGCAGCTG	GGCTGTTCGG	CTCGCCAGGC	CAGGGCAACT
55201	ATGCCGCAGC	CAACGCGTTC	CTCGACGCCC	TTTCGCATCA	CCGCAGGGCG	CACGGCCTGC
55261	CGGCGCTGAG	CATCGACTGG	GGCATGTTCA	CGGAGGTGGG	GATGGCCGTT	GCGCAAGAAA
55321	ACCGTGGCGC	GCGGCTGATC	TCTCGCGGGA	TGCGGGGCAT	CACCCCCGAT	GAGGGTCTGT
55381	CAGCTCTGGC	GCGCTTGCTC	GAGGGTGATC	GCGTGCAGAC	GGGGGTGATA	CCGATCACTC
55441	CGCGGCAGTG	GGTGGAGTTC	TACCCGGCAA	CAGCGGCCTC	ACGGAGGTTG	TCGCGGCTGG
55501	TGACCACGCA	GCGCGCGGTT	GCTGATCGGA	CCGCCGGGGA	TCGGGACCTG	CTCGAACAGC
55561	TTGCCTCGGC	TGAGCCGAGC	GCGCGGGCGG	GGCTGCTGCA	GGACGTCGTG	CGCGTGCAGG
55621	TCTCGCATGT	GCTGCGTCTC	CCTGAAGACA	AGATCGAGGT	GGATGCCCCG	CTCTCGAGCA
55681	TGGGCATGGA	CTCGCTGATG	AGCCTGGAGC	TGCGCAACCG	CATCGAGGCT	GCGCTGGGCG
55741	TCGCCGCGCC	TGCAGCCTTG	GGGTGGACGT	ACCCAACGGT	AGCAGCGATA	ACGCGCTGGC
55801	TGCTCGACGA	CGCCCTCGCC	GTCCGGCTTG	GCGGCGGGTC	GGACACGGAC	GAATCGACGG
55861	CAAGCGCCGG	ATCGTTCGTC	CACGTCCTCC	GCTTTCGTCC	TGTCGTCAAG	CCGCGGGCTC
55921	GTCTCTTCTG	TTTTCACGGT	TCTGGCGGCT	CGCCCGAGGG	CTTCCGTTCC	TGGTCGGAGA
55981	AGTCTGAGTG	GAGCGATCTG	GAAATCGTGG	CCATGTGGCA	CGATCGCAGC	CTCGCCTCCG
56041	AGGACGCGCC	TGGTAAGAAG	TACGTCCAAG	AGGCGGCCTC	GCTGATTCAG	CACTATGCAG
56101	ACGCACCGTT	TGCGTTAGTA	GGGTTCAGCC	TGGGTGTCCG	GTTCGTCATG	GGGACAGCCG
56161	TGGAGCTCGC	TAGTCGTTCC	GGCGCACCGG	CTCCGCTGGC	CGTTTTTGCG	TTGGGCGGCA
56221	GCTTGATCTC	TTCTTCAGAG	ATCACCCCGG	AGATGGAGAC	CGATATAATA	GCCAAGCTCT
56281	TCTTCCGAAA	TGCCGCGGGT	TTCGTGCGAT	CCACCCAACA	AGTTCAGGCC	GATGCTCGCG
56341	CAGACAAGGT	CATCACAGAC	ACCATGGTGG	CTCCGGCCCC	CGGGGACTCG	AAGGAGCCGC
56401	CCTCGAAGAT	CGCGGTCCCT	ATCGTCGCCA	TCGCCGGCTC	GGACGATGTG	ATCGTGCCTC
56461	CAAGCGACGT	TCAGGATCTA	CAATCTCGCA	CCACGGAGCG	CTTCTATATG	CATCTCCTTC
56521	CCGGAGATCA	CGAGTTTCTC	GTCGATCGAG	GGCGCGAGAT	CATGCACATC	GTCGACTCGC
56581	ATCTCAATCC	GCTGCTCGCC	GCGAGGACGA	CGTCGTCAGG	CCCCGCGTTC	GAGGCAAAAT
56641	GATGGCAGCC	TCCCTCGGGC	GCGCGAGATG	GTTGGGAGCA	GCGTGGGTGC	TGGTGGCCGG
56701	CGGCAGGCAG	CGGAGGCTCA	TGAGCCTTCC	TGGAAGTTTG	CAGCATAGGA	GATTTTATGA
56761	CACAGGAGCA	AGCGAATCAG	AGTGAGACGA	AGCCTGCTTT	CGACTTCAAG	CCGTTCGCGC
56821	CTGGGTACGC	GGAGGACCCG	TTTCCCGCGA	TCGAGCGCCT	GAGAGAGGCA	ACCCCCATCT
56881	TCTACTGGGA	TGAAGGCCGC	TCCTGGGTCC	TCACCCGATA	CCACGACGTG	TCGGCGGTGT
56941	TCCGCGACGA	ACGCTTCGCG	GTCAGTCGAG	AAGAATGGGA	ATCGAGCGCG	GAGTACTCGT
57001	CGGCCATTCC	CGAGCTCAGC	GATATGAAGA	AGTACGGATT	GTTCGGGCTG	CCGCCGGAGG
57061	ATCACGCTCG	GGTCCGCAAG	CTCGTCAACC	CATCGTTTAC	GTCACGCGCG	ATCGACCTGC
57121	TGCGCGCCGA	AATACAGCGC	ACCGTCGACC	AGCTGCTCGA	TGCTCGCTCC	GGACAAGAGG
57181	AGTTCGACGT	TGTGCGGGAT	TACGCGGAGG	GAATCCCGAT	GCGTGCGATC	AGCGCTCTGT
57241	TGAAGGTTCC	GGCCGAGTGT	GACGAGAAGT	TCCGTCGCTT	CGGCTCGGCG	ACTGCGCGCG
57301	CGCTCGGCGT	GGGTTTGGTG	CCCCGGGTCG	ATGAGGAGAC	CAAGACCCTG	GTCGCGTCCG
57361	TCACCGAGGG	GCTCGCGCTG	CTCCATGGCG	TCCTCGATGA	GCGGCGCAGG	AACCCGCTCG
57421	AAAATGACGT	CTTGACGATG	CTGCTTCAGG	CCGAGGCCGA	CGGCAGCAGG	CTGAGCACGA
57481	AGGAGCTGGT	CGCGCTCGTG	GGTGCGATTA	TCGCTGCTGG	CACCGATACC	ACGATCTACC
57541	TTATCGCGTT	CGCTGTGCTC	AACCTGCTGC	GGTCGCCCGA	GGCGCTCGAG	CTGGTGAAGG
57601	CCGAGCCCGG	GCTCATGAGG	AACGCGCTCG	ATGAGGTGCT	CCGCTTCGAC	AATATCCTCA
57661	GAATAGGAAC	TGTGCGTTTC	GCCAGGCAGG	ACCTGGAGTA	CTGCGGGGCA	TCGATCAAGA
57721	AAGGGGAGAT	GGTCTTTCTC	CTGATCCCGA	GCGCCCTGAG	AGATGGGACT	GTATTCTCCA
57781	GGCCAGACGT	GTTTGATGTG	CGACGGGACA	CGAGCGCGAG	CCTCGCGTAC	GGTAGAGGCC
57841	CCCATGTCTG	CCCCGGGGTG	TCCCTTGCTC	GCCTCGAGGC	GGAGATCGCC	GTGGGCACCA
57901	TCTTCCGTAG	GTTCCCCGAG	ATGAAGCTGA	AAGAAACTCC	CGTGTTTGGA	TACCACCCCG
57961	CGTTCCGGAA	CATCGAATCA	CTCAACGTCA	TCTTGAAGCC	CTCCAAAGCT	GGATAACTCG
58021	CGGGGGCATC	GCTTCCCGAA	dCTCATTCTT	TCATGATGCA	ACTCGCGCGC	GGGTGCTGTC
58081	TGCCGCGGGT	GCGATTCGAT	CCAGCGGACA	AGCCCATTGT	CAGCGCGCGA	AGATCGAATC
58141	CACGGCCCGG	AGAAGAGCCC	GATGGCGAGC	CCGTCCGGGT	AACGTCGGAA	GAAGTGCCGG
58201	GCGCCGCCCT	GGGAGCGCAA	AGCTCGCTCG	CTCGCGCTCA	GCGCGCCGCT	TGCCATGTCC
58261	GGCCCTGCAC	CCGCACCGAG	GAGCCACCCG	CCCTGATGCA	CGGCCTCACC	GAGCGGCAGG
58321	TTCTGCTCTC	GCTCGTCGCC	CTCGCGCTCG	TCCTCCTGAC	CGCGCGCGCC	TTCGGCGAGC
58381	TCGCGCGGCG	GCTGCGCCAG	CCCGAGGTGC	TCGGCGAGCT	CTTCGGCGGC	GTGGTGCTGG
58441	GCCCGTCCGT	CGTCGGCGCG	CTCGCTCCTG	GGTTCCATCG	AGTCCTCTTC	CAGGATCCGG
58501	CGGTCGGGGG	CGTGCTCTCC	GGCATCTCCT	GGATAGGCGC	GCTCGTCCTG	CTGCTCATGG
58561	CGGGTATCGA	GGTCGATGTG	AGCATTCTAC	GCAAGGAGGC	GCGCCCCGGG	GCGCTCTCGG
58621	CGCTCGGCGC	GATCGCGCCC	CCGCTGCGCA	CGCCGGGCCC	GCTGGTGCAG	CGCATGCAGG
58681	GCACGTTGAC	GTGGGATCTC	GACGTCTCGC	CGCGACGCTC	TGCGCAAGCC	TGAGCCTCGG
58741	CGCCTGCTCG	TACACCTCGC	CGGTGCTCGC	TCCGCCCGCG	GACATCCGGC	CGCCCCCCGC
58801	GGCCCAGCTC	GAGCCGGACT	CGCCGGATGA	CGAGGCCGAC	GAGGCGCTCC	GCCCGTTCCG
58861	CGACGCGATC	GCCGCGTACT	CGGAGGCCGT	TCGGTGGGCG	GAGGCGGCGC	AGCGGCCGCG
58921	GCTGGAGAGC	CTCGTGCGGC	TCGCGATCGT	GCGGCTGGGC	AAGGCGCTCG	ACAAGGCACC
58981	TTTCGCGCAC	ACGACGGCCG	GCGTCTCCCA	GATCGCCGGC	AGACTTCCCC	AGAAAACGAA
59041	TGCGGTCTGG	TTCGATGTCG	CCGCCCGGTA	CGCGAGCTTC	CGCGCGGCGA	CGGAGCACGC
59101	GCTCCGCGAC	GCGGCGTCGG	CCACGGAGGC	GCTCGCGGCC	GGCCCGTACC	GCGGATCGAG
59161	CAGCGTGTCC	GCTGCCGTAG	GGGAGTTTCG	GGGGGAGGCG	GCGCGCCTTC	ACCCCGCGGA
59221	CCGCGTACCC	GCGTCCGACC	AGCAGATCCT	GACCGCGCTG	CGCGCAGCCG	AGCGGGCGCT
59281	CATCGCGCTC	TACACCGCGT	TCGCCCGTGA	GGAGTGAGCC	TCTCTCGGGC	GCAGCCGAGC
59341	GGCGGCGTGC	CGGTTGTTCC	CTCTTCGCAA	CCATGACCGG	AGCCGCGCCC	GGTCCGCGCA
59401	GCGGCTAGCG	CGCGTCGAGG	CAGAGAGCGC	TGGAGCGACA	GGCGACGACC	CGCCCGAGGG
59461	TGTCGAACGG	ATTGCCGCAG	CCCTCATTGC	GGATCCCCTC	CAGACACTCG	TTCAGCGCCT
59521	TGGCGTCGAT	GCCGCCTGGG	CACTCGCCGA	AGGTCAGCTC	GTCGCGCCAG	TCGGATCGGA
59581	TCTTGTTCGA	GCACGCATCC	TTGCTCGAAT	ACTCCCGGTC	TTGTCCGATG	TTGTTGCACC
59641	GCGCCTCGCG	GTCGCACCGC	GCCGCCACGA	TGCTATCGAC	GGCGCTGCCG	ACTGGCACCG
59701	GCGCCTCGCC	TTGCGCGCCA	CCCGGGGTTT	GCGCCTCCCC	GCCTGACCGC	TTTTCGCCGC
59761	CGCACGCCGC	CGCGAGCAGG	CTCATTCCCG	ACATCGAGAT	CAGGCCCACG	ACCAGTTTCC
59821	CAGCAATCTT	TTGCATGGCT	TCCCCTCCCT	CACGACACGT	CACATCAGAG	ATTCTCCGCT
59881	CGGCTCGTCG	GTTCGACAGC	CGGCGACGGC	CACGAGCAGA	ACCGTCCCCG	ACCAGAACAG
59941	CCGCATGCGG	GTTTCTCGCA	GCATGCCACG	ACATCCTTGC	GACTAGCGTG	CCTCCGCTCG
60001	TGCCGAGATC	GGCTGTCCTG	TGCGACGGCA	ATGTCCTGCG	ATCGGCCGGG	CAGGATCGAC
60061	CGACACGGGC	GCCGGGCTGG	AGGTGCCGCC	ACGGGCTCGA	AATGCGCTGT	GGCAGGCGCC
60121	TCCATGCCCG	CTGCCGGGAA	CGCAGCGCCC	GGCCAGCCTC	GGGGCGACGC	TGCGAACGGG
60181	AGATGCTCCC	GGAGAGGCGC	CGGGCACAGC	CGAGCGCCGT	CACCACCGTG	CGCACTCGTG
60241	AGCGCTAGCT	CCTCGGCATA	GAAGAGACCG	TCACTCCCGG	TCCGTGTAGG	CGATCGTGCT
60301	GATCAGCGCG	TCCTCCGCCT	GACGCGAGTC	GAGCCGGGTA	TGCTGCACGA	CGATGGGCAC
60361	GTCCGATTCG	ATCACGCTGG	CATAGTCCGT	ATCGCGCGGG	ATCGGCTCGG	GGTCGGTCAG
60421	ATCGTTGAAC	CGGACGTGCC	GGGTGCGCCT	CGCTGGAACG	GTCACCCGGT	ACGGCCCGGC
60481	GGGGTCGCGG	TCGCTGAAGT	AGACGGTGAT	GGCGACCTGC	GCGTCCCGGT	CCGACGCATT
60541	CAACAGGCAG	GCCGTCTCAT	GGCTCGTCAT	CTGCGGCTCA	GGTCCGTTGC	TCCGGCCTGG
60601	GATGTAGCCC	TCTGCGATTG	CCCAGCGCGT	CCGCCCGATC	GGCTTGTCCA	TGTGTCCTCC
60661	CTCCTGGCTC	CTCTTTGGCA	GCCTCCCTCT	GCTGTCCAGG	TGCGACGGCC	TCTTCGCTCG
60721	ACGCGCTCGG	GGCTCCATGG	CTGAGAATCC	TCGCCGAGCG	CTCCTTGCCG	ACCGGCGCGC
60781	TGAGCGCCGA	CGGGCCTTGA	AAGCACGCGA	CCGGACACGG	GATGCCGGCG	CGACGAGGCC
60841	GCCCCGCGTC	TGATCCCGAT	CGTGGCATCA	CGACGTCCGC	CGACGCCTCG	GCAGGCCGGC
60901	GTGAGCGCTG	CGCGGTCATG	GTCGTCCTCG	CGTCACCGCC	ACCCGCCGAT	TCACATCCCA
60961	CCGCGGCACG	ACGCTTGCTC	AAACCGCGAC	GACACGGCCG	GGCGGCTGTG	GTACCGGCCA
61021	GCCCGGACGC	GAGGCCCGAG	AGGGACAGTG	GGTCCGCCGT	GAAGCAGAGA	GGCGATCGAG
61081	GTGGTGAGAT	GAAACACGTT	GACACGGGCC	GACGAGTCGG	CCGCCGGATA	GGGCTCACGC
61141	TCGGTCTCCT	CGCGAGCATG	GCGCTCGCCG	GCTGCGGCGG	CCCGAGCGAG	AAGACCGTGC
61201	AGGGCACGCG	GCTCGCGCCC	GGCGCCGATG	CGCACGTCAC	CGCCGACGTC	GACGCCGACG
61261	CCGCGACCAC	GCGGCTGGCG	GTGGACGTCG	TTCACCTCTC	GCCGCCCGAG	CGGATCGAGG
61321	CCGGCAGCGA	GCGGTTCGTC	GTCTGGCAGC	GTCCGAACTC	CGAGTCCCCG	TGGCTACGGG
61381	TCGGAGTGCT	CGACTACAAC	GCTGCCAGCC	GAAGAGGCAA	GCTGGCCGAG	ACGACCGTGC
61441	CGCATGCCAA	CTTCGAGCTG	CTCATCACCG	TCGAGAAGCA	GAGCAGCCCT	CAGTCGCCAT
61501	CGTCTGCCGC	CGTCATCGGG	CCGACGTCCG	TCGGGTAACA	TCGCGCTATC	AGCAGCGCTG
61561	AGCCCGCCAG	CATGCCCCAG	AGCCCTGCCT	CGATCGCTTT	CCCCATCATC	CGTGCGCACT
61621	CCTCCAGCGA	CGGCCGCGTC	AAAGCAACCG	CCGTGCCGGC	GCGGCTCTAC	GTGCGCGACA
61681	GGAGAGCGTC	CTAGCGCGGC	CTGCGCATCG	CTGGAAGGAT	CGGCGGAGCA	TGGAGAAAGA
61741	ATCGAGGATC	GCGATCTACG	GCGCCGTCGC	CGCCAACGTG	GCGATCGCGG	CGGTCAAGTT
61801	CATCGCCGCC	GCCGTGACCG	GCAGCTCTGC	GATGCTCTCC	GAGGGCGTGC	ACTCCCTCGT
61861	CGATACCGCA	GACGGGCTCC	TCCTCCTGCT	CGGCAAGCAC	CGGAGCGCCC	GCCCGCCCGA
61921	CGCCGAGCAT	CCGTTCGGCC	ACGGCAAGGA	GCTCTATTTC	TGGACGCTGA	TCGTCGCCAT
61981	CATGATCTTC	GCCGCGGGCG	GCGGCGTCTC	GATCTACGAA	GGGATCTTGC	ACCTCTTGCA
62041	CCCGCGCTCG	ATCGAGGATC	CGACGTGGAA	CTACGTTGTC	CTCGGCGCAG	CGGCCGTCTT
62101	CGAGGGGACG	TCGCTCGCCA	TCTCGATCCA	CGAGTTCAAG	AAGAAAGACG	GACAGGGCTA
62161	CGTCGCGGCG	ATGCGGTCCA	GCAAGGACCC	GACGACGTTC	ACGATCGTCC	TGGAGGATTC
62221	CGCGGCGCTC	GCCGGGCTCG	CCATCGCCTT	CCTCGGCGTC	TGGCTTGGGC	ACCGCCTGGG
62281	AAACCCCTAC	CTCGACGGCG	CGGCGTCGAT	CGGCATCGGC	CTCGTGCTCG	CCGCGGTCGC
62341	GGTCTTCCTC	GCCAGCCAGA	GCCGTGGACT	CCTCGTAGGG	GAGAGCGCGG	ACAGGGAGCT
62401	CCTCGCCGCG	ATCCGCGCGC	TCGCCAGCGC	AGATCCTGGC	GTGTCGGCGG	TGGGGCGGCC
62461	CCTGACGATG	CACTTCGGTC	CGCACGAAGT	CCTGGTCGTG	CTGCGCATCG	AGTTCGACGC
62521	CGCGCTCACG	GCGTCCGGGG	TCGCGGAGGC	GATCGAGCGA	ATCGAGACAC	GGATACGGAG
62581	CGAGCGACCC	GACGTGAAGC	ACATCTACGT	CGAGGCCAGG	TCGCTCCACC	AGCGCGCGAG
62641	GGCGTGACGC	GCCGTGGAGA	GACCGCTCGC	GGCCTCCGCC	ATCCTCCGCG	GCGCCCGGGC
62701	TCGGGTAGCC	CTCGCAGCAG	GGCGCGCCTG	GCGGGCAAAC	CGTGAAGACG	TCGTCCTTCG
62761	ACGCGAGGTA	CGCTGGTTGC	AAGTTGTCAC	GCCGTATCGC	GAGGTCCGGC	AGCGCCGGAG
62821	CCCGGGCGGT	CCGGGCGCAC	GAAGGCCCGG	CGAGCGCGGG	CTTCGAGGGG	GCGACGTCAT
62881	GAGGAAGGGC	AGGGCGCATG	GGGCGATGCT	CGGCGGGCGA	GAGGACGGCT	GGCGTCGCGG
62941	CCTCCCCGGC	GCCGGCGCGC	TTCGCGCCGC	GCTCCAGCGC	GGTCGCTCGC	GCGATCTCGC
63001	CCGGCGCCGG	CTCATCGCCG	CCGTGTCCCT	CACCGGCGGC	GCCAGCATGG	CGGTCGTCTC
63061	GCTGTTCCAG	CTCGGGATCA	TCGAGCACCT	GCCCGATCCT	CCGCTTCCAG	GGTTCGATTC
63121	GGCCAAGGTG	ACGAGCTCCG	ATATCGCGTT	CGGGCTCACG	ATGCCGGACG	CGCCGCTCGC
63181	GCTCACCAGC	TTCGCGTCCA	ACCTGGCGCT	GGCTGGCTGG	GGAGGCGCCG	AGCGCGCCAG
63241	GAACACCCCC	TGGATCCCCG	TCGCCGTGGC	GGCCAAGGCG	GCCGTCGAGG	CGGCCGTGTC
63301	CGGATGGCTC	CTCGTCCAGA	TGCGACGGCG	GGAGAGGGCC	TGGTGCGCGT	ACTGCCTGGT
63361	CGCCATGGCG	GCCAACATGG	CCGTGTTCGC	GCTCTCGCTC	CCGGAAGGGT	GGGCGGCGCT
63421	GAGGAAGGCG	CGAGCGCGCT	CGTGACAGGG	CCGTGCGGGC	GCCGCGGCCA	TCGGAGGCCG
63481	GCGTGCACCC	GCTCCGTCAC	GCCCCGGCCC	GCGCCGCGGT	GAGCTGCCGC	GGACAGGGCG
63541	CGTACCGTGG	ACCCCGCACG	CGCCGCGTCG	ACGGACATCC	CCGGCGGCTC	GCGCGGCGCG
63601	GCCGGCGCAA	CTCCGGCCCG	CCGCCGGGCA	TCGACATCTC	CCGCGAGCAA	GGGCACTCCG
63661	CTCCTGCCCG	CGTCCGCGAA	CGATGGCTGC	GCTGTTTCCA	CCCTGGAGCA	ACTCCGTTTA
63721	CCGCGTGGCG	CTCGTCGGGC	TCATCGCCTC	GGCGGGCGGC	GCCATCCTCG	CGCTCATGAT
63781	CTACGTCCGC	ACGCCGTGGA	AGCGATACCA	GTTCGAGCCC	GTCGATCAGC	CGGTGCAGTT
63841	CGATCACCGC	CATCACGTGC	AGGACGATGG	CATCGATTGC	GTCTACTGCC	ACACCACGGT
63901	GACCCGCTCG	CCGACGGCGG	GGATGCCGCC	GACGGCCACG	TGCATGGGGT	GCCACAGCCA
63961	GATCTGGAAT	CAGAGCGTCA	TGCTCGAGCC	CGTGCGGCGG	AGCTGGTTCT	CCGGCATGCC
64021	GATCCCGTGG	AACCGGGTGA	ACTCCGTGCC	CGACTTCGTT	TATTTCAACC	ACGCGATTCA
64081	CGTGAACAAG	GGCGTGGGCT	GCGTGAGCTG	CCACGGGCGC	GTGGACGAGA	TGGCGGCCGT
64141	CTACAAGGTG	GCGCCGATGA	CGATGGGCTG	GTGCCTGGAG	TGCCATCGCC	TGCCGGAGCC
64201	GCACCTGCGC	CCGCTCTCCG	CGATCACCGA	CATGCGCTGG	GACCCGGGGG	AACGGAGGGA
64261	CGAGCTCGGG	GCGAAGCTCG	CGAAGGAGTA	CGGGGTCCGG	CGGCTCACGC	ACTGCACAGC
64321	GTGCCATCGA	TGAACGATGA	ACAGGGGATC	TCCGTGAAAG	ACGCAGATGA	GATGAAGGAA
64381	TGGTGGCTAG	AAGCGCTCGG	GCCGGCGGGA	GAGCGCGCGT	CCTACAGGCT	GCTGGCGCCG
64441	CTCATCGAGA	GCCCGGAGCT	CCGCGCGCTC	GCCGCGGGCG	AACCGCCCCG	GGGCGTGGAC
64501	GAGCCGGCGG	GCGTCAGCCG	CCGCGCGCTG	CTCAAGCTGC	TCGGCGCGAG	CATGGCGCTC
64561	GCCGGCGTCG	CGGGCTGCAC	CCCGCATGAG	CCCGAGAAGA	TCCTGCCGTA	CAACGAGACC
64621	CCGCCCGGCG	TCGTGCCGGG	TCTCTCCCAG	TCCTACGCGA	CGAGCATGGT	GCTCGACGGG
64681	TATGCCATGG	GCCTCCTCGC	CAAGAGCTAC	GCGGGGCGGC	CCATCAAGAT	CGAGGGCAAC
64741	CCCGCGCACC	CGGCGAGCCT	CGGCGCGACC	GGCGTCCACG	AGCAGGCCTC	GATCCTCTCG
64801	CTGTACGACC	CGTACCGCGC	GCGCGCGCCG	ACGCGCGGCG	GCCAGGTCGC	GTCGTGGGAG
64861	GCGCTCTCCG	CGCGCTTCGG	CGGCGACCGC	GAGGACGGCG	GCGCTGGCCT	CCGCTTCGTC
64921	CTCCAGCCCA	CGAGCTCGCC	CCTCATCGCC	GCGCTGATCG	AGCGCGTCCG	GCGCAGGTTC
64981	CCCGGCGCGC	GGTTCACCTT	CTGGTCGCCG	GTCCACGCCG	AGCAAGCGCT	CGAAGGCGCG
65041	CGGGCGGCGC	TCGGCCTCAG	GCTCTTGCCT	CAGCTCGACT	TCGACCAGGC	CGAGGTGATC
65101	CTCGCCCTGG	ACGCGGACTT	CCTCGCGGAC	ATGCCGTTCA	GCGTGCGCTA	TGCGCGCGAC
65161	TTCGCCGCGC	GCCGCCGACC	CGCGAGCCCG	GCGGCGGCCA	TGAACCGCCT	CTACGTCGCG
65221	GAGGCGATGT	TCACGCCCAC	GGGGACGCTC	GCCGACCACC	GGCTCCGCGT	GCGGCCCGCC
65281	GAGGTCGCGC	GCGTCGCGGC	CGGCGTCGCG	GCGGAGCTCG	TGCACGGCCT	CGGCCTGCGC
65341	CCGCGCGGGA	TCACGGACGC	CGACGCCGCC	GCGCTGCGCG	CGCTCCGCCC	CCCGGACGGC
65401	GAGGGGCACG	GCGCCTTCGT	CCGGGCGCTC	GCGCGCGATC	TCGCGCGCGC	GGGGGGCGCC
65461	GGCGTCGCCG	TCGTCGGCGA	CGGCCAGCCG	CCCATCGTCC	ACGCCCTCGG	GCACGTCATC
65521	AACGCCGCGC	TCCGCAGCCG	GGCGGCCTGG	ATGGTCGATC	CTGTGCTGAT	CGACGCGGGC
65581	CCCTCCACGC	AGGGCTTCTC	CGAGCTCGTC	GGCGAGCTCG	GGCGCGGCGC	GGTCGACACC
65641	TGATCCTCCT	CGACGTGAAC	CCCGTGTACG	CCGCGCCGGC	CGACGTCGAT	TTCGCGGGCC
65701	TCCTCGCGCG	CGTGCCCACG	AGCTTGAAGG	CCGGGCTCTA	CGACGACGAG	ACCGCCCGCG
65761	CTTGCACGTG	GTTCGTGCCG	ACCCGGCATT	ACCTCGAGTC	GTGGGGGGAC	GCGCGGGCGT
65821	ACGACGGGAC	GGTCTCGTTC	GTGCAACCCC	TCGTCCGGCC	GCTGTTCGAC	GGCCGGGCGG
65881	TGCCCGAGCT	GCTCGCCGTC	TTCGCGGGGG	ACGAGCGCCC	GGATCCCCGG	CTGCTGCTGC
65941	GCGAGCACTG	GCGCGGCGCG	CGCGGAGAGG	CGGATTTCGA	GGCCTTCTGG	GGCGAGGCAT
66001	TGAAGCGCGG	CTTCCTCCCT	GACAGCGCCC	GGCCGAGGCA	GACACCGGAT	CTCGCGCCGG
66061	CCGACCTCGC	CAAGGAGCTC	GCGCGGCTCG	CCGCCGCGCC	GCGGCCGGCC	GGCGGCGCGC
66121	TCGACGTGGC	GTTCCTCAGG	TCGCCGTCGG	TCCACGACGG	CAGGTTCGCC	AACAACCCCT
66181	GGCTGCAAGA	GCTCCCGCGG	CCGATCACCA	GGCTCACCTG	GGGCAACGCC	GCCATGATGA
66241	GCGCGGCGAC	CGCGGCGCGG	CTCGGCGTCG	AGCGCGGCGA	TGTCGTCGAG	CTCGCGCTGC
66301	GCGGCCGTAC	GATCGAGATC	CCGGCCGTCG	TCGTCCGCGG	GCACGCCGAC	GACGTGATCA
66361	GCGTCGACCT	CGGCTACGGG	CGCGACGCCG	GCGAGGAGGT	CGCGCGCGGG	GTGGGCGTGT
66421	CGGCGTATCG	GATCCGCCCG	TCCGACGCGC	GGTGGTTCGC	GGGGGGCCTC	TCCGTGAGGA
66481	AGACCGGCGC	CACGGCCGCG	CTCGCGCTGG	CTCAGATCGA	GCTGTCCCAG	CACGACCGTC
66541	CCATCGCGCT	CCGGAGGACG	CTGCCGCAGT	ACCGTGAACA	GCCCGGTTTC	GCGGAGGAGC
66601	ACAAGGGGCC	GGTCCGCTCG	ATCCTGCCGG	AGGTCGAGTA	CACCGGCGCG	CAATGGGCGA
66661	TGTCCATCGA	CATGTCGATC	TGCACCGGGT	GCTCCTCGTG	CGTCGTGGCC	TGTCAGGCCG
66721	AGAACAACGT	CCTCGTCGTC	GGCAAGGAGG	AGGTGATGCA	CGGCCGCGAG	ATGCAGTGGT
66781	TGCGGATCGA	TCAGTACTTC	GAGGGTGGAG	GCGACGAGGT	GAGCGTCGTC	AACCAGCCGA
66841	TGCTCTGCCA	GCACTGCGAG	AAGGCGCCGT	GCGAGTACGT	CTGTCCGGTG	AACGCGACGG
66901	TCCACAGCCC	CGATGGCCTC	AACGAGATGA	TCTACAACCG	ATGCATCGGG	ACGCGCTTTT
66961	GCTCCAACAA	CTGTCCGTAC	AAGATCCGGC	GGTTCAATTT	CTTCGACTAC	AATGCCCACG
67021	TCCCGTACAA	CGCCGGCCTC	CGCAGGCTCC	AGCGCAACCC	GGACGTCACC	GTCCGCGCCC
67081	GCGGCGTCAT	GGAGAAATGC	ACGTACTGCG	TGCAGCGGAT	CCGAGAGGCG	GACATCCGCG
67141	CGCAGATCGA	GCGGCGGCCG	CTCCGGCCGG	GCGAGGTGGT	CACCGCCTGC	CAGCAGGCCT
67201	GTCCGACCGG	CGCGATCCAG	TTCGGGTCGC	TGGATCACGC	GGATACAAAG	ATGGTCGCGT
67261	GGCGCAGGGA	GCCGCGCGCG	TACGCCGTGC	TCCACGACCT	CGGCACCCGG	CCGCGGACGG
67321	AGTACCTCGC	CAAGATCGAG	AACCCGAACC	CGGGGCTCGG	GGCGGAGGGC	GCCGAGAGGC
67381	GACCCGGAGC	CCCGAGCGTC	AAACCCGCGC	TCGGGGCGGA	GGGCGCCGAG	AGGCGACCCG
67441	GAGCCCCGAG	CGTCAAACCG	GAGATTGAAT	GAGCCATGGC	GGGCCCGCTC	ATCCTGGACG
67501	CACCGACCGA	CGATCAGCTG	TCGAAGCAGC	TCCTCGAGCC	GGTATGGAAG	CCGCGCTCCC
67561	GGCTCGGCTG	GATGCTCGCG	TTCGGGCTCG	CGCTCGGCGG	CACGGGCCTG	CTCTTCCTCG
67621	CGATCACCTA	CACCGTCCTC	ACCGGGATCG	GCGTGTGGGG	CAACAACATC	CCGGTCGCCT
67681	GGGCCTTCGC	GATCACCAAC	TTCGTCTGGT	GGATCGGGAT	CGGCCACGCC	GGGACGTTCA
67741	TCTCCGCGAT	CCTCCTCCTG	CTCGAGCAGA	AGTGGCGGAC	GAGCATCAAC	CGCTTCGCCG
67801	AGGCGATGAC	GCTCTTCGCG	GTCGTCCAGG	CCGGCCTCTT	TCCGGTCCTC	CACCTCGGCC
67861	GCCCCTGGTT	CGCCTACTGG	ATCTTCCCGT	ACCCCGCGAC	GATGCAGGTG	TGGCCGCAGT
67921	TCCGGAGCGC	GCTGCCGTGG	GACGCCGCCG	CGATCGCGAC	CTACTTCACG	GTGTCGCTCC
67981	TGTTCTGGTA	CATGGGCCTC	GTCCCGGATC	TGGCGGCGCT	GCGCGACCAC	GCCCCGGGCC
68041	GCGTCCGGCG	GGTGATCTAC	GGGCTCATGT	CGTTCGGCTG	GCACGGCGCG	GCCGACCACT
68101	TCCGGCATTA	CCGGGTGCTG	TACGGGCTGC	TCGCGGGGCT	CGCGACGCCC	CTCGTCGTCT
68161	CGGTGCACTC	GATCGTGAGC	AGCGATTTCG	CGATCGCCCT	GGTGCCCGGC	TGGCACTCGA
68221	CGCTCTTTCC	GCCGTTCTTC	GTCGCGGGCG	CGATCTTCTC	CGGGTTCGCG	ATGGTGCTCA
68281	CGCTGCTCAT	CCCGGTGCGG	CGGATCTACG	GGCTCCATAA	CGTCGTGACC	GCGCGCCACC
68341	TCGACGATCT	CGCGAAGATG	ACGCTCGTGA	CCGGCTGGAT	CGTCATCCTC	TCGTACATCA
68401	TCGAGAACTT	CCTCGCCTGG	TACAGCGGCT	CGGCGTACGA	GATGCATCAG	TTTTTCCAGA
68461	CGCGCCTGCA	CGGCCCGAAC	AGCGCCGCCT	ACTGGGCCCA	GCACGTCTGC	AACGTGCTCG
68521	TCATCCAGCT	CCTCTGGAGC	GAGCGGATCC	GGACGAGCCC	CGTCGCGCTC	TGGCTCATCT
68581	CCCTCCTGGT	CAACGTCGGG	ATGTGGAGCG	AGCGGTTCAC	GCTCATCGTG	ATGTCGCTCG
68641	AGCAAGAGTT	CCTCCCGTCC	AAGTGGCACG	GCTACAGCCC	GACGTGGGTG	GACTGGAGCC
68701	TCTTCATCGG	GTCAGGCGGC	TTCTTCATGC	TCCTGTTCCT	GAGCTTTTTG	CGCGTCTTTC
68761	CGTTCATCCC	CGTCGCGGAG	GTCAAGGAGC	TCAACCATGA	AGAGCTGGAG	AAGGCTCGGG
68821	GCGAGGGGGG	CCGCTGATGG	AGACCGGAAT	GCTCGGCGAG	TTCGATGACC	CGGAGGCGAT
68881	GCTCCATGCG	ATCCGAGAGC	TCAGGCGGCG	CGGCTACCGC	CGGGTGGAAG	CGTTCACGCC
68941	CTATCCGGTG	AAGGGGCTCG	ACGAGGCGCT	CGGCCTCCCG	CGCTCGAACC	TCAACCGGAT
69001	GGTGCTGCCC	TTCGCGATCC	TGGGGGTCGT	GGGCGGCTAC	TTCGTCCAGT	GGTTCTGCAA
69061	CGCTTTCCAC	TATCCGCTGA	ACGTGGGCGG	GCGCCCGCTG	AACTCGGCGC	CGGCGTTCAT
69121	CCCGATCACG	TTCGAGATGG	GGGTGCTCTC	CACCTCGATC	TTCGGCGTGC	TCATCGGCTT
69181	TTACCTGACG	AGGCTGCCGA	GGCTCTACCT	CCCGCTCTTC	GACGCCCCGG	GCTTCGAGCG
69241	CGTCACGCTG	GATCGGTTTC	TGGTCGGGCT	CGACGACACG	GAACCTTCCT	TCTCGAGCGC
69301	CCAGGCGGAG	CGCGACCTCC	TCGCGCTCGG	CGCCCGGCGC	GTCGTCGTCG	CGAGGAGGCG
69361	CGAGGAGCCA	TGAGGGCCGG	CGCCCCGGCT	CGCCCTCTCG	GGCGCGCGCT	CGCGCCGTTC
69421	GCCCTCGTCC	TGCTCGCCGG	GTGCCGCGAG	AAGGTGCTGC	CCGAGCCGGA	CTTCGAGCGG
69481	ATGATCCGCC	AGGAGAAATA	CGGACTCTGG	GAGCCGTGCG	AGCACTTCGA	CGACGGCCGC
69541	GCGATGCAGC	ACCCGCCCGA	GGGGACCGTC	GCGCGCGGGC	GCGTCACCGG	GCCGCCCGGC
69601	TATCTCCAGG	GCGTCCTCGA	CGGGGCGTAC	GTCACGGAGG	TGCCGCTCTT	GCTCACGGTC
69661	GAGCTCGTGC	AGCGCGGCCG	GCAGCGCTTC	GAGACCTTCT	GCGCGCCGTG	CCACGGGATC
69721	CTCGGCGACG	GCAGCTCGCG	CGTGGCGACG	AACATGACGC	TGCGCCCGCC	CCCGTCGCTC
69781	ATCGGACCCG	AGGCGCGGAG	CTTCCCGCCG	GGCAGGATCT	ACCAGGTCAT	CATCGAGGGC
69841	TACGGCCTGA	TGCCGCGCTA	CTCGGACGAT	CTGCCCGACA	TCGAAGAGCG	CTGGGCCGTG
69901	GTCGCCTACG	TGAAGGCGCT	TCAGCTGAGC	CGCGGAGTGG	CCGCGGGCGC	CCTCCCGCCA
69961	GCGCTCCGCG	GCCGGGCAGA	GCAGGAGCTG	CGATGAACAG	GGATGCCATC	GAGTACAAGG
70021	GCGGCGCGAC	GATCGCGGCC	TCGCTCGCGA	TCGCGGCGCT	CGGCGCGGTC	GCCGCGATCG
70081	TCGGCGGCTT	CGTCGATCTC	CGCCGGTTCT	TCTTCTCGTA	CCTCGCCGCG	TGGTCGTTCG
70141	CGGTGTTTCT	GTCCGTGGGC	GCGCTCGTCA	CGCTCCTCAC	CTGCAACGCC	ATGCGCGCGG
70201	GCTGGCCCAC	GGCGGTGCGC	CGCCTCCTCG	AGACGATGGT	GGCGCCGCTG	CCTCTGCTCG
70261	CGGCGCTCTC	CGCGCCGATC	CTGGTCGGCC	TGGACACGCT	GTATCCGTGG	ATGCACCCCG
70321	AGCGGATCGC	CGGCGAGCAC	GCGCGGCGCA	TCCTCGAGCA	CAGGGCGCCC	TACTTCAATC
70381	CAGGCTTCTT	CGTCGTGCGC	TCGGCGATCT	ACTTCGCGAT	CTGGATCGCC	GTCGCCCTCG
70441	TGCTCCGCCG	GCGATCGTTC	GCGCAGGACC	GTGAGCCGAG	GGCCGACGTC	AAGGACGCGA
70501	TGTATGGCCT	GAGCGGCGCC	ATGCTGCCGG	TCGTGGCGAT	CACGATCGTC	TTCTCGTCGT
70561	TCGACTGGCT	CATGTCCCTC	GACGCGACCT	GGTACTCGAC	GATGTTCCCG	GTCTACGTGT
70621	TCGCGAGCGC	CTTCGTGACC	GCCGTCGGCG	CGCTCACGGT	CCTCTCGTAT	GCCGCGCAGA
70681	CGTCCGGCTA	CCTCGCGAGG	CTGAACGACT	CGCACTATTA	CGCGCTCGGG	CGGCTGCTCC
70741	TCGCGTTCAC	GATATTCTGG	GCCTATGCGG	CCTATTTCCA	GTTCATGTTG	ATCTGGATCG
70801	CGAACAAGCC	CGATGAGGTC	GCCTTCTTCC	TCGACCGCTG	GGAAGGGCCC	TGGCGGCCGA
70861	CCTCCGTGCT	CGTCGTCCTC	ACGCGGTTCG	TCGTCCCGTT	CCTGATCCTG	ATGTCGTACG
70921	CGATCAAGCG	GCGCCCGCGC	CAGCTCTCGT	GGATGGCGCT	CTGGGTCGTC	GTCTCCGGCT
70981	ACATCGACTT	TCACTGGCTC	GTGGTGCCGG	CGACAGGGCG	CCACGGGTTC	GCCTATCACT
71041	GGCTCGACCT	CGCGACCCTG	TGCGTCGTGG	GCGGCCTCTC	GACCGCGTTC	GCCGCGTGGC
71101	GGCTGCGAGG	GCGGCCGGTG	GTCCCGGTCC	ACGACCCGCG	GCTCGAAGAG	GCCTTTGCGT
71161	ACCGGAGCAT	ATGATGTTCC	GTTTCCGTCA	CAGCGAGGTT	CGCCAGGAGG	AGGACACGCT
71221	CCCCTGGGGG	CGCGTGATCC	TCGCGTTCGC	CGTCGTGCTC	GCGATCGGCG	GCGCGCTGAC
71281	GCTCTGGGCC	TGGCTCGCGA	TGCGGGCCCG	CGAGGCGGAT	CTGCGGCCCT	CCCTCGCGTT
71341	CCCCGAGAAG	GATCTCGGGC	CGCGGCGCGA	GGTCGGCATG	GTCCAGCAGT	CGCTGTTCGA
71401	CGAGGCGCGC	CTGGGCCAGC	AGCTCGTCGA	CGCGCAGCGC	GCGGAGCTCC	GCCGCTTCGG
71461	CGTCGTCGAT	CGGGAGAGGG	GCATCGTGAG	CATCCCGATC	GACGACGCGA	TCGAGCTCAT
71521	GGTGGCGGGG	GGCGCGCGAT	GAGCCGGGCC	GTCGCCGTGG	CCCTCCTGCT	GGCAGCCGGC
71581	CTCGTGTCGC	GCCCGGGCGC	CGCGTCCGAG	CCCGAGCGCG	CGCGCCCCGC	GCTGGGCCCG
71641	TCCGCGGCCG	ACGCCGCGCC	GGCGAGCGAC	GGCTCCGGCG	CGGAGGAGCC	GCCCGAAGGC
71701	GCCTTCCTGG	AGCCCACGCG	CGGGGTGGAC	ATCGAGGAGC	GCCTCGGCCG	CCCGGTGGAC
71761	CGCGAGCTCG	CCTTCACCGA	CATGGACGGG	CGGCGGGTGC	GCCTCGGCGA	CTACTTCGCC
71821	GACGGCAAGC	CCCTCCTCCT	CGTCCTCGCG	TACTACCGGT	GTCCCGCGCT	GTGCGGCCTC
71881	GTGCTGCGCG	GCGCCGTCGA	GGGGCTGAAG	CTCCTCCCGT	ACCGGCTCGG	CGAGCAGTTC
71941	CACGCGCTCA	CGGTCAGCTT	CGACCCGCGC	GAGCGCCCGG	CGGCCGCDD

EXAMPLE 2

Construction of a Myxococcus xanthus Expression Vector

[0316] The DNA providing the integration and attachment function of phage Mx8 was inserted into commercially available pACYC184 (New England Biolabs). An ˜2360 bp MfeI-SmaI from plasmid pPLH343, described in Salmi et al., February 1998, J. Bact. 180(3): 614-621, was isolated and ligated to the large EcoRI-XmnI restriction fragment of plasmid pACYC184. The circular DNA thus formed was ˜6 kb in size and called plasmid pKOS35-77.

[0317] Plasmid pKOS35-77 serves as a convenient plasmid for expressing recombinant PKS genes of the invention under the control of the epothilone PKS gene promoter. In one illustrative embodiment, the entire epothilone PKS gene with its homologous promoter is inserted in one or more fragments into the plasmid to yield an expression vector of the invention.

[0318] The present invention also provides expression vectors in which the recombinant PKS genes of the invention are under the control of a Myxococcus xanthus promoter. To construct an illustrative vector, the promoter of the pilA gene of M. xanthus was isolated as a PCR amplification product. Plasmid pSWU357, which comprises the pilA gene promoter and is described in Wu and Kaiser, December 1997, J. Bact. 179(24):7748-7758, was mixed with PCR primers Seq1 and Mxpil1 primers:

Seq1:	5′-AGCGGATAACAATTTCACACAGGA	(SEQ ID NC: 3)
	AACAGC-3′;
and
Mxpil1:	5′-TTAATTAAGAGAAGGTTGCAACGG	(SEQ ID NC: 4)
	GGGGC-3′,

[0319] and amplified using standard PCR conditions to yield an ˜800 bp fragment. This fragment was cleaved with restriction enzyme KpnI and ligated to the large KpnI-EcoRV restriction fragment of commercially available plasmid pLitmus 28 (New England Biolabs). The resulting circular DNA was designated plasmid pKOS35-71B.

[0320] The promoter of the pilA gene from plasmid pKOS35-71B was isolated as an ˜800 bp EcoRV-SnaBI restriction fragment and ligated with the large MscI restriction fragment of plasmid pKOS35-77 to yield a circular DNA ˜6.8 kb in size. Because the ˜800 bp fragment could be inserted in either one of two orientations, the ligation produced two plasmids of the same size, which were designated as plasmids pKOS35-82.1 and pKOS35-82.2. Restriction site and function maps of these plasmids are presented in FIG. 3.

[0321] Plasmids pKOS35-82.1 and pKOS35-82.2 serve as convenient starting materials for the vectors of the invention in which a recombinant PKS gene is placed under the control of the Myxococcus xanthus pilA gene promoter. These plasmids comprise a single PacI restriction enzyme recognition sequence placed immediately downstream of the transcription start site of the promoter. In one illustrative embodiment, the entire epothilone PKS gene without its homologous promoter is inserted in one or more fragments into the plasmids at the PacI site to yield expression vectors of the invention.

[0322] The sequence of the pilA promoter in these plasmids is shown below (SEQ ID NO: 5).

CGACGCAGGTGAAGCTGCTTCGTGTGCTCCAGGAGCGGAAGGTGAAGCCGGTCGGCAGCGCCGCGGAGATTCCCTTC
CAGGCGCGTGTCATCGCGGCAACGAACCGGCGGCTCGAAGCCGAAGTAAAGGCCGGACGCTTTCGTGAGGACCTCTT
CTACCGGCTCAACGTCATCACGTTGGAGCTGCCTCCACTGCGCGAGCGTTCCGGCGACGTGTCGTTGCTGGCGAACT
ACTTCCTGTCCAGACTGTCGGAGGAGTTGGGGCGACCCGGTCTGCGTTTCTCCCCCGAGACACTGGGGCTATTGGAG
CGCTATCCCTTCCCAGGCAACGTGCGGCAGCTGCAGAACATGGTGGAGCGGGCCGCGACCCTGTCGGATTCAGACCT
CCTGGGGCCCTCCACGCTTCCACCCGCAGTGCGGGGCGATACAGACCCCGCCGTGCGTCCCGTGGAGGGCAGTGAGC
CAGGGCTGGTGGCGGGCTTCAACCTGGAGCGGCATCTCGACGACAGCGAGCGGCGCTATCTCGTCGCGGCGATGAAG
CAGGCCGGGGGCGTGAAGACCCGTGCTGCGGAGTTGCTGGGCCTTTCGTTCCGTTCATTCCGCTACCGGTTGGCCAA
GCATGGGCTGACGGATGACTTGGAGCCCGGGAGCGCTTCGGATGCGTAGGCTGATCGACAGTTATCGTCAGCGTCAC
TGCCGAATTTTGTCAGCCCTGGACCCATCCTCGCCGAGGGGATTGTTCCAAGCCTTGAGAATTGGGGGGCTTGGAGT
GCGCACCTGGGTTGGCATGCGTAGTGCTAATCCCATCCGCGGGCGCAGTGCCCCCCGTTGCAACCTTCTCTTAATTA
A

[0323] To make the recombinant Myxococcus xanthus host cells of the invention, M. xanthus cells are grown in CYE media (Campos and Zusman, 1975, Regulation of development in Myxococcus xanthus: effect of 3′:5′-cyclic AMP, ADP, and nutrition, Proc. Natl. Acad. Sci. USA 72: 518-522) to a Klett of 100 at 30° C. at 300 rpm. The remainder of the protocol is conducted at 25° C. unless otherwise indicated. The cells are then pelleted by centrifugation (8000 rpm for 10 min. in an SS34 or SA600 rotor) and resuspended in deionized water. The cells are again pelleted and resuspended in {fraction (1/100)}th of the original volume.

[0324] DNA (one to two μL) is electroporated into the cells in a 0.1 cm cuvette at room temperature at 400 ohm, 25 μFD, 0.65 V with a time constant in the range of 8.8-9.4. The DNA should be free of salts and so should be resuspended in distilled and deionized water or dialyzed on a 0.025 μm Type VS membrane (Millipore). For low efficiency electroporations, spot dialyze the DNA, and allow outgrowth in CYE. Immediately after electroporation, add 1 mL of CYE, and pool the cells in the cuvette with an additional 1.5 mL of CYE previously added to a 50 mL Erlenmeyer flask (total volume 2.5 ml). Allow the cells to grow for four to eight hours (or overnight) at 30 to 32° C. at 300 rpm to allow for expression of the selectable marker. Then, plate the cells in CYE soft agar on plates with selection. If kanamycin is the selectable marker, then typical yields are 103 to 105 per μg of DNA. If streptomycin is the selectable marker, then it must be included in the top agar, because it binds agar.

[0325] With this procedure, the recombinant DNA expression vectors of the invention are electroporated into Myxococcus host cells that express recombinant PKSs of the invention and produce the epothilone, epothilone derivatives, and other novel polyketides encoded thereby.

EXAMPLE 3

Construction of a Bacterial Artificial Chromosome (BAC) for Expression of Epothilone in Myxococcus xanthus

[0326] To express the epothilone PKS and modification enzyme genes in a heterologous host to produce epothilones by fermentation, Myxococcus xanthus, which is closely related to Sorangium cellulosum and for which a number of cloning vectors are available, can also be employed in accordance with the methods of the invention. Because both M. xanthus and S. cellulosum are myxobacteria, it is expected that they share common elements of gene expression, translational control, and post translational modification (if any), thereby enhancing the likelihood that the epo genes from S. cellulosum can be expressed to produce epothilone in M. xanthus . Secondly, M. xanthus has been developed for gene cloning and expression. DNA can be introduced by electroporation, and a number of vectors and genetic markers are available for the introduction of foreign DNA, including those that permit its stable insertion into the chromosome. Finally, M. xanthus can be grown with relative ease in complex media in fermentors and can be subjected to manipulations to increase gene expression, if required.

[0327] To introduce the epothilone gene cluster into Myxococcus xanthus, one can build the epothilone cluster into the chromosome by using cosmids of the invention and homologous recombination to assemble the complete gene cluster. Alternatively, the complete epothilone gene cluster can be cloned on a bacterial artificial chromosome (BAC) and then moved into M. xanthus for integration into the chromosome.

[0328] To assemble the gene cluster from cosmids pKOS35-70.1A2, and pKOS35-79.85, small regions of homology from these cosmids have to be introduced into Myxococcus xanthus to provide recombination sites for larger pieces of the gene cluster. As shown in FIG. 4, plasmids pKOS35-154 and pKOS90-22 are created to introduce these recombination sites. The strategy for assembling the epothilone gene cluster in the M. xanthus chromosome is shown in FIG. 5. Initially, a neutral site in the bacterial chromosome is chosen that does not disrupt any genes or transcriptional units. One such region is downstream of the devS gene, which has been shown not to affect the growth or development of M. xanthus. The first plasmid, pKOS35-154, is linearized with DraI and electroporated into M. xanthus. This plasmid contains two regions of the dev locus flanking two fragments of the epothilone gene cluster. Inserted in between the epo gene regions are the kanamycin resistance marker and the galK gene. Kanamycin resistance arises in colonies if the DNA recombines into the dev region by a double recombination using the dev sequence as regions of homology. This strain, K35-159, contains small regions of the epothilone gene cluster that will allow for recombination of pKOS35-79.85. Because the resistance markers on pKOS35-79.85 are the same as that for K35-159, a tetracycline transposon was transposed into the cosmid, and cosmids that contain the transposon inserted into the kanamycin marker were selected. This cosmid, pKOS90-23, was electroporated into K35-159, and oxytetracycline resistant colonies were selected to create strain K35-174. To remove the unwanted regions from the cosmid and leave only the epothilone genes, cells were plated on CYE plates containing 1% galactose. The presence of the galK gene makes the cells sensitive to 1% galactose. Galactose resistant colonies of K35-174 represent cells that have lost the galK marker by recombination or by a mutation in the galK gene. If the recombination event occurs, then the galactose resistant strain is sensitive to kanamycin and oxytetracycline. Strains sensitive to both antibiotics are verified by Southern blot analysis. The correct strain is identified and designated K35-175 and contains the epothilone gene cluster from module 7 through two open reading frames past the epoL gene.

[0329] To introduce modules 1 through module 7, the above process is repeated once more. The plasmid pKOS90-22 is linearized with DraI and electroporated into K35-175 to create K35-180. This strain is electroporated with the tetracycline resistant version of pKOS35-70.1A2, pKOS90-38, and colonies resistant to oxytetracycline are selected. This creates strain K35-185. Recombinants that now have the whole epothilone gene cluster are selected by resistance to 1% galactose. This results in strain K35-188. This strain contains all the epothilone genes as well as all potential promoters. This strain is fermented and tested for the production of epothilones A and B.

[0330] To clone the whole gene cluster as one fragment, a bacterial artificial chromosome (BAC) library is constructed. First, SMP44 cells are embedded in agarose and lysed according to the BIO-RAD genomic DNA plug kit. DNA plugs are partially digested with restriction enzyme, such as Sau3AI or HindIII, and electrophoresed on a FIGE or CHEF gel. DNA fragments are isolated by electroeluting the DNA from the agarose or using gelase to degrade the agarose. The method of choice to isolate the fragments is electroelution, as described in Strong et al., 1997, Nucleic Acids Res. 19: 3959-3961, incorporated herein by reference. The DNA is ligated into the BAC (pBeloBACII) cleaved with the appropriate enzyme. A map of pBeloBACII is shown below. 12

[0331] The DNA is electroporated into DH10B cells by the method of Sheng et al., 1995, Nucleic Acids Res. 23: 1990-1996, incorporated herein by reference, to create an S. cellulosum genomic library. Colonies are screened using a probe from the NRPS region of the epothilone cluster. Positive clones are picked and DNA is isolated for restriction analysis to confirm the presence of the complete gene cluster. This positive clone is designated pKOS35-178.

[0332] To create a strain that can be used to introduce pKOS35-178, a plasmid, pKOS35-164, is constructed that contains regions of homology that are upstream and downstream of the epothilone gene cluster flanked by the dev locus and containing the kanamycin resistance galK cassette, analogous to plasmids pKOS90-22 and pKOS35-154. This plasmid is linearized with DraI and electroporated into M. xanthus, in accordance with the method of Kafeshi et al., 1995, Mol. Microbiol. 15: 483-494, to create K35-183. The plasmid pKOS35-178 can be introduced into K35-183 by electroporation or by transduction with bacteriophage P1 and chloramphenicol resistant colonies are selected. Alternatively, a version of pKOS35-178 that contains the origin of conjugative transfer from pRP4 can be constructed for transfer of DNA from E. coli to K35-183. This plasmid is made by first constructing a transposon containing the oriT region from RP4 and the tetracycline resistance maker from pACYC184 and then transposing the transposon in vitro or in vivo onto pKOS35-178. This plasmid is transformed into S17-1 and conjugated into M. xanthus. This strain, K35-190, is grown in the presence of 1% galactose to select for the second recombination event. This strain contains all the epothilone genes as well as all potential promoters. This strain will be fermented and tested for the production of epothilones A and B.

[0333] Besides integrating pKOS35-178 into the dev locus, it can also be integrated into a phage attachment site using integration functions from myxophages Mx8 or Mx9. A transposon is constructed that contains the integration genes and att site from either Mx8 or Mx9 along with the tetracycline gene from pACYC184. Alternative versions of this transposon may have only the attachment site. In this version, the integration genes are then supplied in trans by coelectroporation of a plasmid containing the integrase gene or having the integrase protein expressed in the electroporated strain from any constitutive promoter, such as the mgl promoter (see Magrini et al., July 1999, J. Bact. 181(13): 4062-4070, incorporated herein by reference). Once the transposon is constructed, it is transposed onto pKOS35-178 to create pKOS35-191. This plasmid is introduced into Myxococcus xanthus as described above. This strain contains all the epothilone genes as well as all potential promoters. This strain is fermented and tested for the production of epothilones A and B.

[0334] Once the epothilone genes have been established in a strain of Myxococcus xanthus, manipulation of any part of the gene cluster, such as changing promoters or swapping modules, can be performed using the kanamycin resistance and galK cassette.

[0335] Cultures of Myxococcus xanthus containing the epo genes are grown in a number of media and examined for production of epothilones. If the levels of production of epothilones (in particular B or D) are too low to permit large scale fermentation, the M. xanthus-producing clones are subjected to media development and strain improvement, as described below for enhancing production in Streptomyces.

EXAMPLE 4

Construction of a Streptomyces Expression Vector

[0336] The present invention provides recombinant expression vectors for the heterologous expression of modular polyketide synthase genes in Streptomyces hosts. These vectors include expression vectors that employ the acti promoter that is regulated by the gene actll ORF4 to allow regulated expression at high levels when growing cells enter stationary phase. Among the vectors available are plasmids pRM1 and pRM5, and derivatives thereof such as pCK7, which are stable, low copy plasmids that carry the marker for thiostrepton resistance in actinomycetes. Such plasmids can accommodate large inserts of cloned DNA and have been used for the expression of the DEBS PKS in S. coelicolor and S. lividans, the picromycin PKS genes in S. lividans, and the oleandomycin PKS genes in S. lividans. See U.S. Pat. No. 5,712,146. Those of skill in the art recognize that S. lividans does not make the tRNA that recognizes the TTA codon for leucine until late-stage growth and that if production of a protein is desired earlier, then appropriate codon modifications can be made. 13

[0337] Another vector is a derivative of plasmid pSET152 and comprises the actII ORF4-PactI expression system but carries the selectable marker for apramycin resistance. These vectors contain the attP site and integrase gene of the actinophage phiC31 and do not replicate autonomously in Streptomyces hosts but integrate by site specific recombination into the chromosome at the attachment site for phiC31 after introduction into the cell. Derivatives of pCK7 and pSET152 have been used together for the heterologous production of a polyketide, with different PKS genes expressed from each plasmid. See U.S. patent application Ser. No. 60/129,731, filed 16 Apr. 1999, incorporated herein by reference. 14

Plasmid pKOS010-153, a pSET152 Derivative

[0338] The need to develop expression vectors for the epothilone PKS that function in Streptomyces is significant. The epothilone compounds are currently produced in the slow growing, genetically intractable host Sorangium cellulosum or are made synthetically. The streptomycetes, bacteria that produce more than 70% of all known antibiotics and important complex polyketides, are excellent hosts for production of epothilones and epothilone derivatives. S. lividans and S. coelicolor have been developed for the expression of heterologous PKS systems. These organisms can stably maintain cloned heterologous PKS genes, express them at high levels under controlled conditions, and modify the corresponding PKS proteins (e.g. phosphopantetheinylation) so that they are capable of production of the polyketide they encode. Furthermore, these hosts contain the necessary pathways to produce the substrates required for polyketide synthesis, e.g. malonyl CoA and methylmalonyl CoA. A wide variety of cloning and expression vectors are available for these hosts, as are methods for the introduction and stable maintenance of large segments of foreign DNA. Relative to the slow growing Sorangium host, S. lividans and S. coelicolor grow well on a number of media and have been adapted for high level production of polyketides in fermentors. A number of approaches are available for yield improvements, including rational approaches to increase expression rates, increase precursor supply, etc. Empirical methods to increase the titers of the polyketides, long since proven effective for numerous other polyketides produced in streptomycetes, can also be employed for the epothilone and epothilone derivative producing host cells of the invention.

[0339] To produce epothilones by fermentation in a heterologous Streptomyces host, the epothilone PKS (including the NRPS module) genes are cloned in two segments in derivatives of pCK7 (loading domain through module 6) and pKOS010-153 (modules 7 through 9). The two plasmids are introduced into S. lividans employing selection for thiostrepton and apramycin resistance. In this arrangement, the pCK7 derivative replicates autonomously whereas the pKOS010-153 derivative is integrated in the chromosome. In both vectors, expression of the epothilone genes is from the acti promoter resident within the plasmid.

[0340] To facilitate the cloning, the two epothilone PKS encoding segments (one for the loading domain through module six and one for modules seven through nine) were cloned as translational fusions with the N-terminal segment of the KS domain of module 5 of the ery PKS. High level expression has been demonstrated from this promoter employing KS5 as the first translated sequence, see Jacobsen et al., 1998, Biochemistry 37: 4928-4934, incorporated herein by reference. A convenient BsaBI site is contained within the DNA segment encoding the amino acid sequence EPIAV that is highly conserved in many KS domains including the KS-encoding regions of epoA and of module 7 in epoE.

[0341] The expression vector for the loading domain and modules one through six of the epothilone PKS was designated pKOS039-124, and the expression vector for modules seven through nine was designated pKOS039-126. Those of skill in the art will recognize that other vectors and vector components can be used to make equivalent vectors. Because preferred expression vectors of the invention, described below and derived from pKOS039-124 and pKOS039-126, have been deposited under the terms of the Budapest Treaty, only a summary of the construction of plasmids pKOS039-124 and pKOS039-126 is provided below.

[0342] The eryKS5 linker coding sequences were cloned as an ˜0.4 kb PacI-Bg1II restriction fragment from plasmid pKOS10-153 into pKOS039-98 to construct plasmid pKOS039-117. The coding sequences for the eryKS5 linker were linked to those for the epothilone loading domain by inserting the ˜8.7 kb EcoRI-XbaI restriction fragment from cosmid pKOS35-70.1A2 into EcoRI-XbaI digested plasmid pLItmus28. The ˜3.4 kb of BsaBI-NotI and ˜3.7 kb NotI-HindIII restriction fragments from the resulting plasmid were inserted into BsaBI-HindIII digested plasmid pKOS039-117 to construct plasmid pKOS039-120. The ˜7 kb PacI-XbaI restriction fragment of plasmid pKOS039-120 was inserted into plasmid pKAO18′ to construct plasmid pKOS039-123. The final pKOS039-124 expression vector was constructed by ligating the ˜34 kb XbaI-AvrII restriction fragment of cosmid pKOS35-70.1A2 with the ˜21.1 kb AvrII-XbaI restriction fragment of pKOS039-123.

[0343] The plasmid pKOS039-126 expression vector was constructed as follows. First the coding sequences for module 7 were linked from cosmids pKOS35-70.4 and pKOS35-79.85 by cloning the ˜6.9 kb BglII-NotI restriction fragment of pKOS35-70.4 and the ˜5.9 kb NotI-HindIII restriction fragment of pKOS35-79.85 into Bg1II-HindIII digested plasmid pLitmus28 to construct plasmid pKOS039-119. The ˜12 kb NdeI-NheI restriction fragment of cosmid pKOS35-79.85 was cloned into NdeI-XbaI digested plasmid pKOS039-119 to construct plasmid pKOS039-122.

[0344] To fuse the eryKS5 linker coding sequences with the coding sequences for module 7, the ˜1 kb BsaBI-Bg1II restriction fragment derived from cosmid pKOS35-70.4 was cloned into BsaBI-Bc1I digested plasmid pKOS039-117 to construct plasmid pKOS039-121. The ˜21.5 kb AvrII restriction fragment from plasmid pKOS039-122 was cloned into AvrII-XbaI digested plasmid pKOS039-121 to construct plasmid pKOS039-125. The ˜21.8 kb PacI-EcoRI restriction fragment of plasmid pKOS039-125 was ligated with the ˜9 kb PacI-EcoRI restriction fragment of plasmid pKOS039-44 to construct pKOS039-126.

[0345] Plasmids pKOS039-124 and pKOS126 were introduced into S. lividans K4-114 sequentially employing selection for the corresponding drug resistance marker. Because plasmid pKOS039-126 does not replicate autonomously in streptomycetes, the selection is for cells in which the plasmid has integrated in the chromosome by site-specific recombination at the attB site of phiC31. Because the plasmid stably integrates, continued selection for apramycin resistance is not required. Selection can be maintained if desired. The presence of thiostrepton in the medium is maintained to ensure continued selection for plasmid pKOS039-124. Plasmids pKOS039-124 and pKOS039-126 were transformed into Streptomyces lividans K4-114, and transformants containing the plasmids were cultured and tested for production of epothilones. Initial tests did not indicate the presence of an epothilone.

[0346] To improve production of epothilones from these vectors, the eryKS5 linker sequences were replaced by epothilone PKS gene coding sequences, and the vectors were introduced into Streptomyces coelicolor CH999. To amplify by PCR coding sequences from the epoA gene coding sequence, two oligonucleotides primers were used:

[0347] N39-73, 5′-

N39-73, (SEQ ID NO: 6)
5′-GCTTAATTAAGGAGGACACATATGCCCGTCGTGGCGGATCGTCC-3′;
and
N39-74, (SEQ ID NO: 7)
5′-GCGGATCCTCGAATCACCGCCAATATC-3′.

[0348] The template DNA was derived from cosmid pKOS35-70.8A3. The ˜0.8 kb PCR product was digested with restriction enzymes PacI and BamHI and then ligated with the ˜2.4 kb BamHI-NotI and the ˜6.4 kb PacI-NotI restriction fragments of plasmid pKOS039-120 to construct plasmid pKOS039-136. To make the expression vector for the epoA, epoB, epoC, and epoD genes, the ˜5 kb PacI-AvrII restriction fragment of plasmid pKOS039-136 was ligated with the ˜50 kb PacI-AvrII restriction fragment of plasmid pKOS039-124 to construct the expression plasmid pKOS039-124R. Plasmid pKOS039-124R has been deposited with the ATCC under the terms of the Budapest Treaty and is available under accession number ______.

[0349] To amplify by PCR sequences from the epoE gene coding sequence, two oligonucleotide primers were used:

N39-67A, (SEQ ID NO: 8)
5′-GCTTAATTAAGGAGGACACATATGACCGACCGAGAAGGCCAGCTCCTGGA-3′,
and
N39-68,  (SEQ ID NO: 9)
5′-GGACCTAGGCGGGATGCCGGCGTCT-3′.

[0350] The template DNA was derived from cosmid pKOS35-70.1A2. The ˜0.4 kb amplification product was digested with restriction enzymes PacI and AvrII and ligated with either the ˜29.5 kb PacI-AvrII restriction fragment of plasmid pKOS039-126 or the ˜23.8 kb PacI-AvrII restriction fragment of plasmid pKOS039-125 to construct plasmid pKOS039-126R or plasmid pKOS039-125R, respectively. Plasmid pKOS039-126R was deposited with the ATCC under the terms of the Budapest Treaty and is available under accession number ______.

[0351] The plasmid pair pKOS039-124R and pKOS039-126R (as well as the plasmid pair pKOS039-124 and pKOS039-126) contain the full complement of epoA, epoB, epoC, epoD, epoE, epoF, epoK, and epoL genes. The latter two genes are present on plasmid pKOS039-126R (as well as plasmid pKOS039-126); however, to ensure that these genes were expressed at high levels, another expression vector of the invention, plasmid pKOS039-141 (FIG. 8), was constructed in which the epoK and epoL genes were placed under the control of the ermE* promoter.

[0352] The epoK gene sequences were amplified by PCR using the oligonucleotide primers:

N39-69, (SEQ ID NO: 10)
5′-AGGCATGCATATGACCCAGGAGCAAGCGAATCAGAGTG-3′;
and
N39-70, (SEQ ID NO: 11)
5′-CCAAGCTTTATCCAGCTTTGGAGGGCTTCAAG-3′.

[0353] The epoL gene sequences were amplified by PCR using the oligonucleotide primers:

N39-71A, (SEQ ID NO: 12)
5′-GTAAGCTTAGGAGGACACATATGATGCAACTCGCGCGCGGGTG-3′;
and
N39-72, (SEQ ID NO: 13)
5′-GCCTGCAGGCTCAGGCTTGCGCAGAGCGT-3′.

[0354] The template DNA for the amplifications was derived from cosmid pKOS35-79.85. The PCR products were subcloned into PCR-script for sequence analysis. Then, the epoK and epoL genes were isolated from the clones as NdeI-HindIII and HindIll-EcoRI restriction fragments, respectively, and ligated with the ˜6 kb NdeI-EcoRI restriction fragment of plasmid pKOS039-134B, which contains the ermE* promoter, to construct plasmid pKOS039-140. The ˜2.4 kb NheI-PstI restriction fragment of plasmid pKOS039-140 was cloned into XbaI-PstI digested plasmid pSAM-Hyg, a plasmid pSAM2 derivative containing a hygromycin resistance conferring gene, to construct plasmid pKOS039-141.

[0355] Another variant of plasmid pKOS039-126R was constructed to provide the epoE and epoF genes on an expression vector without the epoK and epoL genes. This plasmid, pKOS045-12 (FIG. 9), was constructed as follows. Plasmid pXH106 (described in J. Bact., 1991, 173: 5573-5577, incorporated herein by reference) was digested with restriction enzymes Stul and BamHI, and the ˜2.8 kb restriction fragment containing the xylE and hygromycin resistance conferring genes was isolated and cloned into EcoRV-BglII digested plasmid pLitmus28. The ˜2.8 kb NcoI-AvrII restriction fragment of the resulting plasmid was ligated to the ˜18 kb PacI-BspHI restriction fragment of plasmid pKOS039-125R and the ˜9 kb SpeI-PacI restriction fragment of plasmid pKOS039-42 to construct plasmid pKOS045-12.

[0356] To construct an expression vector that comprised only the epoL gene, plasmid pKOS039-141 was partially digested with restriction enzyme NdeI, the ˜9 kb NdeI restriction fragment was isolated, and the fragment then circularized by ligation to yield plasmid pKOS039-150.

[0357] The various expression vectors described above were then transformed into Streptomyces coelicolor CH999 and S. lividans K4-114 in a variety of combinations, the transformed host cells fermented on plates and in liquid culture (R5 medium, which is identical to R2YE medium without agar). Typical fermentation conditions follow. First, a seed culture of about 5 mL containing 50 μg/L thiostrepton was inoculated and grown at 30° C. for two days. Then, about 1 to 2 mL of the seed culture was used to inoculate a production culture of about 50 mL containing 50 μg/L thiostrepton and 1 mM cysteine, and the production culture was grown at 30° C. for 5 days. Also, the seed culture was used to prepare plates of cells (the plates contained the same media as the production culture with 10 mM propionate), which were grown at 30° C. for nine days.

[0358] Certain of the Streptomyces coelicolor cultures and culture broths were analyzed for production of epothilones. The liquid cultures were extracted with three times with equal volumes of ethyl acetate, the organic extracts combined and evaporated, and the residue dissolved in acetonitrile for LC/MS analysis. The agar plate media was chopped and extracted twice with equal volumes of acetone, and the acetone extracts were combined and evaporated to an aqueous slurry, which was extracted three times with equal volumes of ethyl acetate. The organic extracts were combined and evaporated, and the residue dissolved in acetonitrile for LC/MS analysis.

[0359] Production of epothilones was assessed using LC-mass spectrometry. The output flow from the UV detector of an analytical HPLC was split equally between a Perkin-Elmer/Sciex API100LC mass spectrometer and an Alltech 500 evaporative light scattering detector. Samples were injected onto a 4.6×150 mm reversed phase HPLC column (MetaChem 5 m ODS-3 Inertsil) equilibrated in water with a flow rate of 1.0 mL/min. UV detection was set at 250 nm. Sample components were separated using H2O for 1 minute, then a linear gradient from 0 to 100% acetonitrile over 10 minutes. Under these conditions, epothilone A elutes at 10.2 minutes and epothilone B elutes at 10.5 minutes. The identity of these compounds was confirmed by the mass spectra obtained using an atmospheric chemical ionization source with orifice and ring voltages set at 75 V and 300 V, respectively, and a mass resolution of 0.1 amu. Under these conditions, epothilone A shows [M+H] at 494.4 amu, with observed fragments at 476.4, 318.3, and 306.4 amu. Epothilone B shows [M+H] at 508.4 amu, with observed fragments at 490.4, 320.3, and 302.4 amu.

[0360] Transformants containing the vector pairs pKOS039-124R and pKOS039-126R or pKOS039-124 and pKOS039-126R produced detectable amounts of epothilones A and B. Transformants containing these plasmid pairs and the additional plasmid pKOS039-141 produced similar amounts of epothilones A and B, indicating that the additional copies of the epoK and epoL genes were not required for production under the test conditions employed. Thus, these transformants produced epothilones A and B when recombinant epoA, epoB, epoC, epoD, epoE, epoF, epoK, and epoL genes were present. In some cultures, it was observed that the absence of propionate increased the proportion of epothilone B to epothilone A.

[0361] Transformants containing the plasmid pair pKOS039-124R and pKOS045-12 produced epothilones C and D, as did transformants containing this plasmid pair and the additional plasmid pKOS039-150. These results showed that the epoL gene was not required under the test conditions employed to form the C-12-C-13 double bond. These results indicate that either the epothilone PKS gene alone is able to form the double bond or that Streptomyces coelicolor expresses a gene product able to convert epothilones G and H to epothilones C and D. Thus, these transformants produced epothilones C and D when recombinant epoA, epoB, epoC, epoD, epoE, and epoF genes were present.

[0362] The heterologous expression of the epothilone PKS described herein is believed to represent the recombinant expression of the largest proteins and active enzyme complex that have ever been expressed in a recombinant host cell. The epothilone producing Streptomyces coelicolor transformants exhibited growth characteristics indicating that either the epothilone PKS genes, or their products, or the epothilones inhibited cell growth or were somewhat toxic to the cells. Any such inhibition or toxicity could be due to accumulation of the epothilones in the cell, and it is believed that the native Sorangium producer cells may contain transporter proteins that in effect pump epothilones out of the cell. Such transporter genes are believed to be included among the ORFs located downstream of the epoK gene and described above. Thus, the present invention provides Streptomyces and other host cells that include recombinant genes that encode the products of one or more, including all, of the ORFs in this region.

[0363] For example, each ORF can be cloned behind the ermE* promoter, see Stassi et al., 1998, Appl. Microbiol. Biotechnol. 49: 725-731, incorporated herein by reference, in a pSAM2-based plasmid that can integrate into the chromosome of Streptomyces coelicolor and S. lividans at a site distinct from attb of phage phiC31, see Smokvina et al., 1990, Gene 94: 53-59, incorporated herein by reference. A pSAM2-based vector carrying the gene for hygromycin resistance is modified to carry the ermE* promoter along with additional cloning sites. Each ORF downstream is PCR cloned into the vector which is then introduced into the host cell (also containing pKOS039-124R and pKOS039-126R or other expression vectors of the invention) employing hygromycin selection. Clones carrying each individual gene downstream from epoK are analyzed for increased production of epothilones.

[0364] Additional fermentation and strain improvement efforts can be conducted as illustrated by the following. The levels of expression of the PKS genes in the various constructs can be measured by assaying the levels of the corresponding mRNAs (by quantitative RT PCR) relative to the levels of another heterologous PKS MRNA (e.g. picromycin) produced from genes cloned in similar expression vectors in the same host. If one of the epothilone transcripts is underproduced, experiments to enhance its production by cloning the corresponding DNA segment in a different expression vector are conducted. for example, multiple copies of any one or more of the epothilone PKS genes can be introduced into a cell if one or more gene products are rate limiting for biosynthesis. If the basis for low level production is not related to low level PKS gene expression (at the RNA level), an empirical mutagenesis and screening approach that is the backbone of yield improvement of every commercially important fermentation product is undertaken. Spores are subjected to UV, X-ray or chemical mutagens, and individual survivors are plated and picked and tested for the level of compound produced in small scale fermentations. Although this process can be automated, one can examine several thousand isolates for quantifiable epothilone production using the susceptible fungus Mucor hiemalis as a test organism.

[0365] Another method to increase the yield of epothilones produced is to change the KSY domain of the loading domain of the epothilone PKS to a KSQ domain. Such altered loading domains can be constructed in any of a variety of ways, but one illustrative method follows. Plasmid pKOS39-124R of the invention can be conveniently used as a starting material. To amplify DNA fragments useful in the construction, four oligonucleotide primers are employed:

N39-83: (SEQ ID NO: 14)
5′-CCGGTATCCACCGCGACACACGGC-3′,
N39-84: (SEQ ID NO: 15)
5′-GCCAGTCGTCCTCGCTCGTGGCCGTTC-3′,

[0366] and N39-73 and N39-74, which have been described above. The PCR fragment generated with N37-73 and N39-83 and the PCR fragment generated with N39-74 and N39-84 are treated with restriction enzymes PacI and BamHI, respectively, and ligated with the ˜3.1 kb PacI-BamHI fragment of plasmid pKOS39-120 to construct plasmid pKOS039-148. The ˜0.8 kb PacI-BamHI restriction fragment of plasmid pKOS039-148 (comprising the two PCR amplification products) is ligated with the ˜2.4 kb BamnHI-NotI restriction fragment and the ˜6.4 kb PacI-NotI restriction fragment of plasmid pKOS39-120 to construct pKOS39-136Q. The ˜5 kb PacI-AvrII restriction fragment of plasmid pKOS039-136Q is ligated to the ˜50 kb PacI-AvrII restriction fragment of plasmid pKOS039-124 to construct plasmid pKOS39-124Q. Plasmids pKOS039-124Q and pKOS039-126R are then transformed into Streptomyces coelicolor CH999 for epothilone production.

[0367] The epoA through epoF, optionally with epoK or with epoK plus epoL, genes cloned and expressed are sufficient for the synthesis of epothilone compounds, and the distribution of the C-12 H to C-12 methyl congeners appears to be similar to that seen in the natural host (A:B::2:1). This ratio reflects that the AT domain of module 4 more closely resembles that of the malonyl rather than methylmalonyl specifying AT consensus domains. Thus, epothilones D and B are produced at lower quantities than their C-12 unmethylated counterparts C and A. The invention provides PKS genes that produce epothilone D and/or B exclusively. Specifically, methylmalonyl CoA specifying AT domains from a number of sources (e.g. the narbonolide PKS, the rapamycin PKS, and others listed above) can be used to replace the naturally occurring at domain in module 4. The exchange is performed by direct cloning of the incoming DNA into the appropriate site in the epothilone PKS encoding DNA segment or by gene replacement through homologous recombination.

[0368] For gene replacement through homologous recombination, the donor sequence to be exchanged is placed in a delivery vector between segments of at least 1 kb in length that flank the AT domain of epo module 4 encoding DNA. Crossovers in the homologous regions result in the exchange of the epo AT4 domain with that on the delivery vector. Because pKOS039-124 and pKOS039-124R contain AT4 coding sequences, they can be used as the host DNA for replacement. The adjacent DNA segments are cloned in one of a number of E. coli plasmids that are temperature sensitive for replication. The heterologous AT domains can be cloned in these plasmids in the correct orientation between the homologous regions as cassettes enabling the ability to perform several AT exchanges simultaneously. The reconstructed plasmid (pKOS039-124* or pKOS039-124R*) is tested for ability to direct the synthesis of epothilone B and/or by introducing it along with pKOS039-126 or pKOS039-126R in Streptomyces coelicolor and/or S. lividans.

[0369] Because the titers of the polyketide can vary from strain to strain carrying the different gene replacements, the invention provides a number of heterologous methylmalonyl CoA specifying AT domains to ensure that production of epothilone D at titers equivalent to that of the C and D mixture produced in the Streptomyces coelicolor host described above. In addition, larger segments of the donor genes can be used for the replacements, including, in addition to the AT domain, adjacent upstream and downstream sequences that correspond to an entire module. If an entire module is used for the replacement, the KS, methylmalonyl AT, DH, KR, ACP—encoding DNA segment can be obtained from for example and without limitation the DNA encoding the tenth module of the rapamycin PKS, or the first or fifth modules of the FK-520 PKS.

EXAMPLE 5

Heterologous Expression of EpoK and Conversion of Epothilone D to Epothilone B

[0370] This Example describes the construction of E. coli expression vectors for epoK. The epoK gene product was expressed in E. coli as a fusion protein with a polyhistidine tag (his tag). The fusion protein was purified and used to convert epothilone D to epothilone B.

[0371] Plasmids were constructed to encode fusion proteins composed of six histidine residues fused to either the amino or carboxy terminus of EpoK. The following oligos were used to construct the plasmids:

55-101.a-1: (SEQ ID NO: 16)
5′-AAAAACATATGCACCACCACCACCACCACATGACACAGGAGCAAGCGAAT-CAGAGTGAG-3′,
55-101.b:   (SEQ ID NO: 17)
5′-AAAAAGGATCCTTAATCCAGCTTTGGAGGGCTT-3′,
55-101.c:   (SEQ ID NO: 18)
5′-AAAAACATATGACACAGGAGCAAGCGAAT-3′,
and
55-101.d:   (SEQ ID NO: 19)
5′-AAAAAGGATCCTTAGTGGTGGTGGTGGTGGTGTCCAGCTTTGGAGGGCTTC-AAGATGAC-3′.

[0372] The plasmid encoding the amino terminal his tag fusion protein, pKOS55-121, was constructed using primers 55-101.a-1 and 55-101.b, and the one encoding the carboxy terminal his tag, pKOS55-129, was constructed using primers 55-101.c and 55-101.d in PCR reactions containing pKOS35-83.5 as the template DNA. Plasmid pKOS35-83.5 contains the ˜5 kb NotI fragment comprising the epoK gene ligated into pBluescriptSKII+ (Stratagene). The PCR products were cleaved with restriction enzymes BainHI and NdeI and ligated into the BamHI and NdeI sites of pET22b (Invitrogen). Both plasmids were sequenced to verify that no mutations were introduced during the PCR amplification. Protein gels were run as known in the art.

[0373] Purification of EpoK was performed as follows. Plasmids pKOS55-121 and pKOS55-129 were transformed into BL21(DE3) containing the groELS expressing plasmid pREP4-groELS (Caspers et al., 1994, Cellular and Molecular Biology 40(5): 635-644). The strains were inoculated into 250 mL of M9 medium supplemented with 2 mM MgSO4, 1% glucose, 20 mg thiamin, 5 mg FeCl2, 4 mg CaCl2 and 50 mg levulinic acid. The cultures were grown to an OD600 between 0.4 and 0.6, at which point IPTG was added to 1 mM, and the cultures were allowed to grow for an additional two hours. The cells were harvested and frozen at −80° C. The frozen cells were resuspended in 10 ml of buffer 1 (5 mM imidazole, 500 mM NaCl, and 45 mM Tris pH 7.6) and were lysed by sonicating three times for 15 seconds each on setting 8. The cellular debris was pelleted by spinning in an SS-34 rotor at 16,000 rpm for 30 minutes. The supematant was removed and spun again at 16,000 rpm for 30 minutes. The supernatant was loaded onto a 5 mL nickel column (Novagen), after which the column was washed with 50 mL of buffer 1 (Novagen). EpoK was eluted with a gradient from 5 mM to 1M imidazole. Fractions containing EpoK were pooled and dialyzed twice against 1 L of dialysis buffer (45 mM Tris pH7.6, 0.2 mM DTT, 0.1 mM EDTA, and 20% glycerol). Aliquots were frozen in liquid nitrogen and stored at −80° C. The protein preparations were greater than 90% pure.

[0374] The EpoK assay was performed as follows (See Betlach et al., Biochem (1998) 37:14937, incorporated herein by reference). Briefly, reactions consisted of 50 mM Tris (pH7.5), 21 μM spinach ferredoxin, 0.132 units of spinach ferredoxin: NADP+ oxidoreductase, 0.8 units of glucose-6-phosphate dehydrogenase, 1.4 mM NADP, and 7.1 mM glucose-6-phosphate, 100 μM or 200 μM epothilone D (a generous gift of S. Danishefsky), and 1.7 μM amino terminal his tagged EpoK or 1.6 μM carboxy terminal his tagged EpoK in a 100 μL volume. The reactions were incubated at 30° C. for 67 minutes and stopped by heating at 90° C. for 2 minutes. The insoluble material was removed by centrifugation, and 50 μL of the supernatant were analyzed by LC/MS. HPLC conditions: Metachem 5μ ODS-3 Inertsil (4.6×150 mm); 80% H2O for 1 min, then to 100% MeCN over 10 min at 1 mL/min, with UV (λmax=250 nm), ELSD, and MS detection. Under these conditions, epothilone D eluted at 11.6 min and epothilone B at 9.3 min. the LC/MS spectra were obtained using an atmosphere pressure chemical ionization source with orifice and ring voltages set at 20 V and 250 V, respectively, at a mass resolution of 1 amu. Under these conditions, epothilone E shows an [M+H] at m/z 493, with observed fragments at 405 and 304. Epothilone B shows an [M+H] at m/z 509, with observed fragments at 491 and 320.

[0375] The reactions containing EpoK and epothilone D contained a compound absent in the control that displayed the same retention time, molecular weight, and mass fragmentation pattern as pure epothilone B. With an epothilone D concentration of 100 μM, the amino and the carboxy terminal his tagged EpoK was able to convert 82% and 58% to epothilone B, respectively. In the presence of 200 μM, conversion was 44% and 21%, respectively. These results demonstrate that EpoK can convert epothilone D to epothilone B.

EXAMPLE 6

Modified Epothilones from Chemobiosynthesis

[0376] This Example describes a series of thioesters provided by the invention for production of epothilone derivatives via chemobiosynthesis. The DNA sequence of the biosynthetic gene cluster for epothilone from Sorangium cellulosum indicates that priming of the PKS involves a mixture of polyketide and amino acid components. Priming involves loading of the PKS-like portion of the loading domain with malonyl CoA followed by decarboxylation and loading of the module one NRPS with cysteine, then condensation to form enzyme-bound N-acetylcysteine. Cyclization to form a thiazoline is followed by oxidation to form enzyme bound 2-methylthiazole-4-carboxylate, the product of the loading domain and NRPS. Subsequent condensation with methylmalonyl CoA by the ketosynthase of module 2 provides the substrate for module, as shown in the following diagram. 15

[0377] The present invention provides methods and reagents for chemobiosynthesis to produce epothilone derivatives in a manner similar to that described to make 6-dEB and erythromycin analogs in PCT Pat. Pub. Nos. 99/03986 and 97/02358. Two types of feeding substrates are provided: analogs of the NRPS product, and analogs of the module 3 substrate. The module 2 substrates are used with PKS enzymes with a mutated NRPS-like domain, and the module 3 substrates are used with PKS enzymes with a mutated KS domain in module 2.

[0378] The following illustrate module 2 substrates (as N-acetyl cysteamine thioesters) for use as substrates for epothilone PKS with modified inactivated NRPS: 16

[0379] The module 2 substrates are prepared by activation of the corresponding carboxylic acid and treatment with N-acetylcysteamine. Activation methods include formation of the acid chloride, formation of a mixed anhydride, or reaction with a condensing reagent such as a carbodiimide.

[0380] Exemplary module 3 substrates, also as NAc thioesters for use as substrates for epothilone PKS with KS2 knockout are: 17

[0381] These compounds are prepared in a three-step process. First, the appropriate aldehyde is treated with a Wittig reagent or equivalent to form the substituted acrylic ester. The ester is saponified to the acid, which is then activated and treated with N-acetylcysteamine.

[0382] Illustrative reaction schemes for making module 2 and module 3 substrates follow. Additional compounds suitable for making starting materials for polyketide synthesis by the epothilone PKS are shown in FIG. 2 as carboxylic acids (or aldehydes that can be converted to carboxylic acids) that are converted to the N-acylcysteamides for supplying to the host cells of the invention.

A. Thioiphene-3-carboxylate N-acetylcysteamine Thioester

[0383] A solution of thiophene-3-carboxylic acid (128 mg) in 2 mL of dry tetrahydrofuran under inert atmosphere was treated with triethylamine (0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour, N-acetylcysteamine (0.25 mL) was added, and the reaction was allowed to proceed for 12 hours. The mixture was poured into water and extracted three times with equal volumes of ethyl acetate. The organic extracts were combined, washed sequentially with water, 1 N HCl, sat. CuSO4, and brine, then dried over MgSO4, filtered, and concentrated under vacuum. Chromatography on SiO2 using ether followed by ethyl acetate provided pure product, which crystallized upon standing.

B. Furan-3-carboxylate N-acetylcysteamine Thioester

[0384] A solution of furan-3-carboxylic acid (112 mg) in 2 mL of dry tetrahydrofuran under inert atmosphere was treated with triethylamine (0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour, N-acetylcysteamine (0.25 mL) was added and the reaction was allowed to proceed for 12 hours. The mixture was poured into water and extracted three times with equal volumes of ethyl acetate. The organic extracts were combined, washed sequentially with water, 1 N HCl, sat. CUSO4, and brine, then dried over MgSO4, filtered, and concentrated under vacuum. Chromatography on SiO2 using ether followed by ethyl acetate provided pure product, which crystallized upon standing.

C. Pyrrole-2-carboxylate N-acetylcysteamine Thioester

[0385] A solution of pyrrole-2-carboxylic acid (112 mg) in 2 mL of dry tetrahydrofuran under inert atmosphere was treated with triethylamine (0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour, N-acetylcysteamine (0.25 mL) was added and the reaction was allowed to proceed for 12 hours. The mixture was poured into water and extracted three times with equal volumes of ethyl acetate. The organic extracts were combined, washed sequentially with water, 1 N HCl, sat. CUSO4, and brine, then dried over MgSO4,filtered, and concentrated under vacuum. Chromatography on SiO2 using ether followed by ethyl acetate provided pure product, which crystallized upon standing.

D. 2-Methyl-3-(3-thienyl)acrylate N-acetylcysteamine Thioester

[0386] (1) Ethyl 2-methyl-3-(3-thienyl)acrylate: A mixture of thiophene-3-carboxaldehyde (1.12 g) and (carbethoxyethylidene)triphenylphosphorane (4.3 g) in dry tetrahydrofuran (20 mL) was heated at reflux for 16 hours. The mixture was cooled to ambient temperature and concentrated to dryness under vacuum. The solid residue was suspended in 1:1 ether/hexane and filtered to remove triphenylphosphine oxide. The filtrate was filtered through a pad of SiO2 using 1:1 ether/hexane to provide the product (1.78 g, 91%) as a pale yellow oil.

[0387] (2) 2-Methyl-3-(3-thienyl)acrylic acid: The ester from (1) was dissolved in a mixture of methanol (5 mL) and 8 N KOH (5 mL) and heated at reflux for 30 minutes. The mixture was cooled to ambient temperature, diluted with water, and washed twice with ether. The aqueous phase was acidified using 1N HCl then extracted 3 times with equal volumes of ether. The organic extracts were combined, dried with MgSO4, filtered, and concentrated to dryness under vacuum. Crystallization from 2:1 hexane/ether provided the product as colorless needles.

[0388] (3) 2-Methyl-3-(3-thienyl)acrylate N-acetylcysteamine thioester: A solution of 2-Methyl-3-(3-thienyl)acrylic acid (168 mg) in 2 mL of dry tetrahydrofuran under inert atmosphere was treated with triethylamine (0.56 mL) and diphenylphosphoryl azide (0.45 mL). After 15 minutes, N-acetylcysteamine (0.15 mL) is added and the reaction is allowed to proceed for 4 hours. The mixture is poured into water and extracted three times with equal volumes of ethyl acetate. The organic extracts are combined, washed sequentially with water, 1 N HCl, sat. CuSO4, and brine, then dried over MgSO4, filtered, and concentrated under vacuum. Chromatography on SiO2 using ethyl acetate provided pure product, which crystallized upon standing.

[0389] The above compounds are supplied to cultures of host cells containing a recombinant epothilone PKS of the invention in which either the NRPS or the KS domain of module 2 as appropriate has been inactivated by mutation to prepare the corresponding epothilone derivative of the invention.

EXAMPLE 7

Producing Epothilones and Epothilone Derivatives in Soranaium cellulosum SMP44

[0390] The present invention provides a variety of recombinant Sorangium cellulosum host cells that produce less complex mixtures of epothilones than the naturally occurring epothilone producers as well as host cells that produce epothilone derivatives. This Example illustrates the construction of such strains by describing how to make a strain that produce only epothilones C and D without epothilones A and B. To construct this strain, an inactivating mutation is made in epoK. Using plasmid pKOS35-83.5, which contains a NotI fragment harboring the epoK gene, the kanamycin and bleomycin resistance markers from Tn5 are ligated into the Scal site of the epoK gene to construct pKOS90-55. The orientation of the resistance markers is such that transcription initiated at the kanamycin promoter drives expression of genes immediately downstream of epoK. In other words, the mutation should be nonpolar. Next, the origin of conjugative transfer, oriT, from RP4 is ligated into pKOS90-55 to create pKOS90-63. This plasmid can be introduced into S17-1 and conjugated into SMP44. The transconjugants are selected on phleomycin plates as previously described. Alternatively, electroporation of the plasmid can be achieved using conditions described above for Myxococcus xanthus.

[0391] Because there are three generalized transducing phages for Myxococcus xanthus, one can transfer DNA from M. xanthus to SMP44. First, the epoK mutation is constructed in M. xanthus by linearizing plasmid pKOS90-55 and electroporating into M. xanthus. Kanamycin resistant colonies are selected and have a gene replacement of epoK. This strain is infected with Mx9, Mx8, Mx4 ts18 hft him phages to make phage lysates. These lysates are then individually infected into SMP44 and phleomycin resistant colonies are selected. Once the strain is constructed, standard fermentation procedures, as described below, are employed to produce epothilones C and D.

[0392] Prepare a fresh plate of Sorangium host cells (dispersed) on S42 medium. S42 medium contains tryptone, 0.5 g/L; MgSO4, 1.5 g/L; HEPES, 12 g/L; agar, 12 g/L, with deionized water. The pH of S42 medium is set to 7.4 with KOH. To prepare S42 medium, after autoclaving at 121° C. for at least 30 minutes, add the following ingredients (per liter): CaCl2, 1 g; K2HPO4, 0.06 g; Fe Citrate, 0.008 g; Glucose, 3.5 g; Ammonium sulfate, 0.5 g; Spent liquid medium, 35 mL; and 200 micrograms/mL of kanamycin is added to prevent contamination. Incubate the culture at 32° C. for 4-7 days, or until orange sorangia appear on the surface.

[0393] To prepare a seed culture for inoculating agar plates/bioreactor, the following protocol is followed. Scrape off a patch of orange Sorangium cells from the agar (about 5 mm2) and transfer to a 250 ml baffle flask with 38 mm silicone foam closures containing 50 ml of Soymeal Medium containing potato starch, 8 g; defatted soybean meal, 2 g; yeast extract, 2 g; Iron (III) sodium salt EDTA, 0.008 g; MgSO4.7H2O, 1 g; CaCl2.2H2O, 1 g; glucose, 2 g; HEPES buffer, 11.5 g. Use deionized water, and adjust pH to 7.4 with 10% KOH. Add 2-3 drops of antifoam B to prevent foaming. Incubate in a coffin shaker for 4-5 days at 30° C. and 250 RPM. The culture should appear an orange color. This seed culture can be subcultured repeatedly for scale-up to inoculate in the desired volume of production medium.

[0394] The same preparation can be used with Medium 1 containing (per liter) CaCl2.2H2O, 1 g; yeast extract, 2 g; Soytone, 2 g; FeEDTA, 0.008 g; Mg SO4.7H2O, 1 g; HEPES, 11.5 g. Adjust pH to 7.4 with 10% KOH, and autoclave at 121° C. for 30 minutes. Add 8 ml of 40% glucose after sterilization. Instead of a baffle flask, use a 250 ml coiled spring flask with a foil cover. Include 2-3 drops of antifoam B, and incubate in a coffin shaker for 7 days at 37° C. and 250 RPM. Subculture the entire 50 mL into 500 mL of fresh medium in a baffled narrow necked Fembach flask with a 38 mm silicone foam closure. Include 0.5 ml of antifoam to the culture. Incubate under the same conditions for 2-3 days. Use at least a 10% inoculum for a bioreactor fermentation.

[0395] To culture on solid media, the following protocol is used. Prepare agar plates containing (per liter of CNS medium) KNO3, 0.5 g; Na2HPO4, 0.25 g; MgSO4.7H2O, 1 g; FeCl2, 0.01 g; HEPES, 2.4 g; Agar, 15 g; and sterile Whatman filter paper. While the agar is not completely solidified, place a sterile disk of filter paper on the surface. When the plate is dry, add just enough of the seed culture to coat the surface evenly (about 1 mL). Spread evenly with a sterile loop or an applicator, and place in a 32° C. incubator for 7 days. Harvest plates.

[0396] For production in a 5 L bioreactor, the following protocol is used. The fermentation can be conducted in a B. Braun Biostat MD-1 5L bioreactor. Prepare 4 L of production medium (same as the soymeal medium for the seed culture without HEPES buffer). Add 2% (volume to volume) XAD-16 absorption resin, unwashed and untreated, e.g. add 1 mL of XAD per 50 mL of production medium. Use 2.5 N H2SO4 for the acid bottle, 10% KOH for the base bottle, and 50% antifoam B for the antifoam bottle. For the sample port, be sure that the tubing that will come into contact with the culture broth has a small opening to allow the XAD to pass through into the vial for collecting daily samples. Stir the mixture completely before autoclaving to evenly distribute the components. Calibrate the pH probe and test dissolved oxygen probe to ensure proper functioning. Use a small antifoam probe, ˜3 inches in length. For the bottles, use tubing that can be sterile welded, but use silicone tubing for the sample port. Make sure all fittings are secure and the tubings are clamped off, not too tightly, with C-clamps. Do not clamp the tubing to the exhaust condenser. Attach 0.2 μm filter disks to any open tubing that is in contact with the air. Use larger ACRO 50 filter disks for larger tubing, such as the exhaust condenser and the air inlet tubing. Prepare a sterile empty bottle for the inoculum. Autoclave at 121° C. with a sterilization time of 90 minutes. Once the reactor has been taken out of the autoclave, connect the tubing to the acid, base, and antifoam bottles through their respective pump heads. Release the clamps to these bottles, making sure the tubing has not been welded shut. Attach the temperature probe to the control unit. Allow the reactor to cool, while sparging with air through the air inlet at a low air flow rate.

[0397] After ensuring the pumps are working and there is no problem with flow rate or clogging, connect the hoses from the water bath to the water jacket and to the exhaust condenser. Make sure the water jacket is nearly full. Set the temperature to 32° C. Connect pH, D.O., and antifoam probes to the main control unit. Test the antifoam probe for proper functioning. Adjust the set point of the culture to 7.4. Set the agitation to 400 RPM. Calibrate the D.O. probe using air and nitrogen gas. Adjust the airflow using the rate at which the fermentation will operate, e.g. 1 LPM (liter per minute). To control the dissolved oxygen level, adjust the parameters under the cascade setting so that agitation will compensate for lower levels of air to maintain a D.O. value of 50%. Set the minimum and maximum agitation to 400 and 1000 RPM respectively, based on the settings of the control unit. Adjust the settings, if necessary.

[0398] Check the seed culture for any contamination before inoculating the fermenter. The Sorangium cellulosum cells are rod shaped like a pill, with 2 large distinct circular vacuoles at opposite ends of the cell. Length is approximately 5 times that of the width of the cell. Use a 10% inoculum (minimum) volume, e.g. 400 mL into 4 L of production medium. Take an initial sample from the vessel and check against the bench pH. If the difference between the fermenter pH and the bench pH is off by ≧0.1 units, do a 1 point recalibration. Adjust the deadband to 0.1. Take daily 25 mL samples noting fermenter pH, bench pH, temperature, D.O., airflow, agitation, acid, base, and antifoam levels. Adjust pH if necessary. Allow the fermenter to run for seven days before harvesting.

[0399] Extraction and analysis of compounds is performed substantially as described above in Example 4. In brief, fermentation culture is extracted twice with ethyl acetate, and the ethyl acetate extract is concentrated to dryness and dissolved/suspended in ˜500 μL of MeCN-H2O (1:1). The sample is loaded onto a 0.5 mL Bakerbond ODS SPE cartridge pre-equilibrated with MeCN—H2O (1:1). The cartridge is washed with 1 mL of the same solvent, followed by 2 mL of MeCN. The MeCN eluent is concentrated to dryness, and the residue is dissolved in 200 μL of MeCN. Samples (50 μL) are analyzed by HPLC/MS on a system comprised of a Beckman System Gold HPLC and PE Sciex API100LC single quadrapole MS-based detector equipped with an atmospheric pressure chemical ionization source. Ring and orifice voltages are set to 75V and 300V, respectively, and a dual range mass scan from m/z 290-330 and 450-550 is used. HPLC conditions: Metachem 5μ ODS-3 Inertsil (4.6×150 mm); 100% H2O for 1 min, then to 100% MeCN over 10 min a 1 mL/min. Epothilone A elutes at 0.2 min under these conditions and gives characteristic ions at m/z 494 (M+H), 476 (M+H−H2O), 318, and 306.

EXAMPLE 8

Epothilone Derivatives as Anti-Cancer Agents

[0400] The novel epothilone derivatives shown below by Formula (1) set forth above are potent anti-cancer agents and can be used for the treatment of patients with various forms of cancer, including but not limited to breast, ovarian, and lung cancers.

[0401] The epothilone structure-activity relationships based on tubulin binding assay are (see Nicolaou et al., 1997, Angew. Chem. Int. Ed. Engl. 36: 2097-2103, incorporated herein by reference) are illustrated by the diagram below. 18

[0402] A) (3S) configuration important; B) 4,4-ethano group not tolerated; C) (6R, 7S) configuration crucial; D) (8S) configuration important, 8,8-dimethyl group not tolerated; E) epoxide not essential for tubulin polymerization activity, but may be important for cytotoxicity; epoxide configuration may be important; R group important; both olefin geometries tolerated; F) (15S) configuration important; G) bulkier group reduces activity; H) oxygen substitution tolerated; I) substitution important; J) heterocycle important.

[0403] Thus, this SAR indicates that modification of the C1-C8 segment of the molecule can have strong effects on activity, whereas the remainder of the molecule is relatively tolerant to change. Variation of substituent stereochemistry with the C1-C8 segment, or removal of the functionality, can lead to significant loss of activity. Epothilone derivative compounds A-H differ from epothilone by modifications in the less sensitive portion of the molecule and so possess good biological activity and offer better pharmacokinetic characteristics, having improved lipophilic and steric profiles.

[0404] These novel derivatives can be prepared by altering the genes involved in the biosynthesis of epothilone optionally followed by chemical modification. The 9-hydroxy-epothilone derivatives prepared by genetic engineering can be used to generate the carbonate derivatives (compound D) by treatment with triphosgene or 1,1′ carbonyldiimidazole in the presence of a base. In a similar manner, the 9,11-dihydroxy-epothilone derivative, upon proper protection of the C-7 hydroxyl group if it is present, yields the carbonate derivatives (compound F). Selective oximation of the 9 oxo-epothilone derivatives with hydroxylamine followed by reduction (Raney nickel in the presence of hydrogen or sodium cyanoborohydride) yield the 9-amino analogs. Reacting these 9-amino derivatives with p-nitrophenyl chloroformate in the presence of base and subsequently reacting with sodium hydride will produce the carbamate derivatives (compound E). Similarly, the carbamate compound G, upon proper protection of the C7 hydroxyl group if it is present, can be prepared form the 9-amino-11 hydroxy-epothilone derivatives.

[0405] Illustrative syntheses are provided below.

Part A. Epothilone D—7,9-cyclic Carbonate

[0406] To a round bottom flask, a solution of 254 mg epothilone D in 5 mL of methylene chloride is added. It is cooled by an ice bath, and 0.3 mL of triethyl amine is then added. To this solution, 104 mg of triphosgene is added. The ice bath is removed, and the mixture is stirred under nitrogen for 5 hours. The solution is diluted with 20 mL of methylene chloride and washed with dilute sodium bicarbonate solution. The organic solution is dried over magnesium sulfate and filtered. Upon evaporation to dryness, the epothilone D-7, 9-cyclic carbonate is isolated.

Part B. Epothilone D-7,9-cyclic Carbamate

(i) 9-amino-epothilone D

[0407] To a rounded bottom flask, a solution of 252 mg 9-oxo-epothilone D in 5 mL of methanol is added. Upon the addition of 0.5 mL 50% hydroxylamine in water and 0.1 mL acetic acid, the mixture is stirred at room temperature overnight. The solvent is then removed under reduced pressure to yield the 9-oxime-epothilone D. To a solution of this 9 oxime compound in 5 mL of tetrahydrofuran (THF) at ice bath is added 0.25 mL 1M solution of cyanoborohydride in THF. After the mixture is allowed to react for 1 hour, the ice bath is removed, and the solution is allowed to warm slowly to room temperature. One mL of acetic acid is added, and the solvent is then removed under reduced pressure. The residue is dissolved in 30 mL of methylene chloride and washed with saturated sodium chloride solution. The organic layer is separated and dried over magnesium sulfate and filtered. Upon evaporation of the solvent yields the 9-amino-epothilone D.

(ii) Epothilone D-7,9-cyclic Carbamate

[0408] To a solution of 250 mg of 9-amino-epothilone D in 5 mL of methylene is added 110 mg of 4-nitrophenyl chloroformate followed by the addition of 1 mL of triethylamine. The solution is stirred at room temperature for 16 hours. It is diluted with 25 mL of methylene chloride. The solution is washed with saturated sodium chloride and the organic layer is separated and dried over magnesium sulfate. After filtration, the solution is evaporated to dryness at reduced pressure. The residue is dissolved in 10 mL of dry THF. Sodium hydride, 40 mg (60% dispersion in mineral oil), is added to the solution in an ice bath. The ice bath is removed, and the mixture is stirred for 16 hours. One-half mL of acetic acid is added, and the solution is evaporated to dryness under reduced pressure. The residue is re-dissolved in 50 mL methylene chloride and washed with saturated sodium chloride solution. The organic layer is dried over magnesium sulfate and the solution is filtered and the organic solvent is evaporated to dryness under reduced pressure. Upon purification on silica gel column, the epothilone D-7,9-carbamate is isolated.

[0409] The invention having now been described by way of written description and examples, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.

Claims

1. An isolated recombinant nucleic acid compound that comprises a nucleotide sequence encoding at least a domain of an epothilone polyketide synthase (PKS) protein and/or encoding a functional region of an epothilone modification enzyme.

2. The nucleic acid of claim 1, wherein said domain is selected from the group consisting of a loading domain, a thioesterase domain, an NRPS, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain, and an ER domain, a methyl transferase domain and a functional oxidase domain.

3. The nucleic acid of claim 2 that comprises the coding sequence of an epoA gene, and/or the coding sequence of an epoB gene, and/or the coding sequence of an epoC gene, and/or the coding sequence of an epoD gene, and/or the coding sequence of an epoE gene, and/or the coding sequence of an epoF gene, and/or the coding sequence of an epoK gene, and/or the coding sequence of an epoL gene.

4. The nucleic acid of claims 3 that further comprises a promoter positioned to transcribe said encoding nucleotide sequence in host cells in which said promoter is operable.

5. The nucleic acid of claim 4, wherein said promoter is a promoter from a Sorangium gene, or from a Myxococcus gene, or from a Streptomyces gene, or from an epothilone PKS gene, or from a pilA gene, or from an actinorhodin PKS gene.

6. The nucleic acid of any of claims 4 that is a recombinant DNA expression vector.

7. Host cells which contain the nucleic acid of claim 4.

8. The cells of claim 7 which are Sorangium cells, or Myxococcus cells, or Pseudomonas cells, or Streptomyces cells.

9. A method to produce a polyketide which method comprises culturing the cells of claim 7 under conditions wherein the encoding nucleotide sequence is expressed to obtain a functional PKS.

10. A recombinant Sorangium cellulosum host cell that contains a mutated gene for an epothilone PKS protein or epothilone modification enzyme, wherein said mutated gene was inserted in whole or in part into genomic DNA of said cell by homologous recombination with a recombinant vector comprising all or a part of an epothilone PKS gene or epothilone modification gene.

11. The recombinant host cell of claim 10 that makes epothilone C or D but not A or B due to a mutation inactivating or deleting an epoK gene, or makes epothilone A or C but not B or D due to a mutation in epoD altering module 4 AT domain specificity, or makes epothilone B or D but not A or C due to a mutation in epoD altering module 4 AT domain specificity, or makes epothilone C but not epothilone A, B or D due to a mutation in epoD altering module 4 AT domain specificity and a mutation in epoK, or makes epothilone D but not epothilone A, B or C due to a mutation in epoD altering module 4 AT domain specificity and a mutation in epoK.

12. Recombinant Streptomyces or Myxococcus host cells that express an epothilone PKS gene or an epothilone modification enzyme gene, optionally comprising one or more of said epothilone PKS or modification enzyme genes integrated into their chromosomal DNA and/or one or more of said epothilone PKS or modification enzyme genes on an extrachromosomal expression vector.

13. The host cells of claim 12 that are S. coelicolor CH999.

14. A method to produce an epothilone or epothilone derivative which comprises culturing the cells of claims 12.

15. A modified fimctional epothilone PKS wherein said modification comprises at least one of: replacement of at least one AT domain with an AT domain of different specificity; inactivation of the NRPS-like module 1 or of the KS2 catalytic domain; inactivation of at least one activity in at least one β-carbonyl modification domain; addition of at least one of KR, DH and ER activity in at least one β-carbonyl modification domain; and replacement of the NRPS module 1 with an NRPS of different specificity.

16. The modified PKS of claim 15 contained in a cell or contained in a cell-free system, wherein said cell or system contains additional enzymes for modification of the product of said epothilone PKS.

17. The modified PKS of claim 16 wherein said modifying enzymes comprise at least one of a methyltransferase, an oxidase or a glycosylation enzyme.

18. A method to prepare an epothilone derivative which method comprises providing substrates including extender units to the modified PKS of claim 15.

19. A modified functional epothilone PKS wherein said modification comprises inactivation of the NRPS of module 1 or the KS2 of module 2 thereof.

20. A method to make an epothilone derivative which method comprises contacting the modified PKS of claim 19 with a module 2 substrate or a module 3 substrate and extender units.

21. Recombinant host cells which comprise the modified PKS of claim 15.

22. The cells of claim 21 that produce an epothilone derivative selected from the group consisting of 16-desmethyl epothilones, 14-methyl epothilones, 11-hydroxyl epothilones, 10-methyl epothilones, 8,9-anhydro epothilones, 9-hydroxyl epothilones, 9-keto epothilones, 8-desmethyl epothilones, and 6-desmethyl epothilones.

23. A compound selected from the group consisting of 16-desmethyl epothilones, 14-methyl epothilones, 11-hydroxyl epothilones, 10-methyl epothilones, 8,9-anhydro epothilones, 9-hydroxyl epothilones, 9-keto epothilones, 8-desmethyl epothilones, and 6-desmethyl epothilones.

24. A recombinant PKS enzyme that comprises one or more domains, modules, or proteins of a non-epothilone PKS and one or more domains, modules, or proteins of an epothilone PKS, and/or contains a loading domain that comprises a KSQ domain.

25. The PKS enzyme of claim 24, wherein said PKS comprises a DEBS loading domain and 5 modules of DEBS and an NRPS of the epothilone PKS, wherein said PKS comprises all of a non-epothilone PKS with an MT domain of the epothilone PKS

26. A compound of the formula:

27. A compound of the formula

28. A recombinant vector selected from the group consisting of pKOS35-70.8A3, pKOS35-70.1A2, pKOS35-70.4, pKOS35-79.85, pKOS039-124R, and pKOS039-126R.

29. The nucleic acid of claim 3 that comprises the coding sequence of an epoA gene.

30. The nucleic acid of claim 29 that comprises bases 1998 to 6263 of SEQ ID NO: 2.

31. The nucleic acid of claim 3 that comprises the coding sequence of an epoB gene.

32. The nucleic acid of claim 31 that comprises bases 6260 and 10,493 of SEQ ID NO: 2.