Borrelidin-producing Polyketide Synthase And Its Use

Abstract

The present invention relates to the biosynthesis of polyketides and derives from the cloning of nucleic acids encoding a polyketide synthase and other associated proteins involved in the synthesis of the polyketide borrelidin. Materials and Methods including enzyme systems, nucleic acids, vectors and cells are provided for the preparation of polyketides including borrelidin and analogues and derivatives thereof. Novel polyketide molecules are also provided.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to materials and methods for the preparation of polyketides. Enzyme systems, nucleic acids, vectors and cells are provided for the preparation of polyketides, and in particular the polyketide macrolide borrelidin.

BACKGROUND TO THE INVENTION

[0002] Polyketides are natural products produced by a wide range of organisms, and particularly by microorganisms. Polyketides have many important pharmaceutical, veterinary and agricultural uses. Polyketides encompass a huge range of chemical structural space, and have a wide range of associated biological activities. Polyketides with use in medical treatments include antibiotics, immunosuppressants, antitumor agents, other chemotherapeutic agents, and other compounds possessing a broad range of therapeutic and biological properties. The Gram-positive bacteria Streptomyces and their allied genera are prodigious producers of polyketides, and the genetics and biochemistry of polyketide biosynthesis in these organisms are relatively well characterised (Hopwood, 1997). The genes for polyketide biosynthesis in Streptomyces are clustered and the exploitation of DNA technology has made it possible to isolate complete biosynthetic gene clusters by screening gene libraries with DNA probes encoding the genes responsible for their biosynthesis. Thus, increasing numbers of gene clusters for polyketide biosynthesis in Streptomyces and other microorganisms have been isolated and sequenced, including, for example, those for the polyether monensin (WO 01/68867), the polyene nystatin (WO 01/59126) and for rapamycin (Schwecke et al., 1995).

[0003] Polyketides are synthesised through the repeated condensation of building blocks that contain a carboxylic acid function. At each stage of the process this results in the formation of a new .beta.-keto function and an .alpha.-side chain branch into the growing chain. The structural diversity of polyketides derives from a number of aspects of their biosynthetic pathway including: the wide variety of starter units that may be utilised in their biosynthesis; the different lengths of polyketide chains that are possible; the various .alpha.-side chains that are introduced either during or after assembly of the polyketide chain; the various .beta.-substitutions that may be introduced during or after assembly of the polyketide chain; the various degrees of processing that the .beta.-keto groups can undergo (keto, hydroxyl, enoyl, and methylene); and the various stereochemistries that are possible at the .alpha.- and .beta.-centres.

[0004] The synthesis of polyketides is catalysed by an enzyme, or by a complex of enzymes, called the polyketide synthase (PKS) in a manner similar to that of fatty acid biosynthesis. Streptomyces and related genera PKSs fall into three main categories: type-I, type-II and type-III. The type-III PKSs are small proteins related to plant chalcone synthases that have been discovered only recently (Moore & Hopke, 2000). Type-III systems have been implicated in the biosynthesis of a small number of secondary metabolites but may be more generally involved in the biosynthesis of soluble pigments (Cortes et al., 2002). The type-II PKSs consist of several monofunctional proteins that act as a multi-polypeptide complex. Simple aromatic polyketides such as actinorhodin are formed by several rounds of chain assembly, which are performed iteratively on one set of type-II PKS enzymes that are encoded for by one set of PKS genes (Hopwood, 1997). Type-I PKSs are multifunctional proteins and are required for the synthesis of more complex polyketides such as erythromycin and rapamycin. As the focus of this patent, type-I PKS organisation and function are described in detail below:

[0005] Type-I PKSs are organised into modules, whereby each module consists of several catalytic `domains` that are required to carry out one round of chain assembly (Staunton & Wilkinson, 1997). In general a modular PKS contains the correct number of modules (loading plus extension modules) to select and condense the correct number of loading and extension units. For example the erythromycin PKS consists of 7 modules (one loading and six extension modules) to select and condense the one starter and six extension units required for the biosynthesis of the erythromycin precursor 6-deoxyerythronolide B. Thus, there exists a one to one relationship between the number of modules present in the PKS and the number of units incorporated. This one to one relationship is described as `co-linearity`.

[0006] The term `extension module` as used herein refers to the set of contiguous domains, from the .beta.-ketoacyl-acyl carrier protein synthase (KS) domain to the next acyl carrier protein (ACP) domain, which accomplishes one cycle of polyketide chain extension. The term `loading module` as used herein refers to any group of contiguous domains that accomplishes the loading of the starter unit onto the PKS and thus renders it available to the KS domain of the first extension module. Besides condensation of the next extender carboxylic acid (or ketide) unit onto the growing polyketide chain, which is performed by the catalytic activity of the essential KS domain, modules of type-I PKSs may contain domains with .beta.-ketoreductase (KR), dehydratase (DH), and enoyl reductase (ER) activities which are responsible for the further processing of the newly formed .beta.-keto groups during chain extension. The acyl transferase (AT) and the ACP domains present in each module are responsible for the choice of extender unit, and the tethering of the growing chain during its passage on the PKS respectively. The AT domains of a modular PKS can also be found as discrete proteins (Cheng et al., 2003). The completed polyketide chain is generally released from PKSs by the action of a terminal thioesterase (TE) domain that is also generally involved in the cyclisation (lactonisation) of the final product. Other chain terminating/cyclising strategies are also employed such as that for the addition of an amino acid residue and macrolactam formation as observed for rapamycin (Schwecke et al., 1995), for macrolactam formation as for rifamycin (August et al., 1998), and for amino acid incorporation followed by reductive elimination as for myxalamid biosynthesis (Silakowski et al., 2001). In summary, there is a single enzymatic domain present for each successive catalytic step that occurs during biosynthesis on the PKS, and they are used in defined sequence that depends upon their location within the protein and the particular function they perform. This mechanism is termed `processive`.

[0007] The modular arrangement of type-I PKSs was first confirmed by mutation of the erythromycin PKS (also known as 6-deoxyerythronolide B synthase, DEBS) through an in-frame deletion of a region of the KR domain of module 5 (Donadio et al., 1991). This led to the production of the erythromycin analogues, 5,6-dideoxy-3-.alpha.-mycarosyl-5-oxoerythronolide B and 5,6-dideoxy-5-oxoerythronolide B, due to the inability of the mutated KR domain to reduce the .beta.-keto group 5 at this stage of processive biosynthesis. Likewise, alteration of the active site residues in the ER domain of module 4 of DEBS2, by genetic engineering of the corresponding PKS-encoding DNA and its introduction into Saccharopolyspora erythraea, led to the production of 6,7-anhydroerythromycin C (Donadio et al., 1993). In addition, the length of the polyketide chain formed by DEBS has been altered through the specific relocation of the TE domain of DEBS3 to the end of DEBS1; the expected triketide lactone product was produced in good yield (Cortes et al., 1995). It should be noted that the changes described involved modification by deletion of sequence, or by sequence specific inactivation, or by the alternative juxtaposition of DNA sequence from within the same PKS cluster (ie. they are considered `homologous changes`). Other such `homologous` changes to the erythromycin PKS are described in WO 93/13663.

[0008] The modular organisation of type-I PKS genes lends itself to the manipulation of these genes to produce altered polyketide structures. Type I PKSs represent an assembly line for polyketide biosynthesis that can be manipulated by changing the number of modules; by changing their specificities towards different carboxylic acid starter units and extender units; by inactivating, mutating, removing, swapping or inserting domains with different activities and specificities; and by altering the chain or ring size through the repositioning of termination or cyclisation domains (Staunton & Wilkinson, 1997).

[0009] WO 98/01546 describes the production of hybrid PKS gene assemblies comprising the incorporation of heterologous DNA. WO 98/01546 describes methods for generating hybrid PKSs in which the substitution of genes encoding heterologous modules, sub-modules or domains for the native genes generates novel polyketides with altered structures. Specifically, for example the AT domains of heterologous DNA from the rapamycin or monensin PKSs can be exchanged for that native to the erythromycin PKS in order to generate novel polyketides with altered alkyl branching. Such an AT domain swap represented the first example of the production of a truly hybrid PKS (Oliynyk et al., 1996). WO 98/01546 also describes in general terms the production of hybrid PKS assemblies comprising a loading module and at least one extension module. It specifically describes the construction of a hybrid PKS gene assembly by grafting the broad-specificity loading module for the avermectin-producing PKS onto the first protein of the erythromycin PKS (DEBS1) in place of the normal loading module (see also Marsden et al., 1998). Additional examples comprising loading module swaps that are substrate specific have also been described (WO 00/00618; U.S. Pat. No. 5,876,991; Kuhstoss et al., 1996). WO 00/01827 describes methods for varying the .beta.-keto processing capability of a PKS module through the ability to swap `reductive loops`, ie. the ability to rapidly and in a combinatorial manner, alter the number and type of ketoreductase, dehydratase and enoyl reductase domains within a module. In addition to changing the level of .beta.-keto group processing, such changes may also lead to changes in stereochemistry of the .alpha.-alkyl and .beta.-hydroxyl groups thus formed by the altered modules.

[0010] Although modular PKSs operate `normally` in a co-linear and processive manner as described above, examples of a deviation from this mode of operation have been described and are discussed below.

[0011] The picromycin PKS gene cluster in Streptomyces venezuelae is responsible for the biosynthesis of both picromycin (a 14-membered, heptaketide macrolide) and methymycin (a 12-membered, hexaketide macrolide) (Xue et al., 1998). The ability of a single PKS to produce two related macrolides, of different ring sizes, derives from the alternative expression of the final PKS gene pikA4 (Xue & Sherman, 2000). When `normal` expression occurs and full-length PikA4 is formed, a sixth extension unit is incorporated and the picromycin aglycone is produced; when alternative expression occurs and an N-terminally truncated form of PikA4 is produced, no sixth extension unit is incorporated and the growing polyketide chain is passed directly to the TE domain which leads to formation of the methymycin aglycone. Thus, a breakdown of co-linearity occurs and a `ring contracted` product is formed. The biochemical basis for this phenomenon has been investigated and shown to be an ACP5 to ACP6 transfer, missing out covalent attachment to the intervening KS6 domain; such a breakdown of co-linearity has been called `skipping` (Beck et al., 2002).

[0012] Skipping has also been observed to occur when an extra extension module from the rapamycin PKS was interpolated into the erythromycin PKS in order to convert the natural heptketide-producing PKS Into an octaketide-producing one (Rowe et al., 2001). The expected octaketide, 16-membered macrolide was produced, but the major product was the normal heptaketide product 6-deoxyerythronolide. This `skipping` of the interpolated module is believed to occur due to the interpolated module acting on some occasions as a `shuttle`, passing the growing chain from the preceding module to the following downstream module without performing a round of chain extension. It was subsequently shown that the ACP domain of the interpolated module is essential in passing the growing polyketide chain from the preceding ACP domain and passing it to the KS domain of the following module during skipping (Thomas et al., 2002), a mechanism similar to that described for methymycin biosynthesis above. It is shown that skipping can occur without the active site nucleophile of the KS domain. A ring-contracted (skipped) nemadectin (an antiparasitic macrolide) has been reported from a mutant of a Streptomyces soil isolate that was modified by chemical mutation (Rudd et al., 1990); the biosynthesis of the natural PKS product was abolished.

[0013] An alternative manner in which modular PKSs deviate from co-linear operation involves the iterative operation of modules. For example, module 4 of the erythromycin PKS appears to operate iteratively, at a low level, to produce a ring expanded 16-membered, octaketide macrolide related to 6-deoxyerythronolide B (Wilkinson et al., 2000). The ability of the erythromycin PKS to perform this operation has been termed `stuttering`. The `stuttering` of the erythromycin PKS is considered an aberrant process, as the products of this stuttering are formed in low yield and the major product of the erythromycin PKS is the normal heptaketide 6-deoxyerythonolide B formed by co-linear operation. Products that appear to be formed by both stuttering and skipping have also been reported as minor components from the epothilone producer Soranglum cellulosum (Hardt et al., 2001). The stigmatellin biosynthetic cluster of Stigmatella aurantiaca encodes for a PKS that comprises ten (one loading and nine extension) modules (Gaitatzis et al., 2002); however, based on results from structural elucidation and the feeding of stable isotope labelled substrates, stigmatellin is formed from eleven modular derived units. Thus, it would appear that one of the stigmatellin PKS modules operates (twice) iteratively.

[0014] Since the priority filing of the present application, the sequence of the PKS responsible for biosynthesis of the macrolide lankacidin by Streptomyces rochei has been described (Mochizuki et al., 2003). This PKS also appears to contain too few modules in comparison to the number of extension cycles required for lankacidin biosynthesis, although the mechanism by which this would occur is not clear.

[0015] Additional structural diversity can be generated through the modification of polyketides by enzymes other than the PKS, either during the process of chain assembly as seen during the biosynthesis of some ansamycins (Floss, 2001), or after the process of chain assembly following release from the PKS. Such non-PKS mediated reactions may include, but are not limited to the following: reduction, oxidation, hydroxylation, acylation, alkylation, amination, decarboxylation, dehydration, double bond isomerisation/migration, cyclisation, ring cleavage, conjugation, glycosylation, reductive elimination and any combination of these. When these reactions occur after chain assembly they are termed the post-PKS or tailoring steps. Such tailoring steps are generally, but not always, essential for endowing the polyketide natural product with biological activity.

[0016] In addition, the structural diversity of polyketides obtainable biosynthetically can be further enhanced through the use of defined heterologous post-PKS tailoring enzymes as well as through the use of those which naturally modify the natural polyketide (Gaisser et al., 2000). WO 01/79520 describes the heterologous modification of polyketide macrolide structures through glycosylation, epoxidation, hydroxylation, and methylation. The ability to generate analogues of the agricultural compound spinosyn through glycosylation with alternative deoxyhexose substituents has been reported (Gaisser et al., 2002).

[0017] Borrelidin 1 (FIG. 1) is an 18-membered macrolide produced by several bacterial strains including, but not limited to, Streptomyces rochei ATCC23956, Streptomyces parvulus Tu113 and Streptomyces parvulus Tu4055. Borrelidin is herein shown to be derived from a trans-cyclopentane-1,2-dicarboxylic acid starter acid, three malonyl-CoA and five methylmalonyl-CoA extender units (see FIG. 2). From the absolute stereochemistry of borrelidin, based on the crystal structure and recently confirmed through total synthesis, the actual starter acid is predicted to be trans-cyclopentane-(1R,2R)-dicarboxylic acid. Borrelidin isolated after the feeding of stable isotope labelled acetate and propionate substrates clearly indicated the expected incorporation of these building blocks; in addition, it has been demonstrated in the present application that feeding of trans-cyclopentane-1,2-dicarboxylic acid was sufficient to re-establish borrelidin biosynthesis in mutants where specific genes believed to be involved in the formation of the starter unit had been disrupted. Borrelidin contains a nitrile group attached to the C12 position, which is shown herein to arise through the action of tailoring enzymes acting upon a methylmalonyl-CoA derived methyl branch present at this position. The gross structure of borrelidin was first elucidated in 1967 (Keller-Scheirlein, 1967), and was subsequently refined by detailed NMR analysis (Kuo et al., 1989). The absolute configuration of borrelidin was confirmed by X-ray crystallography (Anderson et al., 1989). Its co-identity as the antibiotic treponemycin has been verified (Maehr & Evans, 1987).

[0018] A number of groups have reported the synthesis of fragments of the borrelidin structure, and since the priority filing of the present application, two independent total syntheses of borrelidin have been reported (Hanessian et al., 2003; Duffey et al., 2003).

[0019] Borrelidin was first discovered due to its antibacterial activity (Berger et al., 1949), although this antibacterial activity extends only to a limited number of micrococci, and is not found against all common test bacteria. The mode of action in sensitive microorganisms involves selective inhibition of threonyl tRNA synthetase (Paetz & Nass, 1973). Other activities against spirochetes of the genus Treponema (Singh et al., 1985; U.S. Pat. No. 4,759,928), against viruses (Dickinson et al., 1965), uses for the control of animal pests and weeds (DE 3607287) and use as an agricultural fungicide (DE 19835669; U.S. Pat. No. 6,193,964) have been reported. Additionally, since the priority filing of the present application, borrelidin has been reported to have antimalarial activity against drug resistant Plasmodium falciparum strains (Otoguro et al., 2003). Between all of these reports only two reported any synthetically modified derivatives. The first of these describes the benzyl ester and its bis-O-(4-nitrobenzoyl) derivative (Berger et al., 1949). The second of these describes the borrelidin methyl ester, the methyl ester bis O-acetyl derivative, and the methyl ester .DELTA..sub.14-15-dihydro-, .DELTA..sub.14-15,12-13-tetrahydro-, and .DELTA..sub.14-15,12-13-tetrahydro-C12-amino derivatives (Anderton & Rickards, 1965). No biological activity was reported for any of these compounds.

[0020] A recent disclosure of particular interest is the discovery that borrelidin displays anti-angiogenesis activity (Wakabayashi et al., 1997). Angiogenesis is the process of the formation of new blood vessels. Angiogenesis occurs only locally and transiently in adults, being involved in, for example, repair following local trauma and the female reproductive cycle. It has been established as a key component in several pathogenic processes including cancer, rheumatoid arthritis and diabetic retinopathy. Its importance in enabling tumours to grow beyond a diameter of 1-2 cm was established by Folkman (Folkman, 1986), and is provoked by the tumour responding to hypoxia. In its downstream consequences angiogenesis is mostly a host-derived process, thus inhibition of angiogenesis offers significant potential in the treatment of cancers, avoiding the hurdles of other anticancer therapeutic modalities such as the diversity of cancer types and drug resistance (Matter, 2001). It is of additional interest that recent publications have described the functional involvement of tyrosinyl- and tryptophanyl tRNA synthetases in the regulation of angiogenesis (Wakasugi et al., 2002; Otani et al., 2002).

[0021] In the rat aorta matrix culture model of angiogenesis, borrelidin exhibits a potent angiogenesis-inhibiting effect and also causes disruption of formed capillary tubes in a dose dependent manner by inducing apoptosis of the capillary-forming cells (Wakabayashi et al., 1997). Borrelidin inhibited capillary tube formation with an IC.sub.50 value of 0.4 ng/ml (0.8 nM). In the same study, borrelidin was shown to possess anti-proliferative activity towards human umbilical vein endothelial cells (HUVEC) in a cell growth assay; the IC.sub.50 value was measured at 6 ng/ml, which is 15-fold weaker than the anti-angiogenesis activity measured in the same medium. This anti-proliferative activity of borrelidin was shown to be general towards various cell lines. In addition to these data the authors report that borrelidin inhibits tRNA synthetase and protein synthesis in the cultured rat cells; however the IC.sub.50 value for anti-angiogenesis activity (0.4 ng/ml) was 50-fold lower than that reported for Inhibition of protein synthesis (20 ng/ml), indicating different activities of the compound.

[0022] Borrelidin also displays potent inhibition of angiogenesis in vivo using the mouse dorsal air sac model (Funahashi et al., 1999), which examines VEGF-induced angiogenesis and is an excellent model for studying tumour-angiogenesis. Borrelidin was administered at a dose of 1.8 mg/kg by intraperitoneal injection and shown to significantly reduce the increment of vascular volume induced by WiDr cells, and to a higher degree than does TNP-470, which is a synthetic angiogenesis inhibitor in clinical trials. Detailed controls verified that these data are for angiogenesis inhibition and not inhibition of growth of the tumour cells. The authors also showed that borrelidin is effective for the inhibition of the formation of spontaneous lung metastases of B16-BL6 melanoma cells at the same dosage by inhibiting the angiogenic processes involved in their formation.

[0023] JP 9-227,549 and JP 8-173,167 confirm that borrelidin is effective against WiDr cell lines of human colon cancer, and also against PC-3 cell lines of human prostate cancer. JP 9-227,549 describes the production of borrelidin by Streptomyces rochei Mer-N7167 (Ferm P-14670) and its isolation from the resulting fermentation culture. In addition to borrelidin 1, 12-desnitrile-12-carboxyl borrelidin 2 (presumably a biosynthetic intermediate or shunt metabolite), 10-desmethyl borrelidin 3 (presumably a biosynthetic analogue arising from the mis-incorporation of an alternative malonyl-CoA extender unit in module 4 of the borrelidin PKS), 11-epiborrelidin 4 and the C14,C15-cis borrelidin analogue 5 were described (see FIG. 1). Thus, JP 9-227,549 specifies borrelidin and borrelidin analogues wherein a nitrile or carboxyl group is attached the carbon skeleton at C12, and a hydrogen atom or lower alkyl group is attached to the carbon skeleton at C10.

[0024] WO 01/09113 discloses the preparation of borrelidin analogues that have undergone synthetic modification at the carboxylic acid moiety of the cyclopentane ring. The activity of these compounds was examined using endothelial cell proliferation and endothelial capillary formation assays in a similar manner to that described above. In general, modification of the carboxyl moiety improved the selectivity for inhibiting capillary formation: the major reason for this improvement in selectivity is through a decrease in the cell proliferation inhibition activity whereas the capillary formation inhibitory activity was altered to a much lower degree. Specifically, the borrelidin-morpholinoethyl ester showed a 60-fold selectivity index, the borrelidin-amide showed a 37-fold selectivity index, the borrelidin-(2-pyridyl)-ethyl ester showed a 7.5-fold selectivity index and the borrelidin-morpholinoethyl amide showed a 6-fold selectivity index, for the capillary formation inhibitory activity versus cell proliferation with respect to borrelidin. The capillary formation inhibitory activity of these and other borrelidin derivatives was verified using a micro-vessel formation assay. In addition, the authors showed that borrelidin weakly inhibited the propagation of metastatic nodules, after removal of the primary tumour, when using a Lewis lung adenocarcinoma model. However, the borrelidin-(3-picolylamide) derivative was reported to inhibit very considerably the increase of micrometastases in rats after intraperitoneal and also with per os administration at subtoxic doses. Similarly, using the colon 38 spleen liver model, the metastasis-forming ability of mouse colon adenocarcinoma cells transplanted into mouse spleen was considerably decreased after treatment with a subtoxic dose of this borrelidin derivative. These data confirm the earlier reported ability of borrelidin and its derivatives to inhibit the formation of metastases.

[0025] Borrelidin has also been identified as an inhibitor of cyclin-dependant kinase Cdc28/Cln2 of Saccharomyces cerevisiae with an IC.sub.50 value of 12 .mu.g/ml (24 .mu.M) (Tsuchiya et al., 2001). It was shown that borrelidin arrests both haploid and diploid cells in late G.sub.1 phase (at a time point indistinguishable from .alpha.-mating pheromone), and at concentrations that do not affect gross protein biosynthesis. These data were taken to indicate that borrelidin has potential as a lead compound to develop anti-tumour agents.

[0026] Since the priority filing of the present application, two further reports have been published concerning the biological activity of borrelidin. The first of these indicates that the anti-angiogenic effects of borrelidin are mediated through distinct pathways (Kawamura et al., 2003). High concentrations of threonine were found to attenuate the ability of borrelidin to inhibit both capillary tube formation in the rat aorta culture model and HUVEC cells proliferation; however, it did not affect the ability of borrelidin to collapse formed capillary tubes or to induce apoptosis in HUVEC. Borrelidin was also found to activate caspase-3 and caspase-8, and inhibitors of both of these suppressed borrelidin induced apoptosis in HUVEC. The second of these papers used the method of global cellular mRNA profiling to provide insight into the effects of borrelidin on Saccharomyces cerevisiae (Eastwood and Schaus, 2003). This analysis showed the induction of amino acid biosynthetic enzymes in a time-dependent fashion upon treatment with borrelidin, and it was ascertained that the induction of this pathway involves the GCN4 transcription factor.

[0027] In summary, the angiogenesis-inhibitory effect of borrelidin is directed towards the twin tumour-biological effects of proliferation and capillary formation. In addition, borrelidin, and derivatives thereof, have been shown to inhibit the propagation of metastases. Borrelidin also has indications for use in cell cycle modulation. Thus, borrelidin and related compounds are particularly attractive targets for investigation as therapeutic agents for the treatment of tumour tissues, either as single agents or for use as an adjunct to other therapies. In addition, they may be used for treating other diseases in which angiogenesis is implicated in the pathogenic process, including, but not restricted to, the following list: rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy and various ophthalmic disorders.

SUMMARY OF THE INVENTION

[0028] The present invention provides the entire nucleic acid sequence of the biosynthetic gene cluster responsible for governing the synthesis of the polyketide macrolide borrelidin in Streptomyces parvulus Tu4055. Also provided is the use of all or part of the cloned DNA and the nucleic acid sequences thereof in the specific detection of other polyketide biosynthetic gene clusters, in the engineering of mutant strains of Streptomyces parvulus and other suitable host strains for the production of enhanced levels of borrelidin, or for the production of modified or novel polyketides, and of recombinant genes encoding PKS systems for the biosynthesis of modified or novel polyketides.

[0029] The present invention provides an isolated nucleic acid molecule comprising all or part of a borrelidin biosynthetic gene cluster.

[0030] The complete nucleotide sequence of the borrelidin biosynthetic gene cluster from Streptomyces parvulus Tu4055 is shown in SEQ ID No.1. Its organisation is presented in FIG. 3 and comprises genes and open reading frames designated hereinafter as: borA1, borA2, borA3, borA4, borA5, borA6, borB, borC, borD, borE, borF, borG, borH, borI, borJ, borK, borL, borM, borN, borO, orfB1, orfB2, orfB3, orfB4, orfB5, orfB6, orfB7, orfB8, orfB9, orfB10, orfB11, orfB12, orfB13, orfB14, orfB15, orfB16, orfB17, orfB18, orfB19, orfB20, orfB21 and orfB22.

[0031] The proposed functions of the cloned genes are described in FIG. 4 (proposed biosynthesis of the starter unit), 5 (organisation of the borrelidin PKS and biosynthesis of pre-borrelidin) and 6 (introduction of the C12-nitrile moiety) and are described below.

[0032] The present invention thus provides an isolated nucleic acid molecule comprising:

(a) a nucleotide sequence as shown in SEQ ID No.1, or a portion or fragment thereof; or (b) a nucleotide sequence which is the complement of SEQ ID No.1, or a portion or fragment thereof; or (c) a nucleotide sequence which is degenerate with a coding sequence of SEQ ID No.1, or a portion or fragment thereof.

[0033] As used herein the term "fragment" with respect to nucleotide sequences refers to a stretch of nucleic acid residues that are at least 10, preferably at least 20, at least 30, at least 50, at least 75, at least 100, at least 150 or at least 200 nucleotides in length. A preferred portion or fragment of SEQ ID NO:1 is the sequence extending between nucleotide positions 7603 and 59966 of SEQ ID No.1.

[0034] The sequence may encode or be complementary to a sequence encoding a polypeptide of a polyketide biosynthetic gene cluster, or a portion thereof. By "a polypeptide of a polyketide biosynthetic gene cluster" is meant a polypeptide encoded by one or more open reading frames of a polyketide biosynthetic gene cluster, and particularly the borrelidin biosynthetic gene cluster.

[0035] A polyketide biosynthetic gene cluster is a segment of DNA comprising a plurality of genes encoding polypeptides having activity in the biosynthesis of a polyketide or macrolide moiety. This is not restricted to components of the polyketide synthase (PKS) which function inter alia in the synthesis of the polyketide backbone and reductive processing of side groups, but also encompasses polypeptides having ancillary functions in the synthesis of the polyketide. Thus polypeptides of the biosynthetic gene cluster may also act in macrolide ring or polyketide chain modification (e.g. catalysing a reaction in the formation of the C12 nitrile moiety of borrelidin), in the synthesis of a precursor or starter unit for a polyketide or macrolide moiety (e.g. catalysing a reaction In the synthesis of the trans-cyclopentane-1,2-dicarboxylic acid starter unit for the borrelidin PKS, or responsible for the activation of such molecules as the coenzyme-A thioesters of the starter and extender units of the chain), regulatory activity (e.g. regulation of the expression of the genes or proteins involved in polyketide or macrolide synthesis), transporter activity (e.g. in transport of substrates for the polyketide or macrolide moiety into the cell, or of synthesis products such as the polyketide or macrolide molecule out of the cell), and in conferring resistance of the producing cell to the synthesised products (e.g. through specific binding to the synthesised molecule, or as a replacement for other endogenous proteins to which the synthesised molecule may bind within or outside of the cell).

[0036] The gene cluster also includes non-coding regions, such as promoters and other transcriptional regulatory sequences which are operably linked to the coding regions of the gene cluster. The skilled person is well able to identify such elements based upon the information provided herein, and these are within the scope of the present invention.

[0037] Genes and open reading frames encoded within SEQ ID No.1 represent preferred parts or fragments of SEQ ID No.1. Thus an isolated nucleic acid molecule may comprise a sequence that encodes a polypeptide from a borrelidin biosynthetic gene cluster, wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID Nos.2 to 43 and 113.

[0038] In preferred embodiments, the nucleic acid sequence comprises an open reading frame selected from the group of open reading frames of SEQ ID NO: 1 consisting of borA1, borA2, borA3, borA4, borA5, borA6, borB, borC, borD, borE, borF, borG, borH, borI, borJ, borK, borL, borM, borN, borO, orfB1, orfB2, orfB3, orfB4, orfB5, orfB6, orfB7, orfB8, orfB9, orfB10, orfB11, orfB12, orfB13a, orfB13b, orfB14, orfB15, orfB16, orfB17, orfB18, orfB19, orfB20, orfB21 and orfB22, said open reading frames being described by, respectively, bases 16184*-18814, 18875-23590, 23686-34188, 34185*-39047, 39122*-45514, 45514-50742, 7603-8397c, 8397-9194c, 9244-9996c, 9993-11165c, 11162-11980c, 11992-13611c, 13608-15659*c, 50739*-52019, 52113-53477, 53486-54466, 54506-56176, 56181*-57098, 57112-57858, 57939-59966, 2-313 (incomplete), 501*-3107, 3172-3810c, 3935-4924c, 5123-5953, 5961-6518*c, 6564*-7538, 60153-60533*c, 60620-61003, 61188*-61436, 61526-61738, 61767-62285c, 62750-63067c, 62586-62858c, 63155-65071c, 65374-65871, 65942-68305*c, 68290-68910*c, 69681-70436, 70445-71848, 71851-72957, 73037-73942 and 73995-74534c of SEQ ID No.1.

[0039] In the above list, `c` indicates that the gene is encoded by the complementary strand to that shown in SEQ ID NO: 1. Each open reading frame above represents the longest probable open reading frame present. It is sometimes the case that more than one potential start codon can be Identified. One skilled in the art will recognise this and be able to identify alternative possible start codons, Those genes which have more than one possible start codon are indicated with a `*` symbol. Throughout we have indicated what we believe to be the start codon, however, a person of skill in the art will appreciate that it may be possible to generate active protein using an alternative start codon, proteins generated using these alternative start codons are also considered within the scope of the present invention.

[0040] It should be noted that a number of these open reading frames begin with a codon (GTG, CTG or TTG) other than the more normal ATG initiation codon. It is well known that in some bacterial systems such codons, which normally denote valine (GTG) or leucine (CTG, TTG), may be read as initiation codons encoding methionine at the N terminus of the polypeptide chain. In the amino acid sequences (SEQ ID Nos: 2 to 43 and 113) provided herein, such codons are therefore translated as methionine.

[0041] Also provided are nucleic acid molecules comprising portions of the open reading frames identified herein. For example, such a nucleic acid sequence may comprise one or more isolated domains derived from the open reading frames Identified herein. The polypeptides encoded by these isolated portions of the open reading frames may have independent activity, e.g. catalytic activity. In particular, the polypeptides which make up the borrelidin PKS have modular structures in which individual domains have particular catalytic activities as set out above. Thus any of these domains may be expressed alone or in combination, with other polypeptides from the borrelidin PKS described herein or domains thereof, or with polypeptides from the PKS of other polyketides. In particular, any of these domains may be substituted for the equivalent domains either within the borrelidin PKS or in other polyketide synthases and additionally equivalent domains from other PKSs may be substituted for domains within the borrelidin PKS. In this context an equivalent domain includes domains which have the same type of function but differ in for example, their specificity, an example of substitutions contemplated by the present invention include: the substitution of a malonyl-CoA specific AT domain for a methylmalonyl-CoA specific AT domain, or the substitute of a reductive loop containing a KR domain only for one containing KR, DH and ER. In preferred embodiments the expressed domains represent at least one PKS module as described below.

[0042] The term `PKS domain` as used herein refers to a polypeptide sequence, capable of folding independently of the remainder of the PKS, and having a single distinct enzymatic activity or other function in polyketide or macrolide synthesis including, but not restricted to .beta.-ketoacyl-acyl carrier protein synthase (KS), acyl carrier protein (ACP), acyl transferase (AT), .beta.-ketoreductase (KR), dehydratase (DH), enoyl reductase (ER) or terminal thioesterase (TE).

[0043] Accordingly, the invention further provides:

(a) an isolated nucleic acid molecule comprising a sequence that encodes a PKS domain selected from AT0 and ACP0, said domains being described by, respectively, amino acids 322-664 and 694-763 of SEQ ID No.2. In a preferred embodiment, the PKS domain comprises a sequence selected from the group consisting of bases 17147-18175 and 18263-18472 of SEQ ID No.1; (b) an isolated nucleic acid molecule comprising a sequence that encodes a PKS domain selected from KS1, AT1, KR1 and ACP1, said domains being described by, respectively, amino acids 34-459, 557-885, 1136-1379 and 1419-1486 of SEQ ID No.3. In a preferred embodiment, the PKS domain comprises a sequence selected from the group consisting of bases 18974-20251, 20543-21529, 22280-23011 and 23129-23332 of SEQ ID No.1; (c) an isolated nucleic acid molecule comprising a sequence that encodes a PKS domain selected from KS2, AT2, DH2, KR2, ACP2, KS3, AT3, DH3, KR3 and ACP3, said domains being described by, respectively, amino acids 34-459, 559-887, 903-1050, 1354-1597, 1628-1694, 1724-2149, 2245-2576, 2593-2734, 3060-3307 and 3340-3406 of SEQ ID No.4. In a preferred embodiment, the PKS domain comprises a sequence selected from the group consisting of bases 23785-25062, 25360-26346, 26392-26835, 27745-28476, 28567-28767, 28855-30132, 30418-31413, 31462-31887, 32863-33606 and 33703-33903 of SEQ ID No.1; (d) an isolated nucleic acid molecule comprising a sequence that encodes a PKS domain selected from KS4, AT4, KR4 and ACP4, said domains being described by, respectively, amino acids 34-459, 555-886, 1179-1423 and 1459-1525 of SEQ ID No.5. In a preferred embodiment, the PKS domain comprises a sequence selected from the group consisting of bases 34284-35561, 35847-36842, 37719-38453 and 38559-38759 of SEQ ID No.1; (e) an isolated nucleic acid molecule comprising a sequence that encodes a PKS domain selected from KS5, AT5, DH5, ER5, KR5 and ACP5, said domains being described by, respectively, amino acids 34457, 553-888, 905-1046, 1401-1690, 1696-1942 and 1975-2041 of SEQ ID No.6. In a preferred embodiment, the PKS domain comprises a sequence selected from the group consisting of bases 39221-40492, 40778-41785, 41834-42259, 43322-44191, 44207-44947 and 45044-45244 of SEQ ID No.1; (f) an isolated nucleic acid molecule comprising a sequence that encodes a PKS domain selected from KS6, AT6, KR6, ACP6 and TE, said domains being described by, respectively, amino acids 37-457, 555-883, 1101-1335, 1371-1437 and 1461-1708 of SEQ ID No.7. In a preferred embodiment, the PKS domain comprises a sequence selected from the group consisting of bases 45622-46884, 47176-48162, 48814-49518, 49624-49824 and 49894-50637 of SEQ ID No.1.

[0044] In another of its aspects the invention provides an isolated nucleic acid molecule comprising a sequence that encodes a PKS module, said module being selected from the group consisting of amino acids 322-763 of SEQ ID No.2, 34-1486 of SEQ ID No.3, 34-1694 of SEQ ID No.4, 1724-3406 of SEQ ID No.4, 34-1525 of SEQ ID No.5, 34-2041 of SEQ ID No.6 and 37-1437 or 1708 of SEQ ID No.7. In a preferred embodiment, the module comprises a sequence selected from the group consisting of bases 17147-18472, 18974-23332, 23785-28767, 28855-33903, 34284-38759, 39221-45244, 45622-49824 or 50637 of SEQ ID No.1.

[0045] The term `module` as used herein refers to a single polypeptide comprising a plurality of PKS domains each having a single distinct enzymatic activity in polyketide or macrolide synthesis including, but not restricted to .beta.-ketoacyl-acyl carrier protein synthase (KS), acyltransferase (AT), acyl carrier protein (ACP), .beta.-ketoreductase (KR), dehydratase (DH), or enoyl reductase (ER) or terminal thioesterase (TE). An extension module typically comprises a KS, AT and ACP domain (although some modular PKSs may encode their AT domains as independent proteins). An extension module may further comprise one or more domains capable of reducing a beta-keto group to a hydroxyl, enoyl or methylene group (said group of domains are referred to herein as a "reductive loop"). Thus a module comprising a reductive loop typically contains a KR domain, KR and DH domains, or KR, DH and ER domains.

[0046] A PKS may further comprise a TE domain to perform chain termination and/or cyclisation of the final product, or alternatively it may contain another functionality known to perform a similar function such as that for the addition of an amino acid residue and macrolactam formation as observed for rapamycin (Schwecke et al., 1995), for macrolactam formation as for rifamycin (August et al., 1998), and for amino acid incorporation followed by reductive elimination as for myxalamid biosynthesis (Silakowski et al., 2001).

[0047] Also provided is a nucleic acid molecule encoding a polyketide synthase comprising a sequence encoding one or more of the domains or modules described above.

[0048] The sequences provided herein provide means with which to manipulate and/or to enhance polyketide synthesis. Thus there is provided a method of modifying a parent polyketide synthase, comprising expressing a domain from a borrelidin polyketide synthase or a derivative thereof as described herein in a host cell expressing said parent polyketide synthase, such that the domain is incorporated into said parent polyketide synthase. There is further provided a method of modifying a parent polyketide synthase, comprising introducing into a host cell a nucleic acid encoding a domain from a borrelidin polyketide synthase, or a derivative thereof, wherein the host cell contains nucleic acid encoding said parent polyketide synthase, such that, when expressed, the domain is incorporated into said parent polyketide synthase. The borrelidin PKS domain may be inserted in addition to the native domains of the parent PKS, or may replace a native parent domain. Typically the parent PKS will be a Type I PKS.

The present invention further provides methods of modifying a parent borrelidin PKS. A donor domain (e.g. from a Type I PKS) may be expressed in a host cell expressing said parent borrelidin PKS. There is further provided a method of modifying a parent borrelidin polyketide synthase comprising introducing into a host cell a nucleic acid encoding a domain from a donor polyketide synthase, wherein the host cell contains nucleic acid encoding said parent borrelidin polyketide synthase, such that, when expressed, the domain is incorporated into said parent borrelidin polyketide synthase.

[0049] Additionally or alternatively, a domain of the parent PKS may be deleted or otherwise inactivated; e.g. a parent domain may simply be deleted, or be replaced by a domain from a donor PKS, or a domain from a donor PKS may be added to the parent. Where a domain is added or replaced, the donor domain may be derived from the parent synthase, or from a different synthase.

[0050] These methods may be used to enhance the biosynthesis of borrelidin, to produce new borrelidin derivatives or analogues, or other novel polyketide or macrolide structures. The number and nature of modules in the system may be altered to change the number and type of extender units recruited, and to change the various synthase, reductase and dehydratase activities that determine the structure of the polyketide chain. Such changes can be made by altering the order of the modules that comprise the PKS, by the duplication or removal of modules that comprise the PKS, by the introduction of modules from heterologous sources, or by some combination of these various approaches.

[0051] Thus domains or modules of the borrelidin PKS may be deleted, duplicated, or swapped with other domains or modules from the borrelidin PKS, or from PKS systems responsible for synthesis of other polyketides (heterologous PKS systems, particularly Type I PKS systems), which may be from different bacterial strains or species. Alternatively domains or modules from the borrelidin PKS may be introduced into heterologous PKS systems in order to produce novel polyketide or macrolides. Combinatorial modules may also be swapped between the borrelidin polyketide synthase and other polyketide synthases, these combinatorial modules extend between corresponding domains of two natural-type modules, e.g. from the AT of one module to the AT of the next.

[0052] For example, a particular extender module may be swapped for one having specificity for a different extender unit (as described e.g. in WO98/01571 and WO98/01546), or mutated to display specificity or selectivity for a different extender unit e.g. as described below. Additionally or alternatively, introduction, deletion, swapping or mutation of domains or modules, such as the KR, DH and ER domains responsible for the processing of a given .beta.-keto moiety, may be used to alter the level of reductive processing of an extender unit during polyketide synthesis. Such changes may also lead to changes in stereochemistry of the alpha-alkyl and beta-hydroxyl groups thus formed by altered modules. In a preferred embodiment the BorA5 module may be introduced into a parent PKS to provide iterative addition of extender units to a polyketide backbone, e.g. expanding the ring size of a macrolide polyketide relative to that naturally produced by the parent PKS.

[0053] The borrelidin loading module is the first PKS loading module to be identified having specificity for an alicyclic di-carboxylic acid starter unit. Thus this module or a derivative thereof may be used to introduce alicyclic starter units into heterologous polyketide synthases. This need not be restricted to use of trans-cyclopentane-1,2,-dicarboxylic acid normally used as the borrelidin starter unit. The borrelidin loading module is herein shown also to be capable of directing incorporation of other starter units including trans-cyclobutane-1,2-dicarboxylic acid, 2,3-methylsuccinic acid and 2-methylsuccinic acid. The borrelidin starter unit may also be modified in a borrelidin producing cell, or replaced by a heterologous loading module, to introduce alternative starter units into the borrelidin synthetic pathway.

[0054] The position of the loading module of the PKS may be chosen (e.g. by fusing it to a particular location within the PKS) in order to control the ring size of the resultant polyketide/macrolide molecules.

[0055] The AT domains that determine the carboxylic acid-CoA thioester extender units may be deleted, modified or replaced. The ACP domains may also be deleted, modified or replaced. In addition domains that are not normally present in the borrelidin PKS but which are found in other modular PKS and/or mixed PKS/NRPS systems may be inserted. Examples include, but are not limited to: O-methyl transferase domains, C-methyl transferase domains, epimerisation domains, monooxygenase domains and dehydrogenase domains, aminotransferase domains and non-ribosomal peptide synthetase domains.

[0056] Further, the thioesterase domain of the borrelidin PKS may be altered or repositioned (e.g. fused to a chosen location within the PKS) in order to change its specificity and/or in order to release polyketide/macrolide molecules with a chosen ring size. Alternatively, heterologous thioesterase domains may be inserted into the borrelidin PKS to produce molecules with altered ring size relative to the molecule normally produced by the parent PKS, or to produce a free acid.

[0057] In yet another alternative, the amino acid incorporating and macrolactam forming domains from mixed NRPS/PKS systems such as that for rapamycin, or for related systems such as for rifamycin biosynthesis and myxalamid biosynthesis, or modules from NRPS systems (such as those for bleomycin biosynthesis) may be inserted into the PKS to produce novel polyketide related molecules of mixed origin.

[0058] The open reading frames encoding the PKS described herein may also comprise portions encoding non-enzymatically active portions which nevertheless have a functional role as scaffold regions which space and stabilise the enzymatically active domains and/or modules of the PKS at appropriate distances and orientations, and which may have recognition and docking functions that order the domains and modules of the PKS in the correct spatial arrangement. Thus the nucleic acid sequences of the present invention comprise sequences encoding such scaffold regions, either alone or in combination with sequences encoding domains or modules as described above. It will be appreciated that the various manipulations of PKS coding sequences described above may give rise to hybrid PKS genes or systems. Thus the present invention also provides nucleic acids encoding such hybrid PKS systems. The invention therefore provides a nucleic acid construct comprising at least one first nucleic acid portion encoding at least one domain of a borrelidin PKS and a second nucleic acid portion or portions encoding at least one type I PKS domain which is heterologous to said borrelidin PKS. In preferred embodiments the construct comprises a hybrid polyketide synthase gene, said gene encoding at least one domain of a borrelidin PKS and at least one type I PKS domain which is heterologous to said borrelidin PKS. Further preferred embodiments are as described above.

[0059] In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a polypeptide which catalyses a step in the synthesis of a starter unit or substrate for polyketide synthesis, preferably in the synthesis of the trans-cyclopentane-1,2,-dicarboxylic acid moiety used as a starter unit by the borrelidin PKS. The polypeptide may have activity as a dehydrogenase, 3-oxoacyl-ACP-reductase, cyclase, F420 dependent dehydrogenase, or 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase. Preferably the polypeptide comprises the sequence encoded by one of the group of genes consisting of borC, borD, borE, borF, borG, borH, borK, borL, borM and borN, as shown in SEQ ID NO: 8, 9, 10, 12, 13, 14, 17, 18, 19 or 20.

[0060] These genes may be rendered deleted, disrupted, or otherwise inactivated in a borrelidin-producing cell in order to abolish borrelidin production. Cell lines resulting from such changes may be chemically complemented by the addition of exogenous carboxylic acids which may be incorporated in place of the natural starter unit. Thus, new borrelidin related molecules may be synthesised, which are initiated from the exogenously fed carboxylic acid. Such an approach is termed mutasynthesis. The genes responsible for trans-cyclopentane-1,2,-dicarboxylic acid synthesis may be introduced into a heterologous polyketide producer cell to allow that cell to synthesise the alicyclic dicarboxylic acid as a starter unit for its own PKS.

[0061] Thus the present invention further provides a method for the production of borrelidin and borrelidin analogues at improved titres, said method comprising disrupting borG in the host strain, fermenting the resulting cell line and feeding an exogenous carboxylic acid. In various preferred embodiments the exogenous carboxylic acid is trans-cyclopentane-1,2-dicarboxylic acid or the exogenous carboxylic acid is selected from the group consisting of trans-cyclobutane-1,2-dicarboxylic acid, 2,3-dimethyl succinic acid and 2-methylsuccinic acid and/or the method additional comprises deleting, modifying or replacing one or more borrelidin biosynthetic genes, or borrelidin polyketide synthase domains or modules. A person of skill in the art is aware that polyketide synthases may also be expressed in heterologous hosts, therefore the present invention also contemplates a method for the production of higher titres of borrelidin and borrelidin analogues in a heterologous host, said method comprising transforming a host cell with the entire borrelidin gene cluster with the exception of borG or disrupting the borG gene in situ once the gene cluster has been transferred.

[0062] Alternatively, genes responsible for the synthesis of the starter unit may be over-expressed in order to improve the fermentation titres of borrelidin or borrelidin related molecules. Thus the present invention further provides a method for increasing the titre of borrelidin and borrelidin derivatives or borrelidin related molecules and their derivatives, said method comprising upregulating a borrelidin biosynthetic gene involved in production of the starter unit, said gene selected from the group consisting of borC, borD, borE, borF, borH, borK, borL borM and borN, in a preferred embodiment the upregulated gene is borE or borL.

[0063] In another approach the genes responsible for the synthesis of the starter unit may be modified, or replaced by other synthetic genes directing the production of altered carboxylic acids, leading to the production of borrelidin related molecules. These techniques may be complemented by the modification of the loading module of the PKS as described above.

[0064] In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a polypeptide which catalyses a step in the modification of a side chain of a polyketide moiety, for example in the conversion of a methyl group to a nitrile moiety, e.g. at C12 of pre-borrelidin (14). The polypeptide may have activity as a cytochrome P450 oxidase, amino transferase, or NAD/quinone oxidoreductase. Preferably the polypeptide comprises the sequence encoded by one of the group of genes consisting of borI, borJ, and borK as shown in SEQ ID NO: 15, 16 or 17.

[0065] Various of these genes may be deleted/inactivated such that borrelidin-related molecules, or shunt metabolites thereof, accumulate which represent intermediate stages of the process that introduces the nitrile moiety. The addition of heterologous genes to such systems may allow alternative elaboration of any accumulated biosynthetic intermediates or shunt metabolites thereof. Alternatively, the genes may be mutated in order to alter their substrate specificity such that they function on alternative positions of pre-borrelidin molecules in order to provide borrelidin-related molecules. In addition, the genes responsible for formation of the nitrile group may be over-expressed in order to improve the fermentation titres of borrelidin or borrelidin-related molecules.

[0066] Alternatively, one, some or all of these genes may be introduced into cells capable of producing other polyketides to provide for desired side chain processing of that polyketide, e.g. the introduction of a nitrile moiety. This opens up the possibility of specific biosynthetic introduction of nitrile moieties into polyketides, particularly at side chains derived from methylmalonyl-CoA or ethylmalonyl-CoA extender units. Purified enzymes (see below) may also be used to effect the conversion of polyketide side chains to nitrile moieties in vitro.

[0067] In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a polypeptide involved conferring resistance to borrelidin. The polypeptide may have homology to a threonyl tRNA synthase, and preferably has threonyl tRNA synthase activity. Preferably the polypeptide comprises the sequence encoded by the borO gene as shown in SEQ ID NO: 21. A resistance gene such as borO, carried on a suitable vector (see below) may be used as a selective marker. Thus cells transformed with such a vector may be positively selected by culture in the presence of a concentration of borrelidin which inhibits the growth of, or kills, cells lacking such a gene.

[0068] In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a polypeptide involved in regulation of expression of one or more genes of the borrelidin gene cluster. In a preferred embodiment the polypeptide comprises the sequence encoded by the borL gene as shown in SEQ ID NO: 18, or as encoded by orfB8 or orfB12 as shown in SEQ ID NO: 29 or 33. Regulator genes may be engineered to increase the titre of borrelidin and borrelidin derivatives, or borrelidin related molecules and their derivatives produced by fermentation of the resulting cell lines. For example, repressors may be deleted/inactivated, and/or activators may be up-regulated or overexpressed, e.g. by increasing gene copy number or placing the coding sequence under the control of a strong constitutively active or inducible promoter. The borL gene or a portion thereof may also find use as a hybridisation probe to identify similar regulator genes located in or outside other biosynthetic gene clusters.

[0069] In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a sequence encoding a polypeptide having type II thioesterase activity. In a preferred embodiment the polypeptide comprises the sequence encoded by the borB gene as shown in SEQ ID NO: 8. This nucleic acid may be introduced into a host cell to modulate the titre of a polyketide synthesised by that cell. In particular, the titre may be increased by `editing` of the products of unwanted side reactions (e.g. removal of acyl groups formed by inappropriate decarboxylation of extender units attached to KS domains). However in various aspects it may be desirable to remove such an activity from a producer cell, for example to increase the variety of polyketide products produced by that cell, or to facilitate production of an analogue of a naturally produced polyketide which would normally be blocked by such an editing activity.

[0070] The nucleotide sequences of the invention may be portions of the sequence shown in SEQ ID NO: 1, or the complement thereof, or mutants, variants, derivatives or alleles of these sequences. The sequences may differ from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a coding nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the redundancy of the genetic code. Thus, nucleic acid according to the present invention may include a sequence different from the sequence shown in SEQ ID NO: 1 yet encode a polypeptide with the same amino acid sequence. Preferably mutants, variants, derivatives or alleles of the sequences provided encode polypeptides having the same enzymatic activity as those described herein.

[0071] Where the sequence is a coding sequence, the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequences shown in SEQ ID Nos: 2 to 43 and 113. Nucleic acid encoding a polypeptide which is an amino acid sequence mutant, variant, derivative or allele of any of the sequences shown is further provided by the present invention. Such polypeptides are discussed below. Nucleic acid encoding such a polypeptide may show greater than about 60% identity with the coding sequence of SEQ ID NO: 1, greater than about 70% identity, greater than about 80% identity, or greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% Identity therewith. Percentage identity may be calculated using one of the programs such as BLAST or BestFit from within the Genetics Computer Group (GCG) Version 10 software package available from the University of Wisconsin, using default parameters.

[0072] In preferred embodiments, whether coding or non-coding, the nucleotide sequences of the invention are capable of hybridising specifically with at least a portion of the sequence of SEQ ID NO: 1 or the complement thereof.

[0073] For example, hybridizations may be performed, according to the method of Sambrook et al. (Sambrook et al., 1989), using a hybridization solution comprising: 5.times.SSC, 5.times.Denhardt's reagent, 0.5-1.0% SDS, 100 .mu.g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42.degree. C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2.times.SSC and 1% SDS; (2) 15 minutes at room temperature in 2.times.SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37.degree. C. in 1.times.SSC and 1% SDS; (4) 2 hours at 42-65.degree. C. in 1.times.SSC and 1% SDS, changing the solution every 30 minutes.

[0074] One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989):

T.sub.m=81.5.degree. C.+16.6 Log [Na+]+0.41(% G+C)-0.63(% formamide)-600/#bp in duplex

[0075] As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA duplex decreases by 1-1.5.degree. C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42.degree. C. Such hybridisation would be considered substantially specific to the nucleic acid sequence of the present invention.

[0076] The nucleic acids of the present invention preferably comprise at least 15 contiguous nucleotides of SEQ ID NO: 1. They may comprise 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 500 or more contiguous nucleotides of SEQ ID NO: 1.

[0077] The nucleic acids may be used e.g. as primers or probes for the identification of novel genes or other genetic elements, such as transcriptional regulatory sequences, from polyketide or macrolide biosynthetic gene clusters, e.g. sequences encoding enzymes of the PKS, or domains or modules thereof, enzymes involved in the biosynthesis of a starter unit, enzymes modifying side chains of polyketide moieties, transporters, resistance genes and regulatory molecules as described.

[0078] Thus the present invention provides a method of identifying a novel polyketide biosynthetic gene cluster, or a portion thereof, comprising hybridising a sample of target nucleic acid with a nucleic acid of the present invention capable of hybridising specifically to a nucleic acid having the sequence of SEQ ID NO: 1 or a portion thereof. The target nucleic acid may be any suitable nucleic acid, and is preferably bacterial genomic DNA.

[0079] Typically, the method further comprises the step of detecting hybridisation between the sample of nucleic acid and the nucleic acid of the invention. Hybridisation may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include amplification using PCR, RNAase cleavage and allele specific oligonucleotide probing.

[0080] A method may include hybridization of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridization will generally be preceded by denaturation to produce single-stranded DNA. The hybridization may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridization. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridization events and isolated hybridized nucleic acid.

[0081] Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on, as described above.

[0082] An isolated nucleic acid molecule of the invention may be an isolated naturally occurring nucleic acid molecule (i.e. isolated or separated from the components with which it is normally found in nature) such as free or substantially free of nucleic acid flanking the gene in the bacterial genome, except possibly one or more regulatory sequence(s) for expression. Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.

[0083] The present invention further provides a vector comprising a nucleic acid according to the present invention. The vector is preferably an expression vector comprising a nucleic acid encoding a polypeptide of a polyketide biosynthetic gene cluster (preferably a borrelidin biosynthetic gene cluster), or a portion thereof, as described. Suitable vectors comprising nucleic acid for introduction into bacteria or eukaryotic host cells can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral eg "phage", or "phagemid", as appropriate. For further details see, for example, Sambrook et al., 1989. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. Eds, John Wiley & Sons 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference.

[0084] In another of its aspects the present invention provides an isolated polypeptide encoded by a nucleic acid molecule of the invention as described herein. More particularly, there is provided an isolated polypeptide comprising an amino acid sequence as shown in any one or more of SEQ ID Nos.2 to 43 and 113 or a portion thereof. As set out above, these amino acid sequences represent translations of the longest possible open reading frames present in the sequence of SEQ ID NO: 1 and the complement thereof. The first amino acid is always shown as Met, regardless of whether the initiation codon is ATG, GTG, CTG or TTG.

[0085] As used herein the term "polypeptide(s)" includes peptides, polypeptides and proteins, these terms are used interchangeably unless otherwise specified.

[0086] A polypeptide which is an amino acid sequence variant, allele, derivative or mutant of any one of the amino acid sequences shown may exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the polypeptide of any one of the SEQ ID Nos.2 to 43 and 113, or with a portion thereof. Particular amino acid sequence variants may differ from those shown by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, 50-100, 100-150, or more than 150 amino acids. Percentage identity may be calculated using one of the programs such as FASTA or BestFit from within the Genetics Computer Group (GCG) Version 10 software package available from the University of Wisconsin, using default parameters.

[0087] The present invention also includes active portions, fragments, and derivatives of the polypeptides of the invention.

[0088] An "active portion" means a peptide which is less than the full length polypeptide, but which retains at least some of its essential biological activity. For example, isolated domains or modules of the PKS as described above may be regarded as active portions of the PKS

[0089] A "fragment" means a stretch of amino acid residues of at least five, at least six, or at least seven contiguous amino acids, often at least eight or at least nine contiguous amino acids, typically at least 10, at least 13 contiguous amino acids and, most preferably, at least 20, at last 25, at least 30, at least 50, at least 75, at least 100 or more contiguous amino acids. Fragments of the sequence may comprise antigenic determinants or epitopes useful for raising antibodies to a portion of the relevant polypeptide. Thus the polypeptide need not comprise a complete sequence provided in any one of SEQ ID Nos 2 to 43 and 113, but may comprise a portion thereof having the desired activity, e.g. an isolated domain or module, such as those of the PKS described above. It should be noted that the terms part, portion and fragment are used interchangeably in this specification; no particular significance should be ascribed to the specific use of one of these terms in any particular context.

[0090] A "derivative" of a polypeptide of the invention or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one, two, three, five or more amino acids, without fundamentally altering the essential activity of the wild type polypeptide.

[0091] Polypeptides of the invention are provided in isolated form, e.g. isolated from one or more components with which they are normally found associated in nature. They may be isolated from a host in which they are naturally expressed, or may be synthetic or recombinant.

[0092] The present invention also encompasses a method of making a polypeptide (as disclosed), the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid according to the invention). This may conveniently be achieved by growing a host cell in culture, containing an expression vector as described above, under appropriate conditions which cause or allow expression of the polypeptide. Polypeptides may also be expressed in in vitro systems, such as reticulocyte lysate systems.

[0093] The method may include the step of introducing the nucleic acid into a host cell. The introduction, which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, conjugation, electroporation and transfection using bacteriophage. As an alternative, direct Injection of the nucleic acid could be employed. Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

[0094] Preferred host cells include Actinomycetes, preferably Streptomycetes, and in particular those selected from the group consisting of Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseofuscus, Streptomyces cinnamonensis, Micromonospora griseorubida, Streptomyces hygroscopicus, Streptomyces fradiae, Streptomyces longisporoflavus, Streptomyces lasaliensis, Streptomyces tsukubaensis, Streptomyces griseus, Streptomyces venezuelae, Streptomyces antibioticus, Streptomyces lividans, Streptomyces rimosus and Streptomyces albus. Streptomyces rochei ATCC23956, Streptomyces parvulus Tu113 and Streptomyces parvulus Tu4055, more preferably selected from the group consisting of Streptomyces rochei ATCC23956, Streptomyces parvulus Tu113 and Streptomyces parvulus Tu4055.

[0095] A polypeptide, peptide fragment, allele, mutant or variant according to the present invention may be used as an immunogen or otherwise in obtaining specific antibodies, which may be useful in purification and other manipulation of polypeptides and peptides, screening or other applications.

[0096] In another of its aspects the invention provides for the molecules that may be derived from the objects of the invention and for modified compounds formed therefrom and for methods for their production. The molecules derived from the objects of the invention are shown by formula 1 and extends to pharmaceutically acceptable salts thereof, wherein:

##STR00001##

R.sub.1 is a cycloalkyl group of varying size (n=1-2) and substituted as shown below;

##STR00002##

wherein R.sub.1 can also optionally be substituted with one or more halo atoms, or one or more C.sub.1 to C.sub.3 alkyl groups; R.sub.2, R.sub.3, R.sub.6, R.sub.7, R.sub.8, R.sub.9, or R.sub.11 are each independently H, OCH.sub.3, CH.sub.3 or CH.sub.2CH.sub.3; R.sub.4 is CN, CO.sub.2H, CHO, CH.sub.3, CONH.sub.2, CHNH, R.sub.6, R.sub.10 are OH; or analogues differing from the corresponding "natural" compound in the oxidation state of one or more of the ketide units as shown in FIG. 2 (i.e. selection of alternatives from the group: --CO--, --CH(OH)--, .dbd.CH--, and --CH2-), with the proviso that said compounds are not borrelidin (1), 12-desnitrile-12-carboxyl borrelidin (2), 10-desmethyl borrelidin (3), 11-epiborrelidin (4) or C14,C15-cis borrelidin analogue (5) as shown in FIG. 1. In preferred embodiments: [0097] (a). R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3. [0098] (b). R.sub.4 is CH.sub.3 or COOH [0099] (c). R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3 and R.sub.4 is CH.sub.3 or COOH [0100] (d). R.sub.1 is cyclobutane-1'-carboxylate [0101] (e). R.sub.1 is cyclobutane-1'-carboxylate and R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3. [0102] (f). R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3, R.sub.2 and R.sub.11 are H, R.sub.5 and R.sub.10 are OH, R.sub.4 is either CH.sub.3, COOH or CN and R.sub.1 is cyclopentane-1'-carboxylate or. cyclobutane-1'-carboxylate [0103] (g). R.sub.1 is cyclobutane-1'-carboxylate, R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3 and R.sub.4 is CH.sub.3 or COOH. The present invention also provides compounds of formula 2 and pharmaceutically acceptable salts thereof, wherein:

##STR00003##

[0103] R.sub.2, R.sub.3, R.sub.6, R.sub.7, R.sub.8, R.sub.9, or R.sub.11 are each independently H, OCH.sub.3, CH.sub.3 or CH.sub.2CH.sub.3; R.sub.4 is CN, CO.sub.2H, CHO, CH.sub.3, CONH.sub.2, CHNH, R.sub.5, R.sub.10 are OH; or analogues differing from the corresponding "natural" compound in the oxidation state of one or more of the ketide units as shown in FIG. 2 (i.e. selection of alternatives from the group: --CO--, --CH(OH)--, .dbd.CH--, and --CH.sub.2--), and R.sub.12 and R.sub.13 are independently H or a C1-C4 alkyl group which may be optionally substituted with OH, F, Cl, SH) with the proviso that R.sub.12 and R.sub.13 are not simultaneously H. In preferred embodiments: [0104] (a). R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3. [0105] (b). R.sub.4 is CH.sub.3 or COOH [0106] (c). R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3 and R.sub.4 is CH.sub.3 or COOH [0107] (d). R.sub.12 and R.sub.13 are independently CH.sub.3 or H [0108] (e). R.sub.12 and R.sub.13 are independently CH.sub.3 or H and R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3 [0109] (f). R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3, R.sub.2 and R.sub.11 are H, R.sub.5 and R.sub.10 are OH, R.sub.4 is either CH.sub.3, COOH or CN and R.sub.12 and R.sub.13 are independently CH.sub.3 or H [0110] (g). R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3, R.sub.2 and R.sub.11 are H, R.sub.5 and R.sub.10 are OH, R.sub.4 is either CH.sub.3, COOH or CN and R.sub.12 and R.sub.13 are both CH.sub.3 [0111] (h). R.sub.12 and R.sub.13 are independently CH.sub.3 or H, R.sub.7, R.sub.8 and R.sub.9 are all CH.sub.3 and R.sub.4 is CH.sub.3 or COOH.

[0112] The compounds of the present invention may have tRNA synthetase-inhibitory activity (e.g. they may inhibit threonyl-, tyrosinyl-, or tryptophanyl-tRNA synthetase). They may display anti-microbial activity, including activity against intra- or extracellular parasites and organisms such as bacteria, spirochetes (e.g. Treponema), malaria, viruses and fungi. Additionally or alternatively they may have anti-proliferative activity against mammalian cells, and/or anti-angiogenic activity, either as a result of tRNA synthetase inhibition, or through some other mode of action. This may make the compounds of the present invention particularly suitable as anti-cancer agents (e.g. agents for treatment of bowel cancer, prostate cancer or others), and may also provide application in treatment of other proliferative disorders, such as psoriasis, or conditions in which inappropriate vascularisation occurs, such as psoriasis, rheumatoid arthritis, atherosclerosis and diabetic retinopathy.

[0113] The compounds of the present invention may be formulated into pharmaceutically acceptable compositions, e.g. by admixture with a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such compositions also fall within the scope of the present invention.

[0114] Such pharmaceutically acceptable materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0115] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0116] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0117] The invention further provides the compounds and compositions described above for use in a method of medical treatment. Also provided is the use of the compounds of the invention in the preparation of a medicament for the treatment of microbial conditions (including malaria), for the inhibition of angiogenesis, for the treatment of proliferative disorders, or for the treatment of conditions characterised by inappropriate vascularisation, as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0118] FIG. 1 illustrates the structure of borrelidin and some related metabolites isolated from borrelidin producing organisms.

[0119] FIG. 2 illustrates the incorporation patterns for .sup.13C stable isotope labelled extension substrates and the position of the trans-cyclopentane-1,2-dicarboxylic acid starter unit derived carbons.

[0120] FIG. 3 illustrates the organisation of the borrelidin biosynthetic gene cluster. Restriction sites: B, BamHI; Bc, BclI; E, EcoRI; X, XhoI.

[0121] FIG. 4 illustrates a scheme showing the proposed biosynthetic pathway for the trans-cyclopentane-1,2-dicarboxylic acid starter unit.

[0122] FIG. 5 illustrates the organisation of the borrelidin PKS and the biosynthesis of the pre-borrelidin molecule.

[0123] FIG. 6 illustrates the proposed biosynthetic route for the introduction of the nitrile moiety at the C12 position of borrelidin.

[0124] FIG. 7 illustrates the proposed structure of the molecule 6.

[0125] FIG. 8 illustrates the proposed structure of the molecules 7 & 8.

[0126] FIG. 9 illustrates the molecular characterisation of the 4-hydroxyphenylacetic acid catabolic pathway in E. coli W.

[0127] FIG. 10 illustrates the structures of the molecules 18-20

[0128] FIG. 11 illustrates the structures of the molecules 21-26

DETAILED DESCRIPTION OF THE INVENTION

[0129] A cosmid library of S. parvulus Tu4055 genomic DNA was constructed using fragments obtained from a partial digestion with Sau3AI that were cloned into pWE15 and introduced into E. coli cells using the Gigapack.RTM. III Gold Packaging Extract kit (Stratagene). A library of 3000 E. coli transformants was screened for homology using a labelled probe that was generated using the DIG DNA Labelling and Detection Kit (Roche). The probe used was a 1.7 kbp BglII-BamHI fragment obtained from the gene that encodes module 6 of the third subunit of the oleandomycin PKS from Streptomyces antibioticus (Swan et al., 1994).

[0130] Clones that gave a positive response were selected and cosmid DNA isolated. Cosmid DNA was digested with BamHI and fragments less than 3 kbp in size were sub-cloned into pOJ260 (Bierman et al., 1992). The plasmids were then used to transform S. parvulus Tu4055 protoplasts and resulting mutants were screened for the ability to produce borrelidin. Two mutants were identified as borrelidin non-producers, both of which were derived from plasmids that contained fragments of cosBor32A2. These two fragments were of 1.97 and 2.80 kbp in size, and were later identified as adjacent fragments encoding parts of the borrelidin PKS (borA2 & borA3). Using cosBor32A2 as the probe, a second overlapping cosmid, cosBor19B9 was identified from the original library. These two cosmids are sufficient to cover the entire borrelidin biosynthetic gene cluster (see FIG. 3).

[0131] The complete nucleotide sequence of cosBor32A2 and cosBor19B9 was determined by shotgun sequencing of a Sau3AI-derived subclone library for each cosmid, consisting of 1.5-2.0 kbp fragments in pHSG397 (Takeshita et al., 1987). Specific details are provided in example 3. The complete, overlapping nucleotide-coding sequence for cosBor32A2 and cosBor19B9 is presented as SEQ ID No.1. The region encoded by cosmid cosBor32A2 represents the sequence from nucleotide positions 0-40217 bp of SEQ ID No.1. The region encoded by cosmid cosBor19B9 overlaps this region by 4452 nucleotides, and corresponds to the nucleotide positions 35766-74787 bp of SEQ ID No.1. As described in more detail in the following text, we have performed gene inactivation experiments on many of the orfs identified to be encoded within SEQ ID No.1, and this leads us to identify the limits of the cluster. The borrelidin biosynthetic gene cluster is contained between nucleotide positions 7603 to 59966 of SEQ ID No.1 (borB to borO, which includes the borA region). Thus, these combined efforts have led us to the identification and sequencing of the DNA region encompassing the entire borrelidin biosynthetic gene cluster, and to the identification and description of the functional sequences encoded within this region.

PKS Genes

[0132] Encoded between positions 16184-50742 of SEQ ID No.1 are 6 orfs that display very high homology to the genes that encode the PKSs of known macrolide producing organisms. These genes are designated borA1, borA2, borA3, borA4, borA5 and borA6, and encode the borrelidin PKS as was demonstrated above by disruption of a 1.97 kbp region within borA2. The six orfs are arranged in a head-to-tail manner and each is terminated by an in-frame stop codon. The nucleotide sequence and corresponding polypeptide sequence details are shown below in Table 1:

TABLE-US-00001 TABLE 1 Corresponding PKS Nucleotide position polypeptide encoding gene in SEQ ID No. 1 sequence number borA1 16184-18814 SEQ ID No. 2 borA2 18875-23590 SEQ ID No. 3 borA3 23686-34188 SEQ ID No. 4 borA4 34185-39047 SEQ ID No. 5 borA5 39122-45514 SEQ ID No. 6 borA6 45514-50742 SEQ ID No. 7

[0133] The gene borA1 encodes the starter or loading module (SEQ ID No.1, position 16184-18814). The assignment of the start codon is not obvious for this open reading frame. The start codon given here is what we believe to be the true start codon, but there are at least another three possible start codons between the first and the beginning of the AT0 domain sequence and a person of skill in the art will appreciate that it may be possible to generate active protein using one of these alternative start codons. The start codon given here leaves a significant N-terminal tail of 321 amino acids preceding the AT0 domain. For comparison the N-terminal tail preceding the AT0 of the erythromycin loading module is 108 amino acids and that of the avermectin loading module is 28 amino acids. It is therefore possible that one of the other candidate start codons could be correct; the most likely of these are at positions 16298, 16607 and 16901 of SEQ ID No.1. The length of the N-terminal tail suggests it could possibly represent a catalytic activity, although it does not have any significant homology to other sequences in the databases. The nucleotide sequence position and the corresponding amino acid sequence for each of the functional domains within the starter module are identified below in Table 2:

TABLE-US-00002 TABLE 2 Domain in Bases in Amino acids borA1 SEQ ID No. 1 in SEQ ID No. 2 AT0 17147-18175 322-664 ACP0 18263-18472 694-763

[0134] The gene borA2 encodes the first extension module (SEQ ID No.1, position 18875-23590). The nucleotide sequence position and the corresponding amino acid sequence for each of the functional domains within the first extension module are identified below in Table 3:

TABLE-US-00003 TABLE 3 Domain in Bases in Amino acids borA2 SEQ ID No. 1 in SEQ ID No. 3 KS1 18974-20251 34-459 AT1 20543-21529 557-885 KR1 22280-23011 1136-1379 ACP1 23129-23332 1419-1486

[0135] The gene borA3 encodes the second and third extension modules (SEQ ID No.1, position 23686-34188). The nucleotide sequence position and the corresponding amino acid sequence for each of the functional domains within the second and third extension modules are identified below in Table 4:

TABLE-US-00004 TABLE 4 Domain in Bases in Amino acids borA3 SEQ ID No. 1 in SEQ ID No. 4 KS2 23785-25062 34-459 AT2 25360-26346 559-887 DH2 26392-26835 903-1050 KR2 27745-28476 1354-1597 ACP2 28567-28767 1628-1694 KS3 28855-30132 1724-2149 AT3 30418-31413 2245-2576 DH3 31462-31887 2593-2734 KR3 32863-33606 3060-3307 ACP3 33703-33903 3340-3406

[0136] The gene borA4 encodes the fourth extension module (SEQ ID No.1, position 34185-39047). The nucleotide sequence position and the corresponding amino acid sequence for each of the functional domains within the fourth extension module are identified below in Table 5:

TABLE-US-00005 TABLE 5 Domain in Bases in Amino acids borA4 SEQ ID No. 1 in SEQ ID No. 5 KS4 34284-35561 34-459 AT4 35847-36842 555-886 KR4 37719-38453 1179-1423 ACP4 38559-38759 1459-1525

[0137] The gene borA5 encodes the fifth extension module (SEQ ID No.1, position 39122-45514). The nucleotide sequence position and the corresponding amino acid sequence for each of the functional domains within the fifth extension module are identified below in Table 6:

TABLE-US-00006 TABLE 6 Domain in Bases in Amino acids borA5 SEQ ID No. 1 in SEQ ID No. 6 KS5 39221-40492 34-457 AT5 40778-41785 553-888 DH5 41834-42259 905-1046 ER5 43322-44191 1401-1690 KR5 44207-44947 1696-1942 ACP5 45044-45244 1975-2041

[0138] The gene borA6 encodes the sixth extension module and the chain terminating thioesterase (SEQ ID No.1, position 45514-50742). The nucleotide sequence position and the corresponding amino acid sequence for each of the functional domains within the sixth extension module are identified below in Table 7:

TABLE-US-00007 TABLE 7 Domain in Bases in Amino acids borA6 SEQ ID No. 1 in SEQ ID No. 7 KS6 45622-46884 37-457 AT6 47176-48162 555-883 KR6 48814-49518 1101-1335 ACP6 49624-49824 1371-1437 TE 49894-50637 1461-1708

[0139] The identification of functional domains and their boundaries as described in the aforementioned are determined based on the similarities to the conserved amino acid sequences of other modular PKSs such as those for the rapamycin (Schwecke et al., 1995; Aparicio et al., 1996) and erythromycin (Cortes et al., 1990) biosynthesis. The limits of the catalytic domains are established on the basis of homology to other PKS clusters and the chosen point at which a domain starts or finishes is not absolutely defined, but selected based on the aforementioned considerations. In the case of the .beta.-keto processing domains it is least obvious, as there is typically a large region not assigned to a functional domain that precedes the KR domain. This region may be structurally important, or required for stability of the PKS dimer. An unusual characteristic of the borrelidin PKS is that all of the individual enzymatic domains appear to be catalytically competent based on their oligonucleotide/amino acid sequence, and are all necessary in order to provide the .beta.-keto processing required to produce the functional groups observed in borrelidin. This is rather unusual as the majority of modular PKS sequences so far reported contain one or more inactive domains, an exception being for example the spinosyn PKS (Waldron et al., 2001; U.S. Pat. No. 6,274,50).

[0140] One skilled in the art is familiar with the degeneracy of the genetic code, therefore, the skilled artisan can modify the specific DNA sequences provided by this disclosure to provide proteins having the same or altered or improved characteristics compared to those polypeptides specifically provided herein. One skilled in the art can also modify the DNA sequences to express an identical polypeptide to those provided, albeit expressed at higher levels. Furthermore, one skilled in the art is familiar with means to prepare synthetically, either partially or in whole, DNA sequences which would be useful in preparing recombinant DNA vectors or coding sequences which are encompassed by the current invention. Additionally, recombinant means for modifying the DNA sequences provided may include for example site-directed deletion or site-directed mutagenesis. These techniques are well known to those skilled in the art and need no further explanation here. Consequently, as used herein, DNA which is isolated from natural sources, prepared synthetically or semi-synthetically, or which is modified by recombinant DNA methods, is within the scope of the present invention.

[0141] Likewise, those skilled in the art will recognize that the polypeptides of the invention may be expressed recombinantly. Alternatively, those polypeptides may be synthesised either in whole or in part, by conventional known non-recombinant techniques; for example, solid phase synthesis. Thus, the present invention should not be construed as necessarily limited to any specific vector constructions or means for production of the specific biosynthetic cluster molecules including the polyketide synthase molecules exemplified.

[0142] The loading module of the borrelidin PKS exists as a discrete protein. This is rather unusual as the majority of loading modules are found on the same protein as the first extension module. Exceptions to this include, for example, the nystatin (Brautaset et al., 2000) and amphotericin (Caffrey et al., 2001) PKSs. The loading module, which consists of an AT-ACP didomain, is similar to the broad specificity loading module of the avermectin PKS, which accept a number of alternative starter acids, and are of use in generating libraries of novel polyketides (Marsden et al., 1998; Pacey et al., 1998). The AT domain of the borrelidin PKS loading module diverges from the vast majority of AT domains as the active site serine residue is replaced with a cysteine such that the active site motif is GXCXG (specifically GHCYG). In most available type-I PKS AT domain sequences, the conserved active site motif is GXSXG; the same motif is observed in lipases, fatty acid synthases and most thioesterases. The nucleophilic serine is substituted by cysteine in two NRPS thioesterase domains, specifically the synthetases responsible for the production of mycobactin and pyochelin (Shaw-Reid et al., 1999). A GXCXG motif is also observed in a thioesterase-like domain of ORF1 in the bialaphos cluster (Raibaud et al., 1991). It has been suggested that since it is not possible to move between the two types of serine codons by a single base change, active sites containing an essential serine residue may lie on two lines of descent from an ancient ancestral enzyme that had a cysteine instead of a serine in its active site (Brenner, 1988). The presence of enzymes containing cysteine in the active site may support this view. It may alternatively be the case that cysteine arises in these active sites because it is possible to move from one type of serine codon to the other via a cysteine which would remain active.

[0143] The AT domains of PKSs select a particular carboxylic acid unit as substrate. This selectivity has been shown to correlate with certain motif signatures within the AT domain (Reeves et al., 2001; WO 02/14482). The borrelidin loading module AT domain motif differs from any described so far, which is not surprising as this AT domain is the first to be sequenced that selects an alicyclic dicarboxylic acid. The AT domains for the borrelidin PKS extension modules display the expected active site motif GXSXG, and also each contain the expected motifs for the selection of malonyl-CoA or methylmalonyl-CoA (Reeves et al., 2001; WO 02/14482). The malonyl-CoA selective AT domains (AT1, AT2 and AT6) show very high similarity to one another, both at the protein and at the DNA level. The same is true for the methylmalonyl-CoA selective AT domains (AT3, AT4 and AT5); two of these AT domains (AT3 and AT4) have identical amino acid sequences throughout the conserved region. The high similarity of AT5 to AT3 and AT4 is evidence that the extender unit selected in module 5 is methylmalonyl-CoA, and that the borrelidin C12-methyl group thus incorporated is subsequently modified to a nitrile function after incorporation into the PKS.

[0144] To demonstrate that we can alter the PKS derived structure of borrelidin, the AT domain of module 4 (the AT domain encoded by borA4) is replaced by the AT domain of module 2 of the rapamycin PKS (rapAT2) using a replacement strategy (see example 6). This gives strain S. parvulus Tu4055/467. Upon fermentation and LCMS analysis of culture extracts of this mutant, it can be determined that some borrelidin is produced and a new, more polar compound is also observed with a m/z value 14 units lower than borrelidin. This is consistent with incorporation of a malonate rather that a methylmalonate extender unit by module 4 of the PKS to produce 10-desmethyl borrelidin 3.

[0145] In addition to production by domain swapping methods, 3 is also generated by introducing specific mutations into the module 4 AT domain selectivity motif (Reeves et al., 2001; WO 02/14482) (see example 7). Such a change affects the selectivity of the AT domain such that it selects a substrate molecule of malonyl-CoA preferentially over methylmalonyl-CoA. Thus, the amino acid motif YASH at positions 739 to 742 of SEQ ID No.5 is mutated to HAFH to give strain S. parvulus Tu4055/472. Upon fermentation and LCMS analysis of culture extracts of this mutant it is determined that borrelidin is produced in addition to a new, more polar compound with a m/z value 14 units lower than borrelidin. This new compound is identical to that described above and thus is consistent with incorporation of a malonate rather that a methylmalonate extender unit by module 4 of the PKS to produce 3.

[0146] These results clearly indicate that the borrelidin PKS is amenable to genetic manipulation and to the exchange of native sequence for that of a heterologous strain. It is clear to one skilled in the art that the biosynthetic engineering, by the methods described above, of the borrelidin PKS will lead to the production of novel borrelidin-like molecules.

[0147] The borrelidin loading module is of interest due to the unique structure of its cognate substrate. To examine its potential use in other systems, the loading module native to the erythromycin PKS is replaced with the borrelidin loading module in Saccharopolyspora erythraea; this experiment is analogous to those done previously with the avermectin loading module (WO 98/01546; Marsden et al., 1998). We anticipate that the new strain is capable of producing novel erythromycin like molecules in which the C13-ethyl group is replaced with an exogenously supplied racemic trans-cyclopentane-1,2-dicarboxylic acid moiety. The methodology used to perform this experiment is similar to that described in WO 98/01546, but the transformation is performed using a mutant Saccharopolyspora erythraea DM (Gaisser et al., 2000) which accumulates the aglycone product erythronolide B rather than the fully processed macrolide, as well as using S. erythraea WT. This experiment is described in example 8.

[0148] It is not evident from SEQ ID No.1, which of four candidate start codons is correct for borA1. The four most obvious candidate start codons are at nucleotides 16184, 16298, 16607 and 16901 of SEQ ID No.1. The earliest of these possible start codons was used in giving the amino acid sequence for SEQ ID No.2. A pile-up of this loading module with the erythromycin and avermectin loading modules indicates that the AT0 domain starts at position 321 of SEQ ID No.2, and that there is a long N-terminal tail. No significant homology is found for the first 298 amino acids of borA1. The borrelidin loading module is encoded by a discrete orf, and in order to retain this architecture the splice site chosen for joining the borrelidin PKS loading module sequence to the erythromycin PKS loading module sequence is at the beginning of the homologous region of the KS1 domain of borA2, at amino acids 42-44 of SEQ ID No.3. This approach maintains the putative docking regions at the end of BorA1 and start of BorA2 that are believed to be essential for the production of a functional PKS assembly. To maintain the continuity of this experiment this loading module is fused to the equivalent point at the beginning of the KS1 domain of eryA1. The resulting mutants S. erythraea DM/CJM400-403 are fermented and analysed by negative ion LCMS using standard protocols. This analysis clearly indicates the presence of a new compound 6 with m/z=485.3 as expected (FIG. 7). It is clear to one skilled in the art that the products of these experiments could be biotransformed using an appropriate strain such as S. erythraea JC2 (Rowe et al., 1998) to provide novel, biologically active erythromycin analogues. It is additionally clear to one skilled in the art that the borrelidin loading module has utility for the biosynthetic engineering of other PKSs (i.e. not the borrelidin PKS) to produce further novel polyketides bearing a trans-cyclopentane-1,2-dicarboxylic acid moiety. It is also clear that the diversity of products arising from hybrid PKSs derived from the borrelidin loading module may be further enhanced through the exogenous feeding of carboxylic acids other than the cognate substrate.

[0149] The most striking feature of the borrelidin PKS is the clear divergence from the normal co-linear, processive mode of operation for type-I modular PKSs. Borrelidin is a nonaketide (expected: one loading plus eight extension steps), but only seven modules (one loading and six extension modules) are present in the cluster. Analysis of the PKS domains with respect to the chemical structure of borrelidin correlates with the fifth extension module (BorA5) being used iteratively for three rounds of chain elongation as shown in FIG. 5. Thus, the fifth, sixth and seventh rounds of chain elongation occur on BorA5 with the incorporation of three methylmalonyl-CoA extension units, and with full reductive processing of the .beta.-keto groups to methylene moieties. As described supra, the divergence from co-linear operation for modular PKSs is unusual and limited to a few examples. The present example is interesting as it occurs on a module that reduces the .beta.-keto group fully to a methylene moiety and which is followed by an inter-rather than intra-protein transfer of the growing chain. This is also the case for the two known examples of erroneous iterative use of type-I modules by the erythromycin (Wilkinson et al., 2000) and epothilone (Hardt et al., 2001) PKSs. It is noteworthy that this full reduction makes these modules functionally equivalent to fatty acid synthase (FAS). The type-I PKS modules that can operate iteratively may have retained FAS like activity.

[0150] Although it appears that BorA5 is used iteratively (three times), two other possible scenarios may explain borrelidin biosynthesis given the genes present in the borrelidin biosynthetic cluster. Firstly, two modules may be `missing` from the cluster, but could be present at some other location in the genome. However, in the majority of cases investigated, the genes required for biosynthesis of secondary metabolites in actinomycetes are clustered in a single locus. The second possibility is that three separate BorA5 dimers assemble, and that each catalyses a round of chain elongation; thus the process would be processive. However, this scenario requires that three times the amount of BorA5 is produced with respect to the other PKS proteins, but the organisation of the borrelidin gene cluster does not indicate that the regulation of borA5 differs from that of any of the other PKS genes. In addition, this scenario does not fit with the common thinking as to the roles of inter-protein docking domains, which suggests that there is a specific recognition between the N- and C-terminal ends of the proteins of the biosynthetic complex that need to interact, enabling specific binding between modules encoded on different proteins (Ranganathan et al., 1999; Wu et al., 2001; Broadhurst et al., 2003).

[0151] To address the issues described above, the two proteins encoded by borA4 and borA5 were fused after manipulation at the genetic level to provide strain S. parvulus Tu4055/borA4A5 (see example 9), and separately the two proteins encoded by borA5 and borA6 were fused in an analogous manner to provide strain S. parvulus Tu4055/borA5-A6 (see example 10). Additionally, a double mutant was generated in which the above described fusions were combined to generate a strain in which borA4, borA5 and borA6 were fused to generate strain S. parvulus Tu4055/borA4-A5-A6 (see example 11). Therefore, the new, fused, bi- and tri-modular genes make it impossible to assemble three separate molecules of BorA5, or for another protein(s) encoded by a gene(s) remote from the borrelidin cluster to act in tandem with BorA5. Upon fermentation of strains S. parvulus Tu4055/borA4-A5, /borA5-A6, and /borA4-A5-A6 followed by extraction and analysis, the production of borrelidin was verified at a reduced but significant level (21.+-.4%, 27.+-.4% and 18.+-.5% respectively) when compared to the WT strain. Thus, the production of borrelidin by these mutants indicates that module 5 of the fused BorA4-A5 or BorA5-A6 operates in an iterative manner. Since the priority filing of this application, these limited data have been published (Olano et al., 2003).

[0152] The ability of BorA5 to operate iteratively has great potential for the engineering of heterologous PKSs to provide macrolactones with expanded ring sizes. To examine this possibility BorA5 is swapped into the erythromycin PKS in place of module 4 of DEBS2. This is done by replacement of the appropriate gene fragment in both the erythromycin producer S. erythraea WT and S. erythraea DM. This experiment is chosen as both modules recruit methylmalonyl-CoA extender units and process the .beta.-keto functions formed through to methylene groups. In addition, the stereochemistry of the resulting methyl group in the polyketide chain is the same in both cases. Of most significance is the fact that module 4 of DEBS2 is known to perform erroneous iterative rounds of chain elongation (Wilkinson et al., 2000), indicating that such a process can indeed occur at this location within the PKS and give rise to products that can be fully processed by DEBS3, making it an attractive target to introduce specific iterative use of a heterologous module to make 16- and 18-membered macrolides.

[0153] Briefly, the region of DNA encoding borA5 is swapped for that encoded by module 4 of eryA2, which encodes the C-terminal portion of DEBS2 of the erythromycin PKS (see example 12). The resulting mutant S. erythraea DM/421 is grown and extracted as for the production of metabolites by S. erythraea strains (Wilkinson et al., 2000) and then analysed by LCMS. Two new significant compounds, which are less polar than erythronolide B, are observed. These have an m/z of 435.5 (7, [MNa.sup.+]) and 477.5 (8, [MNa.sup.+]) respectively, which is consistent with the production of two new ring expanded erythronolide B analogues (FIG. 8). Compound 7 with m/z=435.5 is consistent with the presence of the 16-membered ring-expanded erythronolide B related macrolide reported previously as a minor component of S. erythraea WT fermentations (Wilkinson et al., 2000). It is clear to one skilled in the art that such new products can be converted to antibacterial molecules by biotransformation with an appropriate organism. It is also clear to one skilled in the art, that the inclusion of such a module into other positions of the erythromycin PKS, or into other PKSs, may allow the production of novel, ring expanded polyketides in a similar manner. In addition, it is possible to perform this experiment by swapping only the region of the DEBS module 4 from the start of the conserved region of the KS4 to the end of the ACP4 domain; this arrangement retains the C- and N-terminal regions at the end of DEBS2 and DEBS3 respectively, to ensure the mutual recognition and docking of these proteins.

Non-PKS Genes

[0154] Both upstream and downstream of the PKS encoding genes are other orfs involved in the biosynthesis of borrelidin. An orf is designated as consisting of at least 100 contiguous nucleotides, that begins with an appropriate start codon and finishes with an appropriate stop codon, and which has an appropriate codon bias for protein-coding regions of an organism whose DNA is rich in the nucleotides guanine and cytosine. In the DNA sequence both upstream and downstream of the borrelidin PKS genes (borA1-borA6) there are a number of orfs that could be identified by comparison to other sequences in the NCBI database (see FIG. 3). The nucleotide sequence details of these orfs are given below in Table 8:

TABLE-US-00008 TABLE 8 Corresponding polypeptide Gene Bases In SEQ ID No. 1 sequence number borB 7603-8397c SEQ ID No. 8 borC 8397-9194c SEQ ID No. 9 borD 9244-9996c SEQ ID No. 10 borE 9993-11165c SEQ ID No. 11 borF 11162-11980c SEQ ID No. 12 borG 11992-13611c SEQ ID No. 13 borH 13608-15599c * SEQ ID No. 14 borI 50739 *-52019 SEQ ID No. 15 borJ 52113-53477 SEQ ID No. 16 borK 53486-54466 SEQ ID No. 17 borL 54506-56176 SEQ ID No. 18 borM 56181 *-57098 SEQ ID No. 19 borN 57112-57858 SEQ ID No. 20 borO 57939-59966 SEQ ID No. 21 orfB1 2-313 SEQ ID No. 22 orfB2 501 *-3107 SEQ ID No. 23 orfB3 3172-3810c SEQ ID No. 24 orfB4 3935-4924c SEQ ID No. 25 orfB5 5123-5953 SEQ ID No. 26 orfB6 5961-6518 *c SEQ ID No. 27 orfB7 6564 *-7538 SEQ ID No. 28 orfB8 60153-60533c SEQ ID No. 29 orfB9 60620-61003 SEQ ID No. 30 orfB10 61188 *-61436 SEQ ID No. 31 orfB11 61526-61738 SEQ ID No. 32 orfB12 61767-62285c SEQ ID No. 33 orfB13a 62750-63067c SEQ ID No. 34 orfB13b 62586-62858c SEQ ID No. 113 orfB14 63155-65071c SEQ ID No. 35 orfB15 65374-65871 SEQ ID No. 36 orfB16 65942-68305c * SEQ ID No. 37 orfB17 68290-68910c * SEQ ID No. 38 orfB18 69681-70436 SEQ ID No. 39 orfB19 70445-71848 SEQ ID No. 40 orfB20 71851-72957 SEQ ID No. 41 orfB21 73037-73942 SEQ ID No. 42 orfB22 73995-74534c SEQ ID No. 43 [Note 1: c indicates that the gene is encoded by the complement DNA strand; Note 2: for each open reading frame given above, the longest probable open reading frame is described. It is sometimes the case that more than one potential candidate start codon can been identified. One skilled in the art will recognise this and be able to identify alternative possible start codons. We have indicated those genes which have more than one possible start codon with a `*` symbol. Throughout we have indicated what we believe to be the start codon, however, a person of skill in the art will appreciate that it may be possible to generate active protein using an alternative start codon, proteins generated using these alternative start codons are also considered within the scope of the present invention. Note 3 the SEQ ID NO: for orfB13b was originally designated SEQ ID NO: 34 but for clarity a separate sequence and SEQ ID NO has been assigned.]

[0155] Potential functions of the predicted polypeptides (SEQ ID N0. 7 to 43) were obtained from the NCBI database using a BLAST search. The best matches obtained from these searches are described below in Table 9.

TABLE-US-00009 TABLE 9 Accession Proposed Gene Significant protein match Score GenBank function orfB1 hypothetical protein, no full unknown length hits, high GC codon preference orfB2 SCM2.07, hypothetical 998 NP_625154 unknown protein (S. coelicolor) orfB3 SCF76.07, hypothetical 359 NP_624786 unknown protein, (S. coelicolor) orfB4 SCF76.06, araC family 412 NP_624785 unknown transcriptional regulator (S. coelicolor) orfB5 SCF76.05c, non-heme 495 NP_624784 non-heme chloroperoxidase (S. coelicolor) chloroperoxidase orfB6 SCF76.09, hypothetical 159 NP_624788 unknown protein (S. coelicolor) orfB7 SCF76.08c, hypothetical 473 NP_624787 unknown protein (S. coelicolor) borB PteH, polyene macrolide 244 BAB69315 type II thioesterase thioesterase (S. avermitilis) borC XF1726, 2,5-dichloro-2,5- 160 NP_299015 dehydrogenase cyclohexadiene-1,4,-diol dehydrogenase (Xylella fastidiosa strain 9a5c)e borD FabG, 3-oxoacyl-ACP 124 AAK83686 3-oxoacyl-ACP reductase precursor, reductase (Plasmodium falciparum) borE FN1586, O-succinylbenzoyl- 88 NP_602402 cyclase (member of CoA synthase, enolase superfamily) (Fusobacterium nucleatum subsp. nucleatum ATCC 25586) borF putative lysophospholipase 57 NP_565066 unknown homologue, (Arabidopsis thaliana) borG MTH1444, acetolactate 120 NP_276558 Unknown synthase, large subunit, (Methanothermobacter thermautotrophicus) borH PA3592, conserved 116 NP_252282 unknown hypothetical protein, (Pseudomonas aeruginosa) borI TylH1, cytochrome P450, 285 AAD12167 cytochrome P450 (Streptomyces fradiae) oxidase borJ BioA, DAPA 346 BAB39453 amino transferase aminotransferase, (Kurthia sp. 538-KA26) borK Adh1, alcohol 191 NP_213938 NAD/quinone dehydrogenase, (Aquifex oxidoreductase aeolicus) borL putative auxin-regulated 92 NP_176159 unknown protein GH3, (Arabidopsis thaliana) borM SCL6.10, hypothetical protein 108 CAB76875 F420 dependent similar to putative F420- dehydrogenase dependent dehydrogenase (S. coelicolor), borN SC1C2.27, hypothetical 215 NP_629680 2-hydroxyhepta-2,4- protein, 2-hydroxyhepta-2,4- diene-1,7-dioate diene-1,7-dioate isomerase isomerase superfamily (S. coelicolor) borO ThrS, threonyl-tRNA 627 NP_301410 threonyl-tRNA synthetase (Mycobacterium synthetase, self leprae) resistance gene orfB8 conserved hypothetical 37 NP_617908 possible regulator protein (Methanosarcina acetivorans str. C2A). (Pfam pulls out weak MarR family) orfB9 putative anti-sigma factor 113 NP_631789 anti-sigma factor antagonist (Streptomyces antagonist coelicolor) orfB10 conserved hypothetical 95 NP_631790 unknown protein (S. coelicolor) orfB11 hypothetical protein, no full unknown length hits, high GC codon preference orfB12 putative regulator (S. coelicolor) 92 NP_631494 regulator (of a two component system, maybe membrane sensor) orfB13a putative acetyltransferase (S. coelicolor); 58 NP_625155 tentative assignment orfB13b putative acetyltransferase (S. coelicolor) 100 NP_625155 of acetyltransferase in two frames, or sequencing error and should be in a single frame orfB14 putative lipoprotein (S. coelicolor) 386 NP_631245 unknown orfB15 hypothetical protein (S. coelicolor) 41 NP_631424 unknown orfB16 putative formate 915 NP_626265 oxidoreductase dehydrognease (S. coelicolor) (Pfam matches to molybdopterin oxidoreductase/ formate dehydrogenase alpha subunit) orfB17 conserved hypothetical 175 NP_631569 unknown protein, S. coelicolor SCBAC25F8.16 orfB18 product unknown 396 AAD23399 unknown (Streptomyces aureofaciens) orfB19 putative aldehyde 635 AAD23400 aldehyde dehydrogenase (S. aureofaciens) dehydrogenase orfB20 putative alcohol 450 NP_630527 alcohol dehydrogenase (S. coelicolor) dehydrogenase orfB21 hypothetical protein (S. coelicolor) 395 NP_630528 unknown orfB22 putative calcium binding 160 NP_631687 calcium binding protein (S. coelicolor) protein

[0156] Analysis of the functions of the putative gene products indicates that the genes borB to borO most probably form the boundaries of the borrelidin biosynthetic cluster. Evidence to support this came from the disruption of borB2, which produced borrelidin at levels indistinguishable from the wild type parental strain. In addition, borB3 to borB7 have homologues in the Streptomyces coelicolor A3(2) genome encoded on cosmid SCF76; the same orfs are present, but in a different order. The orfs borB8 to borB10 are arranged identically to homologues in the S. coelicolor A3(2) cosmid SC5E3. The orfs borB18 to borB21 have homologues that are arranged similarly in the S. coelicolor A3(2) cosmid SC1A2. The orf borB13 contains a frame-shift and thus any gene product would most probably be inactive. In addition, no function can be readily deduced for the products of these orfs during borrelidin biosynthesis.

Starter Unit Biosynthesis Genes

[0157] In order to identify the genes that are involved in the biosynthesis of the trans-cyclopentane-1,2-dicarboxylic starter unit, each of the genes borB to borN was disrupted (e.g. see examples 13-25). This was done in a manner designed to minimise the possibility of polar effects, which was verified by successful in trans complementation with a full-length copy of the disrupted gene under the control of the ermE* promoter, which gave back approximately WT levels of borrelidin production in each case.

[0158] Each of the disrupted mutants was grown in triplicate as described in example 1, and borrelidin production assessed. Alongside these, each mutant was grown in triplicate and supplemented, after 24 hours, with exogenous starter acid to a final concentration of 1 mM, and borrelidin production assessed. Extraction and analysis for borrelidin provided the data that are described below in Table 10:

TABLE-US-00010 TABLE 10 Borrelidin Borrelidin production Borrelidin production with biosynthetic without feeding (% relative feeding (% relative to unfed gene disrupted to WT) WT) Wild type 100 .+-. 16, (100 .+-. 2) 363 .+-. 65, (269 .+-. 49) (control) borB 75 .+-. 11, (43 .+-. 20) 172 .+-. 51 borC 0, (10 .+-. 3) 933 .+-. 42 borD 7 .+-. 1, (0) 75 .+-. 15 borE 2 .+-. 1 122 .+-. 23 borF 3 .+-. 2 201 .+-. 52 borG 11 .+-. 1, (32 .+-. 3) 1532 .+-. 142 borH 17 .+-. 2, (23 .+-. 13) 203 .+-. 40 borI 0, (0) 0, (0) borJ 0, (0) 0, (0) borK 0, (6 .+-. 1) 319 .+-. 54, (464 .+-. 18) borL 0, (0) 408 .+-. 70, (399 .+-. 69) borM 0, (6 .+-. 3) 461 .+-. 29, (553 .+-. 66) borN 25 .+-. 9, (34 .+-. 3) 68 .+-. 12, (46 .+-. 9) borO N/A N/A [Note 1: The values given in brackets indicate where repeat runs of some experiments were performed; Note 2: N/A = not applicable.]

[0159] Based on the data in table 10, it is clear to one skilled in the art that the gene products BorC-F and K-M are essential or very important for the biosynthesis of trans-cyclopentane-1,2-dicarboxylic acid, as these mutants produced no or very low levels of borrelidin without the addition of exogenous starter acid, whereupon they produced borrelidin at levels approaching, or better than, that of the WT organism. In addition the gene products BorG, H, and N appear to be involved in, but not essential for, the biosynthesis of the starter unit, as they produced significantly lower levels of borrelidin unless exogenous starter acid was added, whereupon they produced borrelidin at levels approaching or better than that of the WT organism; this was particularly notable in the case of the borG.sup.- mutant.

[0160] The normal metabolic function of BorN homologues is the production of 2-oxohepta-3-ene-1,7-dioate 10, a key step in the catabolism of tyrosine via 4-hydroxyphenyl acetic acid 9 (FIG. 9) (Prieto et al., 1996). Therefore, 10 may be an intermediate in the biosynthetic pathway to trans-cyclopentane-1,2-dicarboxylic. The ability of the mutant disrupted in borN to produce borrelidin, albeit at a reduced level, most probably lies in the presence of a homologue elsewhere in the genome utilised in the catabolism of tyrosine during primary metabolism.

[0161] The intermediate 10 contains all the required functionality for the eventual formation of trans-cyclopentane-1,2-dicarboxylic acid. The most probable next step of the biosynthesis is the reduction of the 3-ene position in a reaction similar to that catalysed by an enoyl reductase. Potential enzymes responsible for this step are BorC, BorD, BorK or BorM; these enzymes are all involved in borrelidin starter unit biosynthesis as seen from the data in table 10. The resulting 2-oxohepta-1,7-dioate 11 is one possible substrate for cyclisation through formation of a new C--C bond between C6 and C2. Another possible substrate for this cyclisation would be 2-hydroxyhepta-1,7-dioate 12 or some activated form thereof. This would presumably be formed from 11 by the action of an oxidoreductase such as BorC, BorD or BorM.

[0162] The key cyclisation step is most probably catalysed by BorE, which displays similarity to O-succinylbenzoyl-CoA synthase and chloromuconate cycloisomerase. These enzymes belong to the enolase super-family, the members of which share the common ability to stabilise the formation of an anion on the carbon atom adjacent to a carboxylate group (Schmidt. et al., 2001). It is further notable that the substrate for muconate cycloisomerase is a hexa-1,6-dioate, which is similar in gross structure to 11 and 12. Abstraction of a proton and formation of an anion equivalent at C6 of 11 or 12 (or an activated form thereof, e.g. 13) with subsequent cyclisation to C2 provides the correctly substituted cyclopentane ring structure, although the intermediacy of 11 as substrate would require some further processing of the substituted cyclopentane, most probably via elimination of water to give the symmetric cyclopent-1-ene-1,2-dicarboxylic acid, or possibly the .DELTA..sup.1-unsaturated compound, cyclopent-1-ene-1,2-dicarboxylic acid. However, the feeding of cyclopent-1-ene-1,2-dicarboxylic acid, or ethyl esters thereof, to S. parvulus Tu4055 strains disrupted in any of borC-E, or to WT strains, did not produce any borrelidin, or did not produce borrelidin in any increased amount when compared to the unfed controls. These data indicate that this compound is probably not an intermediate in starter unit biosynthesis, and that the substrate of BorE is possibly the 2-hydroxyhepta-1,7-dioate 12, or an activated form thereof (e.g. 13). A putative pathway for the biosynthetic pathway to trans-cyclopentane-1,2-dicarboxylic acid is shown in FIG. 4.

[0163] The combined, specific genes required for the biosynthetic steps to trans-cyclopentane-1,2-dicarboxylic acid are not clear, but probably are encoded by some combination of borC-H, borK, borM and borN. The lack of certain homologues of genes that are involved in the catabolism of 4-hydroxyphenyl acetic acid 9, and which would act prior to BorN in the pathway, is most probably an indication that primary metabolic genes perform these tasks. The addition of exogenous trans-cyclopentane-1,2-dicarboxylic acid to S. parvulus Tu4055 and related strains increases the titre of borrelidin in the order of 2- to 3-fold under our conditions, indicating that the biosynthesis of starter acid is a limiting factor in borrelidin biosynthesis. These data are consistent with primary metabolic degradation of tyrosine being the source of trans-cyclopentane-1,2-dicarboxylic acid.

[0164] In an attempt to further clarify which genes may be specifically responsible for biosynthesis of the starter unit, a number of co-culture experiments were performed with combinations of the different mutants--these require the knowledge that the gene products of borI and borJ are specifically involved in the formation of the C12-nitrile moiety, which is clarified by the data given in the following section below in combination with the data from table 10. In summary, the co-culture of mutants borE.sup.- & borD.sup.-, and of borE.sup.- & borM.sup.- failed to produce any borrelidin whereas the co-culture of mutants borM.sup.- & borI.sup.-, and borM.sup.- & borK.sup.- produced borrelidin at approximately WT levels. These data, in combination with that in table 10, and below, clearly indicate that borD, borE and borM are involved in starter unit biosynthesis, whereas borI, and possibly borK, are involved in the formation of the nitrile moiety at C12 of borrelidin.

[0165] It is clear from the data in table 10 that exogenous addition of trans-cyclopentane-1,2-dicarboxylic acid is sufficient to re-establish approximately WT levels, or better, of borrelidin production in mutants where genes that are involved in starter unit biosynthesis have been disrupted. These data indicate that there is no problem with the active uptake of added carboxylic acid by S. parvulus Tu4055, and that an activity is present which is capable of converting the carboxylic acid to a CoA thioester equivalent. Thus, given the known technologies of mutasynthesis, it is obvious to one skilled in the art that the addition of exogenous carboxylic acids to one of the aforementioned mutants, for example the borE strain S. parvulus Tu4055/borE:aac3(IV) described in example 16, may lead to the production of borrelidin analogues in which the starter unit carboxylic acid moiety is replaced with a moiety derived from the exogenously added carboxylic acid.

[0166] To examine this possibility, strain S. parvulus Tu4055/borE:aac3(IV) was fed with a trans-cyclobutane-1,2-dicarboxylic acid according to the protocol described in example 1 and then analysed as described in example 4. The structure 18, described in FIG. 10, shows the new borrelidin structure obtained from feeding this carboxylic acid; this compound 18 displayed the anticipated UV chromophore for borrelidin but eluted at an earlier retention time and displayed the expected mass by LCMS (m/z=474.3 [M-H].sup.-XX). Verification of this methodology was provided by the production, isolation and characterisation of 18 (example 33). (RS)-2 It is clear to one skilled in the art that other carboxylic acids could also be used in similar feeding experiments to provide further new borrelidin analogues. Although it is possible that not all carboxylic acids would be incorporated using the exact methodology described herein, a person of skill in the art is aware of a number of available methods to enhance the incorporation of fed starter units.

[0167] In addition to the use of the strain deleted in borE, it was observed (see table 10) that the strain S. parvulus Tu4055/borG:aac3(IV), in which borG has been disrupted, when fed with the natural starter unit of the bor PKS, trans-cyclopentane-1,2-dicarboxylic acid, produced borrelidin at titres significantly higher than those seen when the wild-type organism was fed (4-fold increase) or unfed (15-fold increase). To examine this further, this experiment was repeated using both the natural and an unnatural starter acid as exogenous substrates, fed, in parallel, to wildtype, the borE mutant and the borG mutant. The resulting data are described in table 11.

TABLE-US-00011 TABLE 11 Fed with 1 mM Fed with 1 mM cyclopentane cyclobutane trans-1,2- trans-1,2- Unfed dicarboxylic acid dicarboxylic acid S. parvulus Tu4055 2.3 mg/l 6.6 mg/l -- S. parvulus 0 4.7 mg/l 2.2 mg/l Tu4055/borE: aac3(IV) S. parvulus 0 88.9 mg/l 43.0 mg/l Tu4055/borG: aac3(IV)

As one can see from table 11, using S. parvulus Tu4055/borG:aac3(IV) instead of S. parvulus Tu4055/borE:aac3(IV) for mutasynthesis increases the titre approximately 19-fold, and that S. parvulus Tu4055/borG:aac3(IV) fed with the natural starter acid produces 38-fold more borrelidin A than wild type alone, or 13 fold more borrelidin A than the wild type strain fed with the same amount of cyclopentane trans-1,2-dicarboxylic acid. These data clearly indicate that the use of strain S. parvulus Tu4055/borG:aac3(IV) for mutasynthesis experiments is beneficial for the production of improved titres of borrelidin analogues. This method has general applicability for both the production of borrelidin and borrelidin analogues.

[0168] On the basis of this finding, the feeding experiments with alternative carboxylic acids were repeated in S. parvulus Tu4055/borG:aac3(IV), and extended to include 2,3-dimethyl succinic acid and 2-methylsuccinic acid; the new compounds derived from the incorporation of these alternative starter units, 19 and 20 respectively, are described in FIG. 10.

[0169] In an attempt to improve the titre of borrelidin produced in fermentation cultures of S. parvulus Tu4055 through other means, additional copies of the genes borE and borL were introduced into the organism in vectors that place them under the control of the strong constitutive promoter ermE*. It was anticipated that the over-expression of these genes would increase the intra-cellular levels of the starter acid, which appears to be limiting with respect to borrelidin production.

[0170] The genes borE and borL were amplified by PCR, cloned into the vector pEM4, and then introduced into S. parvulus Tu4055 as described in examples 29 and 30 respectively. In addition, the vector pEM4 alone (not containing any insert) was also introduced in S. parvulus Tu4055 and used as a control. The resulting strains were grown, extracted and analysed as described in examples 1 and 4. Introduction of the vector as a control did not significantly effect the levels of borrelidin production. However, the expression of additional copies of either borE or borL in this manner brought a 4.9.+-.0.3 and 4.3.+-.0.7-fold increase respectively in the titre of borrelidin relative to the wild type strain. Presumably, the steps of biosynthesis catalysed by their gene products are rate limiting, or alternatively their gene products may have a positive regulatory function. For example borL shows greatest homology to auxin response proteins from plants. Auxins are hormones involved in the regulation of various cellular processes in plants, and borL may represent the first example of a related gene having regulatory function in a bacteria. As controls, an additional copy of borJ, borO and borA5, under the control of ermE* in pEM4, were introduced into S. parvulus Tu4055, but did not have any significant effect upon borrelidin titre. This was anticipated as none of the respective gene products are anticipated to be involved in starter unit biosynthesis. In addition, up-regulation of the putative `stuttering` PKS module (borA5) did not increase borrelidin titre, further indicating that iterative use of this module occurs, rather than three independent copies being utilized. The lack of an effect on titre when borO is up-regulated indicates that there is most probably no limitation placed upon borrelidin production due to toxicity in the producing organism and so indicates that there is further scope for titre improvement.

Formation of the Nitrile Moiety at C12

[0171] Sequence analysis of the AT domain of the borrelidin PKS module 3 indicates that the substrate utilised for the third round of chain extension is methylmalonyl-CoA. Thus, the carbon atom of the nitrile moiety most probably arises from the methyl group of methylmalonyl-CoA. This was verified by stable isotope feeding experiments. Feeding [2,3-.sup.13C.sub.2]sodium propionate to S. parvulus Tu113 gave borrelidin which displayed intact labelling of the carbons at C4-C24, C6-C25, C8-C26, C10-C27 and C12-C28, and Identical specific incorporations (as determined within the limits of our experimental methods), as expected (FIG. 2). These data indicate that the conversion of the C12-methyl group occurs either during chain assembly at, or after, the incorporation of the third extension unit, or that it occurs after polyketide chain assembly and release from the PKS. Based on functional assignments given to the borrelidin biosynthetic genes, in conjunction with the gene disruption data described in table 10, both borI and borJ are clearly implicated in formation of the nitrile moiety at C12, while others such as borK may also be.

[0172] The cytochrome P450 hydroxylase BorI shares greatest similarity to TyIHI, which catalyses the hydroxylation of an exocyclic methyl group of the tylosin macrolactone prior to addition of a deoxyhexose moiety (Fouces et al., 1999). BorI is therefore believed to catalyse oxidation of the C12-methyl group during borrelidin biosynthesis. In agreement with this the borI.sup.- mutant S. parvulus Tu4055/borI::aac3(IV) fails to produce borrelidin but accumulates a new product 14 (FIG. 6) that is less polar than borrelidin. 14 is readily transformed to borrelidin when fed to the borE mutant S. parvulus Tu4055/borE::aac3(IV) which lacks the ability to synthesise the PKS starter unit but maintains the rest of the borrelidin biosynthetic genes intact. Fermentation of S. parvulus Tu4055/borI::aac3(IV) followed by extraction and isolation provided .about.30 mg of 14 (example 31). Full structural analysis of 14 identified it as 12-desnitrile-12-methylborrelidin (pre-borrelidin). This is consistent with the proposed role of BorI in borrelidin biosynthesis and provides a route to novel borrelidin analogues with a methyl group attached to C12 of the macrolactone ring.

[0173] The putative PLP dependent aminotransferase BorJ is believed to catalyse the introduction of a nitrogen atom into borrelidin at the activated C28-position, probably via a C12-formyl moiety. In agreement with this the borJ.sup.- mutant S. parvulus Tu4055/borJ::aac3(IV) does not produce borrelidin and accumulates a new compound that is more polar than borrelidin. This new compound is not transformed to borrelidin when fed to mutant S. parvulus Tu4055/borE::aac3(IV) which indicates that it is probably a shunt metabolite rather than an intermediate in borrelidin biosynthesis. Fermentation of S. parvulus Tu4055/borJ::aac3(IV) allowed the isolation of 17 mg of the accumulated compound (example 32). Detailed structural analysis identified the accumulant as 12-desnitrile-12-carboxyl borrelidin 2.

[0174] In addition to the compounds isolated from mutation of the borrelidin biosynthetic genes, 12-desnitrile-12-formyl borrelidin 15 is isolated from the fermentation supernatant of S. parvulus Tu113. The fermentation media and conditions used for these experiments differ from those we have described so far herein, but are designed to maximise the production of borrelidin. We propose that this altered medium, in combination with a drop in the dissolved oxygen concentration that is observed to occur during this specific fermentation, promoted the accumulation of 15. 15 is readily transformed to borrelidin when fed to the mutant S. parvulus Tu4055/borE::aac3(IV) which lacks the ability to synthesise the PKS starter unit but maintains the rest of the borrelidin biosynthetic genes intact.

[0175] The above data lead us to propose a biosynthetic route to the nitrile moiety of borrelidin as presented in FIG. 6. The C12-methyl carbon of pre-borrelidin 14 is first oxidised by BorI to introduce an allylic hydroxyl group at C28 (16). This hydroxyl group is then converted to the formyl moiety attached to C12 (15) using a method selected from the group comprising: spontaneous oxidation (including oxidation mediated by some background enzyme) the action of a specific gene of the borrelidin biosynthetic gene cluster; candidate gene products are thus BorI itself, acting in a multifunctional manner and operating via the formation of a gem-diol structure at C12 followed by dehydration; or alternatively, via one of the oxidoreductase encoding genes such as borC or borK. The next step is anticipated to be BorJ-catalysed transamination of 15 in order to introduce a nitrogen atom at C28, in the form of an amine, through a pyridoxamine phosphate mediated process. The putative product amine 17 then undergoes oxidation, possibly spontaneously, but most probably by an enzymic activity such as BorI (certain parallels can be drawn to the biosynthesis of nitriles in plants (Celenza, 2001; Hahn et al., 1999; Nielson and Moller, 1999)) or by the products of one of the oxidoreductase encoding genes, e.g. borC or borK, or by a general oxidoreductase within the proteome.

[0176] In order to examine this proposed pathway in more detail a number of biotransformation experiments were performed using pre-borrelidin 14 as substrate for investigating the action of borI-K individually and in combination, using pEM4 as vector and S. albus J1074 (Chater & Wilde, 1980) as an expression strain. Expression of borI or borJ individually did not give borrelidin production on addition of 14. The added 14 was only consumed during biotransformation with borI (and not in any of the control experiments); the 14 added was identified as being converted to the shunt metabolite 2. However, co-expression of borI & borJ did convert the added 14 to borrelidin. It thus appears that either BorI or general proteome activities in S. albus are capable of oxidising the proposed amine intermediate 17 in the borrelidin biosynthetic pathway. In addition to the feeding of pre-borrelidin 14, 12-desnitrile-12-carboxyl borrelidin 2 was also fed to the three strains described above. No conversion of 2 to borrelidin was observed in any of these experiments, reinforcing the idea that 2 is a shunt metabolite.

[0177] Detailed investigation of genomic DNA from three borrelidin producing strains, S. rochei ATCC23956, S. parvulus Tu113 and S. parvulus Tu4055, using numerous restriction digests and subsequent Southern Blot analysis, indicates that the borrelidin biosynthetic gene clusters of these three organisms are very closely conserved. It therefore appears that the borrelidin biosynthetic pathways of these strains are very similar. This assumption allows us to consider the data above, which are obtained from different strains, as applicable to a single biosynthetic pathway.

[0178] It is clear to one skilled in the art that manipulation of the genes involved in formation of the C12-nitrile moiety of borrelidin, for example borI, or borJ, is a generally useful method for the production of novel borrelidin related molecules and borrelidin derivatives with altered functionality at C12. In addition, the transfer of these genes to other organisms producing other natural or engineered polyketide products may allow the incorporation of nitrile moieties into such compounds.

[0179] In an extension of this work, disruptions in borI and borJ are separately made in the strain S. parvulus Tu4055/borG:aac3(IV) to give the doubly mutated strains S. parvulus Tu4055/borG:aac3(IV)/borI::hyg and S. parvulus Tu4055/borG:aac3(IV)/borJ::hyg (examples 27 & 28 respectively). These strains are fed alternative carboxylic acids, trans-cyclobutane-1,2-dicarboxylic acid, 2,3-dimethylsuccinic acid and 2-methylsuccinic acid, (as described above) and are found to produce the mutasynthetic borrelidin analogues carrying, either, a methyl (21, 22 and 23 respectively) or a carboxyl function at C12 (24, 25 and 26 respectively) in place of the nitrile group, and which are also derived from alternative starter units corresponding to the exogenously supplied carboxylic acids. This orthogonal library of new compounds is described in FIG. 11 and the observed UV chromophores and mass spectral data for each compound is shown.

Other Genes Involved in Borrelidin Production

[0180] In addition to the type-I terminal thioesterase domain of the borrelidin PKS, a discrete type-II thioesterase is located at the upstream boundary of the biosynthetic gene cluster and is encoded by the gene borB. Such discrete type-II TE proteins are commonly found to be associated with type-I PKSs and are believed to play a role in the `editing` of PKSs by the removal of short chain acyl groups that are formed by unwanted decarboxylation of extender units attached to KS domains (Heathcote et al., 2001). The disruption of such discrete type-I TEs in the picromycin (Xue et al., 1998) and tylosin (Butler et al., 1999) biosynthetic clusters leads to a significant reduction in titre of both macrolides. In accordance with these results, disruption of borB (example 13) gave a mutant that produced between 43-75% of the parental wild type titre.

[0181] The self-resistance of S. parvulus strains to borrelidin is most probably due to the product of borO, which encodes a threonyl tRNA synthetase homologue. Threonyl-tRNA synthetase is the molecular target of borrelidin in sensitive strains (Paetz & Ness, 1973). It is predicted that BorO is resistant to the action of borrelidin, and acts to produce threonyl-tRNAs in cells that make borrelidin, effectively complementing the normal threonyl-tRNA which are inhibited. To verify this hypothesis borO was amplified by PCR and cloned in to the expression vector pEM4A, which puts borO under the control of the strong constitutive promoter ermE* (example 26). The resulting vector pborOR was then transformed into the borrelidin-sensitive strain Streptomyces albus J1074 (Chater & Wilde, 1980). Comparison of this strain with that containing only the expression vector pEM4A, using a soaked disk bioassay, clearly indicated that expression of borO confers resistance to borrelidin.

EXAMPLES

General Methods

[0182] Restriction enzymes, other molecular biology reagents, antibiotics and chemicals were purchased from standard commercial sources. Restriction endonuclease digestion and ligation followed standard methods (Sambrook, J. et al., 1989).

Example 1

Fermentation of S. parvulus Strains

[0183] The following method is generally useful for culturing S. parvulus for the production of borrelidin and/or borrelidin analogues:

[0184] A seed flask containing NYG medium (30 ml in a 250 ml Erlenmeyer flask) was inoculated from a working stock (0.5 ml). NYG medium contains, in deionised water: beef extract (0.3%), Bacto peptone (0.5%), glucose (1%) and yeast extract (0.5%). After 2 days shaking in a rotary incubator (2-inch throw; 30.degree. C.; 250 rpm) the resulting cream culture was used to inoculate PYDG production medium (30 ml in a 250 ml Erlenmyer flask; 10% innoculum). PYDG medium contains per litre of deionised water: peptonised milk nutrient (1.5%), yeast autolysate (0.15%), dextrin (4.5%) and glucose (0.5%) adjusted to pH 7.0. After 5 days shaking on a rotary incubator (2-inch throw; 30.degree. C.; 250 rpm) the culture was harvested for analysis as described in example 4, or for isolation purposes as required. For quantitative analysis these experiments were performed in triplicate.

The following method is useful for the feeding of exogenous carboxylic acids to S. parvulus strains:

[0185] The S. parvulus strain was grown as described above. After 24 hours growth in PYDG production medium, the carboxylic acid of choice was added as a 50 .mu.l single aliquot (0.6 M solution in 70% methanol after neutralization with 5 N NaOH). The resulting culture was harvested after 5 days total fermentation and analysed as described in example 4. For quantitative studies these experiments were performed in triplicate, and the equivalent fed and unfed WT strains served as controls.

Example 2

Cryopreservation of S. parvulus Strains

Working Stocks

[0186] Working stocks of vegetative mycelia were prepared by mixing a 2 day old seed culture grown in NGY medium (0.5 ml) with cryopreservative (0.5 ml). Cryopreservative consists of 20% glycerol and 10% lactose in deionised water.

Spore Stocks

[0187] Strains of S. parvulus were incubated on HA agar plates at 30.degree. C. After 14 days the resulting spores from a single plate were harvested and suspended in of cryopreservative (1 ml). HA agar contains in deionised water: 0.4% yeast extract, 1% malt extract, 0.4% dextrose and 1.5% agar adjusted to pH 7.3.

Example 3

Cloning of the Borrelidin Biosynthetic Gene Cluster and Disruption of borA2 & borA3

Cosmid Library Generation

[0188] A cosmid library was constructed in pWE15 cosmid vector using the Gigapack.RTM.III Gold Packaging Extract kit according to the manufacturer's handbook (Stratagene). Chromosomal DNA was extracted from S. parvulus Tu4055 according to standard protocols (Kieser et al., 2000) and treated with Sau3AI prior to cloning into pWE15. A number of the resulting E. coli transformants (3300) were picked and transferred to 96 well microtitre plates containing Luria Broth (LB) medium (0.1 ml per well) with ampicillin (100 .mu.g/ml). The resulting clones were replica-plated to Luria agar (LA) plates containing ampicillin (100 .mu.g/ml). After incubation overnight at 37.degree. C. colonies were transferred to nylon membrane filters for in situ colony hybridization analysis according to published protocols (Sambrook et al., 1989).

Library Screening

[0189] The cosmid library was screened using a probe that was generated using the DIG DNA Labelling and detection kit (Roche) according to the manufacturers instructions. The probe used was a BglII-BamHI fragment (1.7 kbp) obtained from the gene that encodes module 6 of the third subunit of the oleandomycin PKS from Streptomyces antibioticus (Swan et al., 1994).

Disruption of the Borrelidin Biosynthetic Gene Cluster

[0190] Cosmids that gave a positive response when screened as described above were digested with BamHI and fragments of less than 3 kbp were subcloned into pOJ260 (Bierman et al., 1992). These were then used to transform protoplasts of S. parvulus Tu4055 as described in example 5. The resulting transformants were then assessed for the ability to produce borrelidin. Two clones were borrelidin non-producers; both were obtained from cosBor32A2 and contain sequence typical of a modular PKS. The remaining cosmids were then screened using probes obtained from the two BamHI fragments, which led to the identification of the overlapping cosmid cosBor19B9 that contained the remainder of the borrelidin biosynthetic cluster.

Sequencing of cosBor32A2 and cosBor19B9

[0191] The cosmids cosBor32A2 and cosBor19B9 were transformed into E. coli DH10B and the resulting clones grown at 37.degree. C. in 2.times.TY media (30 ml) containing ampicillin. After 15 hours the cells were harvested and Qiagen Tip 100 kits were used to prepare cosmid DNA. Approximately 5 .mu.g of the cosmid DNA was digested with Sau3AI (1 U). Samples were taken at 2, 4, 6, 8 & 10 minute intervals after the enzyme was added and quenched into an equal volume of ice cold 0.5M EDTA. The samples were mixed and then analysed by gel electrophoresis, and those fragments between 1.5-2.0 kbp recovered from the gel. The fragments were cloned into linearised and dephosphorylated pHSG397 (Takeshita et al., 1987), and transformed into E. coli DH10B. The resulting clones that contained insert were grown in 2.times.TY medium (2 ml) containing chloramphenicol (30 .mu.g/ml) and purified using Wizard kits (Promega).

[0192] DNA sequencing was carried out using an Applied Biosystems 800 Molecular Biology CATALYST robot to perform the dideoxy terminator reactions, which were then loaded into an ABI Prism 3700 automated sequencer (Applied Biosystems). The raw sequence data was processed using the Staden software package. Assembly and contig editing was performed using GAP (Genome Assembly Program) version 4.2 (Bonfield et al., 1995). The GCG package (Devereux et al., 1984) version 10.0 was used for sequence analysis.

Example 4

Chemical Analysis of S. parvulus Strains

[0193] The following method is useful for analysing fermentations (see example 1) for the production of natural borrelidins and of engineered borrelidin analogues:

[0194] In a 2 ml Eppendorf tube, an aliquot of 5 day old fermentation broth (1 ml) was adjusted to pH.about.3 by the addition of 90% formic acid (ca. 20 .mu.l). Ethyl acetate (1 ml) was added to the sample and mixed vigorously for 10 min using a vortex tray. The mixture was separated by centrifugation in a microfuge and the upper phase removed to a clean 2 ml Eppendorf tube. The ethyl acetate was removed by evaporation using a Speed-Vac. Residues were dissolved into methanol (250 .mu.l) and clarified using a microfuge. Analysis was performed on an Agilent HP1100 HPLC system as described below; [0195] Injection volume: 50 .mu.l [0196] Column stationary phase: 150.times.4.6 mm column, base-deactivated reversed phase silica gel, 3 .mu.m particle size (Hypersil C.sub.18-BDS). [0197] Mobile phase A: 10% acetonitrile:90% water, containing 10 mM ammonium acetate and 0.1% TFA. [0198] Mobile phase B: 90% acetonitrile:10% water, containing 10 mM ammonium acetate and 0.1% TFA. [0199] Mobile phase gradient: T=0 min, 25% B; T=15, 100% B; T=19, 100% B; T=19.5, 25% B; T=25, 25% B. [0200] Flow rate: 1 ml/min. [0201] Detection: UV at 258 nm (DAD acquisition over 190-600 nm); MS detection by electrospray ionisation over m/z range 100-1000 amu, with +/-ve ion mode switching.

Example 5

Protoplast Transformation Protocol for S. parvulus Tu4055

[0202] A seed flask containing tryptone soy broth (TSB) medium (10 ml in a 100 ml Erlenmyer flask) was inoculated from a working stock (0.15 ml). After 3 days shaking on a rotary incubator (30.degree. C., 250 rpm), 5 ml of the culture was used to inoculate R5 medium (Kieser et al., 2000) (50 ml in a 250 ml Erlenmeyer flask) that was then shaken on a rotary incubator for 24 hours (30.degree. C., 250 rpm). The PEG mediated transformation of protoplasts was then performed according to standard published protocols (Kieser et al., 2000).

Example 6

Replacement of borAT4 with rapAT2--Production of C10-desmethyl Borrelidin

[0203] The borrelidin PKS AT4 domain is replaced with the AT2 domain of the rapamycin polyketide synthase as follows:

[0204] CosBor32A2 is digested with EcoRI and the 5429 bp band isolated. This is used as a template for PCR using the oligos CM410 (5'-AAAATGCATTCGGCCTGAACGGCCCCGCTGTCA-3') (SEQ ID No.44) and CM411 (5'-AAATGGCCAGCGAACACCAACACCACACCACCA-3') (SEQ ID No.45). CM410 introduces an NsiI restriction site for cloning purposes and CM411 introduces an MscI site for use in the introduction of a heterologous AT. The .about.1.1 kbp product is cloned into pUC18 digested with SmaI and dephosphorylated. The insert can ligate in two orientations and the reverse orientation is screened for by restriction enzyme analysis and the insert sequenced. One correct plasmid is designated pCJM462. Methylation deficient DNA (specifically dcm.sup.-1) of pCJM462 and pCJR26 (Rowe et al. 1998) is isolated by passaging the plasmids through E. coli ET12567. Each plasmid is then digested with MscI and XbaI and the .about.7.8 kbp fragment from pCJR26, containing the rapamycin AT2 and sequences downstream in pCJR26, is ligated to the .about.3.8 kbp backbone generated by digestion of pCJM462. Plasmid pCJM463 is identified by restriction analysis.

[0205] CosBor32A2 is digested with EcoRI and EcoRV and the 2871 bp band isolated. This is used as a template for PCR using the oligos CM412 (5'-AAAGTCCTAGGCGGCGGCCGGCGGGTCGACCT-3') (SEQ ID No.46) and CM413 (5'-TTTAGATCTCGCGACGTCGCACGCGCCGAACGTCA-3') (SEQ ID No.47). CM412 introduces an AvrII restriction site that joins, in frame, the downstream borrelidin homology to the heterologous AT, and CM413 introduces a BglII site for cloning purposes. The .about.1.1 kbp product is cloned into pUC18 digested with SmaI and dephosphorylated. The insert can ligate in two orientations and the reverse orientation is screened for by restriction enzyme analysis and the insert sequenced. One correct plasmid is designated pCJM464.

[0206] Plasmids pCJM463 and pCJM464 are digested with AvrII and XbaI and the .about.1.1 kbp fragment from pCJM464 is ligated into the .about.4.7 kbp backbone of pCJM463 to give pCJM465, which is identified by restriction enzyme analysis. pCJM465 contains the hybrid rapamycin AT2 with flanking regions of borrelidin sequence which provide homology for integration and secondary recombination.

[0207] Plasmid pCJM465 is digested with NsiI and BglII and the .about.3 kbp fragment is cloned into pSL1180 previously digested with NsiI and BamHI to give pCJM466. Plasmid pCJM466 is then digested with NsiI and the apramycin cassette is incorporated on a PstI fragment from pEFBA (Lozano et al., 2000) to give the replacement vector pCJM467. pCJM467 is introduced into #S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin (25 .mu.g/ml) are initially identified, and then passaged several times through MA media without antibiotic selection in order to promote the second recombination (Fernandez et al., 1998). Several apramycin-sensitive colonies are isolated and analysed by PCR and Southern blot. The new mutant is named S. parvulus Tu4055/467.

[0208] S. parvulus Tu4055/467 is analysed as described in example 1 and shown to produce a mixture compounds with the correct UV spectrum. One of the new major components that is more polar than borrelidin has the correct retention time for 10-desmethyl borrelidin 3. LCMS analysis indicates an m/z ratio for a compound that is 14 mass units lower than borrelidin as expected, and with an appropriate mass fragmentation pattern. Borrelidin itself is also produced, but at levels lower than the WT organism.

Example 7

Mutation of the Methylmalonyl-CoA Selective Motif of borAT4 to Generate 10-desmethyl Borrelidin

[0209] Site directed mutagenesis of acyl transferase domains may also be used to alter the specificity of an AT. In this example the specificity of borAT4 is directed from methyl-malonyl-CoA towards malonyl-CoA. An amino acid motif has been Identified (Reeves et al., 2001; WO 02/14482) which directs the specificity of an AT. The motif YASH, as observed in borAT4, is found in methylmalonyl-CoA specific ATs and in this example it is altered to HAFH which is found in malonyl-CoA specific ATs.

[0210] CosBor32A2 is digested with NcoI and the 5167 bp band isolated. This is used as a template for PCR using the primers CM414 (5'-5 AAACTGCAGAGTCGAACATCGGTCACACGCAGGC-3') (SEQ ID No.48) and CM415 (5'-AAAATGCATGATCCACATCGATACGACGCGCCCGA-3') (SEQ ID No.49). CM414 introduces a PstI restriction site for cloning purposes, and CM415 is a mutagenic primer covering the motif encoding region of the AT which will effect the amino acid changes and contains an NsiI site for cloning purposes. The .about.1.1 kbp fragment is cloned into pUC18 digested with SmaI and dephosphorylated. The insert can ligate in either orientation and the forward orientation is screened for by restriction enzyme analysis and the insert sequenced. One correct plasmid is designated pCJM468.

[0211] A second PCR reaction is performed using the 5167 bp NcoI fragment of CosBor32A2 and the primers CM416 (5'-TAAATGCATTCCATTCGGTGCAGGTGGAGTTGATCC-3') (SEQ ID No.50) and CM417 (5'-ATAGGATCCCCTCCGGGTGCTCCAGACCGGCCACCC-3') (SEQ ID No.51). CM416 introduces an NsiI restriction site and is also a mutagenic primer covering the motif encoding region of the AT, and CM417 introduces a BamHI site for cloning purposes. The .about.1.1 kbp fragment is cloned into pUC18 previously digested with SmaI and dephosphorylated. The insert can ligate in two orientations and the forward orientation is screened for by restriction enzyme analysis and the insert sequenced. One correct plasmid is designated pCJM469.

[0212] Plasmids pCJM468 and pCJM469 are digested with NsiI and XbaI and the .about.1.1 kbp fragment from pCJM468 is ligated into the 3.8 kbp backbone of pCJM469 to give pCJM470, which is identified by restriction enzyme analysis. pCJM470 contains the mutated motif of borAT4 with .about.1.1 kbp of homologous DNA on either side which provide homology for integration and secondary recombination.

[0213] Plasmid pCJM470 is digested with PstI and BamHI and the .about.2.2 kbp fragment is cloned into pSL1180 (Amersham Biosciences) previously digested with PstI and BamHI to give pCJM471. Plasmid pCJM471 is then digested with PstI and the apramycin cassette is incorporated on a PstI fragment from pEFBA (Lozano et al., 2000) to provide the replacement vector pCJM472.

[0214] The replacement vector pCJM472 is introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin are initially identified, and then passaged several times through MA media without antibiotic selection in order to promote the second recombination (Fernandez et al., 1998). Several apramycin-sensitive colonies are isolated and analysed by PCR and Southern blot, and one is selected that contains the new AT4 sequence containing the mutated motif and the NsiI site. The new mutant is named S. parvulus Tu4055/472.

[0215] S. parvulus Tu4055/472 is grown and analysed as described in example 1 and shown to produce a mixture of compounds with the correct UV profile for borrelidin. One of the new major components, that is more polar than borrelidin, has the correct retention time for authentic 3. LCMS analysis indicates an m/z ratio for a compound that is 14 mass units lower than borrelidin as expected, and with an appropriate mass fragmentation pattern. Borrelidin itself is also produced, but at levels lower than the WT organism.

Example 8

Introduction of the Borrelidin Loading Module into the Erythromycin PKS

[0216] The borrelidin loading module was amplified for each of the four putative start codons. The PCR template was a 3376 bp BamHI fragment of cosBor32A2 covering the region from nucleotides 15858 to 19234 of SEQ ID No.1. The reverse primer CM368 (5'-TTTCCTGCAGGCCATCCCCACGATCGCGATCGGCT-3') (SEQ ID No:52) introduces a SbfI site at the sequence corresponding to the start of KS1 of borA2 (conserved MACRL motif) and is used with each of the forward primers CM369 (5'-TTTCATATGACAGGCAGTGCTGTTTCGGCCCCATT-3') (SEQ ID No.53), CM370 (5'-TTTCATATGGCGGATGCCGTACGTGCCGCCGGCGCT-3') (SEQ ID No.54), CM371 (5'-TTTCATATGCCCCAGGCGATCGTCCGCACCAC-3') (SEQ ID No.55) and CM372 (5'-TTTCATATGGTCTCGGCCCCCCACACAAGAGCCCTCCGGGC-3') (SEQ ID No:56). The four PCR products (of 2834, 2720, 2411 and 2117 bp respectively) were cloned into pUC18 that had previously been digested with SmaI and dephosphorylated. The resulting plasmids were designated pCJM370, which contains the largest insert, pCJM371, pCJM372 and pCJM373, which contains the smallest insert.

[0217] The four borrelidin loading module fragments were introduced into the vector pKS1W, which contains a PstI site at the start of eryKS1 of DEBS1-TE in the conserved MACRL motif (Rowe et al., 2001); PstI gives the same overhang as SbfI. pKS1W is a pT7-based plasmid containing DEBS1-TE on an NdeI/XbaI fragment, with unique sites flanking the loading module, a unique PstI site at nucleotide position 1698 of the DEBS1-TE encoding gene and a unique NdeI site at the start codon. The borrelidin loading module fragments were excised as follows: pCJM370 was digested with NdeI and SbfI, pCJM371 and pCJM373 were digested with NdeI and PstI, and pCJM372 was digested with NdeI, PstI and DraI. Each loading module containing fragment was cloned into pKS1W previously digested with NdeI and PstI. The resulting plasmids were designated pCJM384, which contains the largest insert, then pCJM386, pCJM388 and pCJM390, which contains the smallest insert.

[0218] The hybrid PKS fragments were transferred into pCJR24, which is a suitable vector for transformation of S. erythraea WT and S. erythraea DM, and for expression of the resulting hybrid PKS (WO 98/01546). Each loading module construct was excised along with a 2346 bp fragment of DNA from DEBS1 in order to allow integration into the chromosome. In order to achieve this, pCJR24 is digested with XbaI and end-filled using Klenow fragment of DNA polymerase I. This is then digested with NdeI to give the backbone fragment. Into this, the four hybrid PKS fragments containing the borrelidin loading modules plus the region of DEBS1 sequence for integration are cloned as NdeI/EcoRV fragments from pCJM384, pCJM386, pCJM388 and pCJM390 to give pCJM400, pCJM401, pCJM402 and pCJM403 respectively.

[0219] Plasmids pCJM400, pCJM401, pCJM402 and pCJM403 were introduced into S. erythraea by transformation of S. erythraea DM protoplasts as described elsewhere (Gaisser et al., 2000). The resulting mutants were analysed by PCR and Southern blot to confirm the presence of the plasmid on the chromosome and to establish that correct integration had occurred. A number of mutants that appeared correct by these methods were grown, extracted and analysed according to standard methods for polyketide production from S. erythraea strains (Wilkinson et al., 2000). When compared to control strains using LCMS methods, the extracts from several of these mutants contained new compounds at reasonable levels. Analysis of their MS spectra showed the presence of a compound with m/z=485.3 ([M-H].sup.-, 6) in negative ion mode. This is in agreement with the expected product compound (M=486.3).

Example 9

Fusion of PKS Modules 4 and 5 (S. parvulus Tu4055/borA4-A5)

[0220] To examine the iterative action of module 5, the two separate proteins encoding modules 4 and 5 were fused together through manipulation at the genetic level. The fusion was performed by a gene replacement in which the last .about.1 kbp of borA4 and the first .about.1 kbp of borA5, were fused by converting the overlapping stop and start codons respectively into an arginine residue, introducing a new XbaI site and converting the two separate orfs into one.

[0221] In the first step of the mutagenesis, two separate PCR amplifications were performed. In the first PCR reaction, the template DNA was cosBor19B9, and the primers were B1819A (5'-GTCATGCATGCGGCGGGCTC-3') (SEQ ID No.57) and B1819B (5'-GGTCTAGAACGGCCGAACTT-3') (SEQ ID No.58). The 1063 bp product was purified, digested NsiI-XbaI and cloned into pSL1180 (Amersham Biosciences) digested similarly to give plasmid pSL18-19AB. The second PCR reaction amplified the borA5 fragment and used the primers B1819C (5'-GTTCTAGAACCTCGGTCGGC-3') (SEQ ID No.59) and B1819D (5'-CTGGATCCCACGCTGCTGCG-3') (SEQ ID No.60). The 1033 bp product was purified, digested with XbaI-BamHI and cloned into pSL18-19AB that had been digested similarly, to give plasmid pSL18-ABCD. Finally, the apramycin cassette from pEFBA (Lozano et al., 2000) was excised as a PstI fragment and cloned into pSL18-19ABCD digested with NsiI to give the replacement vector pSL18-19Apra.

[0222] The replacement vector pSL18-19Apra was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin (25 .mu.g/ml) were initially selected, and then passaged several times through MA media without selection. Several apramycin-sensitive colonies were obtained, two of which produced borrelidin while the others did not.

[0223] Chromosomal DNA was extracted from all of the apramycin sensitive colonies and checked initially by PCR using the primers BLDA (5'-GGAGACTTACGGGGGATGC-3') (SEQ ID No.61) and BLDB (5'-CTCCAGCAGCGACCAGAAC-3') (SEQ ID No.62) that are selective for the loading module (borA1). A 2.9 kbp fragment was observed for the control and the two borrelidin-producing mutants, but not for the non-producing strains. This result is symptomatic and characteristic of non-specific deletions in the chromosome.

[0224] The two borrelidin-producing colonies were analysed further by PCR using the primers B19A (5'-CCCATGCATCACCGACATAC-3') (SEQ ID No.63) and B19B (5'-GCGATATCCCGAAGAACGCG-3') (SEQ ID No.64) in order to check the fusion site. The method was as described above. Both the colonies and the controls gave a PCR product of 1010 bp, but upon digestion with XbaI only those that carried the fusion-producing mutation gave digestion to 600 and 400 bp fragments. Only one of the borrelidin-producing colonies harboured the fusion, while the other had reverted to wild type. Final confirmation came from Southern analysis using a BamHI-XhoI internal fragment from borA5 as probe over chromosomal DNA digested with XbaI and BclI. The control and wild type revertant colony showed a fragment of 11.5 kbp as expected, while the fusion mutant showed a fragment of 7.8 kbp as expected. This new mutant was named S. parvulus Tu4055/borA4A5. S. parvulus Tu4055/borA4-A5 was shown to produce borrelidin at 26.+-.5% of the WT titre, following the protocol described in example 1.

Example 10

Fusion of PKS Modules 5 and 6 (S. parvulus Tu4055/borA5-A.sctn.)

[0225] This experiment was performed for the same reason as, and in an analogous manner to, that of example 9 above. The fusion of these orfs introduced an additional leucine residue into the new protein at the fusion point, in addition to a new SpeI site at the genetic level. In the first step of the process two PCR fragments were generated using cosBor19B9 as template. The first PCR reaction amplified the borA5 region and used the primers B1920A (5'-GCCAAGCTTCCTCGACGCGC-3') (SEQ ID No.65) and B1920B (5'-CACTAGTGCCTCACCCAGTT-3') (SEQ ID No.66). The 804 bp product was purified and digested with HindIII-SpeI. The second PCR reaction amplified the borA6 region and used the primers B1920C (5'-CACTAGTGACGGCCGAAGCG-3') (SEQ ID No.67) and B1920D (5'-TCGGATCCGTCAGACCGTTC-3') (SEQ ID No.68). The 960 bp product was purified and digested with SpeI-BamHI. The two purified and digested gene products were then cloned together into pOJ260 that had been digested with HindIII-BamHI to give the replacement vector pOJF19-20. pOJF19-20 was introduced into S. parvulus Tu4055 by protoplast transformation to give apramycin resistant colonies. One such colony was passaged several times through MA media without selection in order to promote double recombination. Two apramycin sensitive colonies were obtained, and chromosomal DNA from these was examined by Southern hybridisation to check for the presence of a 3.2 kbp BamHI fragment (to control for unwanted deletions in the loading module) and a 3.4 kbp SpeI-BamHI fragment to verify correct introduction of the borA5-A6 fusion (5.8 kbp BamHI fragment in the WT). One of the apramycin colonies carried the correct mutation without deletion and was named S. parvulus Tu4055/borA5A6. S. parvulus Tu4055/borA5-A6 was shown to produce borrelidin at 25.+-.4% of the WT titre, following the protocol as described in example 1.

Example 11

Fusion of PKS Modules 4, 5 and 6 (S. parvulus Tu4055/borA4-A5A6)

[0226] To generate the strain S. parvulus Tu4055/borA4-A5-A6 we took advantage of the previously obtained strain S. parvulus Tu4055/borA4-A5 (Example 9) and plasmid pOJF19-20 (Example 10). pOJF19-20 was introduced into S. parvulus Tu4055/borA4-A5 by protoplast transformation to give apramycin resistant colonies. One such colony was passaged several times through MA media without selection in order to promote double recombination. One apramycin sensitive colony was obtained, and chromosomal DNA from it was examined by Southern hybridisation to check for the presence of a 3.2 kbp BamHI fragment (to control for unwanted deletions in the loading module), a 3.4 kbp SpeI-BamHI fragment to verify correct introduction of the borA5-A6 fusion (5.8 kbp BamHI fragment in the WT) and a 6.4 kbp SpeI-XbaI to verify the presence of both fusions, borA4-A5 and borA5-A6, within the same strain. The chosen colony carried the correct mutation without deletion and was named S. parvulus Tu4055/borA4-A5-A6. S. parvulus Tu4055/borA4-A5-A6 was shown to produce borrelidin at 18.+-.5% of the WT titre, following the protocol as described in example 1.

Example 12

Replacement of the Erythromycin PKS Module 4 with Module 5 of the Borrelidin PKS--Production of Ring Expanded Macrolides

[0227] Example 12 describes the replacement of erythromycin module 4 with borrelidin module 5. Borrelidin module 5 is believed to be responsible for three rounds of condensation of methylmalonyl-CoA, in an iterative fashion, within the borrelidin PKS. Previously, erythromycin module 4 has been shown to occasionally act in an iterative fashion `mis`-incorporating a second methylmalonyl-CoA to make very small amounts of a 16-membered macrolide from the erythromycin PKS. A strain in which the erythromycin module 4 is replaced by borrelidin module 5 is engineered by a replacement strategy as follows, and is based on a derivative process as described for module insertion into the erythromycin PKS (Rowe et al., 2001):

[0228] Initially a series of plasmids are made in order to generate a plasmid in which the borrelidin module 5 is flanked by appropriate regions of homology from the erythromycin PKS. In order to facilitate this, the SbfI site is first removed from the polylinker of pUC18 by digestion with PstI, end-polishing with T4 polymerase and religation. The new plasmid, pCJM409 is identified by restriction enzyme digestion.

[0229] Borrelidin module 5 is isolated on an SbfI fragment by ligating together 4 PCR fragments. PCRA is generated by amplification of .about.1.4 kb of the beginning of borrelidin module 5 using the 6062 bp XcmI fragment of cosBor19B9 as the template and primers CM384 (5'-AACCTGCAGGTACCCCGGTGGGGTGCGGTCGCCCGA-3') (SEQ ID No.69) and CM385 (5'-CGCCGCACGCGTCGAAGCCAACGA-3') (SEQ ID No.70). CM384 introduces an SbfI site in the conserved amino acid sequence MxCR at the beginning of borrelidin module 5. CM385 incorporates a naturally occurring MluI site that is used in the cloning strategy. PCRA is treated with T4 polynucleotide kinase (T4 PNK, NEB) and cloned into pCJM409 previously digested with SmaI and dephosphorylated with Shrimp Alkaline Phosphatase (SAP, Roche). Inserts cloned in the forward direction are screened for by restriction enzyme digestion, and for one correct clone the insert is verified by sequencing. This plasmid is designated pCJM410.

[0230] PCRB is generated by amplification of the adjacent .about.1.4 kb of borrelidin module 5 using the 6062 bp XcmI fragment of cosBor19B9 as the template and primers CM386 (5'-TGTGGGCTGGTCGTTGGCTTCGAC-3') (SEQ ID No.71) and CM387 (5'-GGTGCCTGCAGCGTGAGTTCCTCGACGGATCCGA-3') (SEQ ID No.72). CM386 binds upstream of the same MluI site as CM385 contains, which is used in the cloning strategy. CM387 is used to remove the SbfI site within the borrelidin PKS module 5 whilst leaving the overlapping PstI site for cloning. PCRB is treated with T4 PNK and cloned into pCJM409 previously digested with SmaI and dephosphorylated with SAP. Inserts cloned in the forward direction are screened for by restriction enzyme digestion, and for one correct clone the insert is verified by sequencing. This plasmid is designated pCJM411.

[0231] PCRC is generated by amplification of the downstream adjacent .about.1.5 kb of borrelidin module 5 using the 6062 bp XcmI fragment of cosBor19B9 as the template and oligonucleotides CM388 (5'-GAGGAACTCACCCTGCAGGCACCGCT-3') (SEQ ID No.73) and CM395 (5'-CGAACGTCCAGCCCTCGGGCATGCGT-3') (SEQ ID No.74). CM388 binds at the same SbfI site as CM387, but is not mutagenic and retains the SbfI site. CM395 incorporates an SphI site for cloning purposes. PCRC is treated with T4 PNK and cloned into pCJM409 previously digested with SmaI and dephosphorylated with SAP. Inserts cloned in the forward direction are screened for by restriction enzyme digestion and for one correct clone the insert is verified by sequencing. This plasmid is designated pCJM412.

[0232] PCRD is generated by amplification of the downstream adjacent .about.2.1 kb of borrelidin module 5 using the 7211 bp BbvCI fragment of cosBor19B9 as the template and primers CM396 (5'-TGGCACGCATGCCCGAGGGCTGGACGTT-3') (SEQ ID No.75) and CM397 (5'-TTTCCTGCAGGCCATGCCGACGATCGCGACAGGCT-3') (SEQ ID No.76). CM396 contains the SphI site for cloning purposes, and CM397 introduces an SbfI site in the conserved amino acid sequence MxCR at the end of borrelidin module 5. PCRD is treated with T4 PNK and cloned into pCJM409 previously digested with SmaI and dephosphorylated with SAP. Inserts cloned in the forward direction are screened for by restriction enzyme digestion, and for one correct clone the insert is verified by sequencing, this plasmid is designated pCJM413.

[0233] The four PCR products (PCRA-D) are used to construct the borrelidin module 5 on an SbfI fragment as follows:

[0234] pCJM412 is digested with SphI and the .about.1.5 kb fragment isolated is cloned into pCJM413 previously digested with SphI and dephosphorylated with SAP. This gives plasmid pCJM414, which is identified by restriction enzyme digestion.

[0235] pCJM414 is digested with SbfI and the .about.3.6 kb fragment isolated is cloned into pCJM411 previously digested with PstI and dephosphorylated with SAP. This gives pCJM415 which is Identified by restriction enzyme digestion.

[0236] pCJM410 is digested with MluI and HindIII and the .about.1.4 kb fragment isolated is cloned into pCJM415 previously digested with MluI and HindIII. This gives pCJM416, which is identified by restriction enzyme digestion. pCJM416 is a pUC18-based plasmid containing the borrelidin module 5 as an SbfI fragment.

[0237] In order to introduce the Borrelidin module 5 into the erythromycin PKS by a replacement strategy, flanking regions of homology from the erythromycin PKS are incorporated for recombination as follows:

[0238] PCRE is generated by amplification of .about.3.3 kb of the erythromycin PKS directly upstream of the module 4 KS using the 6428 bp XmnI fragment of pIB023 as the template and primers CM398 (5'-AAACATATGGTCCTGGCGCTGCGCAACGGGGAACTG-3') (SEQ ID No.77) and CM399 (5'-TTTCCTGCAGGCGATGCCGACGATGGCGATGGGCT-3') (SEQ ID No.78). CM398 contains an NdeI site for cloning purposes and CM399 introduces an SbfI site in the conserved amino acid sequence M/IxCR at the beginning of erythromycin module 4. PCRE is treated with T4 PNK and cloned into pCJM409 previously digested with SmaI and dephosphorylated with SAP. Inserts cloned in the forward direction are screened for by restriction enzyme digestion, and for one correct clone the insert is verified by sequencing, this plasmid is designated pCJM417.

[0239] PCRF is generated by amplification of .about.3.4 kb of the erythromycin PKS directly downstream of the module 5 KS using the 7875 bp XmnI/NheI fragment of pIB023 as the template and primers CM400 (5'-AAACCTGCAGGTTCCCCGGCGACGTGGACTCGCCGGAGTCGTT-3') (SEQ ID No.79) and CM401 (5'-TTTTCTAGAGCGACGTCGCAGGCGGCGATGGTCACGCCCGT-3') (SEQ ID No.80). CM400 introduces an SbfI site in the conserved amino acid sequence M/IxCR at the beginning of erythromycin module 4, and primer CM401 contains an XbaI site for cloning purposes. PCRF is treated with T4 PNK and cloned into pCJM409 previously digested with SmaI and dephosphorylated with SAP. Inserts cloned in the forward direction are screened for by restriction enzyme digestion, and for one correct clone the insert is verified by sequencing. This plasmid is designated pCJM418.

[0240] pCJM417 is digested with NdeI and SbfI and the .about.3.3 kb fragment is cloned into pCJM418 digested with NdeI and SbfI (.about.5.8 kbp) to give pCJM419 which is identified by its restriction digest pattern. pCJM419 contains a unique SbfI site which can be used to accept any complete module with SbfI (or PstI) flanking sites appropriate to place, in-frame, the in-coming module exactly into the conserved region of the KS domain.

[0241] The borrelidin module 5 with flanking SbfI sites is cloned from pCJM416 as an SbfI fragment into the unique SbfI site of pCJM419 (which has been dephosphorylated with SAP) to give pCJM420, which is identified by restriction enzyme analysis to confirm the presence and correct orientation of the insert. pCJM420 thus contains borrelidin module 5 with flanking regions of homology to introduce it in-frame between modules 3 and 5 of the erythromycin PKS. The complete insert is removed as an NdeI/XbaI fragment from pCJM420 and cloned into pCJM24 digested with NdeI and XbaI to give the final plasmid pCJM421. pCJR24, and consequently pCJM421, contain an appropriate resistance marker for selection of S. erythraea transformants.

[0242] Plasmid pCJM421 is used to transform S. erythraea strains NRRL2338 (wild type), and S. erythraea DM (eryCIII.sup.-, eryBV.sup.-) protoplasts (Yamamoto et al., 1986; Rowe et al., 1998). Integrants are selected for resistance to thiostrepton (50 mg/L) and a number of integrants (typically 5-8) are analysed further by Southern blot to confirm that the strains are correct and to identify the site of integration. Two correct integrants in each case are sub-cultured in TSB liquid media without antibiotic selection in order to promote the second recombination. Several thiostrepton-sensitive colonies are isolated and analysed by PCR and Southern blot, and in each case one selected that contains the new module correctly inserted. This leads to strains S. erythraea WT/421 and S. erythraea DM/421.

[0243] Strain S. erythraea DM/421 is cultured under conditions appropriate for the production of erythronolides (Wilkinson et al., 2000). Analysis of fermentation broth extracts using LCMS methods indicates the presence of two new significant peaks when compared to the control strain, and which are less polar than erythronolide B. These have an m/z of 435.5 (MNa.sup.+) and 477.5 (MNa.sup.+) respectively, which is consistent with the production of new ring expanded erythronolide B analogues. The compound with m/z=435.5 (7) is consistent with the presence of the 16-membered ring-expanded erythronolide B related macrolide reported previously as a minor component of S. erythraea WT fermentations (Wilkinson et al., 2000); the compound with m/z=477.5 (8) is consistent with the presence of an 18-membered, doubly ring-expanded erythronolide B related macrolide (see FIG. 8). It is clear to one skilled in the art that such new products can be converted to antibacterial molecules by biotransformation with an appropriate organism, or through the fermentation of the strain S. erythraea WT/421. It is further clear to one skilled in the art that the inclusion of such a module into other positions of the erythromycin PKS or into other PKSs may allow the production of novel, ring expanded polyketides in a similar manner.

[0244] An alternative strategy for generating this hybrid PKS is to incorporate the borrelidin module 5 in place of erythromycin module 4 within a large plasmid that contains the entire hybrid PKS, followed by transformation of an eryA.sup.- S. erythraea strain. Such an appropriate existing eryA.sup.- is S. erythraea JC2 (Rowe et al., 1998) and the plasmid containing the eryA genes under the actI promoter, pIB023 that also contains a thiostrepton resistance gene and the actII-ORF4 activator. This strategy is accomplished as follows:

[0245] pIB023 is digested with NdeI and BsmI and the 13.4 kbp fragment is cloned into pCJM419 digested with NdeI and BsmI to give plasmid pCJM425. pIB023 is digested with BbvCI and XbaI and the approx. 6 kbp fragment is cloned into pCJM425 digested with BbvCI and XbaI to give plasmid pCJM426. The NdeI/XbaI fragment from pCJM426 is cloned into pCJM395 digested with NdeI and XbaI. pCJM395 is a plasmid made by digesting pCJR24 with SbfI, end-polishing with T4 polymerase and religating, to give a version of pCJR24 that does not cut with SbfI. The resulting plasmid, pCJM427, contains an engineered version of the erythromycin PKS in which module 4 is removed. This backbone is then ready to accept any complete module with appropriate flanking sites (SbfI or PstI) to generate a hybrid PKS. Introduction of the single borrelidin module 5 is accomplished by digesting pCJM427 with SbfI, dephosphorylating the backbone with SAP, and ligating in the SbfI fragment from pCJM416, to give pCJM430.

[0246] Plasmid pCJM430 is used to transform S. erythraea JC2. Integrants are selected for resistance to thiostrepton (50 mg/L) and a number of integrants (typically 5-8) are analysed further by Southern blot to confirm that the strains are correct and to identify the site of integration. The resulting correct strain S. erythraea JC2/430 is cultured under conditions appropriate for the production of erythromycins (Wilkinson et al., 2000) and analysed for the production of novel compounds 7 & 8.

Example 13

Disruption of borB (S. parvulus Tu4055/borB::aac3(IV))

[0247] In order to disrupt borB, an region of 2751 bp containing borB was amplified by PCR using primers B5B (5-'AACTAGTCCGCAGTGGACCG-3') (SEQ ID No.91) and B5A (5'-TCGATATCCTCACCGCCCGT-3') (SEQ ID No.92) and cosmid Bor32A2 as template. The PCR product was purified and then digested at the flanking sites SpeI-EcoRV and subcloned into pSL1180 digested with the same restriction enzymes to generate pSLB. A SpeI-AgeI fragment (the latter site internal to the insert) from pSLB containing the 5'-end of borB was subcloned into the SpeI-XmaI sites of pEFBA, upstream of the apramycin resistance gene aac(3)IV, to produce pEB1. A BsaAI-EcoRV fragment (the former site internal to the insert) from pSLB containing the 3'-end of borB was then subcloned in the correct orientation into the EcoRV site of pEB1 downstream of aac(3)IV, to generate pEB2. In this way a 741 bp AgeI-BsaAI fragment internal to borB was deleted and replaced by aac(3)IV. Finally, the SpeI-EcoRV fragment was rescued from pEB2 and subcloned, together with a PstI-SpeI fragment containing the hyg gene from pLHyg, into the PstI-EcoRV sites of pSL1180 to generate pSLBr1. This approach was used in order to avoid possible polar effects.

[0248] The vector pSLBr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borB::aac3(IV). Strain S. parvulus Tu4055/borB::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was observed and compared to a wild type control. In addition S. parvulus Tu4055/borB::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

Example 14

Disruption of borC (S. parvulus Tu4055/borC::aac3(IV))

[0249] In order to disrupt borC, an region of 3553 bp containing borC was amplified by PCR using primers B6B (5'-AACTAGTGTGGCAGACGGTC-3') (SEQ ID No.93) and B5A (5'-TCGATATCCTCACCGCCCGT-3') (SEQ ID No.94) and cosmid Bor32A2 as template. The PCR product was purified and then digested with SpeI-EcoRV and subcloned into the same restriction sites of pSL1180 to produce pSLC. The SpeI-SphI and Ball-EcoRV fragments from this plasmid pSLC, containing the 5'-end and the 3'-end of borC respectively, were then cloned stepwise into the SpeI-SphI and EcoRV sites of pEFBA and in the correct orientations. In this way a 302 bp SphI-Ball internal fragment of borC was replaced by the aac(3)IV gene. The resulting plasmid was then digested with SpeI and EcoRV and the resulting fragment was subcloned together with the hyg gene as described above, into pSL1180 leading to the final construct pSLCr1. This approach was used in order to avoid possible polar effects.

[0250] The vector pSLCr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borC::aac3(IV). Strain S. parvulus Tu4055/borC::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borC::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

[0251] To verify that no polar effects were introduced a full-length copy of borC under the control of the ermE* promoter was introduced in trans to the disrupted mutant. Full-length borC was amplified by PCR using the primers B6T1 (5'-CGGATGCATCACCGGCACGG-3') (SEQ ID No.95) and B6T2 (5-TGGGATCCGCGGGGCGGTAC-3') (SEQ ID No.96) using cosmid Bor32A2 as template. The 943 bp product was purified and then digested with NsiI-BamHI and subcloned, together with a BamHI-SpeI fragment from pLHyg (carrying the hyg gene), into pIJ2925 previously digested with PstI-XbaI. A BglII fragment (using this site from the vector) was then isolated and subcloned into pEM4, and in the correct orientation to locate borC under the control of the promoter ermE*. Plasmid pborCH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borC::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borC::aac(3)IV/pborCH was analysed as described in example 1 and shown to produce borrelidin at a titre similar to a WT control.

Example 15

Disruption of borD (S. parvulus Tu4055/borD::aac3(IV))

[0252] In order to disrupt borD, a fragment of 2777 bp was amplified by PCR using the primers BBB (5'-AACTAGTGCGATCCCGGGGA-3') (SEQ ID No.97) and BBA (5'-CGTCGATATCCTCCAGGGGC-3') (SEQ ID No.98) and cosmid Bor32A2 as template. The PCR product was purified and then digested with SpeI-EcoRV and subcloned into pSL1180 to generate pSLD. This was then digested with NdeI-StuI to delete an internal 679 bp region of borD which was replaced by a SmaI-NdeI fragment isolated from pEFBA containing the aac(3)IV gene. The resulting construct was digested with SpeI-EcoRV and the 4.3 kb fragment subcloned together with a SpeI-PstI fragment from pLHyg containing the hyg gene, into pSL1180 digested with PstI-EcoRV. This step leads to the final plasmid pSLDr1. This approach was used in order to avoid possible polar effects.

[0253] The vector pSLDr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borD::aac3(IV). Strain S. parvulus Tu4055/borD::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borD::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

[0254] To verify that no polar effects were introduced a full-length copy of borD under the control of the ermE* promoter was introduced in trans to the disrupted mutant. Full-length borD was amplified by PCR using the primers BBT1 (5'-TACTGCAGCACACCCGGTGC-3') (SEQ ID No.99) and BBT2 (5'-TGGGATCCGCTGTGTCATAT-3') (SEQ ID No.100) using cosmid Bor32A2 as template. The 816 bp PCR product was purified and then digested with PstI-BamHI and subcloned together with a BamHI-SpeI fragment containing the hyg gene from pLHyg, into pIJ2925 digested with PstI-XbaI, to give pIJDH. The BglII fragment from pIJDH (using these sites from the vector) was then subcloned into pEM4 (predigested with BamHI) and in the correct orientation to generate pborDH. Plasmid pborDH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borD::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borD::aac(3)IV/pborDH was analysed as described in example 1 and shown to produce borrelidin at a titre similar to a WT control.

Example 16

Disruption of borE (S. parvulus Tu4055/borE::aac3(IV))

[0255] In order to disrupt borE, an Internal 761 bp fragment of the gene was amplified by PCR using primers B25A (5'-TTCTGCAGCCGCGGCCTTCG-3') (SEQ ID No.81) and B25B (5'-AGAATTCGCCGGCGCCGCTG-3') (SEQ ID No.82) using cosBor32A2 as template. The product was purified, digested PstI-EcoRI and cloned into pOJ260ermE* which had been digested similarly, to provide pOJEd1. This approach was used in order to avoid possible polar effects. The vector pOJEd1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5, and colonies were selected for apramycin resistance on R5 and then on MA agar. The disruption was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borE::aac3(IV). Strain S. parvulus Tu4055/borE::aac3(IV) was grown, extracted and analysed as described in example 1. No borrelidin production was observed whereas a wild type control produced borrelidin as expected.

[0256] To verify that no polar effects were introduced a full-length copy of borE under the control of the ermE* promoter was introduced in trans to the disrupted mutant. Full-length borE was amplified by PCR using the primers B7T1 (5'-GGCTGCAGACGCGGCTGAAG-3') (SEQ ID No.83) and B7T2 (5'-CCGGATCCCAGAGCCACGTC-3') (SEQ ID No.84) using cosBor32A2 as template. The 1216 bp product was purified, digested with PstI-BamHI and cloned into PstI-XbaI digested pIJ2925 (Janssen & Bibb, 1993), along with a BamHI-SpeI digested fragment from pLHyg containing the hygromycin resistance cassette, to generate pIJEH. A 2.8 kbp BamHI fragment was excised from pIJEH and cloned into pEM4 (Quiros et al., 1998), which had been digested similarly, to give pborEH (in which the borE gene was cloned in the correct orientation for gene expression). pborEH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borE::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borE::aac(3)IV/pborEH was analysed as described in example 1 and shown to produce borrelidin at a titre similar to a WT control; the control strain S. parvulus Tu4055/borE::aac(3)IV/pEM4 did not produce borrelidin.

[0257] Chemical complementation of S. parvulus Tu4055/borE::aac3(IV) with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1, demonstrated that the strain thus grown was capable of borrelidin production at 122.+-.23% of the WT parent control. Thus, borE is required for biosynthesis of trans-cyclopentane-1,2-dicarboxylic acid.

Example 17

Disruption of borF (S. parvulus Tu4055/borF::aac3(IV))

[0258] In order to disrupt borF, a region containing borF was amplified by PCR using the primers BCB (5'-CACTAGTCCTCGCCGGGCAC-3') (SEQ ID No.101) and BCA (5'-GAGGATCCCGGTCAGCGGCA-3') (SEQ ID No.102) and cosmid Bor32A2 as template. The resulting 2132 bp product was purified and then digested with SpeI-BamHI and subcloned into the same sites of pSL1180 leading to pSLF. The aac(3)IV gene from pEFBA was then subcloned as a SphI fragment into the SphI site of pSLF, which is located inside the borF coding region. Finally the BamHI-SpeI fragment was subcloned into pLHyg digested with BamHI-NheI to generate pLHFr1.

[0259] The vector pLHFr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borF::aac3(IV). Strain S. parvulus Tu4055/borF::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borF::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

[0260] To verify that no polar effects were introduced a full-length copy of borF under the control of the ermE* promoter was introduced in trans to the disrupted mutant. Full-length borF was amplified by PCR using the primers BCT1 (5'-GCCTGCAGCGACCTCGCCGG-3') (SEQ ID No.103) and BCT2 (5'-CGGGATCCCGTGGCGTGGTC-3') (SEQ ID No.104) using cosmid Bor32A2 as template. The 1048 bp PCR product was purified and then digested with PstI-BamHI and subcloned together with the hyg gene as described above, into pIJ2925. A BglII fragment was then isolated and subcloned into pEM4 to generate pborFH. This was used to complement strain SPMF. Plasmid pborFH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borF::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borF::aac(3)IV/pborFH was analysed as described in example 1 and shown to produce borrelidin at a titre similar to a WT control.

Example 18

Disruption of borG (S. parvulus Tu4055/borG::aac3(IV))

[0261] In order to disrupt borG, an internal region of 885 bp was amplified by PCR using the primers B23A (5'-ATCTGCAGCGGCATCGGTGT-3') (SEQ ID No.105) and B23B (5'-AGAATTCTCCACTGCGGTCG-3') (SEQ ID No.106) and cosmid Bor32A2 as template. The resulting product was purified and the digested at the flanking sites PstI-EcoRI and then subcloned into pOJ260P, downstream of the promoter ermE*, to generate pOJGd1.

[0262] The vector pOJGd1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected on MA agar. The disruption was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borG::aac3(IV). Strain S. parvulus Tu4055/borG::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borG::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

Example 19

Disruption of borH (S. parvulus Tu4055/borH::aac3(IV))

[0263] In order to disrupt borH, and internal region of 697 bp was amplified by PCR using the primers B9A (5'-ACCTGCAGGCCGGGCTCATC-3') (SEQ ID No.107) and B9B (5'-AGAATTCGGGCGAGCCGCCG-3') (SEQ ID No.108) and cosmid Bor32A2 as template. The resulting PCR product was purified and then digested with PstI-EcoRI and then subcloned into pOJ260P, downstream of the promoter ermE*, to generate pOJHd2.

[0264] The vector pOJHd2 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected on MA agar. The disruption was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borH::aac3(IV). Strain S. parvulus Tu4055/borH::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borH::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

Example 20

Disruption of borI (S. parvulus Tu4055/borI::aac3(IV))

[0265] The gene borI and surrounding DNA was amplified from cosBor19B9 using the PCR primers BP4501 (5'-CGTATGCATGGCGCCATGGA-3') (SEQ ID No.85) and BP4502 (5'-AGCCAATTGGTGCACTCCAG-3') (SEQ ID No.86). The 2.32 kbp product was purified, digested with NsiI-MfeI and cloned into pSL1180 digested NsiI-EcoRI, to give plasmid pSLI. The apramycin resistance cassette was excised from pEFBA as an EcoRI fragment and cloned into pSLI digested with EcoRI, to give the plasmid pSLIA. Finally, the hygromycin resistance cassette was excised SpeI-PstI from pLHyg and cloned into pSLIA which had been digested with NsiI-SpeI to give plasmid pSLIr1.

[0266] The replacement vector pSLIr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin (25 .mu.g/ml) were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borI::aac3(IV).

[0267] S. parvulus Tu4055/borI::aac3(IV) was grown and analysed as described in example 1. No borrelidin production was observed whereas several new compounds were observed at significantly lower levels. One of the less polar compounds displayed a UV absorbance maximum of 240 nm, and LCMS analysis indicated an m/z ratio 11 mass units lower than that for borrelidin, which is consistent with the presence of a methyl- rather than a nitrile-group at C12.

[0268] To verify that no polar effects were introduced a full-length copy of borI under the control of the ermE* promoter was introduced in trans to the disrupted mutant. A 2.1 kb NsiI-AvrII fragment containing borI was recovered from pSLI and subcloned into the PstI-XbaI sites of pEM4, together with the NheI-SpeI fragment from pLHyg containing the hyg gene. Both fragments were subcloned in the same orientation generating pborIH. Plasmid pborIH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borI::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borI::aac(3)IV/pborIH was analysed as described in examples 1 & 4, and shown to produce borrelidin at a titre similar to a WT control.

Example 21

Disruption of borJ (S. parvulus Tu4055/borJ::aac3(IV))

[0269] The gene borJ and surrounding DNA was amplified from cosBor19B9 using the PCR primers BNHT1 (5'-GTCATGCATCAGCGCACCCG-3') (SEQ ID No.87) and BNHT2 (5'-GTGCAATTGCCCTGGTAGTC-3') (SEQ ID No.88). The 2.75 kbp product was purified, digested with NsiI-MfeI and cloned into pSL1180 that had been digested with NsiI-EcoRI, to give plasmid pSL. The hygromycin resistance cassette was excised from pLHyg as a PstI-SpeI fragment and cloned into pSL digested with NsiI-SpeI, to give pSLJH. Finally, the apramycin resistance cassette was excised from pEFBA with SpeI-BamHI and cloned into pSLJH that had been pre-digested with AvrII-BglII in order to remove a 453 bp fragment from borJ, to give plasmid pSLJr1.

[0270] The replacement vector pSLJr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin (25 .quadrature.g/ml) were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation. The new mutant was named S. parvulus Tu4055/borJ::aac3(IV).

[0271] S. parvulus Tu4055/borJ::aac3(IV) was grown and analysed as described in example 1. No borrelidin production was observed whereas a new compound more polar than borrelidin was observed with a UV maximum at 262 nm. LCMS analysis indicated a parent compound of 508 amu, which is consistent with a carboxylic acid rather than a nitrile function at C12.

[0272] To verify that no polar effects were introduced a full-length copy of borJ under the control of the ermE* promoter was introduced in trans to the disrupted mutant. A 2.4 kb NsiI-SphI fragment from pSLJ containing borJ was subcloned into the PstI-XbaI sites of pEM4, together with the hyg gene as a SphI-SpeI fragment from pLHyg; both fragments were subcloned in the same orientation as the transcription of the genes. The final construct was designed pborJH. Plasmid pborJH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borJ::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borJ::aac(3)IV/pborJH was analysed as described in examples 1 & 4, and shown to produce borrelidin at a titre similar to a WT control.

Example 22

Disruption of borK (S. parvulus Tu4055/borK::aac3(IV))

[0273] In order to disrupt borK, a fragment of 2680 bp was amplified by PCR using the primers B231 (5'-ATCAAGCTTCGTGTCCATGG-3') (SEQ ID No.109) and B232 (5'-GTCATGCATCAGGCGTTCGG-3') (SEQ ID No.110) and cosmid Bor19B9 as template. The resulting PCR product was purified and then digested with HindIII-NsiI and subcloned into the same sites of pSL1180 to produce pSLK. After MluI digestion of pSLK and treatment with the Klenow fragment, the aac(3)IV gene from pEFBA was subcloned as a SmaI-EcoRV fragment leading to pSLKa. Finally a PstI-SpeI fragment from pLHyg containing the hyg gene was subcloned into pSLKa digested NsiI-XbaI to obtain pSLKr1.

[0274] The vector pSLKr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borK::aac3(IV). Strain S. parvulus Tu4055/borK::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borK::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

[0275] To verify that no polar effects were introduced a full-length copy of borK under the control of the ermE* promoter was introduced in trans to the disrupted mutant. A 2.2 kb BglII (blunt-ended)-NsiI fragment from pSLK was subcloned, together with a 1.6 kb PstI-SpeI fragment from pLHyg containing the hyg gene, into pEM4 digested with PstI (treated with the Klenow fragment) and then XbaI. The final vector was named pborKH. Plasmid pborKH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borK::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borK::aac(3)IV/pborKH was analysed as described in examples 1 & 4, and shown to produce borrelidin at a titre similar to a WT control.

Example 23

Disruption of borL (S. parvulus Tu4055/borL::aac3(IV))

[0276] In order to disrupt borL a 3.95 kbp BglII fragment of cosBor19B9, which contained the full-length borL, was sub-cloned into pSL1180 digested similarly. The resulting clones were analysed by restriction digest and one that displayed the correct orientation was chosen to provide pSL395. Digestion of pSL395 with NheI and SpeI, and subsequent re-ligation to eliminate a fragment of borM that included a BglII site, gave pSLL. The apramycin resistance cassette was excised with KpnI from pEFBA (Lozano et al., 2000) and cloned into pSL that had been digested with KpnI, to give pSLLA. pSLLA was digested with BglII and then subjected to Klenow treatment following the manufacturers instructions (Roche); an EcoRV fragment isolated from pLHyg containing the hygromycin resistance cassette was then cloned into this prepared vector to give pSLLr1.

[0277] The replacement vector pSLLr1 was introduced into S. parvulus Tu4055 by protoplast transformation. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation. The new mutant was named S. parvulus Tu4055/borL::aac3(I V).

[0278] Strain S. parvulus Tu4055/borL::aac3(IV) was grown, extracted and analysed as described in example 1. No borrelidin production was observed whereas a wild type control produced borrelidin as expected. Chemical complementation of S. parvulus Tu4055/borL::aac(IV) using the natural starter acid as described in example 1 showed that the strain thus grown was capable of borrelidin production at 408.+-.70% of the WT parent control titre.

[0279] To verify that no polar effects were introduced a full-length copy of borL under the control of the ermE* promoter was introduced in trans to the disrupted mutant. The vector containing full-length borL was generated as described in example 30. Plasmid pborLH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borL::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borL::aac(3)IV/pborLH was analysed as described in example 1.

Example 24

Disruption of borM (S. parvulus Tu4055/borM::aac3(IV))

[0280] In order to disrupt borM, a 2870 bp fragment containing borM was amplified by PCR using the primers B251 (5'-CTTCTAGATGAACCCCTCCA-3') (SEQ ID No.111) and B252 (5'-GGGCAATTGCGCGGCAGCTT-3') (SEQ ID No.112) and cosmid Bor19B9 as template. The resulting product was purified and then digested with XbaI-MfeI and subcloned into the XbaI-EcoRI sites of pSL1180, leading to pSLM. An internal 780 bp SphI-NheI fragment of borM was then replaced by the aac(3)IV gene which was subcloned from pEFBA as a SpeI-XbaI fragment, leading to pSLMA. pSLMA was digested with NsiI-XbaI and the hyg gene subcloned as a SpeI fragment from pLHyg to generate pSLMr1.

[0281] The vector pSLMr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borM::aac3(IV). Strain S. parvulus Tu4055/borM::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borM::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

[0282] To verify that no polar effects were introduced a full-length copy of borM under the control of the ermE* promoter was introduced in trans to the disrupted mutant. Full-length borM was cloned as a XbaI-AgeI fragment of 2.0 kb from pSLM and subcloned into the EcoRI (end-filled with Klenow)-XbaI sites of pEM4 together with the hyg gene as a XmaI-EcoRV fragment from pLHyg, to give pborMH. Plasmid pborMH and the control plasmid pEM4 were introduced into S. parvulus Tu4055/borM::aac(3)IV by protoplast transformation as described in example 5. The resulting strain S. parvulus Tu4055/borM::aac(3)IV/pborMH was analysed as described in example 1 and shown to produce borrelidin at a titre similar to a WT control.

Example 25

Disruption of borN (S. parvulus Tu4055/borN::aac3(IV))

[0283] In order to disrupt borN, a 1201 bp BamHI fragment from pSLM (containing the 3'-end of borM and the first 161 codons of borN) was subcloned into the BglII-BamHI sites of pSL1180 and in the correct orientation, to generate pSLMN. A BamHI-EcoRI fragment (using these sites from the polylinker) containing borO from pborOR (see below) was subcloned into the BamHI-EcoRI sites of pSLMN, generating pSLNO. After EcoRI digestion of pSLNO and end-filling with Klenow fragment, the hyg gene was subcloned from pLHyg as a EcoRV fragment, leading to pSLNOH. Finally the aac3(IV) gene was subcloned as a NcoI-BamHI fragment from pEFBA into pSLNOH digested with the same restriction enzymes, generating pSLNr1.

[0284] The vector pSLNr1 was introduced into S. parvulus Tu4055 by protoplast transformation as described in example 5. Colonies resistant to apramycin were selected, and then passaged several times through MA media without selection. The replacement was verified by Southern hybridisation and the new mutant was named S. parvulus Tu4055/borN::aac3(IV). Strain S. parvulus Tu4055/borN::aac3(IV) was grown, extracted and analysed as described in example 1. Borrelidin production was compared to a wild type control. In addition, S. parvulus Tu4055/borN::aac3(IV) was chemically complemented with trans-1,2-dicyclopentane dicarboxylic acid, following the protocol described in example 1.

Example 26

Heterologous Expression of borO in Streptomyces albus J1074

[0285] In order to examine whether the putative resistance protein BorO confers resistance to a borrelidin-sensitive organism, borO was expressed in Streptomyces albus J1074. The gene borO was amplified by PCR using the primers BTRNAS1 (5'-TGTCTAGACTCGCGCGAACA-3') (SEQ ID No.89) and BTRNAS2 (5'-TGAATTCCGAAGGGGGTGGT-3') (SEQ ID No.90) with cosBor19B9 as template. The product was purified, digested XbaI-EcoRI and cloned into pEM4A that had been similarly digested to give plasmid pborOR which puts borO under the control of the promoter ermE*. The vector pborOR was introduced into S. albus J1074 by protoplast transformation (Chater & Wilde, 1980) and selected for apramycin resistance. The new strain was named S. albus J1074/pborOR.

[0286] Resistance to borrelidin was assayed on Bennett's agar containing apramycin at 25 .mu.g/ml. Spores of S. albus J1074/pborOR and the control S. albus J1074/pEM4A were spread onto plates and then disks containing borrelidin at 100 & 200 .mu.g/ml were laid upon the lawn of spores and incubated overnight at 30.degree. C. Haloes indicating inhibition of growth were observed for the control strain harbouring pEM4A but not for S. albus J1074/pborOR.

Example 27

Disruption of borG and borI (S. parvulus Tu4055/borG::aac3(IV)/borI::hyg)

[0287] The hyg gene is isolated from pLHyg as an EcoRV fragment and cloned into pSLI (example 20) digested with EcoRI and treated with Klenow fragment to give pSLIH; the hyg gene is cloned in the same orientation as borI. pSLIH is introduced into S. parvulus Tu4055/borG::aac3(IV) by protoplast transformation, as described in example 5, and selected for both apramycin and hygromycin resistance, and is then passaged several times through MA media without selection in order to promote double recombination. Apramycin and hygromycin resistant colonies are analysed by Southern hybridisation and PCR to verify the replacement.

Example 28

Disruption of borG and borJ (S. parvulus Tu4055/borG::aac3(IV)/borJ::hyg)

[0288] The hyg gene is isolated from pLHyg as an EcoRV fragment and cloned into pSLJ (example 21) digested with AvrII-BglII and treated with Klenow, to give pSLJH; the hyg gene is cloned in the same orientation as borI. pSLJH is introduced into S. parvulus Tu4055/borG::aac3(IV) by protoplast transformation, as described in example 5, and selected for both apramycin and hygromycin resistance, and is then passaged several times through MA media without selection in order to promote double recombination. Apramycin and hygromycin resistant colonies are analysed by Southern hybridisation and PCR to verify the replacement.

Example 29

Effects of borE Up-Regulation in S. parvulus Tu4055

[0289] To examine the possibility that biosynthesis of the trans-1,2-cyclopentane dicarboxylic acid starter unit may have a limiting effect upon borrelidin production, borE was up-regulated in the parental strain and the effect upon borrelidin titre was analysed. The vector used, pborEH was described in example 16.

[0290] The vectors pborEH and pEM4 (control) were used to transform protoplasts of S. parvulus Tu4055 to give strains S. parvulus Tu4055/pborEH and S. parvulus Tu4055/pEM4 respectively. Several colonies from each transformation were picked, grown in triplicate and then analysed as described in example 1. Compared to the control strain, up-regulation of borE brought about a 4.2.+-.0.3-fold increase in the titre of borrelidin.

Example 30

Effects of borL Up-Regulation in S. parvulus Tu4055

[0291] To examine the possibility that borL may have a regulatory, or some other related function involved in borrelidin production, the gene was up-regulated in the parental strain and the effect upon borrelidin titre was analysed.

[0292] The expression vector pborLH was generated as follows: pSLL was digested with NotI, treated with Klenow fragment and then digested with BamHI to obtain a fragment of 2190 bp containing borL. This fragment was sub-cloned together with the BamHI-SpeI hyg gene from pLHyg, into pEM4 digested with PstI (treated with Klenow)-XbaI, to obtain pborLH.

[0293] The vectors pborLH and pEM4 (control) were used to transform protoplasts of S. parvulus Tu4055 to give strains S. parvulus Tu4055/pborLH and S. parvulus Tu4055/pEM4 respectively. Several colonies from each transformation were picked, grown in triplicate and then analysed as described in example 1. Compared to the control strain, up-regulation of borL brought about a 4.3.+-.0.7-fold increase in the titre of borrelidin.

Example 31

Production of 12-desnitrile-12-methyl Borrelidin 14 (Pre-Borrelidin)

[0294] Working stocks of S. parvulus Tu4055/borI::aac3(IV) (0.5 ml) were inoculated into primary vegetative pre-cultures of NYG as described in example 1. Secondary pre-cultures were prepared (as example 1 but with 250 ml NYG in 2 l Erlenmeyer flasks). PYDG production medium (4 l), prepared as in example 1 and with 0.01% Plutronic L0101 added to control foaming, was inoculated with secondary pre-culture (12.5% inoculum). A second fermenter containing centre-point medium (4 l) and 0.01% Plutronic L0101 to control foaming, was set up in parallel and was also inoculated with secondary pre-culture (12.5% inoculum). Centre-point production medium contains per litre of deionised water: Tesco's skimmed milk powder (1.5%), Avidex W-80 (4.5%), glucose (0.5%) and yeast autolysate (0.15%) adjusted to pH 7.0 with 5 M NaOH.

[0295] These batches were each allowed to ferment in a 7 l Applikon fermenter for 6.5 days at 30.degree. C. Airflow was set at 0.75 vvm (volume per volume per minute), with tilted baffles and the impeller speed controlled between 400 and 800 rpm to maintain dissolved oxygen tension at or above 30% of air saturation. No further antifoam was added. At 22 hours into the fermentation the starter acid, trans-cyclopentane-1,2-dicarboxylic acid, was added as a neutralised solution of 1:1 MeOH/5 M NaOH, through an in-line filter (0.22 .mu.m). The final concentration in the fermenter vessel of exogenous starter acid was 0.5 mM.

[0296] After 6.5 days of fermentation the broths were combined and acidified to pH 3.5 with concentrated HCl (.about.6 ml), then clarified by centrifugation at 3,500 rpm for 10 minutes. The supernatant was extracted into ethyl acetate (3.times.1 volume equivalent for 4 hours each) and the cell pellet left to steep in methanol (2.times.1.5 litres for 4 hours each). The organics were combined and removed under reduced pressure to yield a tarry gum. The gum was re-suspended in 0.1 M Borax buffer (500 ml at pH 9.4) and washed with hexanes (500 ml) and ethyl acetate (500 ml). The aqueous layer was then acidified with concentrated HCl to pH 3.5 and extracted with ethyl acetate (3.times.500 ml), which were combined and taken to dryness. The resultant gum was dissolved in methanol (15 ml), diluted with water (285 ml) and loaded under gravity onto a C.sub.18-reversed-phase cartridge (50 g, prepared in 5% aqueous methanol). The cartridge was washed with 20% and 50% aqueous methanol (300 ml each) and eluted with 100% methanol (500 ml). This last fraction was taken to dryness under reduced pressure to yield a black gummy-oil (600 mg) that was taken up in methanol. This residue was finally purified by sequential preparative reversed-phase HPLC (eluted with the mobile phases used in example 4, without added TFA, running isocratically at 40% B). Active fractions were combined and desalted on a C.sub.18-cartridge (1 g), to yield 28 mg of a dark oil (3.5 mg/l isolated yield). Table 12 summarises the .sup.1H and .sup.13C NMR chemical shift data for 12-desnitrile-12-methyl borrelidin 14 in CDCl.sub.3.

TABLE-US-00012 TABLE 12 Position .delta..sub.H (ppm) Multiplicity Coupling (Hz) .delta..sub.c (ppm) 1 -- -- -- 174.5 2a 2.29 m -- 37.8 2b 2.26 m -- -- 3 3.85 dt 9.0, 3.0 71.9 4 1.83 m -- 35.1 5a 1.19 bt 13.5 43.6 5b 0.91 m -- -- 6 1.75 m -- 27.0 7a 1.08 m -- 49.2 7b 0.88 m -- -- 8 1.69 m -- 26.5 9a 0.97 m -- 38.3 9b 0.45 t 12.5 -- 10 1.62 m -- 34.1 11 3.53 d 9.0 85.7 12 -- -- -- 138.4 13 5.84 d 11.0 127.7 14 6.28 ddd 14.5, 11.0, 1.0 129.6 15 5.48 ddd 14.5, 10.5, 3.5 129.9 16a 2.53 m -- 39.1 16b 2.22 m -- -- 17 5.07 ddd 11.0, 8.0, 3.0 76.5 18 2.52 m -- 48.0 19a 1.92 m -- 30.4 19b 1.32 m -- -- 20a 1.74 m -- 26.2 20b 1.71 m -- -- 21a 1.96 m -- 32.0 21b 1.84 m -- -- 22 2.45 m 8.0 49.3 23 -- -- -- 182.3 4-CH.sub.3 0.78 d 6.5 18.5 6-CH.sub.3 0.77 d 6.5 18.8 8-CH.sub.3 0.75 d 6.5 20.6 10-CH.sub.3 0.94 d 6.5 16.3 12-CH.sub.3 1.64 s -- 11.4 Chemical shifts are referenced to CDCl.sub.3 (for .sup.1H at 7.26 ppm and for .sup.13C at 77.0 ppm)

Example 32

Production of 12-desnitrile-12-carboxy Borrelidin 2

[0297] Working stocks of S. parvulus Tu4055/borJ::aac3(IV) (0.5 ml) were inoculated into primary vegetative pre-cultures of NYG as described in example 1. Secondary pre-cultures were prepared (as example 1 but with 250 ml NYG in 2 l Erlenmeyer flasks). PYDG production media (4 L), prepared as in example 1 and with 0.01% Plutronic L0101 added to control foaming, was inoculated with the entire secondary pre-culture (10% inoculum). This was allowed to ferment in a 7 L Applikon fermenter for 6 days at 30.degree. C. Airflow was set at 0.75 vvm, with tilted baffles and the impeller speed controlled between 250 and 600 rpm to maintain dissolved oxygen tension at or above 30% of air saturation. No further antifoam was added. A second fermentation was performed exactly as above, but which was batch fed with 0.2 mol of glucose as an aqueous solution every 12 hours from 60 hours post-inoculation.

[0298] After 6 days the fermentations were harvested and combined. The broth was clarified by centrifugation (3,500 rpm, 10 minutes) and the resultant supernatant acidified with 10 M HCl (aq) to pH .about.3.5. This solution was then extracted into ethyl acetate by stirring (3.times.1 volume equivalent for 4 hours each). The cell pellet was extracted twice by steeping the cells in 1:1 methanol/ethyl acetate (500 ml). All the organics were combined and removed under reduced pressure to yield an aqueous slurry. The slurry was diluted to 500 ml with water, acidified to pH .about.3.5 with 10 M HCl and extracted into ethyl acetate (3.times.300 ml). The organics were concentrated under reduced pressure to .about.300 ml and extracted with 0.1 M borax (3.times.150 ml, pH=9.4). The combined borax solutions were acidified with 10 M HCl to pH .about.3.5 and extracted with 6.times.300 ml of ethyl acetate. Analytical HPLC demonstrated that some of the accumulant still resided in the borax solution and so this was loaded, under gravity, onto a C.sub.18-reverse-phase cartridge (50 g). The cartridge was washed with water and the accumulant eluted in 100% methanol. The organics containing the accumulant were combined and reduced to a 40 ml methanolic solution. This was loaded onto a Sephadex LH-20 column (70 g, swelled overnight in methanol, column 60 cm.times.2.5 cm), which was developed with 100% methanol; the active fractions were combined and taken to dryness. The material was then further processed by preparative reversed-phase HPLC (eluted with the mobile phases used in example 4, without added TFA, running isocratically at 40% B). The combined active fractions were taken to dryness, dissolved in methanol (4 ml) and diluted with water (200 ml). This mixture was split into 2 equal fractions and each loaded, under gravity, onto a C.sub.18-reverse-phase cartridge (20 g). The columns were then eluted with 3 column volumes of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 75%, 90% and 100% aqueous methanol. The accumulant eluted in all fractions from 60% to 100% methanol, which were combined and taken to dryness. The accumulant (dissolved in DMSO) was then finally purified by sequential preparative reversed-phase HPLC (eluted with the mobile phases used in example 4, without added TFA, running isocratically at 40% B). Active fractions were combined and desalted on a C.sub.18-cartridge (1 g), to yield 17 mg of a brown oil (2.1 mg/l isolated yield). Table 13 summarises the .sup.1H and .sup.13C NMR chemical shift data for 12-desnitrile-12-carboxy borrelidin 2 in d.sub.4-methanol.

TABLE-US-00013 TABLE 13 Position .delta..sub.H (ppm) Multiplicity Coupling (Hz) .delta..sub.C (ppm) 1 -- -- -- 173.27 2a 2.40 dd 15.8, 4.1 39.31 2b 2.29 dd 15.8, 8.2 3 3.87 m 71.64 4 1.80 m 36.51 5a 1.29 m 44.24 5b 0.90 m 6 1.59 m 27.48 7a 1.09 m ~49.0* 7b 1.03 m 8 1.72 m 28.17 9a 1.12 m 38.42 9b 0.79 m 10 2.03 m 36.43 11 3.90 m 81.95 12 -- -- -- 132.35 13 6.43 d 11.0 140.83 14 6.96 dd 14.5, 11.5 130.91 15 5.91 ddd 15.0, 9.5, 5.0 138.93 16a 2.61 m 15.0 38.57 16b 2.36 m 17 5.04 m 77.40 18 2.50 m 49.80 19a 1.90 m 30.59 19b 1.32 m 20a 1.85 m 26.34 20b 1.41 m 21a 1.97 m 32.40 21b 1.75 m 22 2.52 m ~48.0* 23 -- -- -- 180.27 4-CH.sub.3 0.83 d 7.0 18.76 6-CH.sub.3 0.80 d 6.0 17.06 8-CH.sub.3 0.81 d 6.5 20.60 10-CH.sub.3 0.93 d 6.5 16.61 12-CO.sub.2H -- -- -- 170.49 Chemical shifts are referenced to methanol (for .sup.1H at 3.35 ppm (quintet) and for .sup.13C at 49.0 ppm (septet)); *Obscured by solvent signal, d.sub.4-methanol.

Example 33

Production by Mutasynthesis of 17-des-(cyclopentane-2'-carboxylic acid)-17-(cyclobutane-2'-carboxylic acid)borrelidin 18

[0299] Working stocks of S. parvulus Tu4055/borE::aac3(IV) (0.5 ml) were inoculated into primary vegetative pre-cultures of NYG as described in example 1. Secondary pre-cultures were prepared (as example 1 but with 250 ml NYG in 2 l Erlenmeyer flasks). PYDG production medium (4 l), prepared as in example 1 and with 0.01% Plutronic L0101 added to control foaming, was inoculated with secondary pre-culture (12.5% inoculum). Two further bioreactors were set up in the same manner. These batches were each allowed to ferment in a 7 l Applikon fermenter for 5 days at 30.degree. C. Airflow was set at 0.75 vvm (volume per volume per minute), with tilted baffles and the impeller speed controlled between 400 and 700 rpm to maintain dissolved oxygen tension at or above 30% of air saturation. No further antifoam was added. At 22 hours into the fermentation the starter acid, trans-cyclobutane-1,2-dicarboxylic acid, was added as a neutralised solution of 1:1 MeOH 15 M NaOH. The final concentration in the fermenter vessel of exogenous starter acid was 0.5 mM.

After 5 days of fermentation the broths were combined and acidified to pH 4.0 with concentrated HCl, then clarified by centrifugation at 3,500 rpm for 10 minutes. The supernatant was absorbed onto diaion HP-20SS resin (1 l), which had been pretreated with methanol (2 l) and then 5% aqueous methanol (2 l), by filtration at a rate of approximately 100 ml/min. The resin was then eluted with 20% aqueous methanol (2.5 l) and then 80% aqueous acetone (4.5 l). The organic solvent was removed from the aqueous acetone and the resultant aqueous slurry (1 litre) extracted into ethyl acetate (3.times.1 l). The organics were combined and reduced in vacuo to yield a yellow/brown oil (1.7 g). Meanwhile, the cell pellet left to steep in methanol-ethyl acetate, 1:1 (3.times.1 l for 4 hours each), and the resultant organic supernatants reduced in vacuo to yield an aqueous slurry (400 ml). The particulate matter was dissolved in methanol (50 ml), and added back to the aqueous slurry, which was made up to 500 ml with water. This slurry was absorbed onto diaion HP-20SS resin (300 ml), that had been pretreated with methanol (500 ml) and then 5% aqueous methanol (500 ml). The resin was then eluted with 20% aqueous methanol (1 l) and then 80% aqueous acetone (1.5 l). The organic solvent was removed from the aqueous acetone and the resultant aqueous slurry (made up to 750 ml) extracted into ethyl acetate (3.times.750 ml). The organics were combined and reduced in vacuo to yield a yellow/brown oil (1.7 g). The crude extracts were combined (3.4 g), dissolved in ethyl acetate (10 ml), then adsorbed onto a silica column (5 cm ID.times.10 cm, treated with EtOAc), and eluted with EtOAc. The active fractions were combined and the solvent removed in vacuo to yield a brown gum (1.08 g). This residue was finally purified by sequential preparative reversed-phase HPLC (eluted with the mobile phases used in example 4, without added TFA, running from 25% B to 75% B over 25 minutes with a linear gradient). Active fractions were combined and desalted on a C.sub.18-cartridge (5 g), to yield 83.9 mg (or 7.0 mg/l isolated yield). The .sup.13C-NMR spectrum of 18 is shown in table 14

TABLE-US-00014 TABLE 14 .delta..sub.C (ppm) Position 177.1 COOH (C22) 172.2 1 144.0 13 138.7 15 126.9 14 118.3 12 115.8 CN 75.5 17 73.1 11 69.7 3 47.6 5 43.1 7 40.1 19 40.0 2 37.3 9 35.7 4 35.1 10 34.4 16 30.9 18 27.3 6 26.2 8 21.7 20 21.0 21 20.1 8-Me 18.1 6-Me 16.9 4-Me 14.9 10-Me

.sup.13C-NMR assignment for 18, in CDCl.sub.3, using that carbon signal as reference at .delta..sub.C=77.7 ppm

REFERENCES

[0300] Anderson, B. F., Herit, A. J., Rickards, R. W., and Robertson, G. B. (1989) Crystal and molecular structures of two isomorphous solvates of the macrolide antibiotic borrelidin: absolute configuration determination by incorporation of a chiral solvent in the in the crystal lattice. Aust J. Chem. 42:717-730. [0301] Anderton, K., and Rickards, R. W. (1965) Some structural features of borrelidin, an anti-viral antibiotic. Nature 206:269. [0302] Aparicio, J. F., Molnar, I., Konig, A., Haydock, S. H., Khaw., L. E., Staunton, J., and Leadlay, P. F. (1996) Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169:9-16. [0303] August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R., Yu, T.-W., Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X. H., Hutchinson, C. R., and Floss, H. G. (1998) Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem. Biol. 5:69-79. [0304] Beck, J. B., Yoon, Y. J., Reynolds, K. A., and Sherman, D. H. (2002) The hidden steps of domain skipping: ring size determination in the pikromycin modular polyketide synthase. Chem. Biol. 9:575-583. [0305] Berger, J., Jampolsky, L. M., and Goldberg, M. W. (1949) Borrelidin, a new antibiotic with anti-Borrelia activity and penicillin enhancement properties. Arc. Biochem. 22:476-478. [0306] Bierman, M., Logan, R., O'Brian, K., Seno, E. T., Rao, N., and Schoner, B. E. (1992) Plasmid vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43-49. [0307] Bonfield, J. K., Smith, K. F., and Staden, R. (1995) A new DNA sequence assembly program. Nucleic Acids Research 23:4992-4999. [0308] Brautaset, T., Sekurova, O. N., Sletta, H., Ellingsen, T. E., Strom, A. R., Valla, S., and Zotchev, S. B. (2000) Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem. Biol. 7:395A403. [0309] Brenner, S. (1998) The molecular evolution of genes and proteins: a tale of two serines. Nature 334:528-530. [0310] Broadhurst, R. W., Nietlispach, D., Wheatcroft, M. P., Leadlay, P. F., and Weissman, K. J. (2003) The structure of docking domains in modular polyketide synthases. Chem. Biol. 10:723-731. [0311] Brosius, J. (1989) Super-polylinkers in cloning and expression vectors. DNA 8:759-777. [0312] Butler, A. R., Bate, N., and Cundliffe, E. (1999) Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae. Chem. Biol. 6:287-292. [0313] Caffrey, P., Lynch, S., Flood, E., Finnan, S., and Oliynyk, M. (2001) Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes. Chem. Biol. 8:713-723. [0314] Celenza, J. L. (2001) Metabolism of tyrosine and tryptophan--new genes for old pathways. Curr. Opin. Plant Biol. 4:234-240 [0315] Chater, K. F. and Wilde, L. C. (1980) Streptomyces albus G mutants defective in the SalG1 restriction modification system. J. Gen. Microbiol. 116:323-334. [0316] Cheng, Y. Q., Tang, G. L., and Shen B. (2003) Type I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc. Natl. Acad. Sci. USA. 100:3149-3154. [0317] Cortes J., Haydock, S. F., Roberts, G. A., Bevitt, D. J., and Leadlay, P. F. (1990) An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea. Nature 348:176-178. [0318] Cortes, J., Weissman, K. E. H., Roberts, G. A., Brown, M. J. B., Staunton, J., and Leadlay, P. F. (1995) Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage. Science 268:1487-1489. [0319] Cortes, J., Velasco, J., Foster, G., Blackaby, A. P., Rudd, B. A. M., and Wilkinson, B. (2002) Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea. Mol. Micro. 44:1213-1224. [0320] Devereux, J., Heaberli, P., and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12:387-395. [0321] Dickinson, L., Griffiths, A. J., Mason, C. G., and Mills, R. F. (1965) Anti-viral activity of two antibiotics isolated from a species of Streptomyces. Nature 206:265-268. [0322] Donadio, S., Staver, M. J., McAlpine, J. B., Swanson, S. J., and Katz, L. (1991) Modular organization of genes required for complex polyketide biosynthesis. Science 252:675-679. [0323] Donadio, S., McAlpine, J. B., Sheldon, P. J., Jackson, M., and Katz, L. (1993) An erythromycin analog produced by reprogramming of polyketide synthesis Proc. Nat. Acad. Sci. USA 90:7119-7123. [0324] Duffey, M. O., LeTiran, A., and Morken, J. P. (2003) Enantioselective total synthesis of borrelidin. J. Am. Chem. Soc. 125:1458-1459. [0325] Eastwood, E. L., and Schaus, S. E. (2003) Borrelidin induces the transcription of amino acid biosynthetic enzymes via a GCN4-dependent pathway. Bioorg. Med. Chem. Lett. 13:2235-2237. [0326] Fernandez, E., Weissbach, U., Sanchez-Reillo, C., Brana, A. F., Mendez, C., Rohr, J., and Salas, J. A. (1998) Identification of two genes from Streptomyces argillaceus encoding glycosyltransferases involved in transfer of a disaccharide during the biosynthesis of the antitumor drug mithramycin. J. Bacteriol. 180:4929-4937. [0327] Floss, H. G. (2001) Antibiotic biosynthesis: from natural to unnatural compounds. J. Ind. Micro. Biotech. 27:183-194. [0328] Fouces, R., Mellado, E., Diez, B., and Barredo, J. L. (1999) The tylosin biosynthetic cluster from Streptomyces fradiae: genetic organisation of the left region. Microbiology 145:855-868. [0329] Folkman, J. (1986) How is blood vessel growth regulated in normal and neoplastic tissue? G. H. A. Cloves Memorial Lecture. Cancer Res. 51:467-473. [0330] Funahashi, Y., Wakabayashi, T., Semba, T., Sonoda, J., Kitoh, K., and Yoshimatsu, K. (1999) Establishment of a quantitative mouse dorsal air sac model and its application to evaluate a new angiogenesis inhibitor. Oncol. Res. 11:319-329. [0331] Gaisser, S., Reather, J., Wirtz, G., Kellenberger, L., Staunton, J., and Leadlay, P. F. (2000) A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea. Mol. Microbiol. 36:391-401. [0332] Gaisser, S., Martin, C. J., Wilkinson, B., Sheridan, R. M., Lill, R. E., Weston, A. J., Ready, S. J., Waldron, C., Crouse, G. C., Leadlay, P. F., and Staunton, J. (2002) Engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents. Chem. Commun. 618-619. [0333] Gaitatzis, N., Silakowski, B., Kunze, B., Nordsiek, G., Blocker, H., Hofle, G., and Muller, R. (2002) The biosynthesis of the aromatic myxobacterial electron transport inhibitor stigmatellin is directed by a novel type of modular polyketide synthase. J. Biol. Chem. 277:13082-13090. [0334] Hanessian, S., Yang, Y., Giroux, S., Mascitti, V., Ma, J., and Raeppel, F. (2003) Application of conformation design in acyclic stereoselection: total synthesis of borrelidin as the crystalline benzene solvate. J. Am. Chem. Soc. 125:13784-13792. [0335] Hardt, I. H., Steinmetz, H., Gerth, K., Sassa, F., Reichenbach, H., and Hofle, G. (2001) New natural epothilones from Sorangium cellulosum, strains So ce90/B2 and So ce90/D13: isolation, structure elucidation, and SAR studies. J. Nat. Prod. 64:847-856. [0336] Heathcote, M. L., Staunton, J., and Leadlay, P. F. (2001) Role of type II thioesterases: evidence for the removal of short acyl chains produced by aberrant decarboxylation of chain extender units. Chem. Biol. 8:207-220. [0337] Hopwood, D. (1997) Genetic contributions to understanding polyketide biosynthesis. Chem. Rev. 97:2465-2497. [0338] Hunziker, D., Yu, T.-W., Hutchinson, C. R., Floss, H. G., and Khosla, C. (1998) Primer unit specificity in biosynthesis principally resides in the later stages of the biosynthetic pathways. J. Am. Chem. Soc. 120:1092-1093. [0339] Janssen, G. R., Bibb, M. J., (1993) Derivatives of pUC18 that have BglII sites flanking a modified cloning site and that retain the ability to identify recombinant clones by visual screening of E. coli colonies. Gene 124:133-134. [0340] Kahn, R. A., Fahrendorf, T., Halkier, B. A., and Moller, B. L. (1999) Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolour (L.) Moench. Arch. Biochem. Biophys. 363:9-18. [0341] Kawamura, T., Liu, D., Towle, M. J., Kageyama, R., Tsukahara, N., Wakabayashi, T., and Littlefield, B. A. (2003) Anti-angiogenesis effects of borrelidin are mediated through distinct pathways: Threonyl-tRNA synthetase and caspases are independently involved in suppression of proliferation and induction of apoptosis in endothelial cells. J. Antibiot. 56:709-715. [0342] Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (2000) Practical Streptomyces Genetics. The John Innes Foundation. Norwich. [0343] Keller-Scheirlein, W. (1967) Composition of the antibiotic borrelidin. Helv. Chim. Acta. 60:731-753. [0344] Kuo, M. S., Yurek, D. A., and Kloosterman, D. A. (1989) Assignment of .sup.1H and .sup.13C NMR signals and the alkene geometry at C-7 in borrelidin. J. Antibiot. 42:1006-1007. [0345] Kuhstoss, S., Huber, M., Turner, J. R., Paschal, J. W., and Rao, R. N. (1996) Production of a novel polyketide through the construction of a hybrid polyketide synthase. Gene 183:231-236. [0346] Lozano, M. J., Remsing, L. L., Quiros, L. M., Brana, A. F., Fernandez, E., Sanchez, C., Mendez, C., Rohr, J., and Salas, J. A. (2000) Characterization of two polyketide methyltransferases involved in the biosynthesis of the antitumor drug mithramycin by Streptomyces argillaceus. J. Biol. Chem. 275:3065-3074. [0347] Maehr, H., and Evans, R. H. (1987) Identity of borrelidin with treponemycin. J. Antibiot. 40:1455-1456. [0348] Marsden, A. F., Wilkinson, B., Cortes, J., Dunster, N. J., Staunton, J., and Leadlay, P. F. (1998) Engineering broader specificity into an antibiotic-producing polyketide synthase. Science 279:199-202. [0349] Matter, A., (2001) Tumor angiogenesis as a therapeutic target. Drug Dis. Today 6:1005-1024. [0350] Mochizuki, S., Hiratsu, K., Suwa, M., Ishii, T., Sugino, F., Yamada, K., and Kinashi, H. (2003) The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism. Mol Microbiol. 48:1501-1510. [0351] Moore, B. S., and Hopke, J. N. (2000) Discovery of a new bacterial polyketide biosynthetic pathway. Chembiochem 2:35-38. [0352] Nielsen, J. S., and Moller, B. L. (1999) Biosynthesis of cyanogenic glucosides in Triglochin maritime and the involvement of cytochrome P450 enzymes. Arch. Biochem. Biophys. 368:121-130. [0353] Olano, C., Wilkinson, B., Moss, S. J., Brana, A. F., Mendez, C., Leadlay, P. F., and Sala, J. A. (2003) Evidence from engineered gene fusions for the repeated use of a module in a modular polyketide synthase. Chem. Commun. 2780-2782. [0354] Olynyk, M., Brown, M. J. B., Cortes, J., Staunton., J., and Leadlay, P. F. (1996) Chem. Biol. 3:833-839. [0355] Otani, A., Slike, B. M., Dorrell, H. I., Hood, J., Kinder, K., Cheresh, D. A., Schimmel, P., and Friedlander, M. (2002) A fragment of human TrpRS as a potent antagonist of ocular angiogenesis. Proc. Nat. Acad. Sci. USA 99:178-183. [0356] Otoguru, K., Ui, H., Ishiyama, A., Kobayashi, M., Togashi, H., Takahashi, Y., Masuma, R., Tanaka, H., Tomado, H., Yamada, H., and Omura, S. (2003) In vitro and in vivo antimalarial activities of a non-glycosidic 18-membered macrolide antibiotic, borrelidin, against drug-resistant strains of Plasmodia. J. Antibiot. 56:727-729. [0357] Pacey, M. S., Dirlam, J. P., Geldart, L. W., Leadlay, P. F., McArthur, H. A. I., McCormick, E. L., Monday, R. A., O'Connell, T. N., Staunton, J., and Winchester, T. J. (1998) Novel erythromycins from a recombinant Saccharopolyspora erythraea strain NRRL 2338 pIG1 I. Fermentation, isolation and biological activity. J. Antibiot. 81:1029-1034. [0358] Paetz, W., and Nass, G. (1973) Biochemical and immunological characterization of threonyl-tRNA synthetase of two borrelidin-resistant mutants of Escherichia coli K12. Eur. J. Biochem. 35:331-337. [0359] Prieto, M. A., Diaz, E., and Garcia, J. L. (1996) Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster. J. Bacteriol. 178:111-120. [0360] Quiros, L. M., Aguirrezabalaga, I., Olano, C., Mandez, C., and Salas, J. A. (1998) Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus. Mol. Microbiol. 28:1177-1185. [0361] Raibaud, A., Zalacain, M., Holt, T. G., Tizard, R., and Thompson, C. J. (1991) Nucleotide sequence analysis reveals linked N-acetyl hydrolase, thioesterase, transport, and regulatory genes encoded by the bialophos biosynthetic gene cluster of Streptomyces hygroscopicus. J. Bacteriol. 173:4454-4463. [0362] Ranganathan, A., Timoney, M., Bycroft, M., Cortes, J., Thomas, I. P., Wilkinson, B., Kellenberger, L., Hanefeld, U., Galloway, I. S., Staunton, J., and Leadlay, P. F. (1999) Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues. Chem. Biol. 6:731-741. [0363] Reeves, C. D., Murli, S., Ashley, G. W., Piagentini, M., Hutchinson, C. R., and McDaniel, R. (2001) Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry 40:15464-15470. [0364] Rowe, C. J., Bohm, I. U., Thomas, I. P., Wilkinson, B., Rudd, B. A. M., Foster, G., Blackaby, A. P., Sidebottom, P. J., Roddis, Y., Buss, A. D., (2001) Chem. Biol. 8:475-485. [0365] Rowe, C. J., Cortes, J., Gaisser, S., Staunton, J., and Leadlay, P. F. (1998) Construction of new vectors for high-level expression in actinomycetes. Gene 216:215-223. [0366] Rudd, B. A. M., Noble, D., Foster, S. J., Webb, G., Haxell, M. (1990) The biosynthesis of a family of novel antiparasitic macrolides. Proceedings of the 6.sup.th International Symposium on the Genetics of Industrial Microorganisms. Strausbourg, France. Abstract A70. p. 96. ISBN 2-87805-004-5. [0367] Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual. 2.sup.nd ed. Cold Spring Harbour, Laboratory Press. New York. [0368] Schmidt, D. M. Z., Hubbard, B. K., and Gerlt, J. A. (2001) Evolution of enzymatic activities in the enolase superfamily: functional assignment of unknown proteins in Bacillus subtilis and Escherichia coli as L-Ala-D/L-Glu epimerases. Biochemistry 40:15707-15715. [0369] Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw, L. E., Haydock, S. F., Oliynyk, M., Caffrey, P., Cort

es, J., Lester, J. B., Bohm, G. A., Staunton, J., and Leadlay, P. F. (1995) The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc. Nat. Acad. Sci. USA 92:7839-7843. [0370] Shaw-Reid, C. A., Kelleher, N. L., Losey, H. C., Gehring, A. M., Berg, C., and Walsh, C. T. (1999) Assembly line enzymology by multimodular nonribosomal peptide synthetases: the thioesterase domain of E. coli EntF catalyzes both elongation and cyclolactonization. Chem. Biol. 6:385-400. [0371] Silakowski, B., Nordsiek, G., Kunze, B., Blocker, H., and Muller, R (2001) Novel features in a combined polyketide synthase/non-ribosomal peptide synthetase: the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatelia aurantica Sga15. Chem. Biol. 8:59-69. [0372] Singh, S. K., Gurusiddaiah, S., and Whalen, J. W. (1985) Treponemycin, a nitrile antibiotic active against Treponema hyodysenteriae. Antimicrob. Agents Chemother. 27:239-245. [0373] Staunton, J., and Wilkinson, B. (1997) Biosynthesis of erythromycin and rapamycin. Chem. Rev. 97:2611-2629. [0374] Swan, D. G., Rodriguez, A. M., Vilches, C., Mendez, C., and Salas, J. A. (1994) Characterization of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence. Mol. Gen. Genet. 242:258-362. [0375] Takeshita, S., Sato, M., Toba, M., Masahashi, W., and Hashimoto-Gotoh, T. (1987) High-copy number and low-copy number plasmid vectors for lacZ alpha-complementation and chloroamphenicol- or kanamycin-resistance selection. Gene 61:63-74. [0376] Thomas, I., Martin (nee Rowe), C. J., Wilkinson, C. J., Staunton, J., and Leadlay, P. F. (2002) Skipping in a hybrid polyketide synthase: evidence for ACP to ACP chain transfer. Chem. Biol. 9:781-787. [0377] Tsuchiya, E., Yukawa, M., Miyakawa, T., Kimura, K. I., and Takahashi, H. (2001) Borrelidin inhibits a cyclin-dependent kinase (CDK), Cdc28/Cln2, of Saccharomyces cerevisiae. J. Antibiot. 54:84-90. [0378] Wakasugi, K., Slike, B. M., Hood, J., Otani, A., Ewalt, K. L., Friedlander, M., Cheresh, D. A., and Schimmel, P. (2002) A human aminoacyl-tRNA synthetase as a regulator of angiogenesis. Proc. Nat. Aced. Sci. USA 99:173-177. [0379] Wakabayashi, T., Kageyama, R., Naruse, N., Tsukahara, N., Funahashi, Y., Kitoh, K., and Watanabe, Y. (1997) Borrelidin is an angiogenesis inhibitor; disruption of angiogenic capilla vessels in a rat aorta matrix culture model. J. Antibiot. 50:671-676. [0380] Waldron, C., Matsushima, P., Rosteck, P. R., Broughton, M. C., Turner, J., Madduri, K., Crawford, K. P., Merlo, D. J. and Baltz, R. H. (2001) Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem. Biol. 8:487-499. [0381] Wilkinson, B., Foster, G., Rudd, B. A. M., Taylor, N. L., Blackaby, A. P., Sidebottom, P. J., Dawson, M. J., Buss, A. D., Gaisser, S., Bohm, I. U., Rowe, C. J., Cortes, J., Leadlay, P. F., and Staunton, J. (2000) Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase. Chem. Biol. 7:111-117. [0382] Wu, N., Tsuji, S. Y., Cane, D. E., and Khosla, C. (2001) Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. J. Am. Chem. Soc. 123: 6465-6474. [0383] Xue, Y. Q., Zhao, L. S., Liu, H.-W., and Sherman, D. H. (1998) A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: architecture of metabolic diversity. Proc. Nat. Acad. Sci. USA 95:12111-12116. [0384] Xue, Y. Q., and Sherman, D. H. (2000) Alternative modular polyketide synthase expression controls macrolactone structure. Nature 403:571-575. [0385] Yamamoto, H., Maurer, K. H., Hutchinson, C. R. (1986) Transformation of Streptomyces erythraeus. J. Antibiot. 39:1304-1313.

Sequence CWU 1

1

113174787DNAStreptomyces parvulus Tu4055 1gatcccgcgc ggcatcgccg tcgacgtgct gcgggccggc gaccgctggc cccacagcgc 60ggcaccgcgc caccggggac tcctcaacgc ctggtggggc gcctgggtct gggccacggt 120cttcgaccgc tacgcgtcga ggacctacga cgacgcccag gacgtcgacg cgatccacga 180cgcggcggga ctggtcatgg ccggtgccgg attcgacatc ctcgccgccg tgctcgcgat 240cctcttcgtg cgccggctga ccgccgcaca gcacgcgaag gccctcgcgg ggcccacccc 300gccgacgcac tgagccgccc gcacccgtga tcccgccccg cgatccccgg gcccgataaa 360tgcgttggcc ccggcgcgcg cctgtggtgg gatgagcggc gacgggggcg gctccccggc 420gtgcatcctt ctcaccttcc tgcaaagatc ccgcgcgccc actctccgcc cccgttcttc 480cgtcccgagc cgtcgccgcc gtggaggctt tcctgttgct cgccgccgag tccgtactgc 540tgcgccgtga ccagagcgtc tacgtgaccc cggggtccga gccggacggt ccgccgaggg 600ccgcactgcg ccggctcgag gccgaactgc tcggccgcgg ccacgccgtc tccgcgccgc 660tgcacgcggt cctcgcctcc ttggactccg aggaactggc ggccgcccac gtacgcctcg 720tcggactcgt cgacgacctg ctcggctccg accgcaccca caccccgctc ttccgccgct 780tcccgcgcac cgtgccgcgc gacaccgagg cgctgtacgt ggaccgcgtc ttcgccttcc 840tgctgcagca gcccgagcag ccctgcgtgc tgtgcggcga ggcgcgcacc gtcctgcccg 900tgtcaccctg cgcgcacctg gtctgccggc tgtgctggga cggctccgac tacgcgggat 960gcccgctgtg ccaccgcagg atcgacgggg acgacccctt cctgcgtccg gtccgtgccg 1020tcggcgccgc cagggcgacc gtaccgggcc cgctgcgact gctgcgcctg ggcaccgaca 1080tgaccgccga cgccaccacg gcggtggacg ccctgctggc ccgccgcacc ccgctctccc 1140cgcaggaccg ggacgacctg ctcaccctgt tgccgctcac accggccggc cggggcgacc 1200tgccgcagga catcccggtc cgcgagacca aggcgctggt cctgggcgcg ctggtgcgcc 1260gggcaccgtc gcggccggcc ctgcggaggc tgctcgccga gcggctcacc accgccaccg 1320acgtgctgcg gctgctcgcc gtgctctcgg gcggcgacgc cgggctggtg acaccggcac 1380ggttcacgaa cgttccccgt tccctgcggc gtgacctgct cgccgtcctc gacggactgc 1440cggcgccgta cctggtcgag gacatgctgc ggcaccccac ggcgtggaag cgggccgcgg 1500aggtgctgca ccccttcgag gggcacaccc ggcacccgcg cgccgcgctc gccaccgccg 1560tgctgcgcgc cacaccgttg gacccggaca ccgccttcgg cgccgccctg ctgaccacgg 1620ccgccgcgca cccggacgcc gtgcgcccgg acggcacccg agtccgcccg gccacctggg 1680cgggacggct ggagcaggcg atggccgagg gggacgccgc tcgggccgcg gccctcgccg 1740gggagcggcc cggcgaactg gtgcgccgcc tggacgtgtt gctgcgcctg cacaccgacg 1800aggcgctcgt gccggagctg gagaaggccc tgcggcacgg gctgccgaag gtgggcccgg 1860gcccgctgct gtcggcgctc ggggcgctgc ggacacgcac cgaggaccgc accgggaccc 1920ggcgcgtgtt cttcccgcgg ggcgacgtca cccgggccct gtccgtcccc gagcggcgcc 1980ccgccctgcc cgccgggccg gtgtccgagg tggtcgccct gctggagggg gaactgctgc 2040gccggttcgc cgccgggcgg ccctacgagc tgtcggtgct ggacgccgga ctgaccgacc 2100tcaccgtgcc gttcaccgag cggaccgccg ccaaggccct ggtgaccgtg ggccgcggca 2160gcgtccaggc actccccgag ggctccgtgc tccgactgtt cctgcactgg acggaacccc 2220ggggcaaccg caccgacctg gacctgtccg tcgccttctt cgacgccgag tggacgttca 2280ccggcctgtg cgactacacg aacctggtgc acggtccgga cgcggcgatc cactccggcg 2340acctcacgtc ggccccggcg ccgcgcggcg ccaccgagta cgtggacctc gacctggagc 2400ggctggcgcg gcggggagac acctacgccg tcccgctggt gttcagctac aacaacgtcc 2460cgttcgagga actgccggac gccttcgccg ggttcatggc gctgcccgcg gaaggcccgc 2520gcgacgcgac ctacgacccg cgcaccgtgc ggcagcgctt cgacctcgcg ggcgactcca 2580aggtgtgcct gccgatgatc gtggacctgg cccgccggcg ggcgttgtgg accgacaccc 2640acctgccgtc cgcgggcggc ttccagagca tcggttcgca cggcggcggt gagctggccg 2700cggtggccgg tgacctctgg cagcagttca cctcgggcgg ccgggcgacc ctgtgggacc 2760tcgccgtcct gcgggcggcc gccctctcgc cggaggtggc ggtggtgtcc cgggagccgg 2820agcccgcggt gctgcgttac cggcggcggg cggccgagag cgaggccgcg ttcgccgtcc 2880gagtcgcgtc ccacaaggac gccgaggaac ggctggcgca caccgacccc gactcggccg 2940cggccgggct cgccgccggc cggcgggtct tcctcgcgac ggtccacggt gacgtccggc 3000cgccgggggc gtcgggcacg tcctaccggc tcttccccgg ggccggggac gcctcaccga 3060ccctgacccg cgtgaccgcc ggggacctgc tcgccgagct gggctgagcc aggcgccggc 3120ccgcgccggc ccgcgccggc ccgtccctgc ccgtgccgga gggctcgccg gtcactccgg 3180ccaggcggag ttctcgatga cctcgacgaa gtccgtacgc cggaagccgg gcgcgaagtg 3240ctccagcaca tccgcgttca ccgtgccgaa ggtggtcgcg ggccggtgct cgaacccctc 3300ggtgaacgcc cgcaggatct gcttcttgaa gtccgggcgg ggatgcgcgg cggtgaccgc 3360gtcgatctgg gcccgggtga gattgcccag ccgcaggccg agcacgtcgg tctccacgcc 3420ggcggtggtc gccgcgatct cgggggccat ccggtacggc acctccggag tggtgtgcag 3480ggcgacggcc gtccacacgg tgtccgcgtc ggcctcgggg atgccgtggg cgagcaggaa 3540ggcgtgggcc tggtcggcac cgtccatctc gaagcgctgg tcgtcaccgc ggtagggcgg 3600caccaggccg gtgtcgtgga agagcgcggc gatgtacagc agctccgggt cggggcggat 3660gcccagggcg gcggcctgga ggctgccgaa gaggtacaca cggcgtgagt ggtggaagat 3720cagcggcgga gtggtgtcgc ggatcaggtc ggtcgcctcc cgcgccggcg cgctgtcggg 3780aatctcgatg ccggcgatct gctcggccat ggctgccctc cggggaatcg gtgccgtcgt 3840tgctgcctcc accctccgcc cggcgcgacc ccggcgtccg ctacccgatg gccgacaacc 3900ccttacaagc ggccatgtgc cccgcgccgc cgcctcagcc gccgtccggg cgcgggccgg 3960cgtccggcac ggtggtggcg aagcgctgcc ggtagcgggt gggcgacagc cccagatgac 4020gggcgaaggc ccggcgcagg ctctcgtagc tggggaaacc cgacagcgcg gcggcctcgg 4080tggcgttgtg cccggagtcg agcagcgcct tggcgatgtc gaaacggatc agctccacgt 4140acttcacggg cgtgacgtcc agctcggccc ggaacatccg ggtcagatgc cgggggctga 4200cccgcacgcg cgccgccaac gccgccagac tgtggtcggc ggccggatcg gcctgtacgg 4260cgtcctggac ctgccgcagc acgggcgtcc gcggcgccgg gccccgcaac gaggcggaga 4320actgcgactg gccgccggcc cgctgcaggt acaccaccag cgagcgcgcg accctgcggg 4380cgagatcggg cccgtggtcc tcctccagca gcgcgagggc caggtcgatg cccgccgtca 4440cgccggcgga cgtgtaggtc gccccgtcct tgacgaagat cgcgtcgggc tccacgcgtg 4500tcgacggaca gcggcgggcc agcgcggtgg tgtgctgcca gtgcgtcgtc gcccgtctgc 4560cctccagcag acccgcggca cccagcacga aggcgccggt gcacaccgag gcgacgcgtc 4620cggcccgggc cgccagcgcc ttcgcggcgt cgatgagccg tgggtcgacg ggcgagccgg 4680gcagcgcgtc accgccgacg acgacgagcg tgtccggcgg gccggcggaa cgcgcgtccg 4740cctcggccgg gaccagcagg ccgatggacg aacgcaccgg cgccccgtcc ggggagacga 4800cgccgagccg gtaccgggcc ccgaaccggt tggcctccgc gaagacctcc gccggccccg 4860acaggtcgag catcttcatg ccgtcgaaga ccaggatgcc cacgctgtgc gctctcgccg 4920tcatgtctcc ctctccgcgg gccggcgggc ccctgcgcgc cattgtcccg ccggccgtcc 4980acgccggcgg ccggcggcgc gggcggccgg cggtcggaat gaggcgcgcc ggacatcggc 5040gtagggtggc gagcgtgtgt tcggccgcgg tcccggagac cgcggaacgc aggacctttg 5100gcaggcacgc ggaaggacag cgatgggtac ggtcaccacc tccgacggca cgagcatctt 5160ctacaaggac tggggcccgc gcgacgcccc gccgatcgtc ttccaccacg gctggccgct 5220caccgcggac gactgggaca accagatgct gttcttcctc tcgcacggct accgtgtgat 5280cgcccacgac cggcgcggcc acggccgctc gggccagccc tcgacgggcc acgagatgga 5340cacctacgcc gccgacgtcg cggcgctgac cgaagcgctc gacctgcggg acgccgtcca 5400catcgggcat tcgaccggcg gcggcgaggt cgcgcgctat gtggcgcgcg ccgaaccggg 5460ccgggtcgcc aaggccgtgc tggtcggcgc cgtgccgccg gtgatggtca agtccgacgc 5520caaccccggc ggcaccccga tcgaggtctt cgacgggttc cgcacggccc tggccgccaa 5580ccgggcccag ttctacatcg acgtgccctc cggccccttc tacggattca accgggaggg 5640cgcgaaggtc tcccagggcc tgatcgacaa ctggtggcgg cagggcatgt cgggcgcggc 5700caacgcccac tacgagtgca tcaaggcgtt ctccgagacc gacttcaccg aggacctcaa 5760ggccatcgac gtgccggtgc tggtcgcgca cggcaccgac gaccaggtcg tgccctacgc 5820ggactcggcg ccgctgtcgg tgaagctcct gaagaacggc accctcaagt cgtacgaagg 5880gctcccgcac ggcatgctct ccacccaccc cgaggtggtc aaccccgacc tcctggactt 5940cgtgaggtcc tagtcggcgc tcacgccggc gacacgggag cgggtgcggc gccgcgcacc 6000gggtgcttgc tcaggacgga gacccggttg aaggcgttga tgctgatcgc cacccagatc 6060acggcggaga cctcgtcgtc cgacaggacg ccccgtgcct gcgcgtaggc ggcgctctgc 6120gcggcggcgt ccgccggacg ggtggtcgcc tccgcgaggg cgagcgccgc ccgctcccga 6180gcggtgaaca gctcggtgtc ccgccaggcg ggcagcaccg ccaggcgctg ggtcgtctcg 6240ccggcccgca gcgccgccct ggtgtgcaga ctgagacagt aggcacaggc attgagttgg 6300gagacgcgga tgttcaccag ttccacgagg aggcggtcca ggccggccgc cgcggcggcc 6360tcccgcaccg attccgccgc ggccacgaac gctttgtacg cgccgggggt ctgcttgtcg 6420acgaagaccc gccgctcgtc cgtcgccacc ggggcctgct gtgtcacgtg gtctccttcg 6480tcgcgctctc ttccggcggg tcctatcatc acccccatgg atgttgaaag tgaaactttc 6540aggtcggggc cggacggggg cgcgtggtga gcaacacgga gacacggccc gcggagatgc 6600ggtgcggcgc cctcgaagac gaggtgcccg ccgcgggcgt cgaagtcctc accgcccgtg 6660acgtccccct cggcggcccg cgcgccatga ccgtgcggcg cacgctgccc cagcgggccc 6720ggacgctgat cggagcctgg tgcttcgccg accactacgg tcccgacgac gtggccgcgt 6780cgggcggcat ggacgtcgcc ccgcacccgc acatcggcct gcagacggtc agctggctgt 6840tcagcgggga gatcgagcac cgggacagcc tcggcaccca cgccttcgtc aggcccggcg 6900aactcaacct gatgaccggc ggcttcggca tcgcccactc cgaggtctcg acccccgaca 6960ccactgtcct gcacggcgtc cagctctggg tggcgctgcc ggaggagcac cgcgacaccg 7020gccgcgactt ccagcaccac gcacccgcgc cggtcgcctt cgacggcggc acggcacgcg 7080tcttcctcgg ctcgctcgcc ggggacacct cgcccgtgag caccttcacg ccgctgctgg 7140gcgccgagtt gacgctggtg ccgggcggca ccgccaccct ggacgtcgac cccggcttcg 7200agcacggcgt cctcgtcgac agcggtgacg tacgcgtcga gggcgccgtc gtgcgaccgg 7260ccgaactggg ctacgtcgcg ccgggtcgcg cgacgctgac cctgaccaac gagtcggccg 7320cacccgcccg gctcatcctc ctcggcggcc ccccgttccc cgaggagatc atcatgtggt 7380ggaacttcat cggccggtcg cacgacgaga tcgtgcgggc ccgcgaggac tggatgaagg 7440gcgaccgctt cggcgaggtg cacggctacg acggggcacc cctgcccgcg ccggaactgc 7500cgaacgcacc cttgaagccg cgacgaaggg cgcgctgatc tgcggggaca tgggttggca 7560ccaagggttt cggcgctgct cgatcaccga acccaccgcg agtcactctc gggtgagtcc 7620cgaacggtcg ccgggagcgc gtgagcacgt gcgcagatgc tcggcgatga tgccgagaat 7680cgcatcccgg tgctccagca ggtagaagtg accacccgcg aaggtgtcga gtgtgaacgg 7740gccgtccgtg tgttcggacc atgcccgggc ctcgaccggg gtgaccatcg ggtcatcatc 7800cccggtcaag gcatggatgg ggcaccgcag cttcgggccc ggtcggtagc ggtaggtctc 7860ggcggccctg tagtcgccgc ggatggcggg gagggccata cgcaccagct cctcgtcgtg 7920gaagacctgc tccgcggtgc cgtcgagggt cctcagctcg gccaccaact cctcgtccga 7980caggaggtgc accgtacccc ccgtcctctg ccgggacggt gcgggcctgg ccgagacgag 8040gagtgcctcc agggagatgc ccgcactctc gaaccgtcgg gccagttcga aggcgagggt 8100ggcgcccatg ctgtgtccga acagcgcgac cggctggtga acacgggccc gcagcacggg 8160gaagagctgg ttcgcgagtt cgtcgatgtc ctccaggggc ttctccgcgc gccggtcctg 8220ccggccgggg tactggaccg cgagcacgtc gcaccggggt gccagcgcgg cagccacggg 8280gtggtagaac gtcgcggagc cgccggcgtg cggcagacag atcaactggg gtgccgtggg 8340atgtgcgggg cggtactgcc tgatccacac gtcgctgtgg gtgttcgtac cggtcatcag 8400cggtgctgcc cttccggcgt ggcgttggtg cgggggatgg ccgatccggc cgtgacgcct 8460ccgtcgaccc cgagggtctg cccggtgacg taggcggcga gggggctgag cagccagacg 8520accgcgttgg ccacctcctc gcacttgccc aggcggccca gcggagcccg gcgcgcccgt 8580tgtgcgaggg cgctgggatc ggcgtacagg ctgcgcagca tgggggtgtc ggtcgaaccg 8640gggctgacca cgttgacgcg gatgccgtcg cccgcgtact gcagggccac cgacttgctc 8700aggccgatga ccgcgtgctt ggtggccgag tagagcgggc tctgggcgtg gccgatgtgc 8760ccggccactg atgcgcagtt cacgatcgcg ccgccgccgg ccgtcagcat ggcctcgatc 8820tgtccgcgca tgcacgacca gaccccacgc aggttggtgg cgatcacgcg gtcgaagttg 8880tcggcggtgt cctggtgcag cggaccgaac gagccgaagg tcccggcgtt gttgaacgcc 8940ccgtccagcc gtccgaaccg gctcaccgcc cgggccacgc agtccgccac ctgcttgtcg 9000tcaccgacgt cgcagggcac caccaagtgg tgcgaggagg gtagtccggc ggttgtctcc 9060gtgagggccg actcggtgcg gcccaccagg acgacgcgtg ctccgtgccc cacgaggagc 9120cgggccgcgg cccggccgat gccgctgccc gctccggtga ccatcatcac gcggtcggtg 9180agttccagac tcatcgttgt tccaacgctc cgtccctgct cgtcggatgt gcgatccgct 9240gtgtcatatg tgcagtccgc cgttgacgtc gacgaccgtg ccggtggtgt atccggcgtc 9300ctcgccgcac agatggcaga ccatgcccgc ggcctcggcg acgctgccga agcggccggc 9360ggggatgtgg ctgacgcggt cggcggtcca ctgcgggggc ttgtcctccc aagcccggcg 9420gatgcgctcg gtgccgatga cgccgtgggc gaccgcgttg accgtcacgc cgtgcggggc 9480cagttcgtag gcgcactgct tggtgaaccc gatgacgccg gccttggcgg cgacgtaggc 9540ggcattgctg aaccgggtgt acgtgcgacc ggccacggac gccaggttga cgaccctgcc 9600ccaccccgcc gcgaccatcg ccgggacgca cagccgggtc atggtgaaca cgctcgccag 9660gttgtgcgtg acggcctcct ggaggtcggc ctcggtcagt tcggtcaccg agcgggcccg 9720ggtgtcgcca ccgacgccgt tgaccaggac gcccggccgg tgctgcgggg cgagcgagtc 9780gacggcggac gccagggcgt gagggtcggt cacgtcggcg accagcgggt cccgggccag 9840ccggtcgccg agcccgtccg cgacccggtg caccgcctcg gcgtccttgt cgagcaggac 9900cacccgcagg ccccgggccg ccaggcctcg ggcgacctcc gcgcagatgc cgctgcccgc 9960tccggtcacc agagccacgt cgtgtcgtgc cgtcatgtgt tcctccgcca gccgccgccg 10020gattcccagg tggccgcgca ccgggtgtgt cgcagtagtt cttcggtcgg ttcgatgccc 10080gtgcccggac cggtcaaggg ttcgacccgg tgcagtgacc ggtcgacggt gaacgccggc 10140gtggtcagtg gcacggggaa ccactcgtcg gcccggcccg cctcgaccgt ctgccacagg 10200tcccatgcgg tggccagggt gcgcccggcc gcccacagcg gccccacctc ggccacgtga 10260acgccgagct ggcaaccgac gccgagctcg tcggcgcgca gtgccaggcg tgccgcggcg 10320aggaacccgc cgcacttcga cagccgtacg ttgatgtggc tggcggcgcc gctggtggcg 10380gcggcgtgga ggtcggccgg tccggtacag gactcgtcga gcatgacggg cagaccggtg 10440gcccgccgca gccggcccaa ctcgggccag gaccgcggcg ggagcggctc ctccacccac 10500cccacgccgt ccagttcgcc cgcgaccttc tccgcttcct cggccgtcca ggcgccgttg 10560acgtccagtg agacacgggt gtcggcgggg aggcggtcct gggccgccgt cagccggtcc 10620accgccccgg ccgggtccgc caccttgatc ttcacgtgcc gcaacgccgc cagcgcccgc 10680ggcgtgagtg cgtccaggac ggtcgcgacg tcgcgcgaga ggtggatcac gaggctgacg 10740gacgtcggtc cgtcccgccg tgatcgggca ggcggggcca ggacccgcag gacgtcggcg 10800agcggccggg cgaaatgccg gcacaccgcg tcgagcaggg cgatctccac ggcggccgcc 10860gccgacgagc cgtcgacgag cccggtcagc ggcagctgtg cgatcgaggc gacggcgctc 10920tcgaagtccc gccactcgat gcgctcggcc agctccccgg gatcgcaggc ctggacggct 10980cgcaccgcgc cgtccagggt ctcaccggtg acgtagtcgc ggggcgctcc ctctccccat 11040ccgcgggtgc ccgccagctc gatctcgacc agcagggacg ccgcgctgcg acgggagcgc 11100gtggcgtggt cgaaggccgc ggccatgggc acgacggcgg tgtgcagccg tacgcgacgg 11160atcacgcttc ctccttcagc cgcgtggcca gccagtccca gtacgccgtc cgggccgacg 11220tgaactccac gtagtgccga tccgtggcga agacctcctc gtgcacggct gacgtcagac 11280gccgcagcat cgctcgcgcg gccgacaggt cgatgatcgg gtcgtgagtg gggagcgcca 11340ggtcgacggg gagccgggtg cggggggcac cgcgggcata gtggtcctcc aggtgcacga 11400gcgtcgcctg cgtggccgag gtgacctcgc gcagcatcag gtgatccccg gtgaggaact 11460cccggtagcg cggcaggtcg gtgtagtcgc cgtcggcgag ccccacgggc cgtagcccgc 11520tgccggtgag cgcgcggcgc tcggcgagcg tgtccgcggt gtggcgcgcc cgctgctgtc 11580ccagcgcggg cgcgcacagg accaacctgc ggacgggcag atcgcgggtg caccagagcg 11640cggccagcac gctgccgccg aggctctgcc ccagggcgac cggcccggca ccgccgacct 11700cggccgtcac ggcgtcgagg gcgcgggcgt agtcgtcgag gacgagatcg gccgacggca 11760ggtggccgcg aggcccctcg ctgcggcccg agcctctgcg gtccagggcg tagacgtcga 11820tgccgcgtgc gttgagctcg ggccccgtct cgaacagcca gcccgcgtgg ctctggatgc 11880cgtggaggta gaagacggcc gaggtggcgc cgggcgtggt ccagtggtgc agggtgagcc 11940cggtgccgtc ggcagcggtc agcatgctcg tggtgggcat gggctgcctc ctcagtaccg 12000gacgagattg acgtcggggt ccagccggac gtcgacgagg agcggtcctt cgagcgtcga 12060caacaggtcg ccgacggcgt cgagttcttc cgccttgcgg acggtgaggg cacgtgcgcc 12120catcgccgtg gccagcccgg cgaggtcggg ccaggcgaac gccgagtacg ccgggtcgta 12180gccgtggttc ctgagtttgt agtgctcggc tccgtacgcc ccgtcgttga gtacgaccac 12240gacgagcggc agccggtacc gtaccgccgt cgtgaactcc gacaggtgca tcatgaagcc 12300gccgtccccc acggcggcga ccacgggccg gccggtcccg gccgtcgccg cgccgatcgc 12360cccggcgacg ccgagcccga tcgagccgaa gccgcccatg acggtgaagt gcagcgggtc 12420cgccacgcgc agatacggcc agacacccac gtcgaagcgg ccgatatcgc tgacgacact 12480gcgctcggcg ggcagtatcc ggtccagccg gatcatggcc gtccggatgt cgacggtctc 12540cgctccactg cggtcgtcga cgtcgtcctg cggcgagaac ccggccagtt gcccggcgac 12600gcgctccgcc caggcgccgt tggccgcggt gactccggcc tgatccagca ggacgttcat 12660ggtctcggcc gtgcggcggg catccccggc cacgggctcg tcgacggggc tgtacgagcc 12720gaaccgtgcc ggatcggtgt ccacgtgcac gactctcttg ccgcggagca gctcgccgtt 12780gagcacggtc cacatgttca ggctcgcccc gaacgcgatc acgcagtccg actcggcgat 12840gaccgtgctc gccacgctgt gcgcgagcga gccgaagatg ccgacgtcgc gggggtgacc 12900ggcgaacatc tccttgccga gcacggtggt ggccagcgct gctccggtac ggtccgccag 12960ctccaccagg gcctctcgcg caccggcgac ggccgcaccg tgcccggcga ggaccagcgg 13020ccgcttggcc gagccgatca gccccagcgc gccgtccagc gcctcggcct ccggagcggc 13080cagaggaccc ggcgccaccg ggagcgtgac cggcgcctgc tcgcccgcct ccgcctgcat 13140gaggtcgatc ggcacattga gtacgacggg ccgccgctcg gccacgatcc gctggacggc 13200ccggttcagg tccgcgacga gcgaggccgg tctgtggacg cgttcgtacc ccgcgcccgc 13260cgcggccgcg accgtcgcga tgtcgaagtg gtggaagtgc gtgggcaccg gtggcggatc 13320acctgtgatc agcaggacct ggctgtggct acgagccgct tccacaagag gggtcaaggc 13380gttggtgaaa gccggcccgt gcgtcacgga cgccacaccg atgccgccgc acatacgtgc 13440gcggccgtcg gccatggcga cggcgcccgc ctcgtgggcg accgccacga accgtccgcc 13500cgcgtcggcg aaggcgggca gatagagcag attggcgttg cccatgagac cgaagacggt 13560atcgacgccg tgtgcggtca gagcgtcggc gagcgcgtgg aaaaccttca ttgctgtccc 13620tcggtcgggg cgggctggag ccagacggga tcgttctggt cgaccggcgc gcaggtgggt 13680ggcgggtcct cgcggagcag ggaacgcagg tggttgccgg tgatgagcac ggtggttccg 13740gcgcagtccc gggcgacggc catccgccgg gcgagaactt gggggtgccg caccgcgcgg 13800gtcaggtcgt gcagcggggc cgcgggcacg ccgacggctg ccgccgcgtc cagtacggcg 13860gcgacggggc cctcggcccg cggtggcggg ccttccgcgt cgacgaggac cgtgccgtcc 13920gccgcccgga cgaggtgggg ggcccgtgcg ggtggggcgg tgaaccagcg gtcctgggtg 13980agccagacag cgctgtcgaa cagcgcgacg tcggcggcgc atccgtgctc ggtgcgcaga 14040cggacgtagg tggacacgac ggcggcggca gcggccaggt aggcaccgag gacgtcggcg 14100ctggacactg gagtcctcag cccggcgccc gggccgccga cgaggcgcat gatgcccgac 14160tcggcctgga tcacggtgtc gacgctgcgg tcggcggcgg ccaggccatg gccggtgacc 14220gtgcagtgca cgacgccgtg ccgggacagg atctgatcgg gggcgaggcc ctgcgcggtg 14280agtgtgtcgg ccgcgaggtt ggtgagcacg atgtcgcatc cggcgagcag ccgctcgaac 14340ccggcccggt cctcggcgtc ggcgaggtcg agccgacagg agcgtttgcc cgcgttgttg 14400acgtagtaga ggtagccgac cccggccacc tgctgggcga gccgccggga cccttcgccg 14460tgcggcggct ccaccttcag tacgtcggcg ccgagttggg ccagcagccg gcccgcgtgc 14520ggtccggccg tgtacgagcc gacctccagc aggcggacgc cgcgcagcgg aggagtgccg 14580cgcgcgatcg gctcccagag gccgccctgc cggggcatcg gaccgtcggt gacggcgggt 14640atgagggaac gcagtgggct gcccggtgta ccggacgggt cggtgaccag gccgcgacgc 14700cgggcggcgg ctccgtcgcg tacctcctcg ggggccgcga cctgggcgca cgggatgccg 14760gcggcccgca gggcggtcac cacgtccacg gcccgctgcc cggcggtcca cttgccgagg 14820atctcgtcga gctcgtcggc gttgcggacg cgggcggcgg tgtcggcgaa gcgagggtcg 14880tcggggaggt cccgtcgtcc caggactgcg gtcagcctgt gccatatcgg ctcgcccatc 14940gtgcagatga cgacaggggc gtcctggcac gtgtagctgt tccagggtgc cgccatgccg 15000tgtcggttgc cggtacgacg tggcggacga

ccggcgagcg cgacgctcgg aagcaaggtg 15060cccgtcagag tgaacaggct gtcgaactcg gcgatgtcca ggtagtcccc gcctcctccg 15120cgctcgcggc cgatgagccc ggccacgacg gcgatcagac cggacagggc cgccgtacgc 15180gacgccaggc ccaccacgga gaggaccgat ggttccccct cggtgccggt cgccgaggtc 15240aggcccgcca gcgcctgcaa ggtccgctcg gtggccgggg cgtcacgcag cgggccggtg 15300agcccgaacg cgctcagccg caccgcgacc aactcggggc tgcgatgcgg aagttccggc 15360gccccgagcc ccagagcggc gagccgctca tcgccctccg cgtcgcacac cagcacatcg 15420gccgtctgca gcagacgcga tgcctggtcc caaccggacg ccgactgcgc ggcggagtgc 15480agccaccgct cgaacgggcc cccgtgatcg ggtgatcggc agagcgtcac gacacgtgcc 15540ccgaggtccg cgagcagtct ccccagcagt gcggctggtg tactgcggcc ggccatgagt 15600acggcgatgc cctcgagcgg ccctgccctt gtcatggaat tctccctcgc tccgcgcacc 15660gatgcgggcg tcggtcgtca ccgctgattg gtcgtggacg tcggccgtga ggcgaccgcc 15720agggaaatca caccggcgcc gcccgcatcc gcgggggatc tggccggcag tcccgatgcg 15780ccattaaagc gcgcatgatt cgttccgtgc cgaccgtagc accgagacgg cggaaaatca 15840tcgcacaccc ctgctccgga tccggaaacc ctgctcaggg ggcaaggggg agggggtccg 15900taatggccaa aacgaaattt tacggagctt tacgtttgct ggacgatcta ttggtgagcg 15960cctcgacggg ctggacatgg cagtagtgaa tgtccgcatt catggctatt agtaccgtga 16020ccctgatcac acgagccctg gttgacgggt gaaatttggg gctggcagag tgatgacgag 16080cttccgtccg caaagtggtt gaataactgt tccgaaatct tcggcaattc aaaggagact 16140tacgggggat gcctctatta atgtattgct gtagggcgaa ataatgacag gcagtgctgt 16200ttcggcccca ttcctgcagc ctcccgaacc cgtctcaggg cactccgaac ggaaaagcga 16260tcccgtcctt ctcgtcggcg ccggacgccg tgcccgcatg gcggatgccg tacgtgccgc 16320cggcgctcag gcgggcatcg acccggccgt cctacggcgc acccgggcca ccttgatcac 16380cgcggggagc gcgggagccg caggccggct cgccgccgcc ctgcgcctga ccggcgccac 16440gatctctctg gacacccgcg agacacccac actgctcgcc ctgcacctcg ccgcccaagc 16500gctgcgggcg ggcgacacct cttacgccgt cgtcggtgcc gaacttcccg acgggaactg 16560cgcgttgatc ctggccaggc agtcagcggc aaccgccgag ggggctgtgc cccaggcgat 16620cgtccgcacc accacggcgg accgcaccac cacggcggat cacgcccctg cgcccgacga 16680ccacggcagc ccggcccgtg aagccccgca tgccacccgc acgttgtccc caggcatcac 16740ccaggccccc gccgagggct tcccgggcct gctggcgacc ctgcacgacg acacacccct 16800gcgccccacc gcggtcaccg agcacggcag cgacgccacc accgtcctcg tcctcctcga 16860ccagccccag gacgccgcac ccgcggcacc gctcccctgg gtggtctcgg ccccccacac 16920ccgcgccctc cgggccacgg ccgcgaccct ggccgtccac ctcgacacca caccggccgc 16980acccgccgac gtcgcgcaca ccctgctcac cgcgcgcccc gaccgccacc gtgccgccgt 17040cgtcggcgcg gaccgggcca ccctcaccga cggactgcgc gcactcgcca ccggaggcga 17100cgcgccccac ctcgtccacg gcaccgccac cggatcgccg cgtcccgtct tcgtcttccc 17160cggccagggg tcgcagtggc ccggtatggc cgccgaactc ctcgaaacca gcgagccctt 17220tcacgacagc gtgcacgctt gcgccgacgc gctggccgag ttcgtcgact ggtcggttct 17280cgacgtcctg cgccaggcac cggacgcgcc acccctgcgc cgggtggacg ttctccagcc 17340caccctgtgg gcgacgatgg tctccctggc cgaggtctgg cgctcgtacg gcgtggaacc 17400ggccgccgtc gtcggccact gctacggcga gatcgccgcc gcgcaggtag ccggcgccct 17460cgacatgcgt gacgccgccc gactgctcgc ccaccgcagc cgggcctggc tgcgactggt 17520gggcaagggc acggtcatct ccgtcgccac ctcgggacag gacatcaccc ggcgcatggc 17580ggcctggccc gactccgtcg aactggccgc gctcaacggc ccgcgctccg tggcgctcgc 17640aggcccgccc gacgtcctgg acggcatcgt caacgacctg accgaccagg gcatccacgc 17700caaacgcatc cccggcgtgg acaccgtcgg ccactgctcc caggtcgagg tcctccgcga 17760ccacctgctg gacgtcctgc gcccggtctc gccccggccc gccgccgtgc cgttctactc 17820caccgtcgac ggaaccgaac gcgacaccac cacgctggac accgactact ggtacctcaa 17880cacccgcagc caggtccgct tccaccaggc cgtgcggaac ctgctcgccg ccggacaccg 17940ctcgttcgtc gaggtgagcc cgcacccgct gctcggagcc tccatcgagg acaccgcggc 18000cgagttcggc ctcgacgacg tggccgccgt cggcaccctg cgtcgaggcc agggcggcac 18060ccgccgggtc ctgacctcgg tggcggaggc gtatgtccac ggcatcgaca tcgacttcac 18120gcccgccttc accggcacga cccccaaccg catcgacctt ccgaccgtcg aggaccacgg 18180catcgagggt cacggcgacg acggcggcga gacatggacc gaccgcgtca gaaccctccc 18240ggacgagcag cgcgaagagg ctttgctgga cctcgtgtgc cgcaccgtcg ccgcggtgct 18300cgaagcggac ccggccggca cggcggacgc cgtcgccccc gacacggcgt tcaaggagat 18360gggcctcggc tcactgagcg cggtccggct gcgcaacggc ctccgcgagg ccaccggcgc 18420ccacctgccg gccaccatcg cctacgacca ccccaccccg gccgctctgg cccgccacct 18480ggcgatgacc ctgttcgacg cgacgggcgc cgccccggcg gtcccggcac cgagccgcga 18540cgacgaaccg atcgacgccg agaccgctgt gctgaccgcg ctggaacggg ccgacgaggc 18600gctggaacgg ttgcgggccc cgcacgcccg cacgccccgg caggagaccg gccggcggat 18660cgacgagctg ctgcggtccc tgaccgacaa ggccaggcgg atgagacagg ccgacgccgt 18720cgatgatgtc gatgatccgg ccaccgaccg gttcgccgca gccaccgacg acgagatgtt 18780cgaactcctc gagaaacgtt tcggcatctc ctgaggcgcg ccgacctccc gcactgcgag 18840tcgcttcccc cacgatcccc gaaggcggca accgatggca catgaagaca aactgcgcca 18900cctcctcaag cgtgtcagtg ctgaactcga cgacacccag cgccgggtgc gtgagatgga 18960ggagagcgag cgcgagccga tcgcgatcgt ggggatgagc tgccgtctgc ccggcggggt 19020gaacagcccg ggggagttct ggtcgctgct ggaggccggg acggacgccg tctcggagtt 19080cccgcgggac cgtggctggg atgtggagaa cctctacgac ccggacccgg acgcccccgg 19140gcggtcgtac gtccgcgagg gcggattcct ggacggggcc ggacagttcg acgccgcctt 19200cttcggaatc tcgccccgtg aggcgctggc gatggatccg cagcagcggc tgctgctgga 19260gtgctcgtgg gaggcgatcg agcggtcgcg gatcgacccg aagaccctgc acggcagccg 19320gaccggcgtc ttcgcgggct ccaactggca ggactacaac accctgttgc tgaacgccga 19380ggagcgctcc cagagctacc tggccaccgg cgcctccgga agcgtgctgt ccgggcgcgt 19440ctcgtacacg ctgggcatgg aagggcccgc gatcaccgtg aacacggcgt gctcgtcctc 19500tctggtcgcc gtccacctgg cggcccgttc cctgcgggcg ggggagtgcg acctcgccct 19560ggccggcgcc gtcacggtca tgtccacacc gcagcttccg gtcgccttct cccggcagcg 19620cggactcgcc cctgacggtc gctcgaaagc cttcgcggtt tcggccgacg gcatgggctt 19680cggcgagggg gtgggcgtgc ttgtgctgga gcggttgtcg gtggcgcggc ggaacggtca 19740tcgggtgttg gcggtggtgc ggggttcggc ggtgaaccag gacggtgcgt cgaacggtct 19800gacggcgccg aacggtccgt cgcagcagcg ggtgatacgt gcggcgttgg cgagtgccgg 19860gctgggtccg gccgatgtgg atgtggtgga ggcgcacggt acggggacgc ggttgggtga 19920tccgatcgag gcgcaggcgt tgctggcgac gtacgggcgg ggccgggacg cggagcgtcc 19980gttgtggctg gggtcggtga agtcgaacat cggtcatgcg caggctgctg ccggtgtcgc 20040cggtgtcatc aagatggtgc tggccatgga gaagggccgt ctccctcgga cgctgcatgt 20100ggatgagccg tcgggtgagg tggactggga ctcgggtgcg gtgcggctgc tgaccgaggc 20160gcgggactgg ccgtcggagg aaggtcgtct gcggcgggcc ggtgtgtcgt cgttcgggat 20220ctcaggcacc aacgcgcacg tgatcatcga ggaagcaccg gaagaggggg aggaaccgga 20280gtccgacgcg ggtggtgtgg tgccgtgggt gctctccgcg cggacggaag gggcactgca 20340agcacaggcg gtgcaactga gcgagttcgt cggcgagtcg agtccggtgg atgtgggttg 20400gtcgttggtt tcgacgcgtg cggcgttcga gcatcgggcc gtggtggtgg ggcgcgggcg 20460ggacgagttg gtgcggggct tgtccgaggt cgcgcagggt cggggcgtga ggggtgtcgc 20520gtcttcggcg tcgggtggtc tcgcgtttgt ttttgctggt cagggcagtc agcggttggg 20580gatggggcgg gggttgtatg agcggttccc ggtgtttgcc gaggcgttcg acgaggtgtg 20640tgggcgggtc ggtccggggg tgcgggaggt tgttttcggt tcggatgcgg gtgagttgga 20700ccggacggtg tgggcgcagg cggggttgtt cgcgttggag gtggcgctgt ttcggttgtt 20760ggagtcctgg ggtgtgcggc cgggttgtct gatcgggcat tcggtcggtg agttgtcggc 20820ggcgtgtgtg gcggggttgt ggtcgttgga ggatgcgtgt cgggtcgtgg ctgcccgggc 20880gcggttgatg caggcgttgc cggcgggtgg ggtgatggtc gcggttcggg ccgaggcggg 20940ggagctggcc ggtttcctcg gtgaggacgt ggtgatcgcg tcggtgaacg cgccggggca 21000ggtggtgatc gctggtcctg aggggggtgt ggagcgtgtg gtggctgctt gtggggcgcg 21060gtcgcgtcgt ctggcggtct cgcatgcttt tcattcgcct ttggtggagc cgatgcttgg 21120ggagttccgt cgggttgtgg agtcggtggc gttcggtgtg ccgtcgttgc gggtggtttc 21180caatgtcacg ggtgcgtggg tggatccgga ggagtggggg acgccggagt actgggtgcg 21240tcaggtccgt gagccggtgc gtttcgccga cggggtcgcc acgttgctcg acgcgggtgt 21300gaggacgttc gtcgagctgg gtcccgccgg ggcgctcact tcgatggtca gccactgcgc 21360ggacgccacc gccacttcgg tgacggctgt acctaccttg cgccccgatc acgatgagtc 21420gcggaccgtg ttgagtgccg cagcgtcctt gtacgtccag ggtcacccgg tcgactgggc 21480cccgctgttc ccgcgggccc gcacggtgga cctgcccacc taccccttcc agcaccagca 21540ctactggctc gacgtacctc ctctgttcac cgcctcctcg gcggcccagg acggtggctg 21600gcgataccgc atccactggc ggcggctcgg cacgagggac tccggggacc ggctctccgg 21660ccgctggttg ctgctggtgc ccgagtcgga cgggacggag ccctgggtgg agggggccga 21720gaagatgctg gccgagcgcg ggtgcgaagt cgtccacgtg ccgatcgcgg cgacggccga 21780ccgggacgcg atggtcggag ccgtgcgtga gagcgtcgag gacggtcggg tcgacggtgt 21840gctcagcctg ctggcgctcg acggccgccc gcaccccgat gcggctgcgg tgccgacagg 21900gttggtcgcc acggcgcagg ttgtgcaggt cagtgacgag ctgggcatcg gcccgctgtg 21960ggtcgccacc cgacaggcgg tctccgtcga cggggccgat gaggctgacg gggccggtag 22020gaccaggaag gccgacgacc ccgccgatgt cgcgcaggcc gctgtgtggg ggctcggccg 22080ggtcgccgcg ctggagaagc ctcggttgtg gggcggcctc gtcgacctgc ccgcacgtgc 22140cgacgaacgg atgcgggacc tggtggctca ggccctcacc gctcccgacg ccgaggacca 22200acttgccgtg cgggccgacg gcatcgccgt tcgccgactg gtacgctccg ccgcgtcggc 22260cccggccgac gactggcagc cgagcggcac cgtgctggtc accggcggca ccggaggcgt 22320cggagccaac gtggcgcgtt ggctggtcac ccaggacatc cagcacctgt tgctggtcag 22380ccggcgcggc ccggacgccc ccggagccgc tgagctgctg gccgaactca gcgcctcagg 22440aacgtccgtg accatcgagc cctgcgacgt caccgacgcg gacgcggtac ggcgcctgat 22500cggcgccgta ccggccgaac ggccgctgag cacggtcgtc cacgccgcgg gcgtactgga 22560cgactgcttg atcgacgccc tgaccccgca gcgcctcgcc gccgcactgg aggtcaaggc 22620caagggcgca ctgaacctcc acgaggcggc cggggaagcc cacttggtgc tcttctcctc 22680gctggccgga acaaccggaa ccaagggaca gggcaactac gccgccgcaa acgcctatct 22740cgacgctctg gccgaacggc ggcgtgctga cggcctgccc gccacttcgg tcgcctgggg 22800cgcctggcag ggcgcgggca tggtggccga cgccgccgta gcccaccgca cgcgccgtta 22860tggcctcccg ctcatgagcc ccgaccgcgc cgtcgccacc ctgcggcagg tcatggccga 22920gccggtggcc acgcaggtgg tggcggacgt cgactggcag cgattcgtcg ccgacttcac 22980cgcggtgcgc cccagccgcc tcctcgccga cctgccggaa gtgcgctccc tgggcgagca 23040gcgaaaggac ggcccgggcg gtcagggcga ggaggacggc ttggccagca agctggcagc 23100cctgcccgaa gccgaccgcc gacgagccgt gctggacctc gtggaggaac tcgtcctcgg 23160ggttctgggc cacgagacgc gcgcggcgat cggcccggac agttccttcc acgccatcgg 23220cttcgactcg ctcaccgccg tcgaactgcg caacctgctg accgtacgcc tcgggatgaa 23280gctgcccgcg accctcgtct acgatcaccc gaccctgtcg tcgctggccg accacctgca 23340cgagcaactg gttatcgacg gcacccccat gacggacacc gcggccgacc tgctcgccga 23400actcgacgca ctcgcggcga gactcgccgc cgtcgggctg gaaccggagg cgcgcgcccg 23460catcggacgc aggctcaagg acatgcagac cgcctgcgaa cccaggtcgg agtcctcacg 23520cgacctgaag tccgcctcac gcaccgaagt gctcgacttc ctcaccaacg aactcggcat 23580ctcccgctga ccagttgacc gaccgcgacg aacggcgcac ctggctgcgg ctcgtccacg 23640ccgaccttcg accttgcccg acgcccccgg gagcggacta ccaccatgcc caacgacgaa 23700gaactcctcg actacctgaa gcggactgcc tcgaacctcc aggaggcgcg gcagcgggtg 23760cacgaactgg aggagagcga gcgcgagccg atcgcgatcg tggggatgag ctgccgtctg 23820cccggcgggg tgaacagccc ggaagagttc tggtcgctgc tggaggccgg gacggacgcc 23880gtctcggagt tcccgcggga ccgtggctgg gacgtggagc ggctgtacga cccggacccg 23940gacgcccccg gcaagtcgta cgtgcgggaa ggcggattcc tcgacggcgc gggccggttc 24000gaccccgcgt tcttcggtat ctccccgcgg gaggccgtgg tcatggatcc gcagcagcgg 24060ctgctgctgg agtgctcgtg ggaggcgatc gagcggtcgc ggatcgaccc gaagaccctg 24120cacggcagcc gcgcgggcgt gttcgtgggc tcgaacggcc aggactacgg gacgcttctc 24180ctgcgtgccg acgaccgctc ccacgcctac ctcgccacgg gcgcctccgc gagcgtgctc 24240tccggccgca tctcctacac gctcggactg gagggccctg cggtcacgat cagtacggcc 24300tgctcgtcct cactggtcgc cctccacctg gcggcccgcg ccctgcgggc gggggagtgc 24360gagctggcgc tcgccggcgg tgtgacggtc atgccgacga cccgcctgtt cgaggtcttc 24420tcccggcagc gtggcctggc cggtgacggc cgctgcaagg ccttcgcggc cggggccgac 24480ggcactggct ggggcgaggg cgtgggcgta ctcgtcctgg agcggttgtc ggtggcgcgg 24540cggaacggtc atcgggtgtt ggcggtggtg cggggttcgg cggtgaacca ggacggtgcg 24600tcgaacggtc tgacggcgcc gaacggtccg tcgcagcagc gggtgatccg cgcggccttg 24660gccagtgcac gcctggcccc cgaggacgtg gacgccgtag aggcacacgg cacggggacc 24720tccctgggcg acccgatcga ggcgcaggcg ttgctggcga cgtacgggcg gggccgggac 24780gcggagcgtc cgttgtggct ggggtcggtg aagtcgaaca tcggtcacgc gcaggccgct 24840gccggtgtcg ccggtgtcat caagatggtc aaggcgatgc aggcgggcac gctgccccgg 24900acgctgcatg tggatgagcc gtcgggtgag gtggactggg actcgggtgc ggtgcggctg 24960ctgaccgagg cgcgggactg gccgtcggag gaaggtcgtc tgcggcgggc cggtgtgtcg 25020tcgttcggga tctccggcac caacgcgcac gtgattctcg aggagccgcc ggcggaggac 25080gcggtaccgg agcctgaagc gggtgatgtg gtgccgtggg ttctttcggc gcggtcggct 25140gaggcgttgc gggagcaggc tgcccggctg gcgtcggtgg ctggtgggtt gaacgtggtg 25200gatgtgggct ggtcgttggc ttcgacgcgt gcggcgttcg agcaccgggc cgtagtggtg 25260gggcgggagc gggaagagct gctcgcgggt ctgttcgctg tggctgcggg acgcccggct 25320gcgaacgtgg tgacggggcc cgtcagctcc ggtcggcccg cctttgtttt tgctggtcag 25380ggcagtcagc ggttggggat ggggcggggg ttgtatgagc ggttcccggt gtttgccgag 25440gcgttcgacg aggtgtgtgg gcgggtcggt ccgggggtgc gggaggttgt tttcggttcg 25500gatgcgggtg agttggaccg gacggtgtgg gcgcaggcgg ggttgttcgc gttggaggtg 25560gcgctgtttc ggttgttgga gtcctggggt gtgcggccgg gttgtctgat cgggcattcg 25620gtcggtgagt tgtcggcggc gtgtgtggcg gggttgtggt cgttggagga tgcgtgtcgg 25680gtcgtggctg cccgggcgcg gttgatgcag gcgttgccgg cgggtggggt gatggtcgcg 25740gttcgggccg aggcggggga gctggccggt ttcctcggtg aggacgtggt gatcgcgtcg 25800gtgaacgcgc cggggcaggt ggtgatcgct ggtcctgagg ggggtgtgga gcgtgtggtg 25860gctgcttgtg gggcgcggtc gcgtcgtctg gcggtctcgc atgcttttca ttcgcctttg 25920gtggagccga tgcttgggga gttccgtcgg gttgtggagt cggtggcgtt cggtgtgccg 25980tcgttgcggg tggtttccaa tgtcacgggt gcgtgggtgg atccggagga gtgggggacg 26040ccggagtact gggtgcgtca ggtccgtgag ccggtgcgtt tcgccgacgg ggtcgccacg 26100ttgctcgacg cgggtgtgag gacgttcgtc gagctgggtc ccgctgggac gctcacttcg 26160atggtcagcc actgcgcgga cgccaccgcc acttcggtga cggctgtacc taccttgcgc 26220cccgatcacg atgagtcgcg gaccgtgttg agtgccgcag cgtccttgta cgtccagggt 26280cacccggtcg actgggcccc gctgttcccg cgggcccgca cggtggacct gcccacctac 26340cccttccagc accagcacta ctggatgatg aacaccggaa gtgccgccga gccggcggag 26400ctggggctcg gcgatgcccg tcatccgctg ctcggttccg tcgtcaccgt cgcgggggac 26460gacaaggtcg tcttcgccgg gcggctggcg ctgcgcacac acccctggct ggccgaccac 26520accgtgctcg acgcggtctt gctgcccgct acggccttcc tcgaactggc cgtgcgcgcc 26580ggtgaggagg tgagctgtcc ggtcgtacac gacctgacgc tgcaccgacc gctggtcgta 26640cccgagcggg gcgccgtgca ggtacagatg gctgtgggcg caccggaagc cgatgggcga 26700cgtgaggtcc gggtgtactc ccgccccgac gacgacgcgg agcacgagtg gacgctgcac 26760gccgctggac tgctggcgtc ggccgccacg gcggagcccg ccgtggcggc cggtgcctgg 26820ccgccgccgg aggcgcaggc cgtggacctc gacggcttct acgccggact cgccgagcac 26880ggctaccact acggcccgct gttccagggc gtccgggccg cgtggcggct gggcgacgac 26940gttctcgccg agatcgtgct gcccgaggcg gccggcgccg acgccgcccg gtacggcatg 27000catccggccc tgctcgacgc cgtcctgcac gcggcacggc tgggcgcctt ccgtgagcgg 27060tcggaggaga agtacctgcc gttcgcctgg gaaggcgtga ccctgcgtac caggggagcg 27120accgccgtac gtgctcgaat ctcccgggcc ggtaccgacg ccatccggct ggacgtcacc 27180gacaccgcgg accggccggt cctcacggcc gaatcgctca cgctgcgacc ggtctccgcc 27240ggtcagctca tggccgtccc gcgcgactca ctgttccggg tcgactgggt ttccgcgccc 27300gccgcgaacg gtcccggcct gcggctggcc cgtgccgcca ccgtggaggc ggccctcgcg 27360gcggacgccg acatcgtggt cgtgccatgc ctcgacagtg agggtccgca tcaggcgacg 27420taccaggcac tggagctgct acagcgctgg ctggccgccg acaccggtac caccacgctc 27480gccctgctca cccaccgtgc cgtggcggtc ggcgacgacg tccacgacct ccaccacgcg 27540cctctgtggg gcctggtccg caccgcccag accgaacacc ccggctgctt ccggctcgtc 27600gactcggacg accccgaccc gacgacggac gtcctggccg cggcgctcgc caccggggaa 27660ccccaggtcg cgatccgtga cggcgccgtc ctggccccgc ggctgaccgc ggcctccgcg 27720ccgcgggagc cggccgagtg ggacgccgag ggaacagtcc tcatcaccgg cggatcgggc 27780gccctcgcag ggatcgtggc ccagcacctc gtcgcacgtc acggcgtacg ccgactcgtc 27840ctcgcgagcc gcagcggcag gcccgcaccg ggggccgacc tgctcgacgc cgacgtcacg 27900gccgtgtcct gcgacgtctc cgaccgcgac gccgtggccg cgctgctcgc ctccgtgccg 27960gacgaacacc cgctcaccgc cgtcgtgcac accgcaggcg tactggacga cggcgtcctg 28020cacgccctca cgaccgagcg catcgacacc tcgttcgcgg cgaaggtcga cggcgcccgt 28080catctccacg aactcacctc ccacctggat ctcaccgcgt tcgtgctgtt ctcctccgcg 28140tcggccgtgc tgggcgccgc cggacagggc aactacgccg cggccaacgc ctacctcgac 28200gcgctcgccg cccaccgtcg cagcaacgac ctgcccgccg tgtctctcgc gtgggggctg 28260tgggccgagc acgagggcat ggcccgcgga ctcggtgacg ccgagctgac gcgtatttcc 28320cggatcggcg tcaccgcgct gagcgcggag gacggcatgc ggctgttcga cgccggatgc 28380gccggcgatc agtcacagct cgtgccgatg cgggtggaca ccgcggcgct gcgcgcacgg 28440cgtgaccacc ttcccgcacc gatgtggagc ctggtccccg agcggacccg agcggcacgt 28500acacagcctg ccgcctcgct tcgggacagg ctcgccgaac tgaccgcccc cgaacgcaag 28560cgcacggtcc tcaacctggt gcgcaacgcg gtcgccgaca cactcggcca caacgccgcc 28620gacggagtac cgcccgacca gagcctcgac gccgccgggt tcgactcgct caccgccgtc 28680gagttccgca accggctctc cgccgtcacc gacctgcgcc tgcccgccac cctcacctac 28740gatcacccca cccccgcggc catcgccgag cacatcctga cccgcctcac cctgctgaag 28800gagaccgccg ccccggccgt cggcaccgcc ccggttgcgg cgccgaccga agacgatgcg 28860atcgtcatcg tgggcatggc gggccgcttc cctggcggcg tgcgcacacc cgaaggtctt 28920tgggacctcg tccactccgg cacggacgcc atctcggagt ggcccaccga ccgcggctgg 28980gacgtggaga acctctacga cccggacccc gacgccgtcg gcaagtccta cgtacggcac 29040ggcggattcc tgcacgacgt cgccggcttc gacgcgggct tcttcgggat ctcgccgcgt 29100gaggcgctgg cgatggaccc gcagcagcgg ctcctgctgg agtgctcgta cgaggccctg 29160gagcgggcgg gcatcgaccc ggccacgctc agaggcagcc ggtcgggcgt gtacgccgga 29220gtgatgtacc acgagtacgc ctcccggctg ggcgccacgc ccgcaggctt cgaaggcaca 29280ctcggcaccg gaagctcggg cagcatcgcc tccgggcgca tctcctacac attcgacctc 29340accgggcccg cggtcaccgt cgacaccgca tgttccacct ccctcgtagg cctgcacctg 29400gccgtgcagg ctctgcgggc cggtgagtgc gaactggccc tcgccggcgg cgtcaccgtc 29460atgcacacgc cgcgcccctt cgtcgagttc tcccgccagc gcggcctggc cgcggacggc 29520cggagcaagg ccttcgcggc ctccgccgac ggggtggcct gggccgaagg cgccggaatc 29580ctcgtcctgg agcggctgtc ggcggcgcgg cggaacggtc atcgggtgtt ggcggtggtg 29640cggggttcgg cggtgaacca ggacggtgcg tcgaacggtc tgacggcgcc gaacggtccg 29700tcgcagcagc gggtgatacg tgcggccttg gcgagtgccg ggctgggtcc ggccgatgtg 29760gatgtcgtcg aggcccacgg caccggcacg gccctcggcg atccgatcga ggcgcaggcg 29820ttgctggcga cgtacgggcg ggggcgtgac gcggatcgtc cgttgtggct ggggtcggtg 29880aagtcgaaca tcggtcacac gcaggcggcc gcgggtgtgg caagcgtgat caagatggtg 29940caggcgatgc aggcgggcgt gctgccgcgg acgctgcatg tggacgagcc gtcgggtgag 30000gtggattggg actcgggtgc ggtgcggctg ctgaccgagg cgcgcgagtg gccgtcgggg 30060gaggggcgtg tgcggcgggc gggtgtgtcg

tcgttcggga tctccgggac gaacgcgcac 30120gtgatccttg aggagccgcc ggcggaggac gcgctgccgg agcctgaagc gggtgatgtg 30180gtgccgtggg ttctttcggc gcggtcggca gaggcgttgc gggagcaggc tgcccggctg 30240gcgtcggtgg ctggtgggtt gaacgtggtg gatgtgggct ggtcgttggc ttcgacgcgt 30300gcggcgttcg agcaccgggc cgtcgtcgtg ggaggcgatc gggaagagct cctggggaag 30360ctttcctcgg tttcgggggt cgaggtcggg gtcggggtcg gtgccggtgg tggtgtggtg 30420ttggtgttcg ccggtcaggg gtgtcagtgg gtcggtatgg ggcgggagtt gctgggttcc 30480tcgctggtgt tcgcggagtc gatgcgggag tgcgcggcgg ctctgtcgcc gtttgtggac 30540ttttctgtgg tggatgttct gggttcggct ggggagttgg gtcgggtcga ggtggttcag 30600cctgcgttgt gggcggtgat ggtgtcgctg gcgcgggtgt ggcggtcgtg gggtgttccg 30660gttgctgcgg tggtgggtca ttcgcagggt gagattgccg cggcgacggt ggcgggtgcg 30720ttgagtgtgg gtgatgcggc gcgggtggtg gcgttgcgga gccgtttgat cgcggagcgt 30780ctgtcggggc tgggtgggat ggtttcggtg gcgttgtcgc gtgagcgggt ggtgtcgttg 30840atcgcgggtg tgccgggtgt gtcggtggcg gcggtgaacg gttcttcgtc gacggtggtc 30900tcgggtgagg ccgcggggct ggagagggtg ctggccgcgt gtgtgtcgtc gggggttcgg 30960gcgcgtcgta tcgatgtgga ttacgcctcg cattcggtgc aggtggagtt gatccgtgag 31020gagttgttgg gggttctgga cgggatcgtc ccgcgctcgg gtgagattcc gttcgtgtcc 31080acggtgacgg gtgagcggat cgacactgtc gagctggggg cggagtactg gtaccgcaat 31140ctccgtcaga cagtggaatt ccagtcggtg gtggagggtc tggtcgctca ggggtgtcgg 31200gtgttcctgg agtccagtcc gcatccggtg ttgacggtcg gcatcgagga gtccgcggat 31260cgggtcgtgg cgttggagtc gctgcgtcgt ggcgagggtg gtctgcggcg gttggtggat 31320gcggccggtg aggcgtgggt gcgtggggtg ccgatcgact gggcggggat gctcgccggc 31380ggccggcggg tcgacctgcc cacctatccc ttccaacacc agccctactg gctcgactca 31440ccacgacacc ctgccggaga cgtgaccgcc gtcggtctca cagaggccgg tcacgcgttc 31500gtgccggcgg cggtcgacct gccggacggg cagcgggtct ggacgggacg actgtcgctt 31560ccctcctacc cgtggctggc cgatcatcag gtgctcgggc aggtgctgct ccccggcgtg 31620gtctgggtcg aactcgccct gcacgcgggg caccaggccg gatgcgactc tgtcgatgag 31680ctcaccctac agtcgccgct cgtgctcggt gcgtccgaca ccgtacaggt gagggtcgtc 31740gtcacggaga ccgaagagcc cggcacccgc accgtgtcga tgcactcgcg ccgtgacgac 31800ggcagctggg tgactcacgc cgaggggatc ctcggggcgg gcgggccgcc gccggagccg 31860ctgccggaat ggccgccgac cggcgccatg cccctcgatg tcgagggctt ctacgacgag 31920ctcgcggcgg gcggctacca ctacgggcct cagttccgct gcctgcggcg cgcctggcgt 31980gccggtgagg atctcgtcgc cgagatctcg ctgccggagg gcaccgacgt cgatgcgtac 32040ggcctgcacc ctggactctt cgacgcggcg gtgcacagcg tggcctgcgc ccggacgagc 32100gcgggggccg gcgatgacgg tccccggctg ccgttcgcct tctcggacgt ccggctcttc 32160gcgaccgggg tgacctcgct acgggtccgg atcgatccgc agaactcctc gtggcaggcg 32220tgggacgaat ccgggctgcc ggtcctcacc atcgggcggc tcgccggccg gcctgtcgac 32280gccgatcagt tcgccgtgcg gcgggcgggc cacctcttcc gcgtcgaaac gcggcacgaa 32340gccctggccg gcccggcccc cgcctcctgg gcggtcatcg gagcggaccc ggccgggtac 32400gccgcagccc tggaggccac gggcgcgcag gtgacgacgg ctgccgacct ggccggtctc 32460acatcggcac ccgaagccgc cctgttcacg ctccccggca caaaggacgc gggggtcacc 32520gaggaggtgc cgaccgctgt ccgggaggcg accgctcagg tgctggaggt gctgcaggac 32580tggctcaccg acggacgttt cgacgatgcc cgactggtcg tcgtaagccg cgaagcggaa 32640gacggcgatc tcctccacgg aacggcgcgc ggactgctgc gcgccgcaca ggccgagcac 32700ccggaccgca tcacccttgt cgacctcgat gctcatcccg cctcgctcac ggcccttccc 32760ggtttcgccc tcggtcccga accggaggtc gtcgtacgcg cgggagacgg cagggcaccg 32820cgcctggccc gggcgcaggc ccccaccgga gcgggctcac tgggcacggg cacggtcctg 32880atcaccggag gcacgggcac cctcggggga ctgctcgccc ggcacctggt ggagacgcac 32940ggagtcaccc ggctgctgct ggtcagccga cgaggaccgg ccgccgacgg cgcggaccgg 33000ctgcacgccg agctcaccgg gcatggcgca cacgtcgaca tcgtggcggc cgacctcggc 33060gaccgcacga gcgtggccgc gctcctcgcc acggtcgacg ccgaccaccc cctgtcggcc 33120gtcgtgcacg ccgccggagc gctggacgac ggcgtgctcg gcacccggtc cgccgactgg 33180ctcgacccgg tcctgcgccc caaggcggac gccgcttggc acctgcacga actcaccgcc 33240gaactgcctc tgaccgcctt cgtcatgttc tcctcggccg catccgtgct cggcgcggcg 33300ggacaggcca actacgccgc ggccaacgga tttctggacg cactggccgc ccatcgtgcc 33360gcccggggac tgcccgggac ctcgctggcc tgggggctgt gggagcaccg cagcgaactg 33420acccggcaca cgggctcccc ctcccgcagc atcgcggccg tcggcgctct gtccaccgcg 33480gaggcccttg ccgccttcga cgccggcctg gcctccgggg agccgctggc agtgccgatc 33540cggctggagt cgacatccag tgaggaggta ccgccgatgc tgcgcggcct ggtccgcgta 33600cgccgccggg ccgccaccgg cacggaaccc gcggcgagcg cgggcgccgc gcaggaggtc 33660cggcagctgg ccgagttggg cgccgacgag cgacagcggc gcgtgcagcg gatcgtgctc 33720gacaccgcgg cggccgtcct cggccatgac agccacgacg ccatccccct cacccggggc 33780ttcctggagc tggggttcga ctccctgaca gcggtacggc tgcgcaaccg gctcgcccgc 33840cgactggggc tgcgcctgcc ggccacggtg gtgttcgacc accccagccc ggccgccctg 33900gccgcccacc tggtcgagca tctcgtgggc accgtcgacc cgaccgcgca ggccatggag 33960cagctggagg ctctgcgccg cagcgtgcac gcagccacac ccgccggtgg cctggaccgc 34020gccctggtga cccaacgcct gacggccctg ctcgacgaaa tgcggcacgt cgacggcccc 34080ggcggcaccg aaggccccga cggctccggg gacgacctgg agaacgcgac agcggacgag 34140atctacgccc tcatcgacaa cgaactgggc atcgggggta cgcagtgaac ggcgacgaca 34200aagcactggc ctatctcaag cgggtgaccg cggacctgcg gtcggcgaga gccaggctgc 34260aggaactgga gtccgccgac accgacccca tcgccatcat cggcatgggc tgccgtctgc 34320ccggtggcgt gcgcaccccc gaggacctgt gggacctcgt ggagaagaag catgacgcga 34380tcggcccctt ccccgccgac cgcggatggg acctcgagaa cctgtacgac cccgacccgg 34440acgcgccggg caaggcctac gtccgcgaag gtgggttcgt ccacgacgtc gccggcttcg 34500acgcgggctt cttcggaatc tcgccgcgtg aggcgctggc gatggacccg caacaccggc 34560ttctgctgga gtgctcgtgg gaggccctgg agcgggcggg catcgaccct tcctccctcg 34620agggcacccg caccggcgtc tacaccgggc tcatgaccca tgaatacgcg acccgactgc 34680cctcgatcga cgaggagttg gagggtgtca tcggcatcgg caacgccgga agcgttgcct 34740cgggccgcgt ctcctacacg ctcggcctga acggccccgc tgtcaccgtc gacacggcct 34800gctcctcctc gctcgtcgcc ctgcacctcg ccgcccaagc cctgcgccag ggccagtgca 34860cccttgcgct ggccggaggt gcctccgtca tcgcggcgcc gaccgtgttc gccaccttca 34920gccgacagcg gggcctcgcc cccgacggcc gctgcaaggc gttctcgtcc acgaccgacg 34980gcacgggctt cggcgagggg gtgggcgtac tggtcctgga gcgcctctcg gacgcccgtc 35040gcaacggaca cgaggtcctg gccgtcgtac ggggctcggc ggtcaaccag gacggagcca 35100gcagcggatt caccgccccg aacggaccgt cccagcagga cgtcatccgc gaggccttgg 35160ccgacggtcg actgacccct gcggacgtgg acgtcgtgga gggtcacggt acggggacgc 35220ggttgggtga tccgatcgag gcgcaggcgt tgctggcgac gtacgggcgg gggcgtgacg 35280cggatcgtcc gttgtggctg gggtcggtga agtcgaacat cggtcacacg caggcggccg 35340cgggtgtggc aagcgtgatc aagatggtgc aggcgatgca ggcgggcgtg ctgccgcgga 35400cgctgcatgt ggacgagccg tcgggtgagg tggattggga ctcgggtgcg gtgcggctgc 35460tgaccgaggc gcgcgagtgg ccgtcggggg aggggcgtgt gcggcgggcg ggtgtgtcgt 35520cgttcgggat ctccgggacg aacgcgcacg tgatccttga ggagccgccg gcggaggacg 35580cgctgccgga gcctgaagcg ggtgatgtgg tgccgtgggt tctttcggcg cggtcggcag 35640aggcgttgcg ggagcaggct gcccggctgg cgtcggtggc tggtgggttg aacgtggtgg 35700atgtgggctg gtcgttggct tcgacgcgtg cggcgttcga gcaccgggcc gtcgtcgtgg 35760gaggcgatcg ggaagagctc ctggggaagc tttcctcggt ttcgggggtc gaggtcgggg 35820tcggggtcgg tgccggtggt ggtgtggtgt tggtgttcgc cggtcagggg tgtcagtggg 35880tcggtatggg gcgggagttg ctgggttcct cgctggtgtt cgcggagtcg atgcgggagt 35940gcgcggcggc tctgtcgccg tttgtggact tttctgtggt ggatgttctg ggttcggctg 36000gggagttggg tcgggtcgag gtggttcagc ctgcgttgtg ggcggtgatg gtgtcgctgg 36060cgcgggtgtg gcggtcgtgg ggtgttccgg ttgctgcggt ggtgggtcat tcgcagggtg 36120agattgccgc ggcgacggtg gcgggtgcgt tgagtgtggg tgatgcggcg cgggtggtgg 36180cgttgcggag ccgtttgatc gcggagcgtc tgtcggggct gggtgggatg gtttcggtgg 36240cgttgtcgcg tgagcgggtg gtgtcgttga tcgcgggtgt gccgggtgtg tcggtggcgg 36300cggtgaacgg ttcttcgtcg acggtggtct cgggtgaggc cgcggggctg gagagggtgc 36360tggccgcgtg tgtgtcgtcg ggggttcggg cgcgtcgtat cgatgtggat tacgcctcgc 36420attcggtgca ggtggagttg atccgtgagg agttgttggg ggttctggac gggatcgtcc 36480cgcgctcggg tgagattccg ttcgtgtcca cggtgacggg tgagcggatc gacactgtcg 36540agctgggggc ggagtactgg taccgcaatc tccgtcagac agtggaattc cagtcggtgg 36600tggagggtct ggtcgctcag gggtgtcggg tgttcctgga gtccagtccg catccggtgt 36660tgacggtcgg catcgaggag tccgcggatc gggtcgtggc gttggagtcg ctgcgtcgtg 36720gcgagggtgg tctgcggcgg ttggtggatg cggccggtga ggcgtgggtg cgtggggtgc 36780cgatcgactg ggcggggatg ctcgccggcg gccggcgggt cgacctgccc acctatccct 36840tccaacacca gccctactgg ctcgactcac cacgacaccc tgccggagac gtgaccggcc 36900cgggcgacga cgagttctgg gcggccgtgg agcacggtga ggcgaccgag ttggcggacc 36960tgctccggag gtcggcggcg gagccggggc aggatcttca cgcacccgtc gcggccctgc 37020tgccgacgct tgcaacgtgg cgtcgggacc ggcagcgcag ggcggctgtg gactcctggc 37080ggtaccggat cgtatggcgt ccggtcgcca cgccctcgta cgacagggtg ctgtcggggc 37140gctgggctgt cgtcgtgccc gccggtcacg aggacgaccc cgtcgtcgac tgggtctgct 37200cggcgctgcg ggaccacggg ggcgagcccg aacgcatggt gctgggcccg cgggagagcc 37260gttcggcgct ggccacgcgg ctggccgccg atccccccgg gggcgtggtc tccctgctcg 37320gactgagcgg ggcggcgcac cccgaccacg aggtgctgcc cagtgccgtc gccggtaccg 37380tcctgcttgc ccaggccctc tccgacggcg ccgtacgagc accggtgtgg accctgaccc 37440gcaacggtgt gtccgcgacg gcgacggacc cggtggctcc cacgcacgcc gcgcaggtgt 37500gggccgtggc acgggtggcc ggtctggagc acccggaggc gtggggtggt ctgctcgacc 37560tgccggaccg tctcgacgac cgcgcggccg cccggttcgc cgcggtcctg tccgcgggcg 37620aggacgagga ccaactggca ttacgcgacg ctgggttgct ggcacgaagg ctggtgcgtg 37680cccccgttcc gcgcgacgcg gtgaccgccg gctggcagcc ccgcgacaca gcgctcgtca 37740cgggcggcac cggcggtctc ggcgggcagg tcgcccgctg gctggcggcc gcgggcgtac 37800ggcacctcgt gctggtcagc cgtcgggggg cggaggcgga gggcgcagac cgtctgcgcg 37860acgacctcac cgccctcggc gtacaggtga cgttcggcgc gtgcgacgtc gcggaccgcg 37920ccgcgctctc ggcgctcctc gaccgggttc aggaggacgg cccgccgatc cgcacggtcg 37980tgcacgcggc gggctccggt cgcgccgcca ggctgctgga caccgacgcc gaggagaccg 38040cggcggtgct gcgggcgaag tcggccggag cccggaacct gcacgaactc ctcgatgacg 38100tggacgcgtt cgtgctgttc tcctccggag cgggtgtgtg gggaagcagc gcccagggcg 38160cctacgcggc ggccaacgcc tacctggacg cactggccga acagcgcagg ggccaggggc 38220ggccggcgac ctccgtcgcc tggggcgcct gggccggtga cggcatgaca gccgccgccg 38280gcgaggaatg gtggagcagg cagggtctgc ggttcatggc ccctgaggcc gccctcgacg 38340cgctgcgcca ggccgtcgac cgcgccgaga gcacgctcgt cgtcgcagac atcgactgga 38400agacgttcgc tcccctcttc acgtcggccc gcagccgccc cctcatcacc gacatacctg 38460aagcccgccc cgaaccgagg ccggaaggcg cggaccagcc tacgcagggc ctcgtggcca 38520agctggcggt gctgtccgcg gacgaacggc ggcgcgccct gctcgccgag gtgcgggcgc 38580aggcagcggt ggtgctcggc caccccggcg cggacgccgt accggtcgac cggccgttcc 38640gcgagctcgg attcgactcc ctcagcgcgg tgaaactgcg caacaggatc gttgctgcca 38700ccgggctcga gcttccggcc accctggtct tcgaccaccc cacgtccacg gcgctcgccg 38760cctacctggg cgcccggctc ggaatcgacg gcgcccccgc ggggtccact ctgctggaag 38820acctcgcgcg gctcgagtcc accgtcgcca ccctgaccgc ggcacctctc gcagagaccg 38880tgccggacgc ccgggaccgc gcggcgctca ccacacggct gcgggcgttg ctggagcggt 38940gggaccaggc cgatggcgag gaccaggccg ccgcccgaga agaactcgac gatctgagcg 39000acgacgacct cttcgacttc atcgacgcga agttcggccg ttcgtgacct cggtcggccg 39060ccgccaactc cacgtacacc ccgaagacca cgatcaccac gcgaaaagga cgggcctctc 39120catgggggac gagcagaaac tccgcaccta cctccggcgc gtcactgccg acctggccga 39180cgtgacggag cggttgcagc gagcagagga caagaacgcc gagccgatcg cgatcgtcgg 39240catggggtgc cgctaccccg gtggggtgcg gtcgcccgag gagttctgga acctgctcga 39300cgaaggcgtc gacgcagtgg ccggcttccc cgaggaccgt ggctgggacc tggagaacct 39360gtacgacccc gaccccgacg agccgggtaa gtgctatgcc cgcgaaggcg ggttcctcta 39420cgacgcgggc gagttcgacg ccgcgttctt cgggatatcg ccccgcgagg ccctgtccat 39480ggacccgcag cagcggctgc tgctggagtg ctcctggagt gccctcgagc gggcgggcat 39540cgacccgggc tcgctgcgcg gcaaagacgt cggcgtgtac gtcggcgcat ggaacagcaa 39600ctacggcagg ggcggcgggg cggagagctc cgagggccac ctgctgaccg gcaacgcctc 39660cagcgtggtc tcgggtcgcg tggcgtacgt gctggggctc gaaggccccg ccgtcaccat 39720cgacaccgcc tgttcctcct ccctggtcgg cctgcacctg gccgcccagg ccctcaggtc 39780cggcgagtgc ggtcttgcgc tggccggcgg cgtcaccgtg atgtccaccc ctctgtcgct 39840ggtgtccttc tcccggcagc gggggctcgc acaggacggt cgttccaagg cgttctcggc 39900ggacgccgat ggcatgggca tggccgaagg tgtaggcgta ctggtcctgg agcgcctctc 39960ggaggcgcgc cgcaacgggc acgaggtcct ggccgtcctg cggagctcgg ccgtgaacca 40020ggatggtgcc tcgaacggtc tgagcgcccc gaacgggccg gcgcagcagc gtgtcatcca 40080gtccgccctg accgtcggcc gtctcgcccc ctccgacatc gacgtcgtcg aggcccacgg 40140caccggcacg gccctcggcg atccgatcga ggcgcaggcg ttgctggcga cgtacgggcg 40200ggggcgtgat gcggatcgtc cgttgtggct ggggtcggtg aagtcgaaca tcggtcacac 40260gcaggcggcc gcgggtgtgg ccggggtcat caagatggtg ctggccctgc gcaagggcgt 40320actgccgcgg acgttgcatg tggatgagcc aaccggtgag gtggattggg actcgggtgc 40380ggtgcggctg ctgaccgagg cgcgcgagtg gccgtcgggg gaggggcgtg tgcggcgggc 40440gggtgtgtcg tcgttcggga tctccgggac gaacgcgcat gtgatcgtcg aggaggctcc 40500ggaggaggag ccccggccgg aggctccttc cgtcgacgtg gtgccgtggg ttctttcggc 40560gcggtcggca gaggcgttgc gggagcaggc tgcccggctg gcgtcggtgg ctggtgggtt 40620gaacgtggtg gatgtgggct ggtcgttggc ttcgacgcgt gcggcgttcg agcaccgggc 40680cgtggtggtg gggcgggact ccgaggaatt ggtgtcgggg ctttcctcgg tttcgggggt 40740cgaggtcggg gtcggggtcg gtgccggtgg tggtgtggtg ttggtgttcg ccggtcaggg 40800gtgtcagtgg gtcggtatgg ggcgggagtt gctgggttcc tcgctggtgt tcgcggagtc 40860gatgcgggag tgtgcggcgg ctctgtcgcc gtttgtggac ttttctgtgg tggatgttct 40920gggttcggct ggggagttgg gtcgggtcga ggtggttcag cctgcgttgt gggcggtgat 40980ggtgtcgctg gcgcgggtgt ggcggtcgtg gggtgttccg gttgctgcgg tggtgggtca 41040ttcgcagggt gagattgccg cggcgacggt ggcgggtgcg ttgagtgtgg gtgatgcggc 41100gcgggtggtg gcgttgcgga gccgtttgat cgcggagcgt ctgtcggggc tgggtgggat 41160ggtttcggtg gcgttgtcgc gtgagcgggt ggtgtcgttg atcgcgggtg tgccgggtgt 41220gtcggtggcg gcggtgaacg gttcttcgtc gacggtggtc tcgggtgagg ccgcggggct 41280ggagagggtg ctggccgcgt gtgtgtcgtc gggggttcgg gcgcgtcgta tcgatgtgga 41340ttacgcctcg cattcggtgc aggtggagtt gatccgtgag gagttgttgg gggttctgga 41400cgggatcgtc ccgcgctcgg gtgagattcc gttcgtgtcc acggtgacgg gtgagcggat 41460cgacactgtc gagctggggg cggagtactg gtaccgcaat ctccgtcaga cagtggaatt 41520ccaagcatcc gtgcagacgc tcctcgccca ggggcaccag gtcttcctgg agtccagtcc 41580gcacccggtt ctcaccgtcg gcatcgagga gaccgttcac gagagcgccg cacaggccgt 41640cgttctggga agcctgcggc gggacgaggg tgccctcacc cggctcgtca cctccgccgg 41700tgaggcatgg gcgcgcggtg tgcccgtcga ctgggcgggc atgctcgccg gcggcaggcg 41760ggtcgagttg cccacgtatc ccttcctccg ggagcggctg tggctggagc cgtcgcgctc 41820ccgcaccggg aacctcaaca tggccgggct ggtcgaagcc ggacatgaaa tcctgcccgc 41880cgcagtggag ttgcccggag agcagtgggt gtggaccggc gagctgtcgc tctccgcgta 41940cccgtggctg gccgatcacc aggtgctcgg gcagaccctg gtgccgggcg tggcgtgggt 42000cgaactcgcc ctgcacgcgg gccaccagct cggtttcgga tccgtcgagg aactcaccct 42060gcaggcaccg ctcgtgctcg gcgagtccga cgccgtgcag gtcagagtcg ttgtctccga 42120tctcggggag agtgatcgcc gggcagtgtc ggtgcactcg cgtggtgacg accagacgtg 42180ggtgacccat gcggagggat tcctcaccgc gaaaggggcg cagccggaga ccatggccgt 42240gtggccgccg tccggtgcgg agccggtgga ggctgacggg ttctacgaac gcctcgccga 42300tgcggggtac cactatggcc cggtcttcca gggcgtgagc aaggtctggc gagctggcga 42360ggagatctac gccgaggtcg ggctgctcga cgacgccgac gtggacggct tcggcatcca 42420ccccgccctg ctcgacgccg ccctgcagac cgcctacgtc gcccaacggg gccccgcaga 42480gacgaagttg cctttcgcgt tcggcgatgt acagctgttc gccaccggtg cccggtcgct 42540ccgcgtacgg gtctcgccgg ccgctcagca ggggatggcg tgggaggcct gggaccccac 42600cggacttccg gtgttctccc tcgggtacct ggcgacccgg ccggtcgacc gcggccagct 42660gaccgtgaag cggcccgagt cgctgttcaa ggtggcctgg gacgagaccg tccccgtcgt 42720cgggaatgcg accgccgcgc atggcgtcgt gctgggcgac gacccgttcg ccctcggtgc 42780cgcgctgcgc gcggcgggct gggaggtcgg ggccgccccg gaacccgcgt ccgccgacac 42840cgccgccgaa gtactgctgc tgccctgcac cgcgcccggc gagccggacg cggacctgcc 42900caccgcggtc agggccgtga ctgctcgggt gctcggcgtc ctacaggagt ggctcgccga 42960cgaacggctc gccggcaccc gactggccgt cgtgacccgc aacgccctgc cgggtgacct 43020cctgcacagc cccgtctggg gtctcgtgcg ctccgcccag accgagaacc ccgggcgcat 43080caccctcgtc gacctcgacg accaccccga ctcggcggcc gtccttgccg aggccgtcca 43140gtccgacgag ccgcgcatca tggtccgcga gggccggccc accgccgccc gcctggtccg 43200tgccaccgca cccgagctgg tgccgcccgc cggagccgat gcctggcgcc tcgagatcac 43260cgaaccgggc acgttcgaca acctcacgct gggcgtctac ccgcacgccg agaagaccct 43320cgccgacaac gaggtccggg tcgccgtcca cgcgggcggc ctcaacttcc acgacgtggt 43380cgccgcactc ggcatggtcg aggacgacct gaccctcggc cgtgaggcgg ccggcgtcgt 43440cgtcgaggtc ggagacgccg tgccggatct gacccccggc gaccacgtga tgggcatcct 43500gtcctccggc ttcgggccgc tcgccgtcac cgatcaccgc tacctggcac gcatgcccga 43560gggctggacg ttcgcccagg cggcttcggt gcccgccgcg ttcctgacgg cctactacgg 43620gctgtgcgac ctcggcggca tccgcgcggg cgaccgcgtc ctcatccacg cggccgccgg 43680cggtgtcggc atggccgccg tacagatcgc ccggcacctc ggggcggagg tgttcggcac 43740cgccagcccg cgcaagtggg gcgcgctgcg cgccctgggg ctcgacgacg cccacctgtc 43800ctcctcccgc accctcgact tcgagcagga gttcctggac gccaccgacg gcaggggagt 43860cgacctcgtt ctgaactcgc tggcccggga gttcgtcgac gcctcgctgc ggctgatgcc 43920cggcggcggc cggttcgtgg acatgggcaa gaccgacatc cggcggccgg aacaggtggc 43980ggaggaccac ggcggagtcg cctaccaggc attcgacctc gtcgaggccg ggccgcagcg 44040cacgggggag atgctcgccg agatcgtccg gctcttccaa gccggcgcgt tccggccgct 44100gccgatcacc cagtgggacg tgcgccgggc gccggaggcc ttccgacaca tcagccaggc 44160caagcacata ggcaagatcg tcctcaccgt gccccggccc atcgacaccg acggcaccgt 44220catggtcacc ggcgccaccg ggaccctggg cggcttcgtc gcccggcacc tggtcaccca 44280tcacggcata cgacgactgc tgctggtcag ccgcagcgcg gagcgcaccg acctggtgcg 44340ggaactcacc gagctgggcg ccgacgtcac ctgggcctcc tgcgacctag ccgacgccac 44400cgccgtcgaa gagaccgttc ggtccgtcga cgaacggcat ccgctcgtgg ccgtcgtcca 44460ctctgcggga gtactcgacg acggcgtcat cgacaagcag agccccgaac ggctcgacac 44520cgtgatgcgt cccaaggtcg acgccgcctg gaatctgcac cgactcctcg acaacgcccc 44580gctggccgac ttcgtgctct tctcctccgc cagcggcgtg ctcggtggcg ccggacagtc 44640caactacgcg gccgccaacg ccttcctcga cgcgctcgcc gagcaccgcc gtgcacaggg 44700cctcgccgga caggcgctcg cctggggact gtggtccgac cgcagcacga tgacgggaca 44760gctcggctcc accgaactcg cccggatcgc ccgcaacggc gtcgccgaga tgtccgagac 44820ggagggcctg gccctcttcg acgccgcccg ggacaccgcc gaggcggtgt tgctgcccat 44880gcacctggac gtcgcgaggc tccgcagccg caacggagag gtacccgcgg tgttccgccg 44940gctgatccac gccacggccc gccgcaccgc gagcaccgcg gtccgcagcg ccggcctcga 45000acagcagctc gcctcgctgt ccggccccga acgcacggag ctgctcctgg gactggtgcg 45060cgaccatgcc gccgcggtgc tcggccacgg cacctccgac gccgtctcgc cggaccggcc 45120cttccgcgac ctgggtttcg actccctgac

tgccgtggag ctgcgtaaca ggttcgccgc 45180cctcaccggc ctgcgtctgc cggccacgct cgtcttcgac cacccgagcc cgacggccct 45240cgccgggcac ctcgccggcc tgctgggcgc cgcgacgccc tccgcggccg agccggtcct 45300ggccgccgtc ggacggctgc gcgccgacct ccggtcgctc accccggacg ccgagggcgc 45360cgaggacgtg acgatccagc tggaggccct cctcgccgag tggcgggagg ccgcggagaa 45420gcgggctccg gaggcggtcg gtgacgagga cctgtccacc gccaccgacg acgagatctt 45480cgcgctcgtc gacagcgaac tgggtgaggc ctgatgacgg ccgaagcgtc tcaggacaag 45540ctgcgtgact atctgcgaaa gaccctcgcc gacctgcgga ccaccaagca acggctacgc 45600gacaccgaac gcagggcgac cgagcctgtc gcgatcgtcg gcatgagctg ccgactgccc 45660ggcgacgtac ggacaccgga gcggttctgg gaactcctcg acactggaac cgacgccctg 45720acgcccttgc ccaccgaccg cggctggaat ctcgacacgg cgttcgacga cgaacggccg 45780taccggcgcg aaggcggatt cctttacgac gccggacggt tcgacgccga gttcttcggc 45840atctcgcccc gtgaggcgct ggccatggac cctcagcagc ggctgctcct cgaaagctcg 45900tgggaggcga tcgagcacgc ccgcatcgac cccaggtccc tgcacggcag tcgcaccggc 45960gtctggttcg gcacgatcgg ccaggactac ttctccctct tcgccgcatc cggcggcgag 46020cacgccaact acttggccac cgcctgctcg gccagcgtga tgtccggccg cgtctcgtac 46080gtgctcggcc tggaggggcc cgctgtcacg gtcgacacgg cgtgctcgtc ctccctggtc 46140gccctccact ccgccgtaca ggccctgagg tccggcgagt gcgaactggc tctcgccggg 46200ggcgccacgg tcatggccac cccgacggtg ttcaccgcct tctcccatca gcgtggcctg 46260gccggtgacg gccgctgcaa ggccttcgcg gcgggtgccg acggggcggg cttcgccgag 46320ggggtgggcg tgctggtgct ggagcggttg tcggtggcgc ggcggaacgg tcatcgggtg 46380ttggcggtgg tgcggggttc ggcggtgaac caggacggtg cgtcgaacgg tctgacggcg 46440ccgaacggtc cgtcgcagca gcgggtgatc cgcgcggcgc tggccaacgc gcgcttggcg 46500ccggaggacg tggacgctgt cgaaggccac ggcacgggga cttcgctggg cgacccgatc 46560gaggcgcagg cgttgctggc gacgtacggg cggggccggg acgcggagcg tccgttgtgg 46620ctggggtcgg tgaagtcgaa catcggtcat gcgcaggctg ctgccggtgt cgccggtgtc 46680atcaagatgg tgctggccat ggagaagggc cgtctccctc ggacgctgca tgtggatgag 46740ccgtcgggtg aggtggactg ggactcgggt gcggtacggc tgctgaccga ggcgcgggac 46800tggccgtcgg gggaggggcg ggtgcggcgg gcgggagtgt cgtcgttcgg gatctccggg 46860acgaacgcgc acgtgatcat cgaggagccg caggaggagg aagcggcacc ggattcctct 46920gcttcgggtg ccgtgccgtg ggtgctctcg gcgcgatcgg ccgaagcgtt gcaggctctg 46980gcttcacaac tcgccgacca cagcgccaaa tcgagtccgg tggatgtggg ttggtcgttg 47040gtttcgacgc gtgcggcgtt cgagcatcgg gccgtggtgg tggggcgcgg gcgggacgag 47100ttggtgcggg gcttgtccga ggtcgcgcag ggtcggggcg tgaggggtgt cgcgtcttcg 47160gcgtcgggtg gtctcgcgtt tgtttttgct ggtcagggca gtcagcggtt ggggatgggg 47220cgggggttgt atgagcggtt cccggtgttt gccgaggcgt tcgacgaggt gtgtgggcgg 47280gtcggtccgg gggtgcggga ggttgttttc ggttcggatg cgggtgagtt ggaccggacg 47340gtgtgggcgc aggcggggtt gttcgcgttg gaggtggcgc tgtttcggtt gttggagtcc 47400tggggtgtgc ggccgggttg tctgatcggg cattcggtcg gtgagttgtc ggcggcgtgt 47460gtggcggggt tgtggtcgtt ggaggatgcg tgtcgggtcg tggctgcccg ggcgcggttg 47520atgcaggcgt tgccggcggg tggggtgatg gtcgcggttc gggccgaggc gggggagctg 47580gccggtttcc tcggtgagga cgtggtgatc gcgtcggtga acgcgccggg gcaggtggtg 47640atcgctggtc ctgagggggg tgtggagcgt gtggtggctg cttgtggggc gcggtcgcgt 47700cgtctggcgg tctcgcatgc ttttcattcg cctttggtgg agccgatgct gggggagttc 47760cgtcgggttg tggagtcggt ggcgttcggt gtgccgtcgt tgcgggtggt ttccaatgtc 47820acgggtgcgt gggtggatcc ggaggagtgg gggacgccgg agtactgggt gcgtcaggtc 47880cgtgagccgg tgcgtttcgc cgacggggtc gccacgttgc tcgacgcggg tgtgaggacg 47940ttcgtcgagc tgggtcccgc tgggacgctc acttcgatgg tcagccactg cgcggacgcc 48000accgccactt cggtgacggc tgtacctacc ctgcgccccg atcacgacga gtcgcggacc 48060gtgttgagtg ccgcagcgtc cttgtacgtc cagggtcacc cggtcgactg ggccccgctg 48120ttcccgcggg cccgcacggt ggacctgccc acctacccct tccagcacca gcactactgg 48180atggaaagcg ccgcccggcc caccgtcgag gacaccccgc gcgagcccct cgacggctgg 48240acgcaccgca tcgactgggt gccgctggtg gacgaggaac cggcgcccgt cctggccggt 48300acctggctgc tcgttcgtcc cgaagaaggt ccccgcccgc tcgccgacgc cgtcgcggac 48360gcgctgaccc ggcacggcgc ctccgtcgtc gaggccgctc gtgtcccgca ccaatccgac 48420accgagctga ccggagtcgt ctctctgctg ggcccgggcg ccgacggcga cggcggcctg 48480gacgcgaccc tgcggctggt acaggacttg gccaccgccg ggtccaccgc gcccttgtgg 48540atcgtcacca gcggagccgt ggccgtcggt acgtccgaca ccgtgccgaa ccccgagcag 48600gcgacgctct gggggttggc ccgggcggcg gccaccgagt ggcccggcct gggggcggcg 48660cgcatcgacc tgcccgccga cctcaccgag caggtcggac gtcggctctg cgcccggctg 48720ctcgaccgga gtgagcagga gacggcggtc cgacaggccg gggtgttcgc caggcggctg 48780gtccgtgccc gtaccagcga cggccggtgg acgccgcgcg gcaccgtgct ggtcaccggc 48840gggaccggcg cgctcgccgg acacgtcgcg cgatggctgg cggaggaggg ggccgagcac 48900atcgtgctgg ccgggcgcag agggcccgac ggtcagggcg ccgaggcgct gcgcgccgac 48960ctggtcgccg caggggtcaa ggcgacgatc gtgcgctgcg acgtcgccga ccgggatgcc 49020gtacgtctgc tcctggacgc acaccggccc agcgccatcg tgcacacggc cggggtcgtc 49080gacgacggac tgctcacctc gctgacgccc gcccaggtcg agcgggtgct gcggcccaag 49140ctgctcggcg ccaggaacct gcatgagctg acccgggacc gggaactgga cgccttcgtg 49200ctgttctcct ccctcgccgg agtcctcggc ggggcagggc aggccaacta cgccgctgcc 49260aacgcctact tggacgccct ggccgcacac cgcaccgcgc atgggctgcc ggcggcctcg 49320ctggcatggg ggccgtggga gggcgacggc atggccgcgg cgcaggaggc cgccgaccgg 49380ctccgccgca gcggtctcac cccgctgccg ccggagcagg ccgtacgggc cctcggccgg 49440ggccacgggc cgctggtggt ggccgacgcg gactgggcgc ggctggccgc cggctcgacg 49500cagcgcctgc tcgacgagct tcccgaggtg cgtgcggtca ggccggcgga gcctgctgtc 49560ggacagcgcc ccgacctacc ggcccggttg gcggggcgtc cggccgagga gcagtccgcg 49620gtactgctgg aggcggtccg ggaggagatc gccgccgtac tgcgttacgc cgatccggcg 49680cggatcggcg ccgatcacga gttcctcgcc ctcggcttcg actcgctgac atcgatcgaa 49740ctgcgcaaca ggcttgccac gcgcatcggt ctgacgcttc ccgcgacgct caccctggaa 49800cagcgcaccc ctgccgggct cgccgcgcac ctgcgcgagc ggatcgcgga ccggcccgtc 49860gggtccggtg ccgtcccggt gcccgggagc gctgatgtcc cggaggcggg cggcggtagc 49920ggcctcggtg agctgtggca ggaagccgac cggcacggcc ggcggctgga gttcatcgac 49980gtactcaccg cggccgccgc cttccggccc gcctaccgtg aaccggccga gctggagctg 50040ccgcctctac ggctcacctc cggcggggac gagccgcccc tgttctgcat cccctcgcac 50100ctcggcaagg ccgacccgca caagttcctg cggttcgccg cggccctgcg gggacggcgg 50160gacgtcttcg tcctgcgcca gcccggcttc gtacccgggc agcccctgcc cgcgggcctc 50220gacgtcctgc tcgacaccca cgcgcgggcc atggccgggc acgaccggcc cgtgctgctc 50280ggctactcgg ccggcggtct tgccgcgcag gcgttggccg cccgactcgc cgagctcggc 50340aggccgccgg cggccgtcgt gctcgtcgac acctatgccc ccgacgagac ggaggtgatg 50400gcccgtatcc agggcgccat ggagcagggc cagcgcgatc gcgacggcag gaccggtgcc 50460gccttcggtg aggcctggct caccgcgatg ggccactact tcggcttcga ctggaccccg 50520tgtccggtcg acgtgccggt gctgcacgta cgcgccggcg accccatgac cggtatgccc 50580gtcgaagggc ggtggcaggc gcgctggaac ctgccgcaca ccgccgtcga cgtgcccgga 50640gaccacttca cgatgatgga ggatcacgcc ccgcgcaccg ccgacaccgt gcacgactgg 50700ctcggcacgg ccgtccgccg ccctgagaga acccgcgact gacgactcgc cggcgacagc 50760ggcatcccgc cctgtcccct tcctgtccgt ccgttccctt tcctctcctc gaaacggagt 50820tcgttctcat gccttccttc cccgtacgcc ggtccgtgcc cgacactccg cccgccgagc 50880acctcgaact gctcaaggag agcggcggcg tctgcccctt caccatggag gacggccgtc 50940cggcctggct cgcggccagc cacgacgccg tgcgctccct gctcgccgac cgccgtatca 51000gcaacaaccc ggcgaagacg ccgcccttct cgcagcggga ggccctgcag aaggagcggg 51060gccagttcag ccgtcacctg ttcaacatgg actcgccgga gcacgacgtg gcccgccgca 51120tgatcgcgga ggacttcact ccccggcacg ccgaggcggt ccggccgtac ttcgaggagg 51180tgttcggcga gatcgtcgac gaagtggtcc acaagggccc accggccgag atgatcgagt 51240cgttcgcctt cccggtcgcc acccgcacca tctgcaaggt gctggacatt ccggaggacg 51300actgcgagta cttccagaag cgcaccgagc agatcatcga gatggaccgc ggcgaggaga 51360acctcgaagc cgtcgtcgaa ctgcgccgct acgtcgacag cgtcatgcag cagcgcaccc 51420gcaagcccgg cgacgacctg ctcagcagga tgatcgtcaa ggcgaaggcg tccaaggaga 51480tcgagctcag cgacgccgac ctggtcgaca acgcgatgtt cctgctggtg gccgggcacg 51540agccgtcggc caacatgctg ggcctcggcg tgctcgccct cgccgaattc ccggacgtgg 51600ccgaggaact gcgggccgag ccgcacctgt ggccgggcgc gatcgacgag atgctccgct 51660actacaccat cgcccgggcc accaagcggg tcgcggccgc cgacatcgag tacgaggggc 51720acacgatcaa ggagggggac gccgtcatcg tgctcctcga caccagcaac cgcgacccga 51780aggtgcacgc cgaaccgaac cggctcgaca tccaccgctc ggcgggcaac cacctggcct 51840tcagccacgg accgcaccag tgcctgggca agcacctcgt ccgggtccaa ctggagatcg 51900cgctgcgggc tgtcgccgag cggctgcccg gcctgcgcct ggacatcgcc aaggaggaca 51960tccccttccg cggtgacgcc ctgtcctacg ggccgcgcca gctgcgcgtc acctggtaac 52020agccaccatc ggcccccgcc gcggaccggg cagcacgacc cggtccgcgg cgggggcacc 52080accaccgtca acatccccag agaggcttcc ccgtggagaa gaccgacgtc gaccggctgc 52140gcacactcga ccgagagcac atgtggtacc cgtggacgcc gatgaccgag tggatggccc 52200gtgatcagct cgtcgtcgaa cgcgccgaag gctgctggct gatcgacgca gacggtaagc 52260gctacctcga cggccgctcg tcgatgggca tgaacctgca cggccacggc cgcagcgaga 52320tagtcgaggc cctggtcgcc caggcgcgca aggccggtga gaccacgctc taccgcgtct 52380cgcacccggc ggcggtggaa ctcgccgccc gcctggcatc gatggcgccg gccgggctcc 52440agcgcgtctt cttcgccgag tccggatcga ccgcggtgga gacggctctc aaggctgcct 52500acgcctactg ggtcgcgaag ggcgaaccgc agcgatccac cttcgtgtcc atggagggcg 52560gttaccacgg cgagacccta ggcacggtca gcctgcgcgg caccaacggc gaacaggtcg 52620acatgatccg caagacctac gagccactgt tgttcccctc cctctccttc caccagcccc 52680actgctaccg gtgtcccgtc ggccagtcgt cggacagcga ctgcgggctg gagtgcaccg 52740attcgctgga gaacctcctc acccgggaga agggccggat cgccgcggtc atcgtcgagc 52800cgcgggtcca ggccctcgcc ggagtgatca ccgccccgga gggacacctc gcgaaggtcg 52860cggagatcac ccgcaggcac ggagtgctcc tcatcgtcga cgaggtcctc accggctggg 52920cccgcaccgg cccgacgttc tcctgcgagg ccgagggcgt cacaccggat ctgatgacgg 52980tgggcaaggc gctgaccggc ggatatctgc cgctgtcggc caccttggcc acggaggaga 53040tcttcggagc cttccgtgag agcgtcttcc tcagcggcag cacctactcc ggatacgcgc 53100tcggggcggc cgtcgccctg gccagcctcg acctgttcga gaaggaggac gtaccggccc 53160gggccaaggc gctcgccgac gtgctcacca ccgcactgga acccttccgc gcgctcaccc 53220acgtcggtga cgtccggcag ctcggcctca tcgccggcgt cgagctggtg gccgaccggg 53280agacccgcgc cccctacccg ccccaggagc gcgtcgtcga tcgcatctgc accctggcca 53340gggacaacgg cgtgctggtc aacgcggtcc ccggggacgt gatcaccatg ctgccctcac 53400cgtcgatgag ccccgacgac ctgcgcttcc tcaccggcac cctgtacacg gccgtccgag 53460aggtgaccga agagtgaaag ggctgatgcg ggcggccgtc atccgtgcct ggggcggccc 53520cgagcggctg accctggacc gggtcgaacg gccgtcaccc ccgcccggat ggatcgccgt 53580acgcgtcgag gcctgcgccc tgaaccacct cgacatccac gtgcgcaacg ggcttccggg 53640cgtacggctg gaactcccgc acgtctccgg cggcgacgtc gtcggcgtcg tcgagcaggc 53700caccgacgag gcgggggaga gactgctcgg cagccgtgtg ctgctcgacc cgatgatcgg 53760gcgcggcatc ctcggcgagc actactgggg cgggctcgcc gagtacgtcg tcgcacccgc 53820ccacaacgcg ctccccgtcc ccgatcagga cgcggacccg gcacgctacg ccgcactgcc 53880catctcctac ggcacggccc agcgcatgct cttcagccgc gcccggctgc gtcccggcga 53940gagcgtgctg ctgttcggcg cgaccggcgg cgtcggcgtc gcctgcgccc agctcgccct 54000gcgtgccggg gcccggatca tcgcctgctc cggatcaccg gccaagctcg cccggctgcg 54060ccgactcggc gtgatcgaca cgatcgacac cggcaccgag gacgtacggc gcagggtccg 54120cgaactcacg gacggcggtg ccgacctggt cgtcgactac cagggcaagg acacctggcc 54180cgtctccctg cgctcggcgc gcgccggcgg ccgcatcgtc acctgcggcg cgaccaccgg 54240gtacgaggcg acgaccgacc tgcgctacgt gtggtcgcgt cagctggaca tcctcggctc 54300caacgcgtgg caccgcgacg atctgcacac gctggtcgac ctggtggcca ccgacgccct 54360ggaaccggtg gtgcacgccg acttcccact ctcccgcgcc cccgaggcgg tcgccgaact 54420ggaggagcgc cgggcgttcg ggaaggtcgt gatccgcacg gcgtgaactc actcatgtcc 54480cggctcgatc ccagggggaa acagcgtgac cggcaacacc acatccgccg ccttcctgcg 54540gcggacacag aacgcgctcg ccatgcagcg caagatatgc gcccagcccg aggagaccgc 54600ggagcgcgtg ttctccgaca tcctctcggt gtcacgagac accggcttcg gccgcgaaca 54660cggcctcgcc ggggtccgca cccgccagga gtggcggcgt gccgtgccca tccgcaccta 54720cgacgaactg gccccctacg tcgagcggca gttctccggc gaacgccgcg tgctcaccac 54780cgacgacccc cgcgccttcc tgcgcacctc gggatcgacc ggccgcgcga agctggtacc 54840caccaccgat cactggcgcc gtgtctaccg cggaccggcg ctgtacgcgc agtgggggct 54900ctacttcgaa cagatcggca cgcatcggct caccggcgac gaggtcctcg acctgtcctg 54960ggagcccggc cccatccggc accgactgcg cggcttcccc gtctacagca tcaccgagcg 55020ccccgtgtcg gacgaccccg acgactggaa cccgccgtgg cgtcacgcga ggtggttcac 55080ccgcgatgcc ggtgccgcga ccatggccga cctgctctac ggcaaactgc tgcggctggc 55140cgcccacgac ctgagactga tcgtctcggt gaacccctcc aagatcgtcc tgctcgccga 55200gacactgaag gagaacgccg aacgcctgat ccaggacctg cacgacggcc acggcacgga 55260ccgggcagcc cgcccggact tcctccgccg cctcaccgcc gccttcgacc gcaccggagg 55320ccgtccgctg ctcaccgacc tgtggcccgg cctgcgtctg ctcgtctgct ggaactccgc 55380ctccgcggcg ctgtacgggc cctggctgtc ccggctcgcg accggcgtgg cggcactgcc 55440gttcagcacc acgggcaccg agggaatcgt cacgctgccc gtcgacgacc acctctcggc 55500ggggccgctc gctgtcgacc aggggcattt cgaattcgtt ccgtggcagg acctggacga 55560cggcagccct ctgcccgagg acacccccac cctcggctat gacgaactcg aactcggcgc 55620cgactaccgg ctcgtcatga gccaggccaa cgggctctac cgctacgacg tgggcgacgt 55680gtaccgcgtc gtcggagcgg tcggcgccac gccacggctg gagtttctgg gacgcgcggg 55740attccagtcc tccttcaccg gcgagaagct caccgaatcc gatgtgcaca ccgccgtgat 55800gcgggtcctc ggcagcgaac gcaccgacca cccgcacttc tccggcatcc cggtctggga 55860caccccgccc cactacctcg tcgccatcga atgggctgac gcccacggca cgttgaacgt 55920gcaggacaca gcccgccgca tcgacgcgac tctccaggaa gtcaacgtgg aatacgccga 55980caagcgccgc agcggacgac tgcggcccct gcagatcctg cccctggtgc ccggcgcttt 56040cggccagatc gccgaacgaa ggttccgcca gggcaccgcg ggagcccaga tcaaacacca 56100ctggctgcag aaggactcgg cgttcctcga cacgctgcgc gacctcgacc tcgtccgcgc 56160ccgcccgggg acgtgacggc atgcgcatcg gattcgccgc acccatgtcc ggcccctggg 56220ccaccccgga caccgccgtg cacgtcgccc gcaccgccga acagctcgga tacgcctcgc 56280tctggaccta ccagcgagtc ctcggcgcgc ccgacgactc ctggggcgag gccaaccgca 56340gcgtccacga ccccctgacc accttggcct tcctggccgc gcacaccacc gggatccggc 56400tcggtgtcgc cgttctgatc atgccgctgc acacccccgc ggtgctggcc aagcagctca 56460ccaccctcga cctgctctcc ggcggccgac tcgacgtggg cctcggcaac ggctgggccg 56520ccgaggagta cgccgccgcc ggcgtgaccc ccaccgggct cagccgccgc gccgaggact 56580tcctcgcctg tctgcgggcc ctgtggggtg agcagaccgt ggtggaacac gacggcccct 56640tctaccgggt cccgcccgcc cgcttcgacc cgaagcccgc ccagtccccg cacccgccgc 56700tgctcctggg cggcgccgcg cccggcgcac tgcgccgcgc cggccgcctg tgcgacggct 56760ggatcgcgag cagcaaggcc ggcccggccg ccatccgcga cgccatcacc gtcgtacgcg 56820acagcgctga gcgaaccgga cgcgaccccg cgaccctgag gttcgtctgc cgcgccccgg 56880tccggctgcg gacccggtcg gcccccaacg agccgccgct gaccggcacc gcggagacga 56940tccgggccga tctcgccgcg ctagccgaca ctggcctgac cgagatcttc ctggacccca 57000acttcgaccc cgagatcggc tcaccggacg cgccgaccgg cgacgtgcga caccgcgttg 57060atctgctgct gcacgaactg gcccccgcaa actggtgaga ggaagagaac agtgctgatc 57120gcgcgcgccg ccgtcggaga agaccgaacg tacgcccgcg tcgacacgga cacagggctg 57180atccacctcc tggccggcac tccctacgac gagatccggc cgaccggcga aaccagaccg 57240cttgccgagg cccgcctgct cgcaccggtc gaacccagca aagtgctggt cgcaggacgc 57300aattacggcg atgtcgtcac accggacctg gtggtcttca tgaagccgtc cacctctgtc 57360gtcggcccca ggagcaccgt cctgctgccg gcggaggcca agcaggtccg gtacgaggga 57420gaactcgccg tggtgatcgg gcgccgctgc aaagacgtcc ccgaagacac cgcggaccag 57480gccgtgttcg gctacacctg cgccaacgac gtcaccgcct gggacgtcgg ggaaccgaag 57540ggccactgga ccaaggcgaa gagcttcgac acattctgcc cgctgggacc atggatccgc 57600accgatctcg accccgctga cctcgtcctg cgcacaaccg tgaacggcac gctgcgccag 57660gacggctcca ccaaggaaat gaacaggaat gtccgcgccc tcgtgtcccg ttgcagctca 57720ctgatgacgc tgctgcccgg agacgtgatc ctcaccggca caccggcggg cgccggcgtg 57780ctgcgtccgg gtgacgaggt cgtcgtcgag attgacggga tcggttcgct cgcgaatccg 57840atcggcgtgg ccaagtagtt cactgactac actcgcgcga acaacacggg cccgtctgcg 57900gcgcttcgag ctgcgccgat ccccgaggag agattccagt gtctgtaatc cgtcccaccg 57960ccgaaaccga acgcgcagtc gtggtggtcc cggctgggac gacgtgcgcc gacgcggtca 58020ccgcggcaaa gctgccgcgc aatggcccca acgcgatcgt cgtggtgcga gacccgtccg 58080gcgccctgcg tgacctcgac tggacccccg attccgacgt cgaggtcgag gccgtcgcgt 58140tgtccagcga ggacggcctc acggtgctgc gccactccac ggcacacgta ctggcccagg 58200cggtccagca actctggccg gaggccaggc tcggtatcgg cccgccgatc gagaacggct 58260tctactacga cttcgacgtg gagcgcccct tccagccaga ggacctcgag cgcgtcgagc 58320agcggatgaa ggagatcatc aagtccggcc agcgcttctg ccgccgcgag ttccccgatc 58380gggaagcggc ccgtgccgag cttgccaagg agccgtacaa gctcgagctc gttgacctca 58440agggcgacgt ggacgccgcc gaggcaatgg aggtcggcgg gagcgacctg acgatctacg 58500acaacctcga cgcgagaact ggagatgtgt gctggtccga cctctgccgc ggcccccact 58560tgccgtcgac ccgcctgatc ccggcgttca agctgctgcg caacgcggca gcctactggc 58620gcggcagcga gaagaacccc caactgcagc gcatctacgg cacggcctgg ccgacccgcg 58680acgagctcaa gtcccatctc gccgccttgg aggaggccgc caagcgtgac caccgccgca 58740tcggcgagga actcgacctc ttcgcgttca acaaggagat cggccgcggc ctgccgctgt 58800ggctgcccaa cggcgcgatc atccgcgacg aactcgagga ctgggcccgc aagaccgaac 58860gcaagctcgg ctacaagcgc gtcgtcaccc cgcacatcac ccaggaggac ctttactacc 58920tctcaggcca tctgccttac tacgcggagg acctgtacgc gccgatcgac atcgacggcg 58980agaagtacta tctcaagccg atgaactgcc cgcaccacca catggtgtac aaggcgcgcc 59040cgcacagcta tcgcgacctg ccctacaagg tcgccgaata cggcacggtg taccgattcg 59100agcgcagcgg tcagctgcac ggcatgatgc gtacgcgcgg tttcagccag aatgacgcgc 59160acatctactg cacggcggac caggccaagg accagttcct ggaagtcatg cgcatgcacg 59220cggactacta ccgcactctg gggatcagcg acttctacat ggtgctcgcg ctgcgtgact 59280cggcgaacaa ggacaagtac cacgacgacg agcagatgtg ggaggacgct gagcggatca 59340cccgggaggc catggaagag tccgacatcc ccttccagat cgacctgggc ggtgccgcgc 59400actacggccc gaaggtcgac ttcatgatcc gagccgtcac cggcaaggag ttcgccgcct 59460ccaccaacca ggtcgacctg tacaccccgc agcgtttcgg gctgacctac cacgactccg 59520acggcaccga gaagcccgtc gtggtgatcc atcgcgctcc gctcggctcg cacgagcgct 59580tcaccgccta tctcaccgag cacttcgcag gtgccttccc ggtgtggttg gcgccggagc 59640aggtccggat cattccgatc gtggaggaac tcacggacta cgccgaggaa gtccgcgaca 59700tgctgctgga cgcggacgtg cgtgccgacg tcgatgccgg cgacggccgg ctgaatgcca 59760aggtacgcgc ggccgtcacc cggaagatcc cgctcgtcgt ggtggtcggc aggcgagagg 59820ctgagcagcg caccgtaacc gtgcgcgacc gctccggcga ggagaccccg atgtccctgg 59880agaagttcgt ggcccatgtc actggactca tcaggaccaa gagcctggac ggcgccggcc 59940acatccgtcc gctgtccaag gcctgaccca cagccacggg gccccggcag gtgtcccgcc 60000tcggaccacc cccttcggtc ctcagccgac ggcgggctca tggcagccgc ccgacctgcc 60060ggtgccgtgg ctgttcggca acccgtgggc gccgcccgcc gaggagaccg cgcgctgccg 60120ggggatgatc tcgtccggcc cgcgccccgg gctcaccggg cggcgctgga gcgggccgga 60180cgggagccgg gaccggccga ccgtccgtgg

tcggtctcgt cccggacgag cagggacagc 60240agtgcggcca cggtgaggcc cgcttccgcc gggtggcgca ggaccttgtc cggttcgacg 60300cggtagatgt tgctgcgccc gtcacgggtg tgggagagat agccgtcctg ctccaggtcc 60360gagatgatcc gctggacggc gcgctcggtg agtctgcagt gggcggcgat gtcgcggatc 60420cgcacgttcg gattgtcggc gatggccgcc agtacacgcg cgtggttggt gacgaacgtc 60480catccggagt gagattcagg cactgcaacc atgcacagca ttgtagggac catctttgcc 60540ggacagccaa tacatgacat acttttcgcg ttaagagtgg catgttctgt cccatgggca 60600actgagaagg gacccgaggg tgtctttgga tgaagcggtc gcggggtgct cgcgccacac 60660cggccggcgt cggctcccgg ccgcggagca acccacgcag gcgcagtacg aagcgcacgg 60720cgcctgggtc gtcagcgcac ggggcgcata cgacatgaac tcggtcgagc ccttggccga 60780cgcgttgaaa gacgcggccg agaagtctcc gaaggtggta ctggacgcct ccggcatcac 60840cttcgccgac tccaccctgc tgagtctgct gatcctcacc caccaggcga cggacttccg 60900ggtggccgcg ccgacgtggc aagtgatgcg gctcatgcaa ctgacgggcg tcgacgcctt 60960cttgaaggta cgggccacgg tggaagaggc cgccaccgct taggggcacg gcgtgccggg 61020cctcggctga cgcaagccga tggcttggag ctgagaattc cgggcattcg acgttctgcc 61080tggcccccgg ccgtcgatgg tggccggcca ggcgtgatga agacagtcac ctcctcgagg 61140cgcgatcgac cgcgcctcgg gggagggcgg ttgacgggag agggaaggtg tccatgattc 61200tgccggcgga gaaggaactg cgtgccgtgc tggctcggtt cgctcaggcg cgcatcgacc 61260acgacgtacg tcccagcggc tgcaccagca ggctcctcga ggacgccacg tacaccctgt 61320gcgtgatgac cggtgcccgt accgccgaac aggctctgcg tacggcggac gaacttctcg 61380cacagttcgc cgagcgcacc gctgcccccg tggaggacga agccctggcc gcgtgagccg 61440acggcacaca cctgcggcgc ctcgcgtggc aggtgtgtgc ctgccggcgg gcggacgcga 61500gcacctgagg aaatgagaga gagtcatgag cgatacccgg cttcggcagc gcgatgagac 61560gtcgaagggg ccggccaccg agatcccggc gccgcagtgg cgggacctct tcctcgcccc 61620cgactggggc ggcactgatg agcaggtgat cgtcgccgaa gaggcgcgcg ggcccgagca 61680cttcaccgga gcgcgccgtc cgcgcggcgg ccgccgatcg agtcgacggg ccgcgtgatg 61740cgcggccctg ccgcgacggc cgcaggtcag gtgagggcga tccgtgcggt gatgcgcttg 61800ccggacggct cgggccggat ctcgacggcc tggctcagcg ccgccacgat ctccagaccg 61860tgctgaccca cccgggcggg atcggcgggc cggggagcgg gcagggtggt gtcaccgtct 61920cgcacggtga cagtcaccgc gtcgtccgtg aggctcaggg tgagttctat ggggtcggcc 61980ccgtacttga ccgcgttcgt gatcagctcg ctcaccacca ggtggacggc ctccgacgct 62040ctctccggca ccggcgaccg aaggtcccgc tgtgaacggg tgaggtagtc ggtcgcgaag 62100tgacgggcgt cggcgatccg cagcgcttcc cgccggtacg tcaccgaggc cgcccgcgga 62160tccgcaccct ccggtgcccg ctgtgtcgac gaaccgtcca gtccggtctc acccatgtca 62220accgccacgt tacccccgag ccacgcacgc gcgccgacgc ctcccgcgcg atgagaacat 62280ctcatgtgtt cctacgatag ttctgctttc cgtcggtcac cgcacccgtc ggccagggag 62340aaagcggggg cctggacgtg atccgggcct caggccgtgc tgagcacgcc tccgcgcaag 62400cgggccggca gccgctccgg aatccgtgcg gtcgtcgtcg ccatgatgtc cctccccgtt 62460ccgatggccc gctcgcgtga cgggccactc ggcggctacc ccctgcgcgg gctcgcatgc 62520acggaggcgg agatcttgtc cgaggccgtt ccgctcccgc ccgcgcgacg tgatcaggtc 62580ggcggttacg ggatcaggaa ttccagcacg ggtcggtccc aggccaccgc gggcgggttc 62640gcgcgggcgg ttccggtggg cagggcgccc acagcgcggt agaagccctc ggccggaggg 62700tgcgacacca cccggacacg gtccagccct gccgcgcggg cccgcctctt catgtgatcg 62760acaagcagcc gtccgatgcc gcggccctgg gtaccgtcct ggacgaacag caggtccagc 62820tccgccggcg cgaggagcag cgcgtagaag ccgagcaccc ggtccgtggc gtcctcggcg 62880tcgacgctac gaagacctgg tggttctcga tgtagtcggg cccgacgcgg tagtcggaga 62940ccatcgccgc gtacgggccc tcgtaggctc gtgagccgcg tacgagacgc gagagccgct 63000tggcgtcccg ggcgaccgcc cggcggatga cgatctcgcc ccgtacggac gcggaactcg 63060attgcacggg gagcagtcta cgcgtccggg acgggccggg acctccgcgg gctccgtccg 63120agccttccgt cagtccgtcc cgatgacgac cacctcagtc cgtcccgatg acgaccacct 63180cgtcctcggc cgtccaccgg cgccgctcgc gtttgggcgg attgatccgc actccgtagt 63240gggggcgcgt cgacgcgtcg gcgtggtcgc ggtaaccgat ggcgcactcg ccgcgacggc 63300gtgccgcggc cacgacggtg gcgaaggacg tggtgctccc tggcaacagg tagtcggtcg 63360ccggccgcag gcggaccccg gcgccctcgg cggagaacag ttcctcgaag accgcggcca 63420ggtgccggtt ctgggagatc tgggacatga gcaggccgat gagcttgccg ctgatgatga 63480cgtcggcccc ggggccgatg ggggccagcg cccggttgcg gtcgtcgatc agttcggtga 63540cgaccggcag ctcgcgcccg gtggcttctt ccagttggcg caggagcaga agggtgacga 63600gcgtgcggtt gtcgggatcg tccggcggtt ggcccggggc ggggtcccgc cccagcacga 63660tcacgctgtc gtaggagtgg acgtccaggc gccgcagcgt ctcgggacgg gtgatgtccc 63720cgtggtgcag agccaggctc aggccgttcc caccgttctc cccgtttccg ctgtccgctt 63780cggcctcgct gatctcgcgg atcgtcgcct cgcccggttc cgccacgacg tcgacggccg 63840aaccgggccg ggcgcgtcgg tgcaactggt cgaccacgag cggcgctcgg cggttccagc 63900ccagtagcag aatccgctcc gccggcgcgg gcgtcggagg gcgggaggcc accgcggcct 63960tctcgaccga ctccgcacag tcgtccagcc gggccgtgtc gtcgtcgccg gtgatgacga 64020cgagcaggtc gtccggggcg accggcgtcg tcggcggcgg gttgagcaag ggggtgcagc 64080cgcgcatcag tccgacgacg ctcgtcgtcg agtaggacag gagaacctcg ccgaacgggc 64140ggccggtcag ggccggctcg ctgatcagat agaactcgtc tccggcgaag tcgaggagtt 64200cccggtggac gagggagatc ccggggcggc gggcggcctg gacgatcagc cgggcggtga 64260cggtgtcact ctccaggacg acgccgtcgg gtccggcggc gagacaggcg gccaggcggt 64320accggtcgtc ccggacggcg gcgacgacgg gcggacgcgg tttcgccccg gccagagcgg 64380cccgcagcgc cagcagtgtc ttcaccacct ccgcgtcggc gtgcggctcg tccggaggca 64440gaaccagcac gacaccggcc gtggccgggc tggtcaacgg caacacggcc gggtcggtgg 64500tggggccgct gcggcagatc aaccgcgtac cgccgcagga gcccaccttc gtgcccaggg 64560actcctccat gacggtcttg tcccggtcgg ccagcaccac caccgccgcg ccgcgctggt 64620tgacgttggc ggccaccagc tcgctcacca ccgtgaagac ctgttccgac catccgagga 64680ccacggcgtg cccctgttcc agcacggtgg aacggccccg ccgcaacgag gtgagccgct 64740ccgtgagcgc cgtcgtgatc aggccgacga gcgtggagac gtagagcagg gtgaccagcg 64800cgagcagcac cgacaacatc gcccgcagcg gcgtacccgt ggcaccgccc agccgtagcg 64860tctccccggt gagacgccac acctccgcca gccgctccgc gagggacggc ggggcatccg 64920ggtcggtcca caccatcacc gcgctggccg gcacgacgac ggccagggac agcagcgcca 64980tccagccgac gagcgcggcg gcaccgcggg ccagggtgct gtcgaaccag taacgggccc 65040tgtcaccgaa cggagtccgc cgctgcgcca cccgtccccc ttccgtctcc ccgtactccc 65100accaggccgc gtcggcaccg cagcggcgcc gtgccgcgcg ggcctgcccc ggagcgcgga 65160cgtgggcacg catgtcgcag tgtgggggat cgatcagggc cgcagacgac gttcgaccag 65220ccttcatccc ttcgggtagc cgcgtcctcc acggcggcgg acctgcgaag acgcccggct 65280cggacacgca gatgaaagcc gccgggcctc attcgcggaa cgccgggtat ccgcgagacc 65340ggatcaccgt cacacacagc gaggagagac cctgtgccgt ccaccgatgt cgtcgaactc 65400atcctgcggg accaccgccg tatggaggaa ctgttccgca ccctgcgcaa cgtcgaagcg 65460gaccgtgccg cggccctgac ggagttcgcg gacctgctca tcgcgcacgc ctcggccgag 65520gaggacgagg tctaccccgc cctgcgtcgg tacaagaacg tcgagggtga ggatgtcgac 65580cacagcgtcc acgagcacca cgaggccaac gaggcgctgc tggccctgct cgaggtggag 65640gacaccgctt ccgacgagtg ggatgacaag ctcgaagagc tggtcacggc ggtcaaccac 65700cacgccgacg aggaggaacg aacactcctc aacgacgccc gggagaacgt cgccgacgac 65760cgccgccggg aactggggca gaagttccag gaggcgcgtt cgcggtatct ggagaccggc 65820tgcggcagtg tcgagaacgt gcgcaagctg gtcgccgccg ccgacgactg acccgcgtcg 65880gcgacgtccg ggcgcggagg ggagccgccg ccgtcgggcc ccctcgccgg gcgtaccgcg 65940gtcaggcggg tgagggctgc gtccggtccg ggacgggttc gaggcggacg acgatgccct 66000tggacgtggg ctgattgctg atgtccgcga cgctgtccag cggtaccagg acgttggtct 66060cgggatagta ggcggcggcc gagcccttgg ccgccgggta gggaacgacc tggaagttct 66120cggcccggcg ctcggtgccg tccgcccaga cgctcacgag gtcgacgcga tcgccctggg 66180cgaggccgag ttcgctcagg tcggccgggt tgacgaggac gacgtggcgg ctgccgtgga 66240tgccgcggta gcgatcgttg tcggtgtagg ggacggtgtt ccactggtcg tgcgaacgca 66300gtgtctgcag cagcagatga ccttcgggcg cccgtgggac cacgctctcg ttgcgagtga 66360acagggcctt gccgacctcg gtgttgaaga cgccttcgtt gaccgggttg ggcagttgga 66420agccaccggg ccgggtcacg cgtgcgttga agtcgtggaa gccgggcacg atgcgcgcga 66480tgcggtcgcg gatggtgtgg tagtcgcctt cgaaggtctc ccaggggatc tccaccctgc 66540cgtccagggt gagccgggcg agccggcaca ggatcgcgat ctcgctcagc agcatggggg 66600aggcgggggc caggcggccc cgggaggtgt gcacctcgct catggagttc tcgacggtga 66660cgaactgctc gccgtcggcc tggacgtcgc gctcggtgcg tcccagcgtc ggcaggatca 66720acgcggtgtc accgcagacg gtgtgcgagc ggttgagctt ggtcgagatg tgggcggtca 66780gccggcacga gcgcatcgcc tcctcggtga cctcgctgtc gggcgccgcc cggacgaagt 66840tcccggccag ggcgaggaag accttgacgc ggccctcgcg cattgccttg atcgagttca 66900ccgagtccag gccgtgggcc cgcggcggct cgaagccgaa ctcgtcccgc agggcgtcca 66960ggaaggtgtc cggcatctgc tcccagatgc ccatggtgcg gtcgccctgg acgttgctgt 67020ggccgcgcac cgggcaggcg ccggtgccgg cgcgtcccag gttgccgcgg agcatcagga 67080agttgacgat ctcccggacg gtgggcacgc cgtgcttgtg ctgggtgatg cccatcgccc 67140agcagacgac gacgcgttcg ctgtcgagga cctcgtcgcg taccttctcg atctcctcgc 67200gggtcagtcc ggtcgccgcg cgcacgtcgt cccagtccac cgtgcgggcg tgccgggcga 67260actcctcgaa gccggtggtg tgggcgtcga tgaagtcgtg gtccaggacg gtgccgggcc 67320gggcgtcctc ggcctccagc agcagtcggt tgagggcctg gaagagggcg aggtcgccac 67380cgggcttgat gtgcaggaaa cggtcggcga tccgggtgcc gcgcccgacg accccgcgcg 67440gctgctgcgg gttcttgaag cgtcgcagcc cggcctcggg aagcgggttc acggccacga 67500tccgggcgcc gttccgcttg gcctcctcca gcgcgctgag ctggcgcggg tggttgctgc 67560cggggttctg cccgaccagg aagatcagat cggcgtggtg gaggtcgtcg agaccgacgg 67620tgcccttgcc ggtccccagg gtctcgctca gggcgaagcc gctggactcg tggcacatgt 67680tgctgcagtc gggcaggttg ttggtgccga aggcgcgggc gaagagctgc agcacgaagg 67740cggcctcgtt gctggcgcgg cccgaggtgt agaacaccgc ctcgtcgggg gaggccagcg 67800acttgagctc ctccgcgagg acccccaggg cgtcgttcca gccgatgggc tcgtagtgcg 67860cggagccggg ccgtttgatc atcggctcgg tgagccggcc ctgctggttg agccacatgt 67920cggagcggcc ggcgaggtcg gcgacgctgt gctcgcggaa gaagtcggcg gtcacccgcc 67980gtgtggtcgc ctcgtcgttg atgtgcttgg cgccgttctc gcagtactcg ttgcggtggc 68040gccgtcccgg ggccgggtcc gcccacgcgc agccggggca gtcgatgccg cccacctggt 68100tcatggtcag cagatccacc ccggtcctgc gcggggacgt ctgctccaag gagtactcca 68160gcgcgtgcac gaccgcgggg acgccggcgg cccacttctt cggcggtgtc acggagaggc 68220tggtctccga ctcctcaccg tgcggcttct gcatgtgttc gcctttctct cgccgtgtcc 68280ggccctcggt cagccgacgg gccaccgggc caccctgctc ctgggcggtt ccggtcgatc 68340gtgttgcacg cggccgttgc ggatggtgcc gcgccaggcg ccgccctctt gtcccagtcc 68400ctccatgaac cgtttgaatc tcgtcagctc gccgtggacc agtcgggtgg tcagccggac 68460cgcccgcgag gaacgggtga ggatcctcgc ggctccccgc ggttccagaa gcacccggac 68520ggtgatcgcg gtgccgccgg actccgtcgg cctgaactcg acctctcccc ggtgccacgg 68580cctctgctcc aggccgcgcc aggccaggta ggcgtcgggg tcctgctcca ggatctcgac 68640cgcgaagcgg cggcgcaggg ggccgtaacc gagggtccag gcggtcacgg tgggccggac 68700ctgctcgacg tcgcggacca cggccgagaa ccgagggaag gacttgaact gcgtccactg 68760gttgtacgcc gtccgtacgg gcaccgccac ctcgaccgtc tggtcgacgt gccgctcgcg 68820cagcggacga cggcccgtgc tcctgtcacc gctggtgcgc gcccccggtg tgtccggcac 68880ggcccgcggg ccctcgtgct gctccgccat cgtggtctct cctcctgtgg ggggaaccag 68940gcgtccaggc tctcgacgtc tcctcgtgcg tcagggtccc ctcgtgcgtc agggttttcc 69000cctcccggtt cccacctccg gcgtttcgag tcacctcggt cccgccgcgt cctcgtgcgc 69060gtcggcctcc gacgccccgc ccggagagat ccggctcgac gtcggcctgg tcgctccggc 69120gggcggtgag cagcaggtac tcccagtcga tggccccgtc ccgcagcgcg ccggccgcga 69180ggtcggtgag ggccgcgtcc agggcggcgg cgcgctcggt gtcgtcgccg atgtaccggt 69240agacggcgat gatcggcccg taggcggcct tgaagaactc aaggaagtcc ccccttcgag 69300cggcgctcgg gaccgggggt gcgccggccg gcctgatgac cagcgtcgac ggggtcgtga 69360gagcgtgatc gaggtgctcg acgagccgga gccaccgcag gagagtcgcg ggcagcgctc 69420tctccagcaa cccagccttc gcacacccgt gcgcggcgca aggggcgtac gtcccctgcg 69480caaaggggcg gctggaggcc ggtgccccac gcctcgtctc cgcacggcgc gtcggacggg 69540gggagaacgc gtccgatgaa cacggtgtgc gccctcccga ttcctctccg tgacgtcaat 69600gatgagccca ccgcgcctgg gtcaggcgat ccgcggtccc ctgccgctcg gtcaggggcc 69660ggtgacggaa ggagtccacg gtgttgcttc tcatctctcc ggacggtgtc gaggaagccc 69720tcgactgcgc gaaggcggcg gagcacctcg acatcgtcga cgtgaagaag cccgacgagg 69780gctcgctggg cgcgaacttc ccgtgggtca tcagggagat ccgcgacgcg gtgccggcgg 69840acaagccggt ctcggccacc gtgggggacg tcccgtacaa gcccggcacg gtggcgcagg 69900ccgcgctggg cgcggtcgtc tcgggggcca cgtacatcaa ggtcggcctc tacggatgca 69960cgacgcccga acagggcatc gcggtcatgc gcgcggtggt ccgggcggtg aaggaccacc 70020gtcccgaagc gctcgtcgtc gcgtccggtt acgccgacgc ccaccggatc ggctgcgtca 70080acccgctcgc cctgcccgac atcgccgccc gctccggcgc cgacgccgcg atgctcgaca 70140ccgcggtcaa ggacgggacg cggctgttcg atcacgttcc gccggacacc tgcgccgagt 70200tcgtccgtcg cgcccacgcc gccggcctgc tcgccgccct cgcgggcagc gtcaggcaga 70260ccgacctcgg ccggctgacc cggatcggca cggacatcgt cggggtgcgc ggagcggtct 70320gcgagggcgg cgaccgcaac gccggacgca tccggccgca cctggtggcc gccttccgga 70380gcgagatgga ccggcacgcc cgcgagcacc gggccggcgt caccaccgcg agctgaccgc 70440cggtatgccg acccccgcac ccgaccacgc ccccgcacag cgggccgcgc ctctcgcggt 70500cgtcgatccg gccaccggaa cggtcttcga cgaggccccc gaccagggac cggacgtgct 70560ggacgccgtc gtcgaccggg cccgccgggc ctggcacggc tggcgcgccg atcccgacgc 70620ccgtaccacc gcgctgcgct cggcggccga cgcggtcgag gccgccgggg acgacctcgc 70680ccgtctcctc acccgggaac agggaaagcc cctggccgaa tcgcatgcgg aggtcgcccg 70740gacggcggcc cgcctgcgct acttcgccgg cctggccccc cggacccggc gcatcaccga 70800cgggcggccg gtgcgcagcg aggtccgctg gcgccccctc ggacccgtcg ccgcgatcgt 70860gccgtggaac ttccccctcc aactcgcgtc ggcgaagttc gcgcccgcgc tcgccgcggg 70920caacaccatg gtcctcaaac cctccccctt caccccgctc gccacccggc tgctcgggtc 70980cgtcctcgcc accgccctgc ccgaggacgt cctgacggtc gtcaccggcc gcgagccact 71040cggcgcccgc ctcgccgcac accccggcat ccgccacgtc accttcaccg gatcggtgcc 71100cacgggccgg gccgtcgccc gagcggcggc ggcctcgctc gcccgggtca ccctggaact 71160gggcggcaac gacgccgcgg tcctgctgga cgacgtcgaa gtggaccgga tcgccgaccg 71220gctgttctgg gccgcgttcc gcaactgcgg gcaggtctgc atggcggtca agcgcgtcta 71280cgcaccggcc cgtctgcacg cacaggtcgt cgaagccctc accgagcgcg ccaaggccgt 71340cgccgtcggg cccggcctcg acccccgcac ccggctggga ccggtcgcca acgcccccca 71400gctggcccgg gtcgagcaga tcacccggcg cgccctggcg gacggcgccc gggcggcggc 71460cggcggccac cggctggacg ggccgggctg cttcttcgcc cccacgatcc tcaccgacgt 71520cccgcccgac agcccggtgg tgaccgagga gcagttcggg ccggtactgc cggtgctgcc 71580gtaccggagc ctggacgaag ccgtcgacgc ggccaacggc acgggattcg ggctgggggg 71640ctccgtatgg ggcaccgacc tcgaccgggc cgaggcggtg gccgaccggc tggaatgcgg 71700cacggcctgg gtcaaccacc acgccgagct gtccctcgcc cagcccttcg ccggcgacaa 71760ggacagcggg gtcggcgtcg cgggcgggcc gtggggactg tacggcaacc tccgtccgtt 71820cgtcgtccac cgaccgcggg gggagtgacg gtgagcttcc gggcggccgt actgcgcggg 71880tacgaggacc ccttcacggt cgaggaggtg accctgggga cggagcccgg cgcaggggag 71940atcctggtcg agatcgccgg ctgcggaatg tgccggaccg atctcgcggt ccggcgctcg 72000gccggccgga gcccgctgcc ggcggtgctc ggccacgagg gctccggggt ggtggtgcgg 72060acgggcggcg gcccggacac cgcgatcggc gtcggtgacc acgtggtgct gagcttcgac 72120tcctgcgggc actgccgcaa ctgccgcgcg gcggcccccg cctactgtga ttccttcgcc 72180tccctcaacc tcttcggggg ccgtgcggag gacccgccgc ggctcaccga cgggtcgggg 72240gcggcactgg ctccccggtg gttcggacag tccgccttcg ccgagtacgc gctcgtctcc 72300gcccgcaacg ccgttcgggt cgaccccgcc ctgcccgtcg aactgctcgg gccgctgggc 72360tgcggcttcc tcaccggagc cggagccgtg ctcaacacct tcgccgccgg gccgggcgac 72420accctcgtcg tgctcggcgc gggcgccgtg ggcctggccg cggtgatggc ggccaccgcc 72480gccggcgcac cgtccgtggc cgtggaccgc aacccccgtc gcctggagct ggccgagcgg 72540ttcggcgcgg tcccgctgcc cgccgcgacg gccggactgg ccgagcggat ccggcggctc 72600acggacggcg gcgcgcggta cgcactggac acgaccgcct ccgtcccact gatcaacgag 72660gcgctgcgcg cactgcgtcc caccggcgct ctcggcctgg tggcacggct ccacaccgcg 72720ctgcccctgg aaccgggcac gctcgaccgg gggcgcagca tccgccacgt ctgcgagggg 72780gacgcggtac ccggtctgct gataccgcag ctgacccggc tctggcaggc cggacgcttc 72840cccttcgacc agctcgtccg tacctacccg ctggccgaca tcaacgaggc ggagcgcgac 72900tgcgacgccg gcctcgtggt caaacccgtg ctgctcccgc ccgcgaggag ccggtgagta 72960cggcgcacgg caccgcggtc cgaccgcatc cgacgagcag gaagctcgcg gccccacttc 73020cgccaacgga ggagacatga ccggcacggc gccgcagtac acggacgtgg aaggcgtgaa 73080cggaggtgtg ggcctgacgg ccttcctggt cgccgccgcg cgggcgatcg agacccatcg 73140cgacgacagt ctggcccagg acgtctacgc ggaacacttc gtgcgcgccg ccccggcgtg 73200cgcggactgg ccggtgcgca tcgagcaggt ccccgacggg gacggcaacc cgctgtgggg 73260acggttcgcc cgctatttcg gcctgcggac ccgggccctc gacgacttcc tgctccggtc 73320ggtccggacg ggcccccgac aggtggtgct gctgggcgcg gggttggaca cccgtgcctt 73380ccggctcgac tggccgtcgc agtgcgcggt cttcgagatc gaccggacgg gcgtgctcgc 73440cttcaaacag caggtgctca cggacctggc ggcaaccccg agagtggagc gcgtccccgt 73500tccggtcgat ctgcgcgcgg actgggccgg cgcgctgacc gcggccggct tcgaccccgc 73560ggcgcccagc gtctggctgg ccgagggact gctcttctat ctgccgggcc ccgccgagtc 73620gcttctcgtc gacacggtgg accggctgac caccgacggc agcgcgctgg ccttcgaggc 73680caagctggag aaggacctgc tggcgtaccg cgacagtgcg atctacacgg cgacgcgcga 73740gcagatcggc atcgacctcc tccgcctctt cgacaagggg ccccgacccg actccgcggg 73800tgagctggcg gccagaggct ggtccacctc gatgcacacg cccttcgtct tcacccaccg 73860gtacggacgc ggtcccctcc ccgagccgaa cgacgcgctg gaggggaacc gatgggtctt 73920cgcccgcaag cccgggccct gacgtgccgg ccgcgcttgc cgcccacgcc cggggacgcc 73980gctgacgagc ggtgtcagac ggtccgggcg gccaccagcc ggtcgcccgg gatcccggcc 74040aggtccggcg cgtagtagtc ctcgatgccg gaggcccacg cggccacggt gatgcggtcc 74100gcggcgtccg gggcgaaggc gtcgaacagc gcgtggatgt tcgcttcctc gaaaccgatc 74160gccttcatca gcgcgacgaa gaggggacgc tcgatcagcc cgtccccgtc gggatcgccg 74220agcgcggaca gtgcccgagc gaactcggcg atggtgggac cgaagcgctc gggatcgagt 74280acgaacggac ggaactcctc cacggtgatc acgccgtcgc cgtcggcgtc cagttcggtg 74340gccagcgtgg tccagtagcg gcggaacgcg gcccggacgg cggccttggc gctgtcgtcc 74400gacccggccg ccgccgcaac gacccggtcg gtcatcaggt cgaagtcgtc ggagtcgatg 74460actccgttgc cgttggcgtc gaagagggag aagaccagct cgacccgctt ggcggcctca 74520tctcgcatgc acatcacctg tcttctacgg cccggtcttc gcgggcccgg ccccatgaat 74580gctctgcgtg accgagcggg gcaggacgaa agcctccgag cggtcgcgtc ccagagaacc 74640accatgaatg tccctgaact gcagatcggg catctgctgg cctggtgcgg gcgggggctg 74700gcccggtgcg gcaggggagt gctctggtgc ctgggcaagg ccgtcacggg gatcatcctg 74760ctcgccatct tcgcgtccgc gatgatc 747872876PRTStreptomyces parvulus Tu4055 2Met Thr Gly Ser Ala Val Ser Ala Pro Phe Leu Gln Pro Pro Glu Pro1 5 10 15Val Ser Gly His Ser Glu Arg Lys Ser Asp Pro Val Leu Leu Val Gly 20 25 30Ala Gly Arg Arg Ala Arg Met Ala Asp Ala Val Arg Ala Ala Gly Ala 35 40 45Gln Ala Gly Ile Asp Pro Ala Val Leu Arg Arg Thr Arg Ala Thr Leu 50 55 60Ile Thr Ala Gly Ser Ala Gly Ala Ala Gly Arg Leu

Ala Ala Ala Leu65 70 75 80Arg Leu Thr Gly Ala Thr Ile Ser Leu Asp Thr Arg Glu Thr Pro Thr 85 90 95Leu Leu Ala Leu His Leu Ala Ala Gln Ala Leu Arg Ala Gly Asp Thr 100 105 110Ser Tyr Ala Val Val Gly Ala Glu Leu Pro Asp Gly Asn Cys Ala Leu 115 120 125Ile Leu Ala Arg Gln Ser Ala Ala Thr Ala Glu Gly Ala Val Pro Gln 130 135 140Ala Ile Val Arg Thr Thr Thr Ala Asp Arg Thr Thr Thr Ala Asp His145 150 155 160Ala Pro Ala Pro Asp Asp His Gly Ser Pro Ala Arg Glu Ala Pro His 165 170 175Ala Thr Arg Thr Leu Ser Pro Gly Ile Thr Gln Ala Pro Ala Glu Gly 180 185 190Phe Pro Gly Leu Leu Ala Thr Leu His Asp Asp Thr Pro Leu Arg Pro 195 200 205Thr Ala Val Thr Glu His Gly Ser Asp Ala Thr Thr Val Leu Val Leu 210 215 220Leu Asp Gln Pro Gln Asp Ala Ala Pro Ala Ala Pro Leu Pro Trp Val225 230 235 240Val Ser Ala Pro His Thr Arg Ala Leu Arg Ala Thr Ala Ala Thr Leu 245 250 255Ala Val His Leu Asp Thr Thr Pro Ala Ala Pro Ala Asp Val Ala His 260 265 270Thr Leu Leu Thr Ala Arg Pro Asp Arg His Arg Ala Ala Val Val Gly 275 280 285Ala Asp Arg Ala Thr Leu Thr Asp Gly Leu Arg Ala Leu Ala Thr Gly 290 295 300Gly Asp Ala Pro His Leu Val His Gly Thr Ala Thr Gly Ser Pro Arg305 310 315 320Pro Val Phe Val Phe Pro Gly Gln Gly Ser Gln Trp Pro Gly Met Ala 325 330 335Ala Glu Leu Leu Glu Thr Ser Glu Pro Phe His Asp Ser Val His Ala 340 345 350Cys Ala Asp Ala Leu Ala Glu Phe Val Asp Trp Ser Val Leu Asp Val 355 360 365Leu Arg Gln Ala Pro Asp Ala Pro Pro Leu Arg Arg Val Asp Val Leu 370 375 380Gln Pro Thr Leu Trp Ala Thr Met Val Ser Leu Ala Glu Val Trp Arg385 390 395 400Ser Tyr Gly Val Glu Pro Ala Ala Val Val Gly His Cys Tyr Gly Glu 405 410 415Ile Ala Ala Ala Gln Val Ala Gly Ala Leu Asp Met Arg Asp Ala Ala 420 425 430Arg Leu Leu Ala His Arg Ser Arg Ala Trp Leu Arg Leu Val Gly Lys 435 440 445Gly Thr Val Ile Ser Val Ala Thr Ser Gly Gln Asp Ile Thr Arg Arg 450 455 460Met Ala Ala Trp Pro Asp Ser Val Glu Leu Ala Ala Leu Asn Gly Pro465 470 475 480Arg Ser Val Ala Leu Ala Gly Pro Pro Asp Val Leu Asp Gly Ile Val 485 490 495Asn Asp Leu Thr Asp Gln Gly Ile His Ala Lys Arg Ile Pro Gly Val 500 505 510Asp Thr Val Gly His Cys Ser Gln Val Glu Val Leu Arg Asp His Leu 515 520 525Leu Asp Val Leu Arg Pro Val Ser Pro Arg Pro Ala Ala Val Pro Phe 530 535 540Tyr Ser Thr Val Asp Gly Thr Glu Arg Asp Thr Thr Thr Leu Asp Thr545 550 555 560Asp Tyr Trp Tyr Leu Asn Thr Arg Ser Gln Val Arg Phe His Gln Ala 565 570 575Val Arg Asn Leu Leu Ala Ala Gly His Arg Ser Phe Val Glu Val Ser 580 585 590Pro His Pro Leu Leu Gly Ala Ser Ile Glu Asp Thr Ala Ala Glu Phe 595 600 605Gly Leu Asp Asp Val Ala Ala Val Gly Thr Leu Arg Arg Gly Gln Gly 610 615 620Gly Thr Arg Arg Val Leu Thr Ser Val Ala Glu Ala Tyr Val His Gly625 630 635 640Ile Asp Ile Asp Phe Thr Pro Ala Phe Thr Gly Thr Thr Pro Asn Arg 645 650 655Ile Asp Leu Pro Thr Val Glu Asp His Gly Ile Glu Gly His Gly Asp 660 665 670Asp Gly Gly Glu Thr Trp Thr Asp Arg Val Arg Thr Leu Pro Asp Glu 675 680 685Gln Arg Glu Glu Ala Leu Leu Asp Leu Val Cys Arg Thr Val Ala Ala 690 695 700Val Leu Glu Ala Asp Pro Ala Gly Thr Ala Asp Ala Val Ala Pro Asp705 710 715 720Thr Ala Phe Lys Glu Met Gly Leu Gly Ser Leu Ser Ala Val Arg Leu 725 730 735Arg Asn Gly Leu Arg Glu Ala Thr Gly Ala His Leu Pro Ala Thr Ile 740 745 750Ala Tyr Asp His Pro Thr Pro Ala Ala Leu Ala Arg His Leu Ala Met 755 760 765Thr Leu Phe Asp Ala Thr Gly Ala Ala Pro Ala Val Pro Ala Pro Ser 770 775 780Arg Asp Asp Glu Pro Ile Asp Ala Glu Thr Ala Val Leu Thr Ala Leu785 790 795 800Glu Arg Ala Asp Glu Ala Leu Glu Arg Leu Arg Ala Pro His Ala Arg 805 810 815Thr Pro Arg Gln Glu Thr Gly Arg Arg Ile Asp Glu Leu Leu Arg Ser 820 825 830Leu Thr Asp Lys Ala Arg Arg Met Arg Gln Ala Asp Ala Val Asp Asp 835 840 845Val Asp Asp Pro Ala Thr Asp Arg Phe Ala Ala Ala Thr Asp Asp Glu 850 855 860Met Phe Glu Leu Leu Glu Lys Arg Phe Gly Ile Ser865 870 87531571PRTStreptomyces Parvulus Tu4055 3Met Ala His Glu Asp Lys Leu Arg His Leu Leu Lys Arg Val Ser Ala1 5 10 15Glu Leu Asp Asp Thr Gln Arg Arg Val Arg Glu Met Glu Glu Ser Glu 20 25 30Arg Glu Pro Ile Ala Ile Val Gly Met Ser Cys Arg Leu Pro Gly Gly 35 40 45Val Asn Ser Pro Gly Glu Phe Trp Ser Leu Leu Glu Ala Gly Thr Asp 50 55 60Ala Val Ser Glu Phe Pro Arg Asp Arg Gly Trp Asp Val Glu Asn Leu65 70 75 80Tyr Asp Pro Asp Pro Asp Ala Pro Gly Arg Ser Tyr Val Arg Glu Gly 85 90 95Gly Phe Leu Asp Gly Ala Gly Gln Phe Asp Ala Ala Phe Phe Gly Ile 100 105 110Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu Leu Leu 115 120 125Glu Cys Ser Trp Glu Ala Ile Glu Arg Ser Arg Ile Asp Pro Lys Thr 130 135 140Leu His Gly Ser Arg Thr Gly Val Phe Ala Gly Ser Asn Trp Gln Asp145 150 155 160Tyr Asn Thr Leu Leu Leu Asn Ala Glu Glu Arg Ser Gln Ser Tyr Leu 165 170 175Ala Thr Gly Ala Ser Gly Ser Val Leu Ser Gly Arg Val Ser Tyr Thr 180 185 190Leu Gly Met Glu Gly Pro Ala Ile Thr Val Asn Thr Ala Cys Ser Ser 195 200 205Ser Leu Val Ala Val His Leu Ala Ala Arg Ser Leu Arg Ala Gly Glu 210 215 220Cys Asp Leu Ala Leu Ala Gly Ala Val Thr Val Met Ser Thr Pro Gln225 230 235 240Leu Pro Val Ala Phe Ser Arg Gln Arg Gly Leu Ala Pro Asp Gly Arg 245 250 255Ser Lys Ala Phe Ala Val Ser Ala Asp Gly Met Gly Phe Gly Glu Gly 260 265 270Val Gly Val Leu Val Leu Glu Arg Leu Ser Val Ala Arg Arg Asn Gly 275 280 285His Arg Val Leu Ala Val Val Arg Gly Ser Ala Val Asn Gln Asp Gly 290 295 300Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln Arg Val305 310 315 320Ile Arg Ala Ala Leu Ala Ser Ala Gly Leu Gly Pro Ala Asp Val Asp 325 330 335Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro Ile Glu 340 345 350Ala Gln Ala Leu Leu Ala Thr Tyr Gly Arg Gly Arg Asp Ala Glu Arg 355 360 365Pro Leu Trp Leu Gly Ser Val Lys Ser Asn Ile Gly His Ala Gln Ala 370 375 380Ala Ala Gly Val Ala Gly Val Ile Lys Met Val Leu Ala Met Glu Lys385 390 395 400Gly Arg Leu Pro Arg Thr Leu His Val Asp Glu Pro Ser Gly Glu Val 405 410 415Asp Trp Asp Ser Gly Ala Val Arg Leu Leu Thr Glu Ala Arg Asp Trp 420 425 430Pro Ser Glu Glu Gly Arg Leu Arg Arg Ala Gly Val Ser Ser Phe Gly 435 440 445Ile Ser Gly Thr Asn Ala His Val Ile Ile Glu Glu Ala Pro Glu Glu 450 455 460Gly Glu Glu Pro Glu Ser Asp Ala Gly Gly Val Val Pro Trp Val Leu465 470 475 480Ser Ala Arg Thr Glu Gly Ala Leu Gln Ala Gln Ala Val Gln Leu Ser 485 490 495Glu Phe Val Gly Glu Ser Ser Pro Val Asp Val Gly Trp Ser Leu Val 500 505 510Ser Thr Arg Ala Ala Phe Glu His Arg Ala Val Val Val Gly Arg Gly 515 520 525Arg Asp Glu Leu Val Arg Gly Leu Ser Glu Val Ala Gln Gly Arg Gly 530 535 540Val Arg Gly Val Ala Ser Ser Ala Ser Gly Gly Leu Ala Phe Val Phe545 550 555 560Ala Gly Gln Gly Ser Gln Arg Leu Gly Met Gly Arg Gly Leu Tyr Glu 565 570 575Arg Phe Pro Val Phe Ala Glu Ala Phe Asp Glu Val Cys Gly Arg Val 580 585 590Gly Pro Gly Val Arg Glu Val Val Phe Gly Ser Asp Ala Gly Glu Leu 595 600 605Asp Arg Thr Val Trp Ala Gln Ala Gly Leu Phe Ala Leu Glu Val Ala 610 615 620Leu Phe Arg Leu Leu Glu Ser Trp Gly Val Arg Pro Gly Cys Leu Ile625 630 635 640Gly His Ser Val Gly Glu Leu Ser Ala Ala Cys Val Ala Gly Leu Trp 645 650 655Ser Leu Glu Asp Ala Cys Arg Val Val Ala Ala Arg Ala Arg Leu Met 660 665 670Gln Ala Leu Pro Ala Gly Gly Val Met Val Ala Val Arg Ala Glu Ala 675 680 685Gly Glu Leu Ala Gly Phe Leu Gly Glu Asp Val Val Ile Ala Ser Val 690 695 700Asn Ala Pro Gly Gln Val Val Ile Ala Gly Pro Glu Gly Gly Val Glu705 710 715 720Arg Val Val Ala Ala Cys Gly Ala Arg Ser Arg Arg Leu Ala Val Ser 725 730 735His Ala Phe His Ser Pro Leu Val Glu Pro Met Leu Gly Glu Phe Arg 740 745 750Arg Val Val Glu Ser Val Ala Phe Gly Val Pro Ser Leu Arg Val Val 755 760 765Ser Asn Val Thr Gly Ala Trp Val Asp Pro Glu Glu Trp Gly Thr Pro 770 775 780Glu Tyr Trp Val Arg Gln Val Arg Glu Pro Val Arg Phe Ala Asp Gly785 790 795 800Val Ala Thr Leu Leu Asp Ala Gly Val Arg Thr Phe Val Glu Leu Gly 805 810 815Pro Ala Gly Ala Leu Thr Ser Met Val Ser His Cys Ala Asp Ala Thr 820 825 830Ala Thr Ser Val Thr Ala Val Pro Thr Leu Arg Pro Asp His Asp Glu 835 840 845Ser Arg Thr Val Leu Ser Ala Ala Ala Ser Leu Tyr Val Gln Gly His 850 855 860Pro Val Asp Trp Ala Pro Leu Phe Pro Arg Ala Arg Thr Val Asp Leu865 870 875 880Pro Thr Tyr Pro Phe Gln His Gln His Tyr Trp Leu Asp Val Pro Pro 885 890 895Leu Phe Thr Ala Ser Ser Ala Ala Gln Asp Gly Gly Trp Arg Tyr Arg 900 905 910Ile His Trp Arg Arg Leu Gly Thr Arg Asp Ser Gly Asp Arg Leu Ser 915 920 925Gly Arg Trp Leu Leu Leu Val Pro Glu Ser Asp Gly Thr Glu Pro Trp 930 935 940Val Glu Gly Ala Glu Lys Met Leu Ala Glu Arg Gly Cys Glu Val Val945 950 955 960His Val Pro Ile Ala Ala Thr Ala Asp Arg Asp Ala Met Val Gly Ala 965 970 975Val Arg Glu Ser Val Glu Asp Gly Arg Val Asp Gly Val Leu Ser Leu 980 985 990Leu Ala Leu Asp Gly Arg Pro His Pro Asp Ala Ala Ala Val Pro Thr 995 1000 1005Gly Leu Val Ala Thr Ala Gln Val Val Gln Val Ser Asp Glu Leu Gly 1010 1015 1020Ile Gly Pro Leu Trp Val Ala Thr Arg Gln Ala Val Ser Val Asp Gly1025 1030 1035 1040Ala Asp Glu Ala Asp Gly Ala Gly Arg Thr Arg Lys Ala Asp Asp Pro 1045 1050 1055Ala Asp Val Ala Gln Ala Ala Val Trp Gly Leu Gly Arg Val Ala Ala 1060 1065 1070Leu Glu Lys Pro Arg Leu Trp Gly Gly Leu Val Asp Leu Pro Ala Arg 1075 1080 1085Ala Asp Glu Arg Met Arg Asp Leu Val Ala Gln Ala Leu Thr Ala Pro 1090 1095 1100Asp Ala Glu Asp Gln Leu Ala Val Arg Ala Asp Gly Ile Ala Val Arg1105 1110 1115 1120Arg Leu Val Arg Ser Ala Ala Ser Ala Pro Ala Asp Asp Trp Gln Pro 1125 1130 1135Ser Gly Thr Val Leu Val Thr Gly Gly Thr Gly Gly Val Gly Ala Asn 1140 1145 1150Val Ala Arg Trp Leu Val Thr Gln Asp Ile Gln His Leu Leu Leu Val 1155 1160 1165Ser Arg Arg Gly Pro Asp Ala Pro Gly Ala Ala Glu Leu Leu Ala Glu 1170 1175 1180Leu Ser Ala Ser Gly Thr Ser Val Thr Ile Glu Pro Cys Asp Val Thr1185 1190 1195 1200Asp Ala Asp Ala Val Arg Arg Leu Ile Gly Ala Val Pro Ala Glu Arg 1205 1210 1215Pro Leu Ser Thr Val Val His Ala Ala Gly Val Leu Asp Asp Cys Leu 1220 1225 1230Ile Asp Ala Leu Thr Pro Gln Arg Leu Ala Ala Ala Leu Glu Val Lys 1235 1240 1245Ala Lys Gly Ala Leu Asn Leu His Glu Ala Ala Gly Glu Ala His Leu 1250 1255 1260Val Leu Phe Ser Ser Leu Ala Gly Thr Thr Gly Thr Lys Gly Gln Gly1265 1270 1275 1280Asn Tyr Ala Ala Ala Asn Ala Tyr Leu Asp Ala Leu Ala Glu Arg Arg 1285 1290 1295Arg Ala Asp Gly Leu Pro Ala Thr Ser Val Ala Trp Gly Ala Trp Gln 1300 1305 1310Gly Ala Gly Met Val Ala Asp Ala Ala Val Ala His Arg Thr Arg Arg 1315 1320 1325Tyr Gly Leu Pro Leu Met Ser Pro Asp Arg Ala Val Ala Thr Leu Arg 1330 1335 1340Gln Val Met Ala Glu Pro Val Ala Thr Gln Val Val Ala Asp Val Asp1345 1350 1355 1360Trp Gln Arg Phe Val Ala Asp Phe Thr Ala Val Arg Pro Ser Arg Leu 1365 1370 1375Leu Ala Asp Leu Pro Glu Val Arg Ser Leu Gly Glu Gln Arg Lys Asp 1380 1385 1390Gly Pro Gly Gly Gln Gly Glu Glu Asp Gly Leu Ala Ser Lys Leu Ala 1395 1400 1405Ala Leu Pro Glu Ala Asp Arg Arg Arg Ala Val Leu Asp Leu Val Glu 1410 1415 1420Glu Leu Val Leu Gly Val Leu Gly His Glu Thr Arg Ala Ala Ile Gly1425 1430 1435 1440Pro Asp Ser Ser Phe His Ala Ile Gly Phe Asp Ser Leu Thr Ala Val 1445 1450 1455Glu Leu Arg Asn Leu Leu Thr Val Arg Leu Gly Met Lys Leu Pro Ala 1460 1465 1470Thr Leu Val Tyr Asp His Pro Thr Leu Ser Ser Leu Ala Asp His Leu 1475 1480 1485His Glu Gln Leu Val Ile Asp Gly Thr Pro Met Thr Asp Thr Ala Ala 1490 1495 1500Asp Leu Leu Ala Glu Leu Asp Ala Leu Ala Ala Arg Leu Ala Ala Val1505 1510 1515 1520Gly Leu Glu Pro Glu Ala Arg Ala Arg Ile Gly Arg Arg Leu Lys Asp 1525 1530 1535Met Gln Thr Ala Cys Glu Pro Arg Ser Glu Ser Ser Arg Asp Leu Lys 1540 1545 1550Ser Ala Ser Arg Thr Glu Val Leu Asp Phe Leu Thr Asn Glu Leu Gly 1555 1560 1565Ile Ser Arg 157043500PRTStreptomyces parvulus Tu4055 4Met Pro Asn Asp Glu Glu Leu Leu Asp Tyr Leu Lys Arg Thr Ala Ser1 5 10 15Asn Leu Gln Glu Ala Arg Gln Arg Val His Glu Leu Glu Glu Ser Glu 20 25 30Arg Glu Pro Ile Ala Ile Val Gly Met Ser Cys Arg Leu Pro Gly Gly 35 40 45Val Asn Ser Pro Glu Glu Phe Trp Ser Leu Leu Glu Ala Gly Thr Asp 50 55 60Ala Val Ser Glu Phe Pro Arg Asp Arg Gly Trp Asp Val Glu Arg Leu65 70 75 80Tyr Asp Pro Asp Pro Asp Ala

Pro Gly Lys Ser Tyr Val Arg Glu Gly 85 90 95Gly Phe Leu Asp Gly Ala Gly Arg Phe Asp Pro Ala Phe Phe Gly Ile 100 105 110Ser Pro Arg Glu Ala Val Val Met Asp Pro Gln Gln Arg Leu Leu Leu 115 120 125Glu Cys Ser Trp Glu Ala Ile Glu Arg Ser Arg Ile Asp Pro Lys Thr 130 135 140Leu His Gly Ser Arg Ala Gly Val Phe Val Gly Ser Asn Gly Gln Asp145 150 155 160Tyr Gly Thr Leu Leu Leu Arg Ala Asp Asp Arg Ser His Ala Tyr Leu 165 170 175Ala Thr Gly Ala Ser Ala Ser Val Leu Ser Gly Arg Ile Ser Tyr Thr 180 185 190Leu Gly Leu Glu Gly Pro Ala Val Thr Ile Ser Thr Ala Cys Ser Ser 195 200 205Ser Leu Val Ala Leu His Leu Ala Ala Arg Ala Leu Arg Ala Gly Glu 210 215 220Cys Glu Leu Ala Leu Ala Gly Gly Val Thr Val Met Pro Thr Thr Arg225 230 235 240Leu Phe Glu Val Phe Ser Arg Gln Arg Gly Leu Ala Gly Asp Gly Arg 245 250 255Cys Lys Ala Phe Ala Ala Gly Ala Asp Gly Thr Gly Trp Gly Glu Gly 260 265 270Val Gly Val Leu Val Leu Glu Arg Leu Ser Val Ala Arg Arg Asn Gly 275 280 285His Arg Val Leu Ala Val Val Arg Gly Ser Ala Val Asn Gln Asp Gly 290 295 300Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln Arg Val305 310 315 320Ile Arg Ala Ala Leu Ala Ser Ala Arg Leu Ala Pro Glu Asp Val Asp 325 330 335Ala Val Glu Ala His Gly Thr Gly Thr Ser Leu Gly Asp Pro Ile Glu 340 345 350Ala Gln Ala Leu Leu Ala Thr Tyr Gly Arg Gly Arg Asp Ala Glu Arg 355 360 365Pro Leu Trp Leu Gly Ser Val Lys Ser Asn Ile Gly His Ala Gln Ala 370 375 380Ala Ala Gly Val Ala Gly Val Ile Lys Met Val Lys Ala Met Gln Ala385 390 395 400Gly Thr Leu Pro Arg Thr Leu His Val Asp Glu Pro Ser Gly Glu Val 405 410 415Asp Trp Asp Ser Gly Ala Val Arg Leu Leu Thr Glu Ala Arg Asp Trp 420 425 430Pro Ser Glu Glu Gly Arg Leu Arg Arg Ala Gly Val Ser Ser Phe Gly 435 440 445Ile Ser Gly Thr Asn Ala His Val Ile Leu Glu Glu Pro Pro Ala Glu 450 455 460Asp Ala Val Pro Glu Pro Glu Ala Gly Asp Val Val Pro Trp Val Leu465 470 475 480Ser Ala Arg Ser Ala Glu Ala Leu Arg Glu Gln Ala Ala Arg Leu Ala 485 490 495Ser Val Ala Gly Gly Leu Asn Val Val Asp Val Gly Trp Ser Leu Ala 500 505 510Ser Thr Arg Ala Ala Phe Glu His Arg Ala Val Val Val Gly Arg Glu 515 520 525Arg Glu Glu Leu Leu Ala Gly Leu Phe Ala Val Ala Ala Gly Arg Pro 530 535 540Ala Ala Asn Val Val Thr Gly Pro Val Ser Ser Gly Arg Pro Ala Phe545 550 555 560Val Phe Ala Gly Gln Gly Ser Gln Arg Leu Gly Met Gly Arg Gly Leu 565 570 575Tyr Glu Arg Phe Pro Val Phe Ala Glu Ala Phe Asp Glu Val Cys Gly 580 585 590Arg Val Gly Pro Gly Val Arg Glu Val Val Phe Gly Ser Asp Ala Gly 595 600 605Glu Leu Asp Arg Thr Val Trp Ala Gln Ala Gly Leu Phe Ala Leu Glu 610 615 620Val Ala Leu Phe Arg Leu Leu Glu Ser Trp Gly Val Arg Pro Gly Cys625 630 635 640Leu Ile Gly His Ser Val Gly Glu Leu Ser Ala Ala Cys Val Ala Gly 645 650 655Leu Trp Ser Leu Glu Asp Ala Cys Arg Val Val Ala Ala Arg Ala Arg 660 665 670Leu Met Gln Ala Leu Pro Ala Gly Gly Val Met Val Ala Val Arg Ala 675 680 685Glu Ala Gly Glu Leu Ala Gly Phe Leu Gly Glu Asp Val Val Ile Ala 690 695 700Ser Val Asn Ala Pro Gly Gln Val Val Ile Ala Gly Pro Glu Gly Gly705 710 715 720Val Glu Arg Val Val Ala Ala Cys Gly Ala Arg Ser Arg Arg Leu Ala 725 730 735Val Ser His Ala Phe His Ser Pro Leu Val Glu Pro Met Leu Gly Glu 740 745 750Phe Arg Arg Val Val Glu Ser Val Ala Phe Gly Val Pro Ser Leu Arg 755 760 765Val Val Ser Asn Val Thr Gly Ala Trp Val Asp Pro Glu Glu Trp Gly 770 775 780Thr Pro Glu Tyr Trp Val Arg Gln Val Arg Glu Pro Val Arg Phe Ala785 790 795 800Asp Gly Val Ala Thr Leu Leu Asp Ala Gly Val Arg Thr Phe Val Glu 805 810 815Leu Gly Pro Ala Gly Thr Leu Thr Ser Met Val Ser His Cys Ala Asp 820 825 830Ala Thr Ala Thr Ser Val Thr Ala Val Pro Thr Leu Arg Pro Asp His 835 840 845Asp Glu Ser Arg Thr Val Leu Ser Ala Ala Ala Ser Leu Tyr Val Gln 850 855 860Gly His Pro Val Asp Trp Ala Pro Leu Phe Pro Arg Ala Arg Thr Val865 870 875 880Asp Leu Pro Thr Tyr Pro Phe Gln His Gln His Tyr Trp Met Met Asn 885 890 895Thr Gly Ser Ala Ala Glu Pro Ala Glu Leu Gly Leu Gly Asp Ala Arg 900 905 910His Pro Leu Leu Gly Ser Val Val Thr Val Ala Gly Asp Asp Lys Val 915 920 925Val Phe Ala Gly Arg Leu Ala Leu Arg Thr His Pro Trp Leu Ala Asp 930 935 940His Thr Val Leu Asp Ala Val Leu Leu Pro Ala Thr Ala Phe Leu Glu945 950 955 960Leu Ala Val Arg Ala Gly Glu Glu Val Ser Cys Pro Val Val His Asp 965 970 975Leu Thr Leu His Arg Pro Leu Val Val Pro Glu Arg Gly Ala Val Gln 980 985 990Val Gln Met Ala Val Gly Ala Pro Glu Ala Asp Gly Arg Arg Glu Val 995 1000 1005Arg Val Tyr Ser Arg Pro Asp Asp Asp Ala Glu His Glu Trp Thr Leu 1010 1015 1020His Ala Ala Gly Leu Leu Ala Ser Ala Ala Thr Ala Glu Pro Ala Val1025 1030 1035 1040Ala Ala Gly Ala Trp Pro Pro Pro Glu Ala Gln Ala Val Asp Leu Asp 1045 1050 1055Gly Phe Tyr Ala Gly Leu Ala Glu His Gly Tyr His Tyr Gly Pro Leu 1060 1065 1070Phe Gln Gly Val Arg Ala Ala Trp Arg Leu Gly Asp Asp Val Leu Ala 1075 1080 1085Glu Ile Val Leu Pro Glu Ala Ala Gly Ala Asp Ala Ala Arg Tyr Gly 1090 1095 1100Met His Pro Ala Leu Leu Asp Ala Val Leu His Ala Ala Arg Leu Gly1105 1110 1115 1120Ala Phe Arg Glu Arg Ser Glu Glu Lys Tyr Leu Pro Phe Ala Trp Glu 1125 1130 1135Gly Val Thr Leu Arg Thr Arg Gly Ala Thr Ala Val Arg Ala Arg Ile 1140 1145 1150Ser Arg Ala Gly Thr Asp Ala Ile Arg Leu Asp Val Thr Asp Thr Ala 1155 1160 1165Asp Arg Pro Val Leu Thr Ala Glu Ser Leu Thr Leu Arg Pro Val Ser 1170 1175 1180Ala Gly Gln Leu Met Ala Val Pro Arg Asp Ser Leu Phe Arg Val Asp1185 1190 1195 1200Trp Val Ser Ala Pro Ala Ala Asn Gly Pro Gly Leu Arg Leu Ala Arg 1205 1210 1215Ala Ala Thr Val Glu Ala Ala Leu Ala Ala Asp Ala Asp Ile Val Val 1220 1225 1230Val Pro Cys Leu Asp Ser Glu Gly Pro His Gln Ala Thr Tyr Gln Ala 1235 1240 1245Leu Glu Leu Leu Gln Arg Trp Leu Ala Ala Asp Thr Gly Thr Thr Thr 1250 1255 1260Leu Ala Leu Leu Thr His Arg Ala Val Ala Val Gly Asp Asp Val His1265 1270 1275 1280Asp Leu His His Ala Pro Leu Trp Gly Leu Val Arg Thr Ala Gln Thr 1285 1290 1295Glu His Pro Gly Cys Phe Arg Leu Val Asp Ser Asp Asp Pro Asp Pro 1300 1305 1310Thr Thr Asp Val Leu Ala Ala Ala Leu Ala Thr Gly Glu Pro Gln Val 1315 1320 1325Ala Ile Arg Asp Gly Ala Val Leu Ala Pro Arg Leu Thr Ala Ala Ser 1330 1335 1340Ala Pro Arg Glu Pro Ala Glu Trp Asp Ala Glu Gly Thr Val Leu Ile1345 1350 1355 1360Thr Gly Gly Ser Gly Ala Leu Ala Gly Ile Val Ala Gln His Leu Val 1365 1370 1375Ala Arg His Gly Val Arg Arg Leu Val Leu Ala Ser Arg Ser Gly Arg 1380 1385 1390Pro Ala Pro Gly Ala Asp Leu Leu Asp Ala Asp Val Thr Ala Val Ser 1395 1400 1405Cys Asp Val Ser Asp Arg Asp Ala Val Ala Ala Leu Leu Ala Ser Val 1410 1415 1420Pro Asp Glu His Pro Leu Thr Ala Val Val His Thr Ala Gly Val Leu1425 1430 1435 1440Asp Asp Gly Val Leu His Ala Leu Thr Thr Glu Arg Ile Asp Thr Ser 1445 1450 1455Phe Ala Ala Lys Val Asp Gly Ala Arg His Leu His Glu Leu Thr Ser 1460 1465 1470His Leu Asp Leu Thr Ala Phe Val Leu Phe Ser Ser Ala Ser Ala Val 1475 1480 1485Leu Gly Ala Ala Gly Gln Gly Asn Tyr Ala Ala Ala Asn Ala Tyr Leu 1490 1495 1500Asp Ala Leu Ala Ala His Arg Arg Ser Asn Asp Leu Pro Ala Val Ser1505 1510 1515 1520Leu Ala Trp Gly Leu Trp Ala Glu His Glu Gly Met Ala Arg Gly Leu 1525 1530 1535Gly Asp Ala Glu Leu Thr Arg Ile Ser Arg Ile Gly Val Thr Ala Leu 1540 1545 1550Ser Ala Glu Asp Gly Met Arg Leu Phe Asp Ala Gly Cys Ala Gly Asp 1555 1560 1565Gln Ser Gln Leu Val Pro Met Arg Val Asp Thr Ala Ala Leu Arg Ala 1570 1575 1580Arg Arg Asp His Leu Pro Ala Pro Met Trp Ser Leu Val Pro Glu Arg1585 1590 1595 1600Thr Arg Ala Ala Arg Thr Gln Pro Ala Ala Ser Leu Arg Asp Arg Leu 1605 1610 1615Ala Glu Leu Thr Ala Pro Glu Arg Lys Arg Thr Val Leu Asn Leu Val 1620 1625 1630Arg Asn Ala Val Ala Asp Thr Leu Gly His Asn Ala Ala Asp Gly Val 1635 1640 1645Pro Pro Asp Gln Ser Leu Asp Ala Ala Gly Phe Asp Ser Leu Thr Ala 1650 1655 1660Val Glu Phe Arg Asn Arg Leu Ser Ala Val Thr Asp Leu Arg Leu Pro1665 1670 1675 1680Ala Thr Leu Thr Tyr Asp His Pro Thr Pro Ala Ala Ile Ala Glu His 1685 1690 1695Ile Leu Thr Arg Leu Thr Leu Leu Lys Glu Thr Ala Ala Pro Ala Val 1700 1705 1710Gly Thr Ala Pro Val Ala Ala Pro Thr Glu Asp Asp Ala Ile Val Ile 1715 1720 1725Val Gly Met Ala Gly Arg Phe Pro Gly Gly Val Arg Thr Pro Glu Gly 1730 1735 1740Leu Trp Asp Leu Val His Ser Gly Thr Asp Ala Ile Ser Glu Trp Pro1745 1750 1755 1760Thr Asp Arg Gly Trp Asp Val Glu Asn Leu Tyr Asp Pro Asp Pro Asp 1765 1770 1775Ala Val Gly Lys Ser Tyr Val Arg His Gly Gly Phe Leu His Asp Val 1780 1785 1790Ala Gly Phe Asp Ala Gly Phe Phe Gly Ile Ser Pro Arg Glu Ala Leu 1795 1800 1805Ala Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Cys Ser Tyr Glu Ala 1810 1815 1820Leu Glu Arg Ala Gly Ile Asp Pro Ala Thr Leu Arg Gly Ser Arg Ser1825 1830 1835 1840Gly Val Tyr Ala Gly Val Met Tyr His Glu Tyr Ala Ser Arg Leu Gly 1845 1850 1855Ala Thr Pro Ala Gly Phe Glu Gly Thr Leu Gly Thr Gly Ser Ser Gly 1860 1865 1870Ser Ile Ala Ser Gly Arg Ile Ser Tyr Thr Phe Asp Leu Thr Gly Pro 1875 1880 1885Ala Val Thr Val Asp Thr Ala Cys Ser Thr Ser Leu Val Gly Leu His 1890 1895 1900Leu Ala Val Gln Ala Leu Arg Ala Gly Glu Cys Glu Leu Ala Leu Ala1905 1910 1915 1920Gly Gly Val Thr Val Met His Thr Pro Arg Pro Phe Val Glu Phe Ser 1925 1930 1935Arg Gln Arg Gly Leu Ala Ala Asp Gly Arg Ser Lys Ala Phe Ala Ala 1940 1945 1950Ser Ala Asp Gly Val Ala Trp Ala Glu Gly Ala Gly Ile Leu Val Leu 1955 1960 1965Glu Arg Leu Ser Ala Ala Arg Arg Asn Gly His Arg Val Leu Ala Val 1970 1975 1980Val Arg Gly Ser Ala Val Asn Gln Asp Gly Ala Ser Asn Gly Leu Thr1985 1990 1995 2000Ala Pro Asn Gly Pro Ser Gln Gln Arg Val Ile Arg Ala Ala Leu Ala 2005 2010 2015Ser Ala Gly Leu Gly Pro Ala Asp Val Asp Val Val Glu Ala His Gly 2020 2025 2030Thr Gly Thr Ala Leu Gly Asp Pro Ile Glu Ala Gln Ala Leu Leu Ala 2035 2040 2045Thr Tyr Gly Arg Gly Arg Asp Ala Asp Arg Pro Leu Trp Leu Gly Ser 2050 2055 2060Val Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ala Gly Val Ala Ser2065 2070 2075 2080Val Ile Lys Met Val Gln Ala Met Gln Ala Gly Val Leu Pro Arg Thr 2085 2090 2095Leu His Val Asp Glu Pro Ser Gly Glu Val Asp Trp Asp Ser Gly Ala 2100 2105 2110Val Arg Leu Leu Thr Glu Ala Arg Glu Trp Pro Ser Gly Glu Gly Arg 2115 2120 2125Val Arg Arg Ala Gly Val Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala 2130 2135 2140His Val Ile Leu Glu Glu Pro Pro Ala Glu Asp Ala Leu Pro Glu Pro2145 2150 2155 2160Glu Ala Gly Asp Val Val Pro Trp Val Leu Ser Ala Arg Ser Ala Glu 2165 2170 2175Ala Leu Arg Glu Gln Ala Ala Arg Leu Ala Ser Val Ala Gly Gly Leu 2180 2185 2190Asn Val Val Asp Val Gly Trp Ser Leu Ala Ser Thr Arg Ala Ala Phe 2195 2200 2205Glu His Arg Ala Val Val Val Gly Gly Asp Arg Glu Glu Leu Leu Gly 2210 2215 2220Lys Leu Ser Ser Val Ser Gly Val Glu Val Gly Val Gly Val Gly Ala2225 2230 2235 2240Gly Gly Gly Val Val Leu Val Phe Ala Gly Gln Gly Cys Gln Trp Val 2245 2250 2255Gly Met Gly Arg Glu Leu Leu Gly Ser Ser Leu Val Phe Ala Glu Ser 2260 2265 2270Met Arg Glu Cys Ala Ala Ala Leu Ser Pro Phe Val Asp Phe Ser Val 2275 2280 2285Val Asp Val Leu Gly Ser Ala Gly Glu Leu Gly Arg Val Glu Val Val 2290 2295 2300Gln Pro Ala Leu Trp Ala Val Met Val Ser Leu Ala Arg Val Trp Arg2305 2310 2315 2320Ser Trp Gly Val Pro Val Ala Ala Val Val Gly His Ser Gln Gly Glu 2325 2330 2335Ile Ala Ala Ala Thr Val Ala Gly Ala Leu Ser Val Gly Asp Ala Ala 2340 2345 2350Arg Val Val Ala Leu Arg Ser Arg Leu Ile Ala Glu Arg Leu Ser Gly 2355 2360 2365Leu Gly Gly Met Val Ser Val Ala Leu Ser Arg Glu Arg Val Val Ser 2370 2375 2380Leu Ile Ala Gly Val Pro Gly Val Ser Val Ala Ala Val Asn Gly Ser2385 2390 2395 2400Ser Ser Thr Val Val Ser Gly Glu Ala Ala Gly Leu Glu Arg Val Leu 2405 2410 2415Ala Ala Cys Val Ser Ser Gly Val Arg Ala Arg Arg Ile Asp Val Asp 2420 2425 2430Tyr Ala Ser His Ser Val Gln Val Glu Leu Ile Arg Glu Glu Leu Leu 2435 2440 2445Gly Val Leu Asp Gly Ile Val Pro Arg Ser Gly Glu Ile Pro Phe Val 2450 2455 2460Ser Thr Val Thr Gly Glu Arg Ile Asp Thr Val Glu Leu Gly Ala Glu2465 2470 2475 2480Tyr Trp Tyr Arg Asn Leu Arg Gln Thr Val Glu Phe Gln Ser Val Val 2485 2490 2495Glu Gly Leu Val Ala Gln Gly Cys Arg Val Phe Leu Glu Ser Ser Pro 2500 2505 2510His Pro Val Leu Thr Val Gly Ile Glu Glu Ser Ala Asp Arg Val Val 2515 2520 2525Ala Leu Glu Ser Leu Arg Arg Gly Glu Gly Gly Leu Arg Arg Leu Val 2530 2535 2540Asp Ala Ala Gly Glu Ala Trp Val Arg Gly

Val Pro Ile Asp Trp Ala2545 2550 2555 2560Gly Met Leu Ala Gly Gly Arg Arg Val Asp Leu Pro Thr Tyr Pro Phe 2565 2570 2575Gln His Gln Pro Tyr Trp Leu Asp Ser Pro Arg His Pro Ala Gly Asp 2580 2585 2590Val Thr Ala Val Gly Leu Thr Glu Ala Gly His Ala Phe Val Pro Ala 2595 2600 2605Ala Val Asp Leu Pro Asp Gly Gln Arg Val Trp Thr Gly Arg Leu Ser 2610 2615 2620Leu Pro Ser Tyr Pro Trp Leu Ala Asp His Gln Val Leu Gly Gln Val2625 2630 2635 2640Leu Leu Pro Gly Val Val Trp Val Glu Leu Ala Leu His Ala Gly His 2645 2650 2655Gln Ala Gly Cys Asp Ser Val Asp Glu Leu Thr Leu Gln Ser Pro Leu 2660 2665 2670Val Leu Gly Ala Ser Asp Thr Val Gln Val Arg Val Val Val Thr Glu 2675 2680 2685Thr Glu Glu Pro Gly Thr Arg Thr Val Ser Met His Ser Arg Arg Asp 2690 2695 2700Asp Gly Ser Trp Val Thr His Ala Glu Gly Ile Leu Gly Ala Gly Gly2705 2710 2715 2720Pro Pro Pro Glu Pro Leu Pro Glu Trp Pro Pro Thr Gly Ala Met Pro 2725 2730 2735Leu Asp Val Glu Gly Phe Tyr Asp Glu Leu Ala Ala Gly Gly Tyr His 2740 2745 2750Tyr Gly Pro Gln Phe Arg Cys Leu Arg Arg Ala Trp Arg Ala Gly Glu 2755 2760 2765Asp Leu Val Ala Glu Ile Ser Leu Pro Glu Gly Thr Asp Val Asp Ala 2770 2775 2780Tyr Gly Leu His Pro Gly Leu Phe Asp Ala Ala Val His Ser Val Ala2785 2790 2795 2800Cys Ala Arg Thr Ser Ala Gly Ala Gly Asp Asp Gly Pro Arg Leu Pro 2805 2810 2815Phe Ala Phe Ser Asp Val Arg Leu Phe Ala Thr Gly Val Thr Ser Leu 2820 2825 2830Arg Val Arg Ile Asp Pro Gln Asn Ser Ser Trp Gln Ala Trp Asp Glu 2835 2840 2845Ser Gly Leu Pro Val Leu Thr Ile Gly Arg Leu Ala Gly Arg Pro Val 2850 2855 2860Asp Ala Asp Gln Phe Ala Val Arg Arg Ala Gly His Leu Phe Arg Val2865 2870 2875 2880Glu Thr Arg His Glu Ala Leu Ala Gly Pro Ala Pro Ala Ser Trp Ala 2885 2890 2895Val Ile Gly Ala Asp Pro Ala Gly Tyr Ala Ala Ala Leu Glu Ala Thr 2900 2905 2910Gly Ala Gln Val Thr Thr Ala Ala Asp Leu Ala Gly Leu Thr Ser Ala 2915 2920 2925Pro Glu Ala Ala Leu Phe Thr Leu Pro Gly Thr Lys Asp Ala Gly Val 2930 2935 2940Thr Glu Glu Val Pro Thr Ala Val Arg Glu Ala Thr Ala Gln Val Leu2945 2950 2955 2960Glu Val Leu Gln Asp Trp Leu Thr Asp Gly Arg Phe Asp Asp Ala Arg 2965 2970 2975Leu Val Val Val Ser Arg Glu Ala Glu Asp Gly Asp Leu Leu His Gly 2980 2985 2990Thr Ala Arg Gly Leu Leu Arg Ala Ala Gln Ala Glu His Pro Asp Arg 2995 3000 3005Ile Thr Leu Val Asp Leu Asp Ala His Pro Ala Ser Leu Thr Ala Leu 3010 3015 3020Pro Gly Phe Ala Leu Gly Pro Glu Pro Glu Val Val Val Arg Ala Gly3025 3030 3035 3040Asp Gly Arg Ala Pro Arg Leu Ala Arg Ala Gln Ala Pro Thr Gly Ala 3045 3050 3055Gly Ser Leu Gly Thr Gly Thr Val Leu Ile Thr Gly Gly Thr Gly Thr 3060 3065 3070Leu Gly Gly Leu Leu Ala Arg His Leu Val Glu Thr His Gly Val Thr 3075 3080 3085Arg Leu Leu Leu Val Ser Arg Arg Gly Pro Ala Ala Asp Gly Ala Asp 3090 3095 3100Arg Leu His Ala Glu Leu Thr Gly His Gly Ala His Val Asp Ile Val3105 3110 3115 3120Ala Ala Asp Leu Gly Asp Arg Thr Ser Val Ala Ala Leu Leu Ala Thr 3125 3130 3135Val Asp Ala Asp His Pro Leu Ser Ala Val Val His Ala Ala Gly Ala 3140 3145 3150Leu Asp Asp Gly Val Leu Gly Thr Arg Ser Ala Asp Trp Leu Asp Pro 3155 3160 3165Val Leu Arg Pro Lys Ala Asp Ala Ala Trp His Leu His Glu Leu Thr 3170 3175 3180Ala Glu Leu Pro Leu Thr Ala Phe Val Met Phe Ser Ser Ala Ala Ser3185 3190 3195 3200Val Leu Gly Ala Ala Gly Gln Ala Asn Tyr Ala Ala Ala Asn Gly Phe 3205 3210 3215Leu Asp Ala Leu Ala Ala His Arg Ala Ala Arg Gly Leu Pro Gly Thr 3220 3225 3230Ser Leu Ala Trp Gly Leu Trp Glu His Arg Ser Glu Leu Thr Arg His 3235 3240 3245Thr Gly Ser Pro Ser Arg Ser Ile Ala Ala Val Gly Ala Leu Ser Thr 3250 3255 3260Ala Glu Ala Leu Ala Ala Phe Asp Ala Gly Leu Ala Ser Gly Glu Pro3265 3270 3275 3280Leu Ala Val Pro Ile Arg Leu Glu Ser Thr Ser Ser Glu Glu Val Pro 3285 3290 3295Pro Met Leu Arg Gly Leu Val Arg Val Arg Arg Arg Ala Ala Thr Gly 3300 3305 3310Thr Glu Pro Ala Ala Ser Ala Gly Ala Ala Gln Glu Val Arg Gln Leu 3315 3320 3325Ala Glu Leu Gly Ala Asp Glu Arg Gln Arg Arg Val Gln Arg Ile Val 3330 3335 3340Leu Asp Thr Ala Ala Ala Val Leu Gly His Asp Ser His Asp Ala Ile3345 3350 3355 3360Pro Leu Thr Arg Gly Phe Leu Glu Leu Gly Phe Asp Ser Leu Thr Ala 3365 3370 3375Val Arg Leu Arg Asn Arg Leu Ala Arg Arg Leu Gly Leu Arg Leu Pro 3380 3385 3390Ala Thr Val Val Phe Asp His Pro Ser Pro Ala Ala Leu Ala Ala His 3395 3400 3405Leu Val Glu His Leu Val Gly Thr Val Asp Pro Thr Ala Gln Ala Met 3410 3415 3420Glu Gln Leu Glu Ala Leu Arg Arg Ser Val His Ala Ala Thr Pro Ala3425 3430 3435 3440Gly Gly Leu Asp Arg Ala Leu Val Thr Gln Arg Leu Thr Ala Leu Leu 3445 3450 3455Asp Glu Met Arg His Val Asp Gly Pro Gly Gly Thr Glu Gly Pro Asp 3460 3465 3470Gly Ser Gly Asp Asp Leu Glu Asn Ala Thr Ala Asp Glu Ile Tyr Ala 3475 3480 3485Leu Ile Asp Asn Glu Leu Gly Ile Gly Gly Thr Gln 3490 3495 350051620PRTStreptomyces parvulus Tu4055 5Met Asn Gly Asp Asp Lys Ala Leu Ala Tyr Leu Lys Arg Val Thr Ala1 5 10 15Asp Leu Arg Ser Ala Arg Ala Arg Leu Gln Glu Leu Glu Ser Ala Asp 20 25 30Thr Asp Pro Ile Ala Ile Ile Gly Met Gly Cys Arg Leu Pro Gly Gly 35 40 45Val Arg Thr Pro Glu Asp Leu Trp Asp Leu Val Glu Lys Lys His Asp 50 55 60Ala Ile Gly Pro Phe Pro Ala Asp Arg Gly Trp Asp Leu Glu Asn Leu65 70 75 80Tyr Asp Pro Asp Pro Asp Ala Pro Gly Lys Ala Tyr Val Arg Glu Gly 85 90 95Gly Phe Val His Asp Val Ala Gly Phe Asp Ala Gly Phe Phe Gly Ile 100 105 110Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln His Arg Leu Leu Leu 115 120 125Glu Cys Ser Trp Glu Ala Leu Glu Arg Ala Gly Ile Asp Pro Ser Ser 130 135 140Leu Glu Gly Thr Arg Thr Gly Val Tyr Thr Gly Leu Met Thr His Glu145 150 155 160Tyr Ala Thr Arg Leu Pro Ser Ile Asp Glu Glu Leu Glu Gly Val Ile 165 170 175Gly Ile Gly Asn Ala Gly Ser Val Ala Ser Gly Arg Val Ser Tyr Thr 180 185 190Leu Gly Leu Asn Gly Pro Ala Val Thr Val Asp Thr Ala Cys Ser Ser 195 200 205Ser Leu Val Ala Leu His Leu Ala Ala Gln Ala Leu Arg Gln Gly Gln 210 215 220Cys Thr Leu Ala Leu Ala Gly Gly Ala Ser Val Ile Ala Ala Pro Thr225 230 235 240Val Phe Ala Thr Phe Ser Arg Gln Arg Gly Leu Ala Pro Asp Gly Arg 245 250 255Cys Lys Ala Phe Ser Ser Thr Thr Asp Gly Thr Gly Phe Gly Glu Gly 260 265 270Val Gly Val Leu Val Leu Glu Arg Leu Ser Asp Ala Arg Arg Asn Gly 275 280 285His Glu Val Leu Ala Val Val Arg Gly Ser Ala Val Asn Gln Asp Gly 290 295 300Ala Ser Ser Gly Phe Thr Ala Pro Asn Gly Pro Ser Gln Gln Asp Val305 310 315 320Ile Arg Glu Ala Leu Ala Asp Gly Arg Leu Thr Pro Ala Asp Val Asp 325 330 335Val Val Glu Gly His Gly Thr Gly Thr Arg Leu Gly Asp Pro Ile Glu 340 345 350Ala Gln Ala Leu Leu Ala Thr Tyr Gly Arg Gly Arg Asp Ala Asp Arg 355 360 365Pro Leu Trp Leu Gly Ser Val Lys Ser Asn Ile Gly His Thr Gln Ala 370 375 380Ala Ala Gly Val Ala Ser Val Ile Lys Met Val Gln Ala Met Gln Ala385 390 395 400Gly Val Leu Pro Arg Thr Leu His Val Asp Glu Pro Ser Gly Glu Val 405 410 415Asp Trp Asp Ser Gly Ala Val Arg Leu Leu Thr Glu Ala Arg Glu Trp 420 425 430Pro Ser Gly Glu Gly Arg Val Arg Arg Ala Gly Val Ser Ser Phe Gly 435 440 445Ile Ser Gly Thr Asn Ala His Val Ile Leu Glu Glu Pro Pro Ala Glu 450 455 460Asp Ala Leu Pro Glu Pro Glu Ala Gly Asp Val Val Pro Trp Val Leu465 470 475 480Ser Ala Arg Ser Ala Glu Ala Leu Arg Glu Gln Ala Ala Arg Leu Ala 485 490 495Ser Val Ala Gly Gly Leu Asn Val Val Asp Val Gly Trp Ser Leu Ala 500 505 510Ser Thr Arg Ala Ala Phe Glu His Arg Ala Val Val Val Gly Gly Asp 515 520 525Arg Glu Glu Leu Leu Gly Lys Leu Ser Ser Val Ser Gly Val Glu Val 530 535 540Gly Val Gly Val Gly Ala Gly Gly Gly Val Val Leu Val Phe Ala Gly545 550 555 560Gln Gly Cys Gln Trp Val Gly Met Gly Arg Glu Leu Leu Gly Ser Ser 565 570 575Leu Val Phe Ala Glu Ser Met Arg Glu Cys Ala Ala Ala Leu Ser Pro 580 585 590Phe Val Asp Phe Ser Val Val Asp Val Leu Gly Ser Ala Gly Glu Leu 595 600 605Gly Arg Val Glu Val Val Gln Pro Ala Leu Trp Ala Val Met Val Ser 610 615 620Leu Ala Arg Val Trp Arg Ser Trp Gly Val Pro Val Ala Ala Val Val625 630 635 640Gly His Ser Gln Gly Glu Ile Ala Ala Ala Thr Val Ala Gly Ala Leu 645 650 655Ser Val Gly Asp Ala Ala Arg Val Val Ala Leu Arg Ser Arg Leu Ile 660 665 670Ala Glu Arg Leu Ser Gly Leu Gly Gly Met Val Ser Val Ala Leu Ser 675 680 685Arg Glu Arg Val Val Ser Leu Ile Ala Gly Val Pro Gly Val Ser Val 690 695 700Ala Ala Val Asn Gly Ser Ser Ser Thr Val Val Ser Gly Glu Ala Ala705 710 715 720Gly Leu Glu Arg Val Leu Ala Ala Cys Val Ser Ser Gly Val Arg Ala 725 730 735Arg Arg Ile Asp Val Asp Tyr Ala Ser His Ser Val Gln Val Glu Leu 740 745 750Ile Arg Glu Glu Leu Leu Gly Val Leu Asp Gly Ile Val Pro Arg Ser 755 760 765Gly Glu Ile Pro Phe Val Ser Thr Val Thr Gly Glu Arg Ile Asp Thr 770 775 780Val Glu Leu Gly Ala Glu Tyr Trp Tyr Arg Asn Leu Arg Gln Thr Val785 790 795 800Glu Phe Gln Ser Val Val Glu Gly Leu Val Ala Gln Gly Cys Arg Val 805 810 815Phe Leu Glu Ser Ser Pro His Pro Val Leu Thr Val Gly Ile Glu Glu 820 825 830Ser Ala Asp Arg Val Val Ala Leu Glu Ser Leu Arg Arg Gly Glu Gly 835 840 845Gly Leu Arg Arg Leu Val Asp Ala Ala Gly Glu Ala Trp Val Arg Gly 850 855 860Val Pro Ile Asp Trp Ala Gly Met Leu Ala Gly Gly Arg Arg Val Asp865 870 875 880Leu Pro Thr Tyr Pro Phe Gln His Gln Pro Tyr Trp Leu Asp Ser Pro 885 890 895Arg His Pro Ala Gly Asp Val Thr Gly Pro Gly Asp Asp Glu Phe Trp 900 905 910Ala Ala Val Glu His Gly Glu Ala Thr Glu Leu Ala Asp Leu Leu Arg 915 920 925Arg Ser Ala Ala Glu Pro Gly Gln Asp Leu His Ala Pro Val Ala Ala 930 935 940Leu Leu Pro Thr Leu Ala Thr Trp Arg Arg Asp Arg Gln Arg Arg Ala945 950 955 960Ala Val Asp Ser Trp Arg Tyr Arg Ile Val Trp Arg Pro Val Ala Thr 965 970 975Pro Ser Tyr Asp Arg Val Leu Ser Gly Arg Trp Ala Val Val Val Pro 980 985 990Ala Gly His Glu Asp Asp Pro Val Val Asp Trp Val Cys Ser Ala Leu 995 1000 1005Arg Asp His Gly Gly Glu Pro Glu Arg Met Val Leu Gly Pro Arg Glu 1010 1015 1020Ser Arg Ser Ala Leu Ala Thr Arg Leu Ala Ala Asp Pro Pro Gly Gly1025 1030 1035 1040Val Val Ser Leu Leu Gly Leu Ser Gly Ala Ala His Pro Asp His Glu 1045 1050 1055Val Leu Pro Ser Ala Val Ala Gly Thr Val Leu Leu Ala Gln Ala Leu 1060 1065 1070Ser Asp Gly Ala Val Arg Ala Pro Val Trp Thr Leu Thr Arg Asn Gly 1075 1080 1085Val Ser Ala Thr Ala Thr Asp Pro Val Ala Pro Thr His Ala Ala Gln 1090 1095 1100Val Trp Ala Val Ala Arg Val Ala Gly Leu Glu His Pro Glu Ala Trp1105 1110 1115 1120Gly Gly Leu Leu Asp Leu Pro Asp Arg Leu Asp Asp Arg Ala Ala Ala 1125 1130 1135Arg Phe Ala Ala Val Leu Ser Ala Gly Glu Asp Glu Asp Gln Leu Ala 1140 1145 1150Leu Arg Asp Ala Gly Leu Leu Ala Arg Arg Leu Val Arg Ala Pro Val 1155 1160 1165Pro Arg Asp Ala Val Thr Ala Gly Trp Gln Pro Arg Asp Thr Ala Leu 1170 1175 1180Val Thr Gly Gly Thr Gly Gly Leu Gly Gly Gln Val Ala Arg Trp Leu1185 1190 1195 1200Ala Ala Ala Gly Val Arg His Leu Val Leu Val Ser Arg Arg Gly Ala 1205 1210 1215Glu Ala Glu Gly Ala Asp Arg Leu Arg Asp Asp Leu Thr Ala Leu Gly 1220 1225 1230Val Gln Val Thr Phe Gly Ala Cys Asp Val Ala Asp Arg Ala Ala Leu 1235 1240 1245Ser Ala Leu Leu Asp Arg Val Gln Glu Asp Gly Pro Pro Ile Arg Thr 1250 1255 1260Val Val His Ala Ala Gly Ser Gly Arg Ala Ala Arg Leu Leu Asp Thr1265 1270 1275 1280Asp Ala Glu Glu Thr Ala Ala Val Leu Arg Ala Lys Ser Ala Gly Ala 1285 1290 1295Arg Asn Leu His Glu Leu Leu Asp Asp Val Asp Ala Phe Val Leu Phe 1300 1305 1310Ser Ser Gly Ala Gly Val Trp Gly Ser Ser Ala Gln Gly Ala Tyr Ala 1315 1320 1325Ala Ala Asn Ala Tyr Leu Asp Ala Leu Ala Glu Gln Arg Arg Gly Gln 1330 1335 1340Gly Arg Pro Ala Thr Ser Val Ala Trp Gly Ala Trp Ala Gly Asp Gly1345 1350 1355 1360Met Thr Ala Ala Ala Gly Glu Glu Trp Trp Ser Arg Gln Gly Leu Arg 1365 1370 1375Phe Met Ala Pro Glu Ala Ala Leu Asp Ala Leu Arg Gln Ala Val Asp 1380 1385 1390Arg Ala Glu Ser Thr Leu Val Val Ala Asp Ile Asp Trp Lys Thr Phe 1395 1400 1405Ala Pro Leu Phe Thr Ser Ala Arg Ser Arg Pro Leu Ile Thr Asp Ile 1410 1415 1420Pro Glu Ala Arg Pro Glu Pro Arg Pro Glu Gly Ala Asp Gln Pro Thr1425 1430 1435 1440Gln Gly Leu Val Ala Lys Leu Ala Val Leu Ser Ala Asp Glu Arg Arg 1445 1450 1455Arg Ala Leu Leu Ala Glu Val Arg Ala Gln Ala Ala Val Val Leu Gly 1460 1465 1470His Pro Gly Ala Asp Ala Val Pro Val Asp Arg Pro Phe Arg Glu Leu 1475 1480 1485Gly Phe Asp Ser Leu Ser Ala Val Lys Leu Arg Asn Arg Ile Val Ala 1490 1495 1500Ala Thr Gly Leu Glu Leu Pro Ala Thr Leu

Val Phe Asp His Pro Thr1505 1510 1515 1520Ser Thr Ala Leu Ala Ala Tyr Leu Gly Ala Arg Leu Gly Ile Asp Gly 1525 1530 1535Ala Pro Ala Gly Ser Thr Leu Leu Glu Asp Leu Ala Arg Leu Glu Ser 1540 1545 1550Thr Val Ala Thr Leu Thr Ala Ala Pro Leu Ala Glu Thr Val Pro Asp 1555 1560 1565Ala Arg Asp Arg Ala Ala Leu Thr Thr Arg Leu Arg Ala Leu Leu Glu 1570 1575 1580Arg Trp Asp Gln Ala Asp Gly Glu Asp Gln Ala Ala Ala Arg Glu Glu1585 1590 1595 1600Leu Asp Asp Leu Ser Asp Asp Asp Leu Phe Asp Phe Ile Asp Ala Lys 1605 1610 1615Phe Gly Arg Ser 162062130PRTStreptomyces parvulus Tu4055 6Met Gly Asp Glu Gln Lys Leu Arg Thr Tyr Leu Arg Arg Val Thr Ala1 5 10 15Asp Leu Ala Asp Val Thr Glu Arg Leu Gln Arg Ala Glu Asp Lys Asn 20 25 30Ala Glu Pro Ile Ala Ile Val Gly Met Gly Cys Arg Tyr Pro Gly Gly 35 40 45Val Arg Ser Pro Glu Glu Phe Trp Asn Leu Leu Asp Glu Gly Val Asp 50 55 60Ala Val Ala Gly Phe Pro Glu Asp Arg Gly Trp Asp Leu Glu Asn Leu65 70 75 80Tyr Asp Pro Asp Pro Asp Glu Pro Gly Lys Cys Tyr Ala Arg Glu Gly 85 90 95Gly Phe Leu Tyr Asp Ala Gly Glu Phe Asp Ala Ala Phe Phe Gly Ile 100 105 110Ser Pro Arg Glu Ala Leu Ser Met Asp Pro Gln Gln Arg Leu Leu Leu 115 120 125Glu Cys Ser Trp Ser Ala Leu Glu Arg Ala Gly Ile Asp Pro Gly Ser 130 135 140Leu Arg Gly Lys Asp Val Gly Val Tyr Val Gly Ala Trp Asn Ser Asn145 150 155 160Tyr Gly Arg Gly Gly Gly Ala Glu Ser Ser Glu Gly His Leu Leu Thr 165 170 175Gly Asn Ala Ser Ser Val Val Ser Gly Arg Val Ala Tyr Val Leu Gly 180 185 190Leu Glu Gly Pro Ala Val Thr Ile Asp Thr Ala Cys Ser Ser Ser Leu 195 200 205Val Gly Leu His Leu Ala Ala Gln Ala Leu Arg Ser Gly Glu Cys Gly 210 215 220Leu Ala Leu Ala Gly Gly Val Thr Val Met Ser Thr Pro Leu Ser Leu225 230 235 240Val Ser Phe Ser Arg Gln Arg Gly Leu Ala Gln Asp Gly Arg Ser Lys 245 250 255Ala Phe Ser Ala Asp Ala Asp Gly Met Gly Met Ala Glu Gly Val Gly 260 265 270Val Leu Val Leu Glu Arg Leu Ser Glu Ala Arg Arg Asn Gly His Glu 275 280 285Val Leu Ala Val Leu Arg Ser Ser Ala Val Asn Gln Asp Gly Ala Ser 290 295 300Asn Gly Leu Ser Ala Pro Asn Gly Pro Ala Gln Gln Arg Val Ile Gln305 310 315 320Ser Ala Leu Thr Val Gly Arg Leu Ala Pro Ser Asp Ile Asp Val Val 325 330 335Glu Ala His Gly Thr Gly Thr Ala Leu Gly Asp Pro Ile Glu Ala Gln 340 345 350Ala Leu Leu Ala Thr Tyr Gly Arg Gly Arg Asp Ala Asp Arg Pro Leu 355 360 365Trp Leu Gly Ser Val Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ala 370 375 380Gly Val Ala Gly Val Ile Lys Met Val Leu Ala Leu Arg Lys Gly Val385 390 395 400Leu Pro Arg Thr Leu His Val Asp Glu Pro Thr Gly Glu Val Asp Trp 405 410 415Asp Ser Gly Ala Val Arg Leu Leu Thr Glu Ala Arg Glu Trp Pro Ser 420 425 430Gly Glu Gly Arg Val Arg Arg Ala Gly Val Ser Ser Phe Gly Ile Ser 435 440 445Gly Thr Asn Ala His Val Ile Val Glu Glu Ala Pro Glu Glu Glu Pro 450 455 460Arg Pro Glu Ala Pro Ser Val Asp Val Val Pro Trp Val Leu Ser Ala465 470 475 480Arg Ser Ala Glu Ala Leu Arg Glu Gln Ala Ala Arg Leu Ala Ser Val 485 490 495Ala Gly Gly Leu Asn Val Val Asp Val Gly Trp Ser Leu Ala Ser Thr 500 505 510Arg Ala Ala Phe Glu His Arg Ala Val Val Val Gly Arg Asp Ser Glu 515 520 525Glu Leu Val Ser Gly Leu Ser Ser Val Ser Gly Val Glu Val Gly Val 530 535 540Gly Val Gly Ala Gly Gly Gly Val Val Leu Val Phe Ala Gly Gln Gly545 550 555 560Cys Gln Trp Val Gly Met Gly Arg Glu Leu Leu Gly Ser Ser Leu Val 565 570 575Phe Ala Glu Ser Met Arg Glu Cys Ala Ala Ala Leu Ser Pro Phe Val 580 585 590Asp Phe Ser Val Val Asp Val Leu Gly Ser Ala Gly Glu Leu Gly Arg 595 600 605Val Glu Val Val Gln Pro Ala Leu Trp Ala Val Met Val Ser Leu Ala 610 615 620Arg Val Trp Arg Ser Trp Gly Val Pro Val Ala Ala Val Val Gly His625 630 635 640Ser Gln Gly Glu Ile Ala Ala Ala Thr Val Ala Gly Ala Leu Ser Val 645 650 655Gly Asp Ala Ala Arg Val Val Ala Leu Arg Ser Arg Leu Ile Ala Glu 660 665 670Arg Leu Ser Gly Leu Gly Gly Met Val Ser Val Ala Leu Ser Arg Glu 675 680 685Arg Val Val Ser Leu Ile Ala Gly Val Pro Gly Val Ser Val Ala Ala 690 695 700Val Asn Gly Ser Ser Ser Thr Val Val Ser Gly Glu Ala Ala Gly Leu705 710 715 720Glu Arg Val Leu Ala Ala Cys Val Ser Ser Gly Val Arg Ala Arg Arg 725 730 735Ile Asp Val Asp Tyr Ala Ser His Ser Val Gln Val Glu Leu Ile Arg 740 745 750Glu Glu Leu Leu Gly Val Leu Asp Gly Ile Val Pro Arg Ser Gly Glu 755 760 765Ile Pro Phe Val Ser Thr Val Thr Gly Glu Arg Ile Asp Thr Val Glu 770 775 780Leu Gly Ala Glu Tyr Trp Tyr Arg Asn Leu Arg Gln Thr Val Glu Phe785 790 795 800Gln Ala Ser Val Gln Thr Leu Leu Ala Gln Gly His Gln Val Phe Leu 805 810 815Glu Ser Ser Pro His Pro Val Leu Thr Val Gly Ile Glu Glu Thr Val 820 825 830His Glu Ser Ala Ala Gln Ala Val Val Leu Gly Ser Leu Arg Arg Asp 835 840 845Glu Gly Ala Leu Thr Arg Leu Val Thr Ser Ala Gly Glu Ala Trp Ala 850 855 860Arg Gly Val Pro Val Asp Trp Ala Gly Met Leu Ala Gly Gly Arg Arg865 870 875 880Val Glu Leu Pro Thr Tyr Pro Phe Leu Arg Glu Arg Leu Trp Leu Glu 885 890 895Pro Ser Arg Ser Arg Thr Gly Asn Leu Asn Met Ala Gly Leu Val Glu 900 905 910Ala Gly His Glu Ile Leu Pro Ala Ala Val Glu Leu Pro Gly Glu Gln 915 920 925Trp Val Trp Thr Gly Glu Leu Ser Leu Ser Ala Tyr Pro Trp Leu Ala 930 935 940Asp His Gln Val Leu Gly Gln Thr Leu Val Pro Gly Val Ala Trp Val945 950 955 960Glu Leu Ala Leu His Ala Gly His Gln Leu Gly Phe Gly Ser Val Glu 965 970 975Glu Leu Thr Leu Gln Ala Pro Leu Val Leu Gly Glu Ser Asp Ala Val 980 985 990Gln Val Arg Val Val Val Ser Asp Leu Gly Glu Ser Asp Arg Arg Ala 995 1000 1005Val Ser Val His Ser Arg Gly Asp Asp Gln Thr Trp Val Thr His Ala 1010 1015 1020Glu Gly Phe Leu Thr Ala Lys Gly Ala Gln Pro Glu Thr Met Ala Val1025 1030 1035 1040Trp Pro Pro Ser Gly Ala Glu Pro Val Glu Ala Asp Gly Phe Tyr Glu 1045 1050 1055Arg Leu Ala Asp Ala Gly Tyr His Tyr Gly Pro Val Phe Gln Gly Val 1060 1065 1070Ser Lys Val Trp Arg Ala Gly Glu Glu Ile Tyr Ala Glu Val Gly Leu 1075 1080 1085Leu Asp Asp Ala Asp Val Asp Gly Phe Gly Ile His Pro Ala Leu Leu 1090 1095 1100Asp Ala Ala Leu Gln Thr Ala Tyr Val Ala Gln Arg Gly Pro Ala Glu1105 1110 1115 1120Thr Lys Leu Pro Phe Ala Phe Gly Asp Val Gln Leu Phe Ala Thr Gly 1125 1130 1135Ala Arg Ser Leu Arg Val Arg Val Ser Pro Ala Ala Gln Gln Gly Met 1140 1145 1150Ala Trp Glu Ala Trp Asp Pro Thr Gly Leu Pro Val Phe Ser Leu Gly 1155 1160 1165Tyr Leu Ala Thr Arg Pro Val Asp Arg Gly Gln Leu Thr Val Lys Arg 1170 1175 1180Pro Glu Ser Leu Phe Lys Val Ala Trp Asp Glu Thr Val Pro Val Val1185 1190 1195 1200Gly Asn Ala Thr Ala Ala His Gly Val Val Leu Gly Asp Asp Pro Phe 1205 1210 1215Ala Leu Gly Ala Ala Leu Arg Ala Ala Gly Trp Glu Val Gly Ala Ala 1220 1225 1230Pro Glu Pro Ala Ser Ala Asp Thr Ala Ala Glu Val Leu Leu Leu Pro 1235 1240 1245Cys Thr Ala Pro Gly Glu Pro Asp Ala Asp Leu Pro Thr Ala Val Arg 1250 1255 1260Ala Val Thr Ala Arg Val Leu Gly Val Leu Gln Glu Trp Leu Ala Asp1265 1270 1275 1280Glu Arg Leu Ala Gly Thr Arg Leu Ala Val Val Thr Arg Asn Ala Leu 1285 1290 1295Pro Gly Asp Leu Leu His Ser Pro Val Trp Gly Leu Val Arg Ser Ala 1300 1305 1310Gln Thr Glu Asn Pro Gly Arg Ile Thr Leu Val Asp Leu Asp Asp His 1315 1320 1325Pro Asp Ser Ala Ala Val Leu Ala Glu Ala Val Gln Ser Asp Glu Pro 1330 1335 1340Arg Ile Met Val Arg Glu Gly Arg Pro Thr Ala Ala Arg Leu Val Arg1345 1350 1355 1360Ala Thr Ala Pro Glu Leu Val Pro Pro Ala Gly Ala Asp Ala Trp Arg 1365 1370 1375Leu Glu Ile Thr Glu Pro Gly Thr Phe Asp Asn Leu Thr Leu Gly Val 1380 1385 1390Tyr Pro His Ala Glu Lys Thr Leu Ala Asp Asn Glu Val Arg Val Ala 1395 1400 1405Val His Ala Gly Gly Leu Asn Phe His Asp Val Val Ala Ala Leu Gly 1410 1415 1420Met Val Glu Asp Asp Leu Thr Leu Gly Arg Glu Ala Ala Gly Val Val1425 1430 1435 1440Val Glu Val Gly Asp Ala Val Pro Asp Leu Thr Pro Gly Asp His Val 1445 1450 1455Met Gly Ile Leu Ser Ser Gly Phe Gly Pro Leu Ala Val Thr Asp His 1460 1465 1470Arg Tyr Leu Ala Arg Met Pro Glu Gly Trp Thr Phe Ala Gln Ala Ala 1475 1480 1485Ser Val Pro Ala Ala Phe Leu Thr Ala Tyr Tyr Gly Leu Cys Asp Leu 1490 1495 1500Gly Gly Ile Arg Ala Gly Asp Arg Val Leu Ile His Ala Ala Ala Gly1505 1510 1515 1520Gly Val Gly Met Ala Ala Val Gln Ile Ala Arg His Leu Gly Ala Glu 1525 1530 1535Val Phe Gly Thr Ala Ser Pro Arg Lys Trp Gly Ala Leu Arg Ala Leu 1540 1545 1550Gly Leu Asp Asp Ala His Leu Ser Ser Ser Arg Thr Leu Asp Phe Glu 1555 1560 1565Gln Glu Phe Leu Asp Ala Thr Asp Gly Arg Gly Val Asp Leu Val Leu 1570 1575 1580Asn Ser Leu Ala Arg Glu Phe Val Asp Ala Ser Leu Arg Leu Met Pro1585 1590 1595 1600Gly Gly Gly Arg Phe Val Asp Met Gly Lys Thr Asp Ile Arg Arg Pro 1605 1610 1615Glu Gln Val Ala Glu Asp His Gly Gly Val Ala Tyr Gln Ala Phe Asp 1620 1625 1630Leu Val Glu Ala Gly Pro Gln Arg Thr Gly Glu Met Leu Ala Glu Ile 1635 1640 1645Val Arg Leu Phe Gln Ala Gly Ala Phe Arg Pro Leu Pro Ile Thr Gln 1650 1655 1660Trp Asp Val Arg Arg Ala Pro Glu Ala Phe Arg His Ile Ser Gln Ala1665 1670 1675 1680Lys His Ile Gly Lys Ile Val Leu Thr Val Pro Arg Pro Ile Asp Thr 1685 1690 1695Asp Gly Thr Val Met Val Thr Gly Ala Thr Gly Thr Leu Gly Gly Phe 1700 1705 1710Val Ala Arg His Leu Val Thr His His Gly Ile Arg Arg Leu Leu Leu 1715 1720 1725Val Ser Arg Ser Ala Glu Arg Thr Asp Leu Val Arg Glu Leu Thr Glu 1730 1735 1740Leu Gly Ala Asp Val Thr Trp Ala Ser Cys Asp Leu Ala Asp Ala Thr1745 1750 1755 1760Ala Val Glu Glu Thr Val Arg Ser Val Asp Glu Arg His Pro Leu Val 1765 1770 1775Ala Val Val His Ser Ala Gly Val Leu Asp Asp Gly Val Ile Asp Lys 1780 1785 1790Gln Ser Pro Glu Arg Leu Asp Thr Val Met Arg Pro Lys Val Asp Ala 1795 1800 1805Ala Trp Asn Leu His Arg Leu Leu Asp Asn Ala Pro Leu Ala Asp Phe 1810 1815 1820Val Leu Phe Ser Ser Ala Ser Gly Val Leu Gly Gly Ala Gly Gln Ser1825 1830 1835 1840Asn Tyr Ala Ala Ala Asn Ala Phe Leu Asp Ala Leu Ala Glu His Arg 1845 1850 1855Arg Ala Gln Gly Leu Ala Gly Gln Ala Leu Ala Trp Gly Leu Trp Ser 1860 1865 1870Asp Arg Ser Thr Met Thr Gly Gln Leu Gly Ser Thr Glu Leu Ala Arg 1875 1880 1885Ile Ala Arg Asn Gly Val Ala Glu Met Ser Glu Thr Glu Gly Leu Ala 1890 1895 1900Leu Phe Asp Ala Ala Arg Asp Thr Ala Glu Ala Val Leu Leu Pro Met1905 1910 1915 1920His Leu Asp Val Ala Arg Leu Arg Ser Arg Asn Gly Glu Val Pro Ala 1925 1930 1935Val Phe Arg Arg Leu Ile His Ala Thr Ala Arg Arg Thr Ala Ser Thr 1940 1945 1950Ala Val Arg Ser Ala Gly Leu Glu Gln Gln Leu Ala Ser Leu Ser Gly 1955 1960 1965Pro Glu Arg Thr Glu Leu Leu Leu Gly Leu Val Arg Asp His Ala Ala 1970 1975 1980Ala Val Leu Gly His Gly Thr Ser Asp Ala Val Ser Pro Asp Arg Pro1985 1990 1995 2000Phe Arg Asp Leu Gly Phe Asp Ser Leu Thr Ala Val Glu Leu Arg Asn 2005 2010 2015Arg Phe Ala Ala Leu Thr Gly Leu Arg Leu Pro Ala Thr Leu Val Phe 2020 2025 2030Asp His Pro Ser Pro Thr Ala Leu Ala Gly His Leu Ala Gly Leu Leu 2035 2040 2045Gly Ala Ala Thr Pro Ser Ala Ala Glu Pro Val Leu Ala Ala Val Gly 2050 2055 2060Arg Leu Arg Ala Asp Leu Arg Ser Leu Thr Pro Asp Ala Glu Gly Ala2065 2070 2075 2080Glu Asp Val Thr Ile Gln Leu Glu Ala Leu Leu Ala Glu Trp Arg Glu 2085 2090 2095Ala Ala Glu Lys Arg Ala Pro Glu Ala Val Gly Asp Glu Asp Leu Ser 2100 2105 2110Thr Ala Thr Asp Asp Glu Ile Phe Ala Leu Val Asp Ser Glu Leu Gly 2115 2120 2125Glu Ala 213071742PRTStreptomyces parvulus Tu4055 7Met Thr Ala Glu Ala Ser Gln Asp Lys Leu Arg Asp Tyr Leu Arg Lys1 5 10 15Thr Leu Ala Asp Leu Arg Thr Thr Lys Gln Arg Leu Arg Asp Thr Glu 20 25 30Arg Arg Ala Thr Glu Pro Val Ala Ile Val Gly Met Ser Cys Arg Leu 35 40 45Pro Gly Asp Val Arg Thr Pro Glu Arg Phe Trp Glu Leu Leu Asp Thr 50 55 60Gly Thr Asp Ala Leu Thr Pro Leu Pro Thr Asp Arg Gly Trp Asn Leu65 70 75 80Asp Thr Ala Phe Asp Asp Glu Arg Pro Tyr Arg Arg Glu Gly Gly Phe 85 90 95Leu Tyr Asp Ala Gly Arg Phe Asp Ala Glu Phe Phe Gly Ile Ser Pro 100 105 110Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Ser 115 120 125Ser Trp Glu Ala Ile Glu His Ala Arg Ile Asp Pro Arg Ser Leu His 130 135 140Gly Ser Arg Thr Gly Val Trp Phe Gly Thr Ile Gly Gln Asp Tyr Phe145 150 155 160Ser Leu Phe Ala Ala Ser Gly Gly Glu His Ala Asn Tyr Leu Ala Thr 165 170 175Ala Cys Ser Ala Ser Val Met Ser Gly Arg Val Ser Tyr Val Leu Gly 180 185 190Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys Ser Ser Ser Leu 195 200 205Val Ala Leu His Ser Ala Val Gln Ala

Leu Arg Ser Gly Glu Cys Glu 210 215 220Leu Ala Leu Ala Gly Gly Ala Thr Val Met Ala Thr Pro Thr Val Phe225 230 235 240Thr Ala Phe Ser His Gln Arg Gly Leu Ala Gly Asp Gly Arg Cys Lys 245 250 255Ala Phe Ala Ala Gly Ala Asp Gly Ala Gly Phe Ala Glu Gly Val Gly 260 265 270Val Leu Val Leu Glu Arg Leu Ser Val Ala Arg Arg Asn Gly His Arg 275 280 285Val Leu Ala Val Val Arg Gly Ser Ala Val Asn Gln Asp Gly Ala Ser 290 295 300Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln Arg Val Ile Arg305 310 315 320Ala Ala Leu Ala Asn Ala Arg Leu Ala Pro Glu Asp Val Asp Ala Val 325 330 335Glu Gly His Gly Thr Gly Thr Ser Leu Gly Asp Pro Ile Glu Ala Gln 340 345 350Ala Leu Leu Ala Thr Tyr Gly Arg Gly Arg Asp Ala Glu Arg Pro Leu 355 360 365Trp Leu Gly Ser Val Lys Ser Asn Ile Gly His Ala Gln Ala Ala Ala 370 375 380Gly Val Ala Gly Val Ile Lys Met Val Leu Ala Met Glu Lys Gly Arg385 390 395 400Leu Pro Arg Thr Leu His Val Asp Glu Pro Ser Gly Glu Val Asp Trp 405 410 415Asp Ser Gly Ala Val Arg Leu Leu Thr Glu Ala Arg Asp Trp Pro Ser 420 425 430Gly Glu Gly Arg Val Arg Arg Ala Gly Val Ser Ser Phe Gly Ile Ser 435 440 445Gly Thr Asn Ala His Val Ile Ile Glu Glu Pro Gln Glu Glu Glu Ala 450 455 460Ala Pro Asp Ser Ser Ala Ser Gly Ala Val Pro Trp Val Leu Ser Ala465 470 475 480Arg Ser Ala Glu Ala Leu Gln Ala Leu Ala Ser Gln Leu Ala Asp His 485 490 495Ser Ala Lys Ser Ser Pro Val Asp Val Gly Trp Ser Leu Val Ser Thr 500 505 510Arg Ala Ala Phe Glu His Arg Ala Val Val Val Gly Arg Gly Arg Asp 515 520 525Glu Leu Val Arg Gly Leu Ser Glu Val Ala Gln Gly Arg Gly Val Arg 530 535 540Gly Val Ala Ser Ser Ala Ser Gly Gly Leu Ala Phe Val Phe Ala Gly545 550 555 560Gln Gly Ser Gln Arg Leu Gly Met Gly Arg Gly Leu Tyr Glu Arg Phe 565 570 575Pro Val Phe Ala Glu Ala Phe Asp Glu Val Cys Gly Arg Val Gly Pro 580 585 590Gly Val Arg Glu Val Val Phe Gly Ser Asp Ala Gly Glu Leu Asp Arg 595 600 605Thr Val Trp Ala Gln Ala Gly Leu Phe Ala Leu Glu Val Ala Leu Phe 610 615 620Arg Leu Leu Glu Ser Trp Gly Val Arg Pro Gly Cys Leu Ile Gly His625 630 635 640Ser Val Gly Glu Leu Ser Ala Ala Cys Val Ala Gly Leu Trp Ser Leu 645 650 655Glu Asp Ala Cys Arg Val Val Ala Ala Arg Ala Arg Leu Met Gln Ala 660 665 670Leu Pro Ala Gly Gly Val Met Val Ala Val Arg Ala Glu Ala Gly Glu 675 680 685Leu Ala Gly Phe Leu Gly Glu Asp Val Val Ile Ala Ser Val Asn Ala 690 695 700Pro Gly Gln Val Val Ile Ala Gly Pro Glu Gly Gly Val Glu Arg Val705 710 715 720Val Ala Ala Cys Gly Ala Arg Ser Arg Arg Leu Ala Val Ser His Ala 725 730 735Phe His Ser Pro Leu Val Glu Pro Met Leu Gly Glu Phe Arg Arg Val 740 745 750Val Glu Ser Val Ala Phe Gly Val Pro Ser Leu Arg Val Val Ser Asn 755 760 765Val Thr Gly Ala Trp Val Asp Pro Glu Glu Trp Gly Thr Pro Glu Tyr 770 775 780Trp Val Arg Gln Val Arg Glu Pro Val Arg Phe Ala Asp Gly Val Ala785 790 795 800Thr Leu Leu Asp Ala Gly Val Arg Thr Phe Val Glu Leu Gly Pro Ala 805 810 815Gly Thr Leu Thr Ser Met Val Ser His Cys Ala Asp Ala Thr Ala Thr 820 825 830Ser Val Thr Ala Val Pro Thr Leu Arg Pro Asp His Asp Glu Ser Arg 835 840 845Thr Val Leu Ser Ala Ala Ala Ser Leu Tyr Val Gln Gly His Pro Val 850 855 860Asp Trp Ala Pro Leu Phe Pro Arg Ala Arg Thr Val Asp Leu Pro Thr865 870 875 880Tyr Pro Phe Gln His Gln His Tyr Trp Met Glu Ser Ala Ala Arg Pro 885 890 895Thr Val Glu Asp Thr Pro Arg Glu Pro Leu Asp Gly Trp Thr His Arg 900 905 910Ile Asp Trp Val Pro Leu Val Asp Glu Glu Pro Ala Pro Val Leu Ala 915 920 925Gly Thr Trp Leu Leu Val Arg Pro Glu Glu Gly Pro Arg Pro Leu Ala 930 935 940Asp Ala Val Ala Asp Ala Leu Thr Arg His Gly Ala Ser Val Val Glu945 950 955 960Ala Ala Arg Val Pro His Gln Ser Asp Thr Glu Leu Thr Gly Val Val 965 970 975Ser Leu Leu Gly Pro Gly Ala Asp Gly Asp Gly Gly Leu Asp Ala Thr 980 985 990Leu Arg Leu Val Gln Asp Leu Ala Thr Ala Gly Ser Thr Ala Pro Leu 995 1000 1005Trp Ile Val Thr Ser Gly Ala Val Ala Val Gly Thr Ser Asp Thr Val 1010 1015 1020Pro Asn Pro Glu Gln Ala Thr Leu Trp Gly Leu Ala Arg Ala Ala Ala1025 1030 1035 1040Thr Glu Trp Pro Gly Leu Gly Ala Ala Arg Ile Asp Leu Pro Ala Asp 1045 1050 1055Leu Thr Glu Gln Val Gly Arg Arg Leu Cys Ala Arg Leu Leu Asp Arg 1060 1065 1070Ser Glu Gln Glu Thr Ala Val Arg Gln Ala Gly Val Phe Ala Arg Arg 1075 1080 1085Leu Val Arg Ala Arg Thr Ser Asp Gly Arg Trp Thr Pro Arg Gly Thr 1090 1095 1100Val Leu Val Thr Gly Gly Thr Gly Ala Leu Ala Gly His Val Ala Arg1105 1110 1115 1120Trp Leu Ala Glu Glu Gly Ala Glu His Ile Val Leu Ala Gly Arg Arg 1125 1130 1135Gly Pro Asp Gly Gln Gly Ala Glu Ala Leu Arg Ala Asp Leu Val Ala 1140 1145 1150Ala Gly Val Lys Ala Thr Ile Val Arg Cys Asp Val Ala Asp Arg Asp 1155 1160 1165Ala Val Arg Leu Leu Leu Asp Ala His Arg Pro Ser Ala Ile Val His 1170 1175 1180Thr Ala Gly Val Val Asp Asp Gly Leu Leu Thr Ser Leu Thr Pro Ala1185 1190 1195 1200Gln Val Glu Arg Val Leu Arg Pro Lys Leu Leu Gly Ala Arg Asn Leu 1205 1210 1215His Glu Leu Thr Arg Asp Arg Glu Leu Asp Ala Phe Val Leu Phe Ser 1220 1225 1230Ser Leu Ala Gly Val Leu Gly Gly Ala Gly Gln Ala Asn Tyr Ala Ala 1235 1240 1245Ala Asn Ala Tyr Leu Asp Ala Leu Ala Ala His Arg Thr Ala His Gly 1250 1255 1260Leu Pro Ala Ala Ser Leu Ala Trp Gly Pro Trp Glu Gly Asp Gly Met1265 1270 1275 1280Ala Ala Ala Gln Glu Ala Ala Asp Arg Leu Arg Arg Ser Gly Leu Thr 1285 1290 1295Pro Leu Pro Pro Glu Gln Ala Val Arg Ala Leu Gly Arg Gly His Gly 1300 1305 1310Pro Leu Val Val Ala Asp Ala Asp Trp Ala Arg Leu Ala Ala Gly Ser 1315 1320 1325Thr Gln Arg Leu Leu Asp Glu Leu Pro Glu Val Arg Ala Val Arg Pro 1330 1335 1340Ala Glu Pro Ala Val Gly Gln Arg Pro Asp Leu Pro Ala Arg Leu Ala1345 1350 1355 1360Gly Arg Pro Ala Glu Glu Gln Ser Ala Val Leu Leu Glu Ala Val Arg 1365 1370 1375Glu Glu Ile Ala Ala Val Leu Arg Tyr Ala Asp Pro Ala Arg Ile Gly 1380 1385 1390Ala Asp His Glu Phe Leu Ala Leu Gly Phe Asp Ser Leu Thr Ser Ile 1395 1400 1405Glu Leu Arg Asn Arg Leu Ala Thr Arg Ile Gly Leu Thr Leu Pro Ala 1410 1415 1420Thr Leu Thr Leu Glu Gln Arg Thr Pro Ala Gly Leu Ala Ala His Leu1425 1430 1435 1440Arg Glu Arg Ile Ala Asp Arg Pro Val Gly Ser Gly Ala Val Pro Val 1445 1450 1455Pro Gly Ser Ala Asp Val Pro Glu Ala Gly Gly Gly Ser Gly Leu Gly 1460 1465 1470Glu Leu Trp Gln Glu Ala Asp Arg His Gly Arg Arg Leu Glu Phe Ile 1475 1480 1485Asp Val Leu Thr Ala Ala Ala Ala Phe Arg Pro Ala Tyr Arg Glu Pro 1490 1495 1500Ala Glu Leu Glu Leu Pro Pro Leu Arg Leu Thr Ser Gly Gly Asp Glu1505 1510 1515 1520Pro Pro Leu Phe Cys Ile Pro Ser His Leu Gly Lys Ala Asp Pro His 1525 1530 1535Lys Phe Leu Arg Phe Ala Ala Ala Leu Arg Gly Arg Arg Asp Val Phe 1540 1545 1550Val Leu Arg Gln Pro Gly Phe Val Pro Gly Gln Pro Leu Pro Ala Gly 1555 1560 1565Leu Asp Val Leu Leu Asp Thr His Ala Arg Ala Met Ala Gly His Asp 1570 1575 1580Arg Pro Val Leu Leu Gly Tyr Ser Ala Gly Gly Leu Ala Ala Gln Ala1585 1590 1595 1600Leu Ala Ala Arg Leu Ala Glu Leu Gly Arg Pro Pro Ala Ala Val Val 1605 1610 1615Leu Val Asp Thr Tyr Ala Pro Asp Glu Thr Glu Val Met Ala Arg Ile 1620 1625 1630Gln Gly Ala Met Glu Gln Gly Gln Arg Asp Arg Asp Gly Arg Thr Gly 1635 1640 1645Ala Ala Phe Gly Glu Ala Trp Leu Thr Ala Met Gly His Tyr Phe Gly 1650 1655 1660Phe Asp Trp Thr Pro Cys Pro Val Asp Val Pro Val Leu His Val Arg1665 1670 1675 1680Ala Gly Asp Pro Met Thr Gly Met Pro Val Glu Gly Arg Trp Gln Ala 1685 1690 1695Arg Trp Asn Leu Pro His Thr Ala Val Asp Val Pro Gly Asp His Phe 1700 1705 1710Thr Met Met Glu Asp His Ala Pro Arg Thr Ala Asp Thr Val His Asp 1715 1720 1725Trp Leu Gly Thr Ala Val Arg Arg Pro Glu Arg Thr Arg Asp 1730 1735 17408264PRTStreptomyces parvulus Tu4055 8Met Thr Gly Thr Asn Thr His Ser Asp Val Trp Ile Arg Gln Tyr Arg1 5 10 15Pro Ala His Pro Thr Ala Pro Gln Leu Ile Cys Leu Pro His Ala Gly 20 25 30Gly Ser Ala Thr Phe Tyr His Pro Val Ala Ala Ala Leu Ala Pro Arg 35 40 45Cys Asp Val Leu Ala Val Gln Tyr Pro Gly Arg Gln Asp Arg Arg Ala 50 55 60Glu Lys Pro Leu Glu Asp Ile Asp Glu Leu Ala Asn Gln Leu Phe Pro65 70 75 80Val Leu Arg Ala Arg Val His Gln Pro Val Ala Leu Phe Gly His Ser 85 90 95Met Gly Ala Thr Leu Ala Phe Glu Leu Ala Arg Arg Phe Glu Ser Ala 100 105 110Gly Ile Ser Leu Glu Ala Leu Leu Val Ser Ala Arg Pro Ala Pro Ser 115 120 125Arg Gln Arg Thr Gly Gly Thr Val His Leu Leu Ser Asp Glu Glu Leu 130 135 140Val Ala Glu Leu Arg Thr Leu Asp Gly Thr Ala Glu Gln Val Phe His145 150 155 160Asp Glu Glu Leu Val Arg Met Ala Leu Pro Ala Ile Arg Gly Asp Tyr 165 170 175Arg Ala Ala Glu Thr Tyr Arg Tyr Arg Pro Gly Pro Lys Leu Arg Cys 180 185 190Pro Ile His Ala Leu Thr Gly Asp Asp Asp Pro Met Val Thr Pro Val 195 200 205Glu Ala Arg Ala Trp Ser Glu His Thr Asp Gly Pro Phe Thr Leu Asp 210 215 220Thr Phe Ala Gly Gly His Phe Tyr Leu Leu Glu His Arg Asp Ala Ile225 230 235 240Leu Gly Ile Ile Ala Glu His Leu Arg Thr Cys Ser Arg Ala Pro Gly 245 250 255Asp Arg Ser Gly Leu Thr Arg Glu 2609265PRTStreptomyces parvulus Tu4055 9Met Ser Leu Glu Leu Thr Asp Arg Val Met Met Val Thr Gly Ala Gly1 5 10 15Ser Gly Ile Gly Arg Ala Ala Ala Arg Leu Leu Val Gly His Gly Ala 20 25 30Arg Val Val Leu Val Gly Arg Thr Glu Ser Ala Leu Thr Glu Thr Thr 35 40 45Ala Gly Leu Pro Ser Ser His His Leu Val Val Pro Cys Asp Val Gly 50 55 60Asp Asp Lys Gln Val Ala Asp Cys Val Ala Arg Ala Val Ser Arg Phe65 70 75 80Gly Arg Leu Asp Gly Ala Phe Asn Asn Ala Gly Thr Phe Gly Ser Phe 85 90 95Gly Pro Leu His Gln Asp Thr Ala Asp Asn Phe Asp Arg Val Ile Ala 100 105 110Thr Asn Leu Arg Gly Val Trp Ser Cys Met Arg Gly Gln Ile Glu Ala 115 120 125Met Leu Thr Ala Gly Gly Gly Ala Ile Val Asn Cys Ala Ser Val Ala 130 135 140Gly His Ile Gly His Ala Gln Ser Pro Leu Tyr Ser Ala Thr Lys His145 150 155 160Ala Val Ile Gly Leu Ser Lys Ser Val Ala Leu Gln Tyr Ala Gly Asp 165 170 175Gly Ile Arg Val Asn Val Val Ser Pro Gly Ser Thr Asp Thr Pro Met 180 185 190Leu Arg Ser Leu Tyr Ala Asp Pro Ser Ala Leu Ala Gln Arg Ala Arg 195 200 205Arg Ala Pro Leu Gly Arg Leu Gly Lys Cys Glu Glu Val Ala Asn Ala 210 215 220Val Val Trp Leu Leu Ser Pro Leu Ala Ala Tyr Val Thr Gly Gln Thr225 230 235 240Leu Gly Val Asp Gly Gly Val Thr Ala Gly Ser Ala Ile Pro Arg Thr 245 250 255Asn Ala Thr Pro Glu Gly Gln His Arg 260 26510250PRTStreptomyces parvulus Tu4055 10Met Thr Ala Arg His Asp Val Ala Leu Val Thr Gly Ala Gly Ser Gly1 5 10 15Ile Cys Ala Glu Val Ala Arg Gly Leu Ala Ala Arg Gly Leu Arg Val 20 25 30Val Leu Leu Asp Lys Asp Ala Glu Ala Val His Arg Val Ala Asp Gly 35 40 45Leu Gly Asp Arg Leu Ala Arg Asp Pro Leu Val Ala Asp Val Thr Asp 50 55 60Pro His Ala Leu Ala Ser Ala Val Asp Ser Leu Ala Pro Gln His Arg65 70 75 80Pro Gly Val Leu Val Asn Gly Val Gly Gly Asp Thr Arg Ala Arg Ser 85 90 95Val Thr Glu Leu Thr Glu Ala Asp Leu Gln Glu Ala Val Thr His Asn 100 105 110Leu Ala Ser Val Phe Thr Met Thr Arg Leu Cys Val Pro Ala Met Val 115 120 125Ala Ala Gly Trp Gly Arg Val Val Asn Leu Ala Ser Val Ala Gly Arg 130 135 140Thr Tyr Thr Arg Phe Ser Asn Ala Ala Tyr Val Ala Ala Lys Ala Gly145 150 155 160Val Ile Gly Phe Thr Lys Gln Cys Ala Tyr Glu Leu Ala Pro His Gly 165 170 175Val Thr Val Asn Ala Val Ala His Gly Val Ile Gly Thr Glu Arg Ile 180 185 190Arg Arg Ala Trp Glu Asp Lys Pro Pro Gln Trp Thr Ala Asp Arg Val 195 200 205Ser His Ile Pro Ala Gly Arg Phe Gly Ser Val Ala Glu Ala Ala Gly 210 215 220Met Val Cys His Leu Cys Gly Glu Asp Ala Gly Tyr Thr Thr Gly Thr225 230 235 240Val Val Asp Val Asn Gly Gly Leu His Ile 245 25011390PRTStreptomyces parvulus Tu4055 11Met Ile Arg Arg Val Arg Leu His Thr Ala Val Val Pro Met Ala Ala1 5 10 15Ala Phe Asp His Ala Thr Arg Ser Arg Arg Ser Ala Ala Ser Leu Leu 20 25 30Val Glu Ile Glu Leu Ala Gly Thr Arg Gly Trp Gly Glu Gly Ala Pro 35 40 45Arg Asp Tyr Val Thr Gly Glu Thr Leu Asp Gly Ala Val Arg Ala Val 50 55 60Gln Ala Cys Asp Pro Gly Glu Leu Ala Glu Arg Ile Glu Trp Arg Asp65 70 75 80Phe Glu Ser Ala Val Ala Ser Ile Ala Gln Leu Pro Leu Thr Gly Leu 85 90 95Val Asp Gly Ser Ser Ala Ala Ala Ala Val Glu Ile Ala Leu Leu Asp 100 105 110Ala Val Cys Arg His Phe Ala Arg Pro Leu Ala Asp Val Leu Arg Val 115 120 125Leu Ala Pro Pro Ala Arg Ser Arg Arg Asp Gly Pro Thr Ser Val Ser 130 135 140Leu

Val Ile His Leu Ser Arg Asp Val Ala Thr Val Leu Asp Ala Leu145 150 155 160Thr Pro Arg Ala Leu Ala Ala Leu Arg His Val Lys Ile Lys Val Ala 165 170 175Asp Pro Ala Gly Ala Val Asp Arg Leu Thr Ala Ala Gln Asp Arg Leu 180 185 190Pro Ala Asp Thr Arg Val Ser Leu Asp Val Asn Gly Ala Trp Thr Ala 195 200 205Glu Glu Ala Glu Lys Val Ala Gly Glu Leu Asp Gly Val Gly Trp Val 210 215 220Glu Glu Pro Leu Pro Pro Arg Ser Trp Pro Glu Leu Gly Arg Leu Arg225 230 235 240Arg Ala Thr Gly Leu Pro Val Met Leu Asp Glu Ser Cys Thr Gly Pro 245 250 255Ala Asp Leu His Ala Ala Ala Thr Ser Gly Ala Ala Ser His Ile Asn 260 265 270Val Arg Leu Ser Lys Cys Gly Gly Phe Leu Ala Ala Ala Arg Leu Ala 275 280 285Leu Arg Ala Asp Glu Leu Gly Val Gly Cys Gln Leu Gly Val His Val 290 295 300Ala Glu Val Gly Pro Leu Trp Ala Ala Gly Arg Thr Leu Ala Thr Ala305 310 315 320Trp Asp Leu Trp Gln Thr Val Glu Ala Gly Arg Ala Asp Glu Trp Phe 325 330 335Pro Val Pro Leu Thr Thr Pro Ala Phe Thr Val Asp Arg Ser Leu His 340 345 350Arg Val Glu Pro Leu Thr Gly Pro Gly Thr Gly Ile Glu Pro Thr Glu 355 360 365Glu Leu Leu Arg His Thr Arg Cys Ala Ala Thr Trp Glu Ser Gly Gly 370 375 380Gly Trp Arg Arg Asn Thr385 39012272PRTStreptomyces parvulus Tu4055 12Met Pro Thr Thr Ser Met Leu Thr Ala Ala Asp Gly Thr Gly Leu Thr1 5 10 15Leu His His Trp Thr Thr Pro Gly Ala Thr Ser Ala Val Phe Tyr Leu 20 25 30His Gly Ile Gln Ser His Ala Gly Trp Leu Phe Glu Thr Gly Pro Glu 35 40 45Leu Asn Ala Arg Gly Ile Asp Val Tyr Ala Leu Asp Arg Arg Gly Ser 50 55 60Gly Arg Ser Glu Gly Pro Arg Gly His Leu Pro Ser Ala Asp Leu Val65 70 75 80Leu Asp Asp Tyr Ala Arg Ala Leu Asp Ala Val Thr Ala Glu Val Gly 85 90 95Gly Ala Gly Pro Val Ala Leu Gly Gln Ser Leu Gly Gly Ser Val Leu 100 105 110Ala Ala Leu Trp Cys Thr Arg Asp Leu Pro Val Arg Arg Leu Val Leu 115 120 125Cys Ala Pro Ala Leu Gly Gln Gln Arg Ala Arg His Thr Ala Asp Thr 130 135 140Leu Ala Glu Arg Arg Ala Leu Thr Gly Ser Gly Leu Arg Pro Val Gly145 150 155 160Leu Ala Asp Gly Asp Tyr Thr Asp Leu Pro Arg Tyr Arg Glu Phe Leu 165 170 175Thr Gly Asp His Leu Met Leu Arg Glu Val Thr Ser Ala Thr Gln Ala 180 185 190Thr Leu Val His Leu Glu Asp His Tyr Ala Arg Gly Ala Pro Arg Thr 195 200 205Arg Leu Pro Val Asp Leu Ala Leu Pro Thr His Asp Pro Ile Ile Asp 210 215 220Leu Ser Ala Ala Arg Ala Met Leu Arg Arg Leu Thr Ser Ala Val His225 230 235 240Glu Glu Val Phe Ala Thr Asp Arg His Tyr Val Glu Phe Thr Ser Ala 245 250 255Arg Thr Ala Tyr Trp Asp Trp Leu Ala Thr Arg Leu Lys Glu Glu Ala 260 265 27013539PRTStreptomyces parvulus Tu4055 13Met Lys Val Phe His Ala Leu Ala Asp Ala Leu Thr Ala His Gly Val1 5 10 15Asp Thr Val Phe Gly Leu Met Gly Asn Ala Asn Leu Leu Tyr Leu Pro 20 25 30Ala Phe Ala Asp Ala Gly Gly Arg Phe Val Ala Val Ala His Glu Ala 35 40 45Gly Ala Val Ala Met Ala Asp Gly Arg Ala Arg Met Cys Gly Gly Ile 50 55 60Gly Val Ala Ser Val Thr His Gly Pro Ala Phe Thr Asn Ala Leu Thr65 70 75 80Pro Leu Val Glu Ala Ala Arg Ser His Ser Gln Val Leu Leu Ile Thr 85 90 95Gly Asp Pro Pro Pro Val Pro Thr His Phe His His Phe Asp Ile Ala 100 105 110Thr Val Ala Ala Ala Ala Gly Ala Gly Tyr Glu Arg Val His Arg Pro 115 120 125Ala Ser Leu Val Ala Asp Leu Asn Arg Ala Val Gln Arg Ile Val Ala 130 135 140Glu Arg Arg Pro Val Val Leu Asn Val Pro Ile Asp Leu Met Gln Ala145 150 155 160Glu Ala Gly Glu Gln Ala Pro Val Thr Leu Pro Val Ala Pro Gly Pro 165 170 175Leu Ala Ala Pro Glu Ala Glu Ala Leu Asp Gly Ala Leu Gly Leu Ile 180 185 190Gly Ser Ala Lys Arg Pro Leu Val Leu Ala Gly His Gly Ala Ala Val 195 200 205Ala Gly Ala Arg Glu Ala Leu Val Glu Leu Ala Asp Arg Thr Gly Ala 210 215 220Ala Leu Ala Thr Thr Val Leu Gly Lys Glu Met Phe Ala Gly His Pro225 230 235 240Arg Asp Val Gly Ile Phe Gly Ser Leu Ala His Ser Val Ala Ser Thr 245 250 255Val Ile Ala Glu Ser Asp Cys Val Ile Ala Phe Gly Ala Ser Leu Asn 260 265 270Met Trp Thr Val Leu Asn Gly Glu Leu Leu Arg Gly Lys Arg Val Val 275 280 285His Val Asp Thr Asp Pro Ala Arg Phe Gly Ser Tyr Ser Pro Val Asp 290 295 300Glu Pro Val Ala Gly Asp Ala Arg Arg Thr Ala Glu Thr Met Asn Val305 310 315 320Leu Leu Asp Gln Ala Gly Val Thr Ala Ala Asn Gly Ala Trp Ala Glu 325 330 335Arg Val Ala Gly Gln Leu Ala Gly Phe Ser Pro Gln Asp Asp Val Asp 340 345 350Asp Arg Ser Gly Ala Glu Thr Val Asp Ile Arg Thr Ala Met Ile Arg 355 360 365Leu Asp Arg Ile Leu Pro Ala Glu Arg Ser Val Val Ser Asp Ile Gly 370 375 380Arg Phe Asp Val Gly Val Trp Pro Tyr Leu Arg Val Ala Asp Pro Leu385 390 395 400His Phe Thr Val Met Gly Gly Phe Gly Ser Ile Gly Leu Gly Val Ala 405 410 415Gly Ala Ile Gly Ala Ala Thr Ala Gly Thr Gly Arg Pro Val Val Ala 420 425 430Ala Val Gly Asp Gly Gly Phe Met Met His Leu Ser Glu Phe Thr Thr 435 440 445Ala Val Arg Tyr Arg Leu Pro Leu Val Val Val Val Leu Asn Asp Gly 450 455 460Ala Tyr Gly Ala Glu His Tyr Lys Leu Arg Asn His Gly Tyr Asp Pro465 470 475 480Ala Tyr Ser Ala Phe Ala Trp Pro Asp Leu Ala Gly Leu Ala Thr Ala 485 490 495Met Gly Ala Arg Ala Leu Thr Val Arg Lys Ala Glu Glu Leu Asp Ala 500 505 510Val Gly Asp Leu Leu Ser Thr Leu Glu Gly Pro Leu Leu Val Asp Val 515 520 525Arg Leu Asp Pro Asp Val Asn Leu Val Arg Tyr 530 53514683PRTStreptomyces parvulus Tu4055 14Met Arg Gly Ala Arg Glu Asn Ser Met Thr Arg Ala Gly Pro Leu Glu1 5 10 15Gly Ile Ala Val Leu Met Ala Gly Arg Ser Thr Pro Ala Ala Leu Leu 20 25 30Gly Arg Leu Leu Ala Asp Leu Gly Ala Arg Val Val Thr Leu Cys Arg 35 40 45Ser Pro Asp His Gly Gly Pro Phe Glu Arg Trp Leu His Ser Ala Ala 50 55 60Gln Ser Ala Ser Gly Trp Asp Gln Ala Ser Arg Leu Leu Gln Thr Ala65 70 75 80Asp Val Leu Val Cys Asp Ala Glu Gly Asp Glu Arg Leu Ala Ala Leu 85 90 95Gly Leu Gly Ala Pro Glu Leu Pro His Arg Ser Pro Glu Leu Val Ala 100 105 110Val Arg Leu Ser Ala Phe Gly Leu Thr Gly Pro Leu Arg Asp Ala Pro 115 120 125Ala Thr Glu Arg Thr Leu Gln Ala Leu Ala Gly Leu Thr Ser Ala Thr 130 135 140Gly Thr Glu Gly Glu Pro Ser Val Leu Ser Val Val Gly Leu Ala Ser145 150 155 160Arg Thr Ala Ala Leu Ser Gly Leu Ile Ala Val Val Ala Gly Leu Ile 165 170 175Gly Arg Glu Arg Gly Gly Gly Gly Asp Tyr Leu Asp Ile Ala Glu Phe 180 185 190Asp Ser Leu Phe Thr Leu Thr Gly Thr Leu Leu Pro Ser Val Ala Leu 195 200 205Ala Gly Arg Pro Pro Arg Arg Thr Gly Asn Arg His Gly Met Ala Ala 210 215 220Pro Trp Asn Ser Tyr Thr Cys Gln Asp Ala Pro Val Val Ile Cys Thr225 230 235 240Met Gly Glu Pro Ile Trp His Arg Leu Thr Ala Val Leu Gly Arg Arg 245 250 255Asp Leu Pro Asp Asp Pro Arg Phe Ala Asp Thr Ala Ala Arg Val Arg 260 265 270Asn Ala Asp Glu Leu Asp Glu Ile Leu Gly Lys Trp Thr Ala Gly Gln 275 280 285Arg Ala Val Asp Val Val Thr Ala Leu Arg Ala Ala Gly Ile Pro Cys 290 295 300Ala Gln Val Ala Ala Pro Glu Glu Val Arg Asp Gly Ala Ala Ala Arg305 310 315 320Arg Arg Gly Leu Val Thr Asp Pro Ser Gly Thr Pro Gly Ser Pro Leu 325 330 335Arg Ser Leu Ile Pro Ala Val Thr Asp Gly Pro Met Pro Arg Gln Gly 340 345 350Gly Leu Trp Glu Pro Ile Ala Arg Gly Thr Pro Pro Leu Arg Gly Val 355 360 365Arg Leu Leu Glu Val Gly Ser Tyr Thr Ala Gly Pro His Ala Gly Arg 370 375 380Leu Leu Ala Gln Leu Gly Ala Asp Val Leu Lys Val Glu Pro Pro His385 390 395 400Gly Glu Gly Ser Arg Arg Leu Ala Gln Gln Val Ala Gly Val Gly Tyr 405 410 415Leu Tyr Tyr Val Asn Asn Ala Gly Lys Arg Ser Cys Arg Leu Asp Leu 420 425 430Ala Asp Ala Glu Asp Arg Ala Gly Phe Glu Arg Leu Leu Ala Gly Cys 435 440 445Asp Ile Val Leu Thr Asn Leu Ala Ala Asp Thr Leu Thr Ala Gln Gly 450 455 460Leu Ala Pro Asp Gln Ile Leu Ser Arg His Gly Val Val His Cys Thr465 470 475 480Val Thr Gly His Gly Leu Ala Ala Ala Asp Arg Ser Val Asp Thr Val 485 490 495Ile Gln Ala Glu Ser Gly Ile Met Arg Leu Val Gly Gly Pro Gly Ala 500 505 510Gly Leu Arg Thr Pro Val Ser Ser Ala Asp Val Leu Gly Ala Tyr Leu 515 520 525Ala Ala Ala Ala Ala Val Val Ser Thr Tyr Val Arg Leu Arg Thr Glu 530 535 540His Gly Cys Ala Ala Asp Val Ala Leu Phe Asp Ser Ala Val Trp Leu545 550 555 560Thr Gln Asp Arg Trp Phe Thr Ala Pro Pro Ala Arg Ala Pro His Leu 565 570 575Val Arg Ala Ala Asp Gly Thr Val Leu Val Asp Ala Glu Gly Pro Pro 580 585 590Pro Arg Ala Glu Gly Pro Val Ala Ala Val Leu Asp Ala Ala Ala Ala 595 600 605Val Gly Val Pro Ala Ala Pro Leu His Asp Leu Thr Arg Ala Val Arg 610 615 620His Pro Gln Val Leu Ala Arg Arg Met Ala Val Ala Arg Asp Cys Ala625 630 635 640Gly Thr Thr Val Leu Ile Thr Gly Asn His Leu Arg Ser Leu Leu Arg 645 650 655Glu Asp Pro Pro Pro Thr Cys Ala Pro Val Asp Gln Asn Asp Pro Val 660 665 670Trp Leu Gln Pro Ala Pro Thr Glu Gly Gln Gln 675 68015426PRTStreptomyces parvulus Tu4055 15Met Thr Thr Arg Arg Arg Gln Arg His Pro Ala Leu Ser Pro Ser Cys1 5 10 15Pro Ser Val Pro Phe Pro Leu Leu Glu Thr Glu Phe Val Leu Met Pro 20 25 30Ser Phe Pro Val Arg Arg Ser Val Pro Asp Thr Pro Pro Ala Glu His 35 40 45Leu Glu Leu Leu Lys Glu Ser Gly Gly Val Cys Pro Phe Thr Met Glu 50 55 60Asp Gly Arg Pro Ala Trp Leu Ala Ala Ser His Asp Ala Val Arg Ser65 70 75 80Leu Leu Ala Asp Arg Arg Ile Ser Asn Asn Pro Ala Lys Thr Pro Pro 85 90 95Phe Ser Gln Arg Glu Ala Leu Gln Lys Glu Arg Gly Gln Phe Ser Arg 100 105 110His Leu Phe Asn Met Asp Ser Pro Glu His Asp Val Ala Arg Arg Met 115 120 125Ile Ala Glu Asp Phe Thr Pro Arg His Ala Glu Ala Val Arg Pro Tyr 130 135 140Phe Glu Glu Val Phe Gly Glu Ile Val Asp Glu Val Val His Lys Gly145 150 155 160Pro Pro Ala Glu Met Ile Glu Ser Phe Ala Phe Pro Val Ala Thr Arg 165 170 175Thr Ile Cys Lys Val Leu Asp Ile Pro Glu Asp Asp Cys Glu Tyr Phe 180 185 190Gln Lys Arg Thr Glu Gln Ile Ile Glu Met Asp Arg Gly Glu Glu Asn 195 200 205Leu Glu Ala Val Val Glu Leu Arg Arg Tyr Val Asp Ser Val Met Gln 210 215 220Gln Arg Thr Arg Lys Pro Gly Asp Asp Leu Leu Ser Arg Met Ile Val225 230 235 240Lys Ala Lys Ala Ser Lys Glu Ile Glu Leu Ser Asp Ala Asp Leu Val 245 250 255Asp Asn Ala Met Phe Leu Leu Val Ala Gly His Glu Pro Ser Ala Asn 260 265 270Met Leu Gly Leu Gly Val Leu Ala Leu Ala Glu Phe Pro Asp Val Ala 275 280 285Glu Glu Leu Arg Ala Glu Pro His Leu Trp Pro Gly Ala Ile Asp Glu 290 295 300Met Leu Arg Tyr Tyr Thr Ile Ala Arg Ala Thr Lys Arg Val Ala Ala305 310 315 320Ala Asp Ile Glu Tyr Glu Gly His Thr Ile Lys Glu Gly Asp Ala Val 325 330 335Ile Val Leu Leu Asp Thr Ser Asn Arg Asp Pro Lys Val His Ala Glu 340 345 350Pro Asn Arg Leu Asp Ile His Arg Ser Ala Gly Asn His Leu Ala Phe 355 360 365Ser His Gly Pro His Gln Cys Leu Gly Lys His Leu Val Arg Val Gln 370 375 380Leu Glu Ile Ala Leu Arg Ala Val Ala Glu Arg Leu Pro Gly Leu Arg385 390 395 400Leu Asp Ile Ala Lys Glu Asp Ile Pro Phe Arg Gly Asp Ala Leu Ser 405 410 415Tyr Gly Pro Arg Gln Leu Arg Val Thr Trp 420 42516454PRTStreptomyces parvulus Tu4055 16Met Glu Lys Thr Asp Val Asp Arg Leu Arg Thr Leu Asp Arg Glu His1 5 10 15Met Trp Tyr Pro Trp Thr Pro Met Thr Glu Trp Met Ala Arg Asp Gln 20 25 30Leu Val Val Glu Arg Ala Glu Gly Cys Trp Leu Ile Asp Ala Asp Gly 35 40 45Lys Arg Tyr Leu Asp Gly Arg Ser Ser Met Gly Met Asn Leu His Gly 50 55 60His Gly Arg Ser Glu Ile Val Glu Ala Leu Val Ala Gln Ala Arg Lys65 70 75 80Ala Gly Glu Thr Thr Leu Tyr Arg Val Ser His Pro Ala Ala Val Glu 85 90 95Leu Ala Ala Arg Leu Ala Ser Met Ala Pro Ala Gly Leu Gln Arg Val 100 105 110Phe Phe Ala Glu Ser Gly Ser Thr Ala Val Glu Thr Ala Leu Lys Ala 115 120 125Ala Tyr Ala Tyr Trp Val Ala Lys Gly Glu Pro Gln Arg Ser Thr Phe 130 135 140Val Ser Met Glu Gly Gly Tyr His Gly Glu Thr Leu Gly Thr Val Ser145 150 155 160Leu Arg Gly Thr Asn Gly Glu Gln Val Asp Met Ile Arg Lys Thr Tyr 165 170 175Glu Pro Leu Leu Phe Pro Ser Leu Ser Phe His Gln Pro His Cys Tyr 180 185 190Arg Cys Pro Val Gly Gln Ser Ser Asp Ser Asp Cys Gly Leu Glu Cys 195 200 205Thr Asp Ser Leu Glu Asn Leu Leu Thr Arg Glu Lys Gly Arg Ile Ala 210 215 220Ala Val Ile Val Glu Pro Arg Val Gln Ala Leu Ala Gly Val Ile Thr225 230 235 240Ala Pro Glu Gly His Leu Ala Lys Val Ala Glu Ile Thr Arg Arg His 245 250 255Gly Val Leu Leu Ile Val Asp Glu Val Leu Thr Gly Trp Ala Arg Thr 260 265 270Gly Pro Thr Phe Ser Cys Glu Ala Glu Gly Val Thr Pro Asp Leu Met 275 280

285Thr Val Gly Lys Ala Leu Thr Gly Gly Tyr Leu Pro Leu Ser Ala Thr 290 295 300Leu Ala Thr Glu Glu Ile Phe Gly Ala Phe Arg Glu Ser Val Phe Leu305 310 315 320Ser Gly Ser Thr Tyr Ser Gly Tyr Ala Leu Gly Ala Ala Val Ala Leu 325 330 335Ala Ser Leu Asp Leu Phe Glu Lys Glu Asp Val Pro Ala Arg Ala Lys 340 345 350Ala Leu Ala Asp Val Leu Thr Thr Ala Leu Glu Pro Phe Arg Ala Leu 355 360 365Thr His Val Gly Asp Val Arg Gln Leu Gly Leu Ile Ala Gly Val Glu 370 375 380Leu Val Ala Asp Arg Glu Thr Arg Ala Pro Tyr Pro Pro Gln Glu Arg385 390 395 400Val Val Asp Arg Ile Cys Thr Leu Ala Arg Asp Asn Gly Val Leu Val 405 410 415Asn Ala Val Pro Gly Asp Val Ile Thr Met Leu Pro Ser Pro Ser Met 420 425 430Ser Pro Asp Asp Leu Arg Phe Leu Thr Gly Thr Leu Tyr Thr Ala Val 435 440 445Arg Glu Val Thr Glu Glu 45017326PRTStreptomyces parvulus Tu4055 17Met Arg Ala Ala Val Ile Arg Ala Trp Gly Gly Pro Glu Arg Leu Thr1 5 10 15Leu Asp Arg Val Glu Arg Pro Ser Pro Pro Pro Gly Trp Ile Ala Val 20 25 30Arg Val Glu Ala Cys Ala Leu Asn His Leu Asp Ile His Val Arg Asn 35 40 45Gly Leu Pro Gly Val Arg Leu Glu Leu Pro His Val Ser Gly Gly Asp 50 55 60Val Val Gly Val Val Glu Gln Ala Thr Asp Glu Ala Gly Glu Arg Leu65 70 75 80Leu Gly Ser Arg Val Leu Leu Asp Pro Met Ile Gly Arg Gly Ile Leu 85 90 95Gly Glu His Tyr Trp Gly Gly Leu Ala Glu Tyr Val Val Ala Pro Ala 100 105 110His Asn Ala Leu Pro Val Pro Asp Gln Asp Ala Asp Pro Ala Arg Tyr 115 120 125Ala Ala Leu Pro Ile Ser Tyr Gly Thr Ala Gln Arg Met Leu Phe Ser 130 135 140Arg Ala Arg Leu Arg Pro Gly Glu Ser Val Leu Leu Phe Gly Ala Thr145 150 155 160Gly Gly Val Gly Val Ala Cys Ala Gln Leu Ala Leu Arg Ala Gly Ala 165 170 175Arg Ile Ile Ala Cys Ser Gly Ser Pro Ala Lys Leu Ala Arg Leu Arg 180 185 190Arg Leu Gly Val Ile Asp Thr Ile Asp Thr Gly Thr Glu Asp Val Arg 195 200 205Arg Arg Val Arg Glu Leu Thr Asp Gly Gly Ala Asp Leu Val Val Asp 210 215 220Tyr Gln Gly Lys Asp Thr Trp Pro Val Ser Leu Arg Ser Ala Arg Ala225 230 235 240Gly Gly Arg Ile Val Thr Cys Gly Ala Thr Thr Gly Tyr Glu Ala Thr 245 250 255Thr Asp Leu Arg Tyr Val Trp Ser Arg Gln Leu Asp Ile Leu Gly Ser 260 265 270Asn Ala Trp His Arg Asp Asp Leu His Thr Leu Val Asp Leu Val Ala 275 280 285Thr Asp Ala Leu Glu Pro Val Val His Ala Asp Phe Pro Leu Ser Arg 290 295 300Ala Pro Glu Ala Val Ala Glu Leu Glu Glu Arg Arg Ala Phe Gly Lys305 310 315 320Val Val Ile Arg Thr Ala 32518556PRTStreptomyces parvulus Tu4055 18Met Thr Gly Asn Thr Thr Ser Ala Ala Phe Leu Arg Arg Thr Gln Asn1 5 10 15Ala Leu Ala Met Gln Arg Lys Ile Cys Ala Gln Pro Glu Glu Thr Ala 20 25 30Glu Arg Val Phe Ser Asp Ile Leu Ser Val Ser Arg Asp Thr Gly Phe 35 40 45Gly Arg Glu His Gly Leu Ala Gly Val Arg Thr Arg Gln Glu Trp Arg 50 55 60Arg Ala Val Pro Ile Arg Thr Tyr Asp Glu Leu Ala Pro Tyr Val Glu65 70 75 80Arg Gln Phe Ser Gly Glu Arg Arg Val Leu Thr Thr Asp Asp Pro Arg 85 90 95Ala Phe Leu Arg Thr Ser Gly Ser Thr Gly Arg Ala Lys Leu Val Pro 100 105 110Thr Thr Asp His Trp Arg Arg Val Tyr Arg Gly Pro Ala Leu Tyr Ala 115 120 125Gln Trp Gly Leu Tyr Phe Glu Gln Ile Gly Thr His Arg Leu Thr Gly 130 135 140Asp Glu Val Leu Asp Leu Ser Trp Glu Pro Gly Pro Ile Arg His Arg145 150 155 160Leu Arg Gly Phe Pro Val Tyr Ser Ile Thr Glu Arg Pro Val Ser Asp 165 170 175Asp Pro Asp Asp Trp Asn Pro Pro Trp Arg His Ala Arg Trp Phe Thr 180 185 190Arg Asp Ala Gly Ala Ala Thr Met Ala Asp Leu Leu Tyr Gly Lys Leu 195 200 205Leu Arg Leu Ala Ala His Asp Leu Arg Leu Ile Val Ser Val Asn Pro 210 215 220Ser Lys Ile Val Leu Leu Ala Glu Thr Leu Lys Glu Asn Ala Glu Arg225 230 235 240Leu Ile Gln Asp Leu His Asp Gly His Gly Thr Asp Arg Ala Ala Arg 245 250 255Pro Asp Phe Leu Arg Arg Leu Thr Ala Ala Phe Asp Arg Thr Gly Gly 260 265 270Arg Pro Leu Leu Thr Asp Leu Trp Pro Gly Leu Arg Leu Leu Val Cys 275 280 285Trp Asn Ser Ala Ser Ala Ala Leu Tyr Gly Pro Trp Leu Ser Arg Leu 290 295 300Ala Thr Gly Val Ala Ala Leu Pro Phe Ser Thr Thr Gly Thr Glu Gly305 310 315 320Ile Val Thr Leu Pro Val Asp Asp His Leu Ser Ala Gly Pro Leu Ala 325 330 335Val Asp Gln Gly His Phe Glu Phe Val Pro Trp Gln Asp Leu Asp Asp 340 345 350Gly Ser Pro Leu Pro Glu Asp Thr Pro Thr Leu Gly Tyr Asp Glu Leu 355 360 365Glu Leu Gly Ala Asp Tyr Arg Leu Val Met Ser Gln Ala Asn Gly Leu 370 375 380Tyr Arg Tyr Asp Val Gly Asp Val Tyr Arg Val Val Gly Ala Val Gly385 390 395 400Ala Thr Pro Arg Leu Glu Phe Leu Gly Arg Ala Gly Phe Gln Ser Ser 405 410 415Phe Thr Gly Glu Lys Leu Thr Glu Ser Asp Val His Thr Ala Val Met 420 425 430Arg Val Leu Gly Ser Glu Arg Thr Asp His Pro His Phe Ser Gly Ile 435 440 445Pro Val Trp Asp Thr Pro Pro His Tyr Leu Val Ala Ile Glu Trp Ala 450 455 460Asp Ala His Gly Thr Leu Asn Val Gln Asp Thr Ala Arg Arg Ile Asp465 470 475 480Ala Thr Leu Gln Glu Val Asn Val Glu Tyr Ala Asp Lys Arg Arg Ser 485 490 495Gly Arg Leu Arg Pro Leu Gln Ile Leu Pro Leu Val Pro Gly Ala Phe 500 505 510Gly Gln Ile Ala Glu Arg Arg Phe Arg Gln Gly Thr Ala Gly Ala Gln 515 520 525Ile Lys His His Trp Leu Gln Lys Asp Ser Ala Phe Leu Asp Thr Leu 530 535 540Arg Asp Leu Asp Leu Val Arg Ala Arg Pro Gly Thr545 550 55519305PRTStreptomyces parvulus Tu4055 19Met Arg Ile Gly Phe Ala Ala Pro Met Ser Gly Pro Trp Ala Thr Pro1 5 10 15Asp Thr Ala Val His Val Ala Arg Thr Ala Glu Gln Leu Gly Tyr Ala 20 25 30Ser Leu Trp Thr Tyr Gln Arg Val Leu Gly Ala Pro Asp Asp Ser Trp 35 40 45Gly Glu Ala Asn Arg Ser Val His Asp Pro Leu Thr Thr Leu Ala Phe 50 55 60Leu Ala Ala His Thr Thr Gly Ile Arg Leu Gly Val Ala Val Leu Ile65 70 75 80Met Pro Leu His Thr Pro Ala Val Leu Ala Lys Gln Leu Thr Thr Leu 85 90 95Asp Leu Leu Ser Gly Gly Arg Leu Asp Val Gly Leu Gly Asn Gly Trp 100 105 110Ala Ala Glu Glu Tyr Ala Ala Ala Gly Val Thr Pro Thr Gly Leu Ser 115 120 125Arg Arg Ala Glu Asp Phe Leu Ala Cys Leu Arg Ala Leu Trp Gly Glu 130 135 140Gln Thr Val Val Glu His Asp Gly Pro Phe Tyr Arg Val Pro Pro Ala145 150 155 160Arg Phe Asp Pro Lys Pro Ala Gln Ser Pro His Pro Pro Leu Leu Leu 165 170 175Gly Gly Ala Ala Pro Gly Ala Leu Arg Arg Ala Gly Arg Leu Cys Asp 180 185 190Gly Trp Ile Ala Ser Ser Lys Ala Gly Pro Ala Ala Ile Arg Asp Ala 195 200 205Ile Thr Val Val Arg Asp Ser Ala Glu Arg Thr Gly Arg Asp Pro Ala 210 215 220Thr Leu Arg Phe Val Cys Arg Ala Pro Val Arg Leu Arg Thr Arg Ser225 230 235 240Ala Pro Asn Glu Pro Pro Leu Thr Gly Thr Ala Glu Thr Ile Arg Ala 245 250 255Asp Leu Ala Ala Leu Ala Asp Thr Gly Leu Thr Glu Ile Phe Leu Asp 260 265 270Pro Asn Phe Asp Pro Glu Ile Gly Ser Pro Asp Ala Pro Thr Gly Asp 275 280 285Val Arg His Arg Val Asp Leu Leu Leu His Glu Leu Ala Pro Ala Asn 290 295 300Trp30520248PRTStreptomyces parvulus Tu4055 20Met Leu Ile Ala Arg Ala Ala Val Gly Glu Asp Arg Thr Tyr Ala Arg1 5 10 15Val Asp Thr Asp Thr Gly Leu Ile His Leu Leu Ala Gly Thr Pro Tyr 20 25 30Asp Glu Ile Arg Pro Thr Gly Glu Thr Arg Pro Leu Ala Glu Ala Arg 35 40 45Leu Leu Ala Pro Val Glu Pro Ser Lys Val Leu Val Ala Gly Arg Asn 50 55 60Tyr Gly Asp Val Val Thr Pro Asp Leu Val Val Phe Met Lys Pro Ser65 70 75 80Thr Ser Val Val Gly Pro Arg Ser Thr Val Leu Leu Pro Ala Glu Ala 85 90 95Lys Gln Val Arg Tyr Glu Gly Glu Leu Ala Val Val Ile Gly Arg Arg 100 105 110Cys Lys Asp Val Pro Glu Asp Thr Ala Asp Gln Ala Val Phe Gly Tyr 115 120 125Thr Cys Ala Asn Asp Val Thr Ala Trp Asp Val Gly Glu Pro Lys Gly 130 135 140His Trp Thr Lys Ala Lys Ser Phe Asp Thr Phe Cys Pro Leu Gly Pro145 150 155 160Trp Ile Arg Thr Asp Leu Asp Pro Ala Asp Leu Val Leu Arg Thr Thr 165 170 175Val Asn Gly Thr Leu Arg Gln Asp Gly Ser Thr Lys Glu Met Asn Arg 180 185 190Asn Val Arg Ala Leu Val Ser Arg Cys Ser Ser Leu Met Thr Leu Leu 195 200 205Pro Gly Asp Val Ile Leu Thr Gly Thr Pro Ala Gly Ala Gly Val Leu 210 215 220Arg Pro Gly Asp Glu Val Val Val Glu Ile Asp Gly Ile Gly Ser Leu225 230 235 240Ala Asn Pro Ile Gly Val Ala Lys 24521675PRTStreptomyces parvulus Tu4055 21Met Ser Val Ile Arg Pro Thr Ala Glu Thr Glu Arg Ala Val Val Val1 5 10 15Val Pro Ala Gly Thr Thr Cys Ala Asp Ala Val Thr Ala Ala Lys Leu 20 25 30Pro Arg Asn Gly Pro Asn Ala Ile Val Val Val Arg Asp Pro Ser Gly 35 40 45Ala Leu Arg Asp Leu Asp Trp Thr Pro Asp Ser Asp Val Glu Val Glu 50 55 60Ala Val Ala Leu Ser Ser Glu Asp Gly Leu Thr Val Leu Arg His Ser65 70 75 80Thr Ala His Val Leu Ala Gln Ala Val Gln Gln Leu Trp Pro Glu Ala 85 90 95Arg Leu Gly Ile Gly Pro Pro Ile Glu Asn Gly Phe Tyr Tyr Asp Phe 100 105 110Asp Val Glu Arg Pro Phe Gln Pro Glu Asp Leu Glu Arg Val Glu Gln 115 120 125Arg Met Lys Glu Ile Ile Lys Ser Gly Gln Arg Phe Cys Arg Arg Glu 130 135 140Phe Pro Asp Arg Glu Ala Ala Arg Ala Glu Leu Ala Lys Glu Pro Tyr145 150 155 160Lys Leu Glu Leu Val Asp Leu Lys Gly Asp Val Asp Ala Ala Glu Ala 165 170 175Met Glu Val Gly Gly Ser Asp Leu Thr Ile Tyr Asp Asn Leu Asp Ala 180 185 190Arg Thr Gly Asp Val Cys Trp Ser Asp Leu Cys Arg Gly Pro His Leu 195 200 205Pro Ser Thr Arg Leu Ile Pro Ala Phe Lys Leu Leu Arg Asn Ala Ala 210 215 220Ala Tyr Trp Arg Gly Ser Glu Lys Asn Pro Gln Leu Gln Arg Ile Tyr225 230 235 240Gly Thr Ala Trp Pro Thr Arg Asp Glu Leu Lys Ser His Leu Ala Ala 245 250 255Leu Glu Glu Ala Ala Lys Arg Asp His Arg Arg Ile Gly Glu Glu Leu 260 265 270Asp Leu Phe Ala Phe Asn Lys Glu Ile Gly Arg Gly Leu Pro Leu Trp 275 280 285Leu Pro Asn Gly Ala Ile Ile Arg Asp Glu Leu Glu Asp Trp Ala Arg 290 295 300Lys Thr Glu Arg Lys Leu Gly Tyr Lys Arg Val Val Thr Pro His Ile305 310 315 320Thr Gln Glu Asp Leu Tyr Tyr Leu Ser Gly His Leu Pro Tyr Tyr Ala 325 330 335Glu Asp Leu Tyr Ala Pro Ile Asp Ile Asp Gly Glu Lys Tyr Tyr Leu 340 345 350Lys Pro Met Asn Cys Pro His His His Met Val Tyr Lys Ala Arg Pro 355 360 365His Ser Tyr Arg Asp Leu Pro Tyr Lys Val Ala Glu Tyr Gly Thr Val 370 375 380Tyr Arg Phe Glu Arg Ser Gly Gln Leu His Gly Met Met Arg Thr Arg385 390 395 400Gly Phe Ser Gln Asn Asp Ala His Ile Tyr Cys Thr Ala Asp Gln Ala 405 410 415Lys Asp Gln Phe Leu Glu Val Met Arg Met His Ala Asp Tyr Tyr Arg 420 425 430Thr Leu Gly Ile Ser Asp Phe Tyr Met Val Leu Ala Leu Arg Asp Ser 435 440 445Ala Asn Lys Asp Lys Tyr His Asp Asp Glu Gln Met Trp Glu Asp Ala 450 455 460Glu Arg Ile Thr Arg Glu Ala Met Glu Glu Ser Asp Ile Pro Phe Gln465 470 475 480Ile Asp Leu Gly Gly Ala Ala His Tyr Gly Pro Lys Val Asp Phe Met 485 490 495Ile Arg Ala Val Thr Gly Lys Glu Phe Ala Ala Ser Thr Asn Gln Val 500 505 510Asp Leu Tyr Thr Pro Gln Arg Phe Gly Leu Thr Tyr His Asp Ser Asp 515 520 525Gly Thr Glu Lys Pro Val Val Val Ile His Arg Ala Pro Leu Gly Ser 530 535 540His Glu Arg Phe Thr Ala Tyr Leu Thr Glu His Phe Ala Gly Ala Phe545 550 555 560Pro Val Trp Leu Ala Pro Glu Gln Val Arg Ile Ile Pro Ile Val Glu 565 570 575Glu Leu Thr Asp Tyr Ala Glu Glu Val Arg Asp Met Leu Leu Asp Ala 580 585 590Asp Val Arg Ala Asp Val Asp Ala Gly Asp Gly Arg Leu Asn Ala Lys 595 600 605Val Arg Ala Ala Val Thr Arg Lys Ile Pro Leu Val Val Val Val Gly 610 615 620Arg Arg Glu Ala Glu Gln Arg Thr Val Thr Val Arg Asp Arg Ser Gly625 630 635 640Glu Glu Thr Pro Met Ser Leu Glu Lys Phe Val Ala His Val Thr Gly 645 650 655Leu Ile Arg Thr Lys Ser Leu Asp Gly Ala Gly His Ile Arg Pro Leu 660 665 670Ser Lys Ala 67522103PRTStreptomyces parvulus Tu4055 22Met Pro Arg Gly Ile Ala Val Asp Val Leu Arg Ala Gly Asp Arg Trp1 5 10 15Pro His Ser Ala Ala Pro Arg His Arg Gly Leu Leu Asn Ala Trp Trp 20 25 30Gly Ala Trp Val Trp Ala Thr Val Phe Asp Arg Tyr Ala Ser Arg Thr 35 40 45Tyr Asp Asp Ala Gln Asp Val Asp Ala Ile His Asp Ala Ala Gly Leu 50 55 60Val Met Ala Gly Ala Gly Phe Asp Ile Leu Ala Ala Val Leu Ala Ile65 70 75 80Leu Phe Val Arg Arg Leu Thr Ala Ala Gln His Ala Lys Ala Leu Ala 85 90 95Gly Pro Thr Pro Pro Thr His 10023868PRTStreptomyces parvulus Tu4055 23Met Glu Ala Phe Leu Leu Leu Ala Ala Glu Ser Val Leu Leu Arg Arg1 5 10 15Asp Gln Ser Val Tyr Val Thr Pro Gly Ser Glu Pro Asp Gly Pro Pro 20 25 30Arg Ala Ala Leu Arg Arg Leu Glu Ala Glu Leu Leu Gly Arg Gly His 35 40 45Ala Val Ser Ala Pro Leu His Ala Val Leu Ala Ser Leu Asp Ser Glu 50 55 60Glu Leu

Ala Ala Ala His Val Arg Leu Val Gly Leu Val Asp Asp Leu65 70 75 80Leu Gly Ser Asp Arg Thr His Thr Pro Leu Phe Arg Arg Phe Pro Arg 85 90 95Thr Val Pro Arg Asp Thr Glu Ala Leu Tyr Val Asp Arg Val Phe Ala 100 105 110Phe Leu Leu Gln Gln Pro Glu Gln Pro Cys Val Leu Cys Gly Glu Ala 115 120 125Arg Thr Val Leu Pro Val Ser Pro Cys Ala His Leu Val Cys Arg Leu 130 135 140Cys Trp Asp Gly Ser Asp Tyr Ala Gly Cys Pro Leu Cys His Arg Arg145 150 155 160Ile Asp Gly Asp Asp Pro Phe Leu Arg Pro Val Arg Ala Val Gly Ala 165 170 175Ala Arg Ala Thr Val Pro Gly Pro Leu Arg Leu Leu Arg Leu Gly Thr 180 185 190Asp Met Thr Ala Asp Ala Thr Thr Ala Val Asp Ala Leu Leu Ala Arg 195 200 205Arg Thr Pro Leu Ser Pro Gln Asp Arg Asp Asp Leu Leu Thr Leu Leu 210 215 220Pro Leu Thr Pro Ala Gly Arg Gly Asp Leu Pro Gln Asp Ile Pro Val225 230 235 240Arg Glu Thr Lys Ala Leu Val Leu Gly Ala Leu Val Arg Arg Ala Pro 245 250 255Ser Arg Pro Ala Leu Arg Arg Leu Leu Ala Glu Arg Leu Thr Thr Ala 260 265 270Thr Asp Val Leu Arg Leu Leu Ala Val Leu Ser Gly Gly Asp Ala Gly 275 280 285Leu Val Thr Pro Ala Arg Phe Thr Asn Val Pro Arg Ser Leu Arg Arg 290 295 300Asp Leu Leu Ala Val Leu Asp Gly Leu Pro Ala Pro Tyr Leu Val Glu305 310 315 320Asp Met Leu Arg His Pro Thr Ala Trp Lys Arg Ala Ala Glu Val Leu 325 330 335His Pro Phe Glu Gly His Thr Arg His Pro Arg Ala Ala Leu Ala Thr 340 345 350Ala Val Leu Arg Ala Thr Pro Leu Asp Pro Asp Thr Ala Phe Gly Ala 355 360 365Ala Leu Leu Thr Thr Ala Ala Ala His Pro Asp Ala Val Arg Pro Asp 370 375 380Gly Thr Arg Val Arg Pro Ala Thr Trp Ala Gly Arg Leu Glu Gln Ala385 390 395 400Met Ala Glu Gly Asp Ala Ala Arg Ala Ala Ala Leu Ala Gly Glu Arg 405 410 415Pro Gly Glu Leu Val Arg Arg Leu Asp Val Leu Leu Arg Leu His Thr 420 425 430Asp Glu Ala Leu Val Pro Glu Leu Glu Lys Ala Leu Arg His Gly Leu 435 440 445Pro Lys Val Gly Pro Gly Pro Leu Leu Ser Ala Leu Gly Ala Leu Arg 450 455 460Thr Arg Thr Glu Asp Arg Thr Gly Thr Arg Arg Val Phe Phe Pro Arg465 470 475 480Gly Asp Val Thr Arg Ala Leu Ser Val Pro Glu Arg Arg Pro Ala Leu 485 490 495Pro Ala Gly Pro Val Ser Glu Val Val Ala Leu Leu Glu Gly Glu Leu 500 505 510Leu Arg Arg Phe Ala Ala Gly Arg Pro Tyr Glu Leu Ser Val Leu Asp 515 520 525Ala Gly Leu Thr Asp Leu Thr Val Pro Phe Thr Glu Arg Thr Ala Ala 530 535 540Lys Ala Leu Val Thr Val Gly Arg Gly Ser Val Gln Ala Leu Pro Glu545 550 555 560Gly Ser Val Leu Arg Leu Phe Leu His Trp Thr Glu Pro Arg Gly Asn 565 570 575Arg Thr Asp Leu Asp Leu Ser Val Ala Phe Phe Asp Ala Glu Trp Thr 580 585 590Phe Thr Gly Leu Cys Asp Tyr Thr Asn Leu Val His Gly Pro Asp Ala 595 600 605Ala Ile His Ser Gly Asp Leu Thr Ser Ala Pro Ala Pro Arg Gly Ala 610 615 620Thr Glu Tyr Val Asp Leu Asp Leu Glu Arg Leu Ala Arg Arg Gly Asp625 630 635 640Thr Tyr Ala Val Pro Leu Val Phe Ser Tyr Asn Asn Val Pro Phe Glu 645 650 655Glu Leu Pro Asp Ala Phe Ala Gly Phe Met Ala Leu Pro Ala Glu Gly 660 665 670Pro Arg Asp Ala Thr Tyr Asp Pro Arg Thr Val Arg Gln Arg Phe Asp 675 680 685Leu Ala Gly Asp Ser Lys Val Cys Leu Pro Met Ile Val Asp Leu Ala 690 695 700Arg Arg Arg Ala Leu Trp Thr Asp Thr His Leu Pro Ser Ala Gly Gly705 710 715 720Phe Gln Ser Ile Gly Ser His Gly Gly Gly Glu Leu Ala Ala Val Ala 725 730 735Gly Asp Leu Trp Gln Gln Phe Thr Ser Gly Gly Arg Ala Thr Leu Trp 740 745 750Asp Leu Ala Val Leu Arg Ala Ala Ala Leu Ser Pro Glu Val Ala Val 755 760 765Val Ser Arg Glu Pro Glu Pro Ala Val Leu Arg Tyr Arg Arg Arg Ala 770 775 780Ala Glu Ser Glu Ala Ala Phe Ala Val Arg Val Ala Ser His Lys Asp785 790 795 800Ala Glu Glu Arg Leu Ala His Thr Asp Pro Asp Ser Ala Ala Ala Gly 805 810 815Leu Ala Ala Gly Arg Arg Val Phe Leu Ala Thr Val His Gly Asp Val 820 825 830Arg Pro Pro Gly Ala Ser Gly Thr Ser Tyr Arg Leu Phe Pro Gly Ala 835 840 845Gly Asp Ala Ser Pro Thr Leu Thr Arg Val Thr Ala Gly Asp Leu Leu 850 855 860Ala Glu Leu Gly86524212PRTStreptomyces parvulus Tu4055 24Met Ala Glu Gln Ile Ala Gly Ile Glu Ile Pro Asp Ser Ala Pro Ala1 5 10 15Arg Glu Ala Thr Asp Leu Ile Arg Asp Thr Thr Pro Pro Leu Ile Phe 20 25 30His His Ser Arg Arg Val Tyr Leu Phe Gly Ser Leu Gln Ala Ala Ala 35 40 45Leu Gly Ile Arg Pro Asp Pro Glu Leu Leu Tyr Ile Ala Ala Leu Phe 50 55 60His Asp Thr Gly Leu Val Pro Pro Tyr Arg Gly Asp Asp Gln Arg Phe65 70 75 80Glu Met Asp Gly Ala Asp Gln Ala His Ala Phe Leu Leu Ala His Gly 85 90 95Ile Pro Glu Ala Asp Ala Asp Thr Val Trp Thr Ala Val Ala Leu His 100 105 110Thr Thr Pro Glu Val Pro Tyr Arg Met Ala Pro Glu Ile Ala Ala Thr 115 120 125Thr Ala Gly Val Glu Thr Asp Val Leu Gly Leu Arg Leu Gly Asn Leu 130 135 140Thr Arg Ala Gln Ile Asp Ala Val Thr Ala Ala His Pro Arg Pro Asp145 150 155 160Phe Lys Lys Gln Ile Leu Arg Ala Phe Thr Glu Gly Phe Glu His Arg 165 170 175Pro Ala Thr Thr Phe Gly Thr Val Asn Ala Asp Val Leu Glu His Phe 180 185 190Ala Pro Gly Phe Arg Arg Thr Asp Phe Val Glu Val Ile Glu Asn Ser 195 200 205Ala Trp Pro Glu 21025329PRTStreptomyces parvulus Tu4055 25Met Thr Ala Arg Ala His Ser Val Gly Ile Leu Val Phe Asp Gly Met1 5 10 15Lys Met Leu Asp Leu Ser Gly Pro Ala Glu Val Phe Ala Glu Ala Asn 20 25 30Arg Phe Gly Ala Arg Tyr Arg Leu Gly Val Val Ser Pro Asp Gly Ala 35 40 45Pro Val Arg Ser Ser Ile Gly Leu Leu Val Pro Ala Glu Ala Asp Ala 50 55 60Arg Ser Ala Gly Pro Pro Asp Thr Leu Val Val Val Gly Gly Asp Ala65 70 75 80Leu Pro Gly Ser Pro Val Asp Pro Arg Leu Ile Asp Ala Ala Lys Ala 85 90 95Leu Ala Ala Arg Ala Gly Arg Val Ala Ser Val Cys Thr Gly Ala Phe 100 105 110Val Leu Gly Ala Ala Gly Leu Leu Glu Gly Arg Arg Ala Thr Thr His 115 120 125Trp Gln His Thr Thr Ala Leu Ala Arg Arg Cys Pro Ser Thr Arg Val 130 135 140Glu Pro Asp Ala Ile Phe Val Lys Asp Gly Ala Thr Tyr Thr Ser Ala145 150 155 160Gly Val Thr Ala Gly Ile Asp Leu Ala Leu Ala Leu Leu Glu Glu Asp 165 170 175His Gly Pro Asp Leu Ala Arg Arg Val Ala Arg Ser Leu Val Val Tyr 180 185 190Leu Gln Arg Ala Gly Gly Gln Ser Gln Phe Ser Ala Ser Leu Arg Gly 195 200 205Pro Ala Pro Arg Thr Pro Val Leu Arg Gln Val Gln Asp Ala Val Gln 210 215 220Ala Asp Pro Ala Ala Asp His Ser Leu Ala Ala Leu Ala Ala Arg Val225 230 235 240Arg Val Ser Pro Arg His Leu Thr Arg Met Phe Arg Ala Glu Leu Asp 245 250 255Val Thr Pro Val Lys Tyr Val Glu Leu Ile Arg Phe Asp Ile Ala Lys 260 265 270Ala Leu Leu Asp Ser Gly His Asn Ala Thr Glu Ala Ala Ala Leu Ser 275 280 285Gly Phe Pro Ser Tyr Glu Ser Leu Arg Arg Ala Phe Ala Arg His Leu 290 295 300Gly Leu Ser Pro Thr Arg Tyr Arg Gln Arg Phe Ala Thr Thr Val Pro305 310 315 320Asp Ala Gly Pro Arg Pro Asp Gly Gly 32526276PRTStreptomyces parvulus Tu4055 26Met Gly Thr Val Thr Thr Ser Asp Gly Thr Ser Ile Phe Tyr Lys Asp1 5 10 15Trp Gly Pro Arg Asp Ala Pro Pro Ile Val Phe His His Gly Trp Pro 20 25 30Leu Thr Ala Asp Asp Trp Asp Asn Gln Met Leu Phe Phe Leu Ser His 35 40 45Gly Tyr Arg Val Ile Ala His Asp Arg Arg Gly His Gly Arg Ser Gly 50 55 60Gln Pro Ser Thr Gly His Glu Met Asp Thr Tyr Ala Ala Asp Val Ala65 70 75 80Ala Leu Thr Glu Ala Leu Asp Leu Arg Asp Ala Val His Ile Gly His 85 90 95Ser Thr Gly Gly Gly Glu Val Ala Arg Tyr Val Ala Arg Ala Glu Pro 100 105 110Gly Arg Val Ala Lys Ala Val Leu Val Gly Ala Val Pro Pro Val Met 115 120 125Val Lys Ser Asp Ala Asn Pro Gly Gly Thr Pro Ile Glu Val Phe Asp 130 135 140Gly Phe Arg Thr Ala Leu Ala Ala Asn Arg Ala Gln Phe Tyr Ile Asp145 150 155 160Val Pro Ser Gly Pro Phe Tyr Gly Phe Asn Arg Glu Gly Ala Lys Val 165 170 175Ser Gln Gly Leu Ile Asp Asn Trp Trp Arg Gln Gly Met Ser Gly Ala 180 185 190Ala Asn Ala His Tyr Glu Cys Ile Lys Ala Phe Ser Glu Thr Asp Phe 195 200 205Thr Glu Asp Leu Lys Ala Ile Asp Val Pro Val Leu Val Ala His Gly 210 215 220Thr Asp Asp Gln Val Val Pro Tyr Ala Asp Ser Ala Pro Leu Ser Val225 230 235 240Lys Leu Leu Lys Asn Gly Thr Leu Lys Ser Tyr Glu Gly Leu Pro His 245 250 255Gly Met Leu Ser Thr His Pro Glu Val Val Asn Pro Asp Leu Leu Asp 260 265 270Phe Val Arg Ser 27527185PRTStreptomyces parvulus Tu4055 27Met Gly Val Met Ile Gly Pro Ala Gly Arg Glu Arg Asp Glu Gly Asp1 5 10 15His Val Thr Gln Gln Ala Pro Val Ala Thr Asp Glu Arg Arg Val Phe 20 25 30Val Asp Lys Gln Thr Pro Gly Ala Tyr Lys Ala Phe Val Ala Ala Ala 35 40 45Glu Ser Val Arg Glu Ala Ala Ala Ala Ala Gly Leu Asp Arg Leu Leu 50 55 60Val Glu Leu Val Asn Ile Arg Val Ser Gln Leu Asn Ala Cys Ala Tyr65 70 75 80Cys Leu Ser Leu His Thr Arg Ala Ala Leu Arg Ala Gly Glu Thr Thr 85 90 95Gln Arg Leu Ala Val Leu Pro Ala Trp Arg Asp Thr Glu Leu Phe Thr 100 105 110Ala Arg Glu Arg Ala Ala Leu Ala Leu Ala Glu Ala Thr Thr Arg Pro 115 120 125Ala Asp Ala Ala Ala Gln Ser Ala Ala Tyr Ala Gln Ala Arg Gly Val 130 135 140Leu Ser Asp Asp Glu Val Ser Ala Val Ile Trp Val Ala Ile Ser Ile145 150 155 160Asn Ala Phe Asn Arg Val Ser Val Leu Ser Lys His Pro Val Arg Gly 165 170 175Ala Ala Pro Ala Pro Val Ser Pro Ala 180 18528324PRTStreptomyces parvulus Tu4055 28Met Val Ser Asn Thr Glu Thr Arg Pro Ala Glu Met Arg Cys Gly Ala1 5 10 15Leu Glu Asp Glu Val Pro Ala Ala Gly Val Glu Val Leu Thr Ala Arg 20 25 30Asp Val Pro Leu Gly Gly Pro Arg Ala Met Thr Val Arg Arg Thr Leu 35 40 45Pro Gln Arg Ala Arg Thr Leu Ile Gly Ala Trp Cys Phe Ala Asp His 50 55 60Tyr Gly Pro Asp Asp Val Ala Ala Ser Gly Gly Met Asp Val Ala Pro65 70 75 80His Pro His Ile Gly Leu Gln Thr Val Ser Trp Leu Phe Ser Gly Glu 85 90 95Ile Glu His Arg Asp Ser Leu Gly Thr His Ala Phe Val Arg Pro Gly 100 105 110Glu Leu Asn Leu Met Thr Gly Gly Phe Gly Ile Ala His Ser Glu Val 115 120 125Ser Thr Pro Asp Thr Thr Val Leu His Gly Val Gln Leu Trp Val Ala 130 135 140Leu Pro Glu Glu His Arg Asp Thr Gly Arg Asp Phe Gln His His Ala145 150 155 160Pro Ala Pro Val Ala Phe Asp Gly Gly Thr Ala Arg Val Phe Leu Gly 165 170 175Ser Leu Ala Gly Asp Thr Ser Pro Val Ser Thr Phe Thr Pro Leu Leu 180 185 190Gly Ala Glu Leu Thr Leu Val Pro Gly Gly Thr Ala Thr Leu Asp Val 195 200 205Asp Pro Gly Phe Glu His Gly Val Leu Val Asp Ser Gly Asp Val Arg 210 215 220Val Glu Gly Ala Val Val Arg Pro Ala Glu Leu Gly Tyr Val Ala Pro225 230 235 240Gly Arg Ala Thr Leu Thr Leu Thr Asn Glu Ser Ala Ala Pro Ala Arg 245 250 255Leu Ile Leu Leu Gly Gly Pro Pro Phe Pro Glu Glu Ile Ile Met Trp 260 265 270Trp Asn Phe Ile Gly Arg Ser His Asp Glu Ile Val Arg Ala Arg Glu 275 280 285Asp Trp Met Lys Gly Asp Arg Phe Gly Glu Val His Gly Tyr Asp Gly 290 295 300Ala Pro Leu Pro Ala Pro Glu Leu Pro Asn Ala Pro Leu Lys Pro Arg305 310 315 320Arg Arg Ala Arg29126PRTStreptomyces parvulus Tu4055 29Met Val Pro Thr Met Leu Cys Met Val Ala Val Pro Glu Ser His Ser1 5 10 15Gly Trp Thr Phe Val Thr Asn His Ala Arg Val Leu Ala Ala Ile Ala 20 25 30Asp Asn Pro Asn Val Arg Ile Arg Asp Ile Ala Ala His Cys Arg Leu 35 40 45Thr Glu Arg Ala Val Gln Arg Ile Ile Ser Asp Leu Glu Gln Asp Gly 50 55 60Tyr Leu Ser His Thr Arg Asp Gly Arg Ser Asn Ile Tyr Arg Val Glu65 70 75 80Pro Asp Lys Val Leu Arg His Pro Ala Glu Ala Gly Leu Thr Val Ala 85 90 95Ala Leu Leu Ser Leu Leu Val Arg Asp Glu Thr Asp His Gly Arg Ser 100 105 110Ala Gly Pro Gly Ser Arg Pro Ala Arg Ser Ser Ala Ala Arg 115 120 12530127PRTStreptomyces parvulus Tu4055 30Met Ser Leu Asp Glu Ala Val Ala Gly Cys Ser Arg His Thr Gly Arg1 5 10 15Arg Arg Leu Pro Ala Ala Glu Gln Pro Thr Gln Ala Gln Tyr Glu Ala 20 25 30His Gly Ala Trp Val Val Ser Ala Arg Gly Ala Tyr Asp Met Asn Ser 35 40 45Val Glu Pro Leu Ala Asp Ala Leu Lys Asp Ala Ala Glu Lys Ser Pro 50 55 60Lys Val Val Leu Asp Ala Ser Gly Ile Thr Phe Ala Asp Ser Thr Leu65 70 75 80Leu Ser Leu Leu Ile Leu Thr His Gln Ala Thr Asp Phe Arg Val Ala 85 90 95Ala Pro Thr Trp Gln Val Met Arg Leu Met Gln Leu Thr Gly Val Asp 100 105 110Ala Phe Leu Lys Val Arg Ala Thr Val Glu Glu Ala Ala Thr Ala 115 120 1253182PRTStreptomyces parvulus Tu4055 31Met Ser Met Ile Leu Pro Ala Glu Lys Glu Leu Arg Ala Val Leu Ala1 5 10 15Arg Phe Ala Gln Ala Arg Ile Asp His Asp Val Arg Pro Ser Gly Cys 20 25 30Thr Ser Arg Leu Leu Glu Asp Ala Thr Tyr Thr Leu Cys Val Met Thr 35 40 45Gly Ala Arg Thr Ala Glu Gln Ala Leu Arg Thr Ala

Asp Glu Leu Leu 50 55 60Ala Gln Phe Ala Glu Arg Thr Ala Ala Pro Val Glu Asp Glu Ala Leu65 70 75 80Ala Ala3270PRTStreptomyces parvulus Tu4055 32Met Ser Asp Thr Arg Leu Arg Gln Arg Asp Glu Thr Ser Lys Gly Pro1 5 10 15Ala Thr Glu Ile Pro Ala Pro Gln Trp Arg Asp Leu Phe Leu Ala Pro 20 25 30Asp Trp Gly Gly Thr Asp Glu Gln Val Ile Val Ala Glu Glu Ala Arg 35 40 45Gly Pro Glu His Phe Thr Gly Ala Arg Arg Pro Arg Gly Gly Arg Arg 50 55 60Ser Ser Arg Arg Ala Ala65 7033172PRTStreptomyces parvulus Tu4055 33Met Arg Cys Ser His Arg Ala Gly Gly Val Gly Ala Arg Ala Trp Leu1 5 10 15Gly Gly Asn Val Ala Val Asp Met Gly Glu Thr Gly Leu Asp Gly Ser 20 25 30Ser Thr Gln Arg Ala Pro Glu Gly Ala Asp Pro Arg Ala Ala Ser Val 35 40 45Thr Tyr Arg Arg Glu Ala Leu Arg Ile Ala Asp Ala Arg His Phe Ala 50 55 60Thr Asp Tyr Leu Thr Arg Ser Gln Arg Asp Leu Arg Ser Pro Val Pro65 70 75 80Glu Arg Ala Ser Glu Ala Val His Leu Val Val Ser Glu Leu Ile Thr 85 90 95Asn Ala Val Lys Tyr Gly Ala Asp Pro Ile Glu Leu Thr Leu Ser Leu 100 105 110Thr Asp Asp Ala Val Thr Val Thr Val Arg Asp Gly Asp Thr Thr Leu 115 120 125Pro Ala Pro Arg Pro Ala Asp Pro Ala Arg Val Gly Gln His Gly Leu 130 135 140Glu Ile Val Ala Ala Leu Ser Gln Ala Val Glu Ile Arg Pro Glu Pro145 150 155 160Ser Gly Lys Arg Ile Thr Ala Arg Ile Ala Leu Thr 165 17034105PRTStreptomyces parvulus Tu4055 34Met Gln Ser Ser Ser Ala Ser Val Arg Gly Glu Ile Val Ile Arg Arg1 5 10 15Ala Val Ala Arg Asp Ala Lys Arg Leu Ser Arg Leu Val Arg Gly Ser 20 25 30Arg Ala Tyr Glu Gly Pro Tyr Ala Ala Met Val Ser Asp Tyr Arg Val 35 40 45Gly Pro Asp Tyr Ile Glu Asn His Gln Val Phe Val Ala Ser Thr Pro 50 55 60Arg Thr Pro Arg Thr Gly Cys Ser Ala Ser Thr Arg Cys Ser Ser Arg65 70 75 80Arg Arg Ser Trp Thr Cys Cys Ser Ser Arg Thr Val Pro Arg Ala Ala 85 90 95Ala Ser Asp Gly Cys Leu Ser Ile Thr 100 10535638PRTStreptomyces parvulus Tu4055 35Met Ala Gln Arg Arg Thr Pro Phe Gly Asp Arg Ala Arg Tyr Trp Phe1 5 10 15Asp Ser Thr Leu Ala Arg Gly Ala Ala Ala Leu Val Gly Trp Met Ala 20 25 30Leu Leu Ser Leu Ala Val Val Val Pro Ala Ser Ala Val Met Val Trp 35 40 45Thr Asp Pro Asp Ala Pro Pro Ser Leu Ala Glu Arg Leu Ala Glu Val 50 55 60Trp Arg Leu Thr Gly Glu Thr Leu Arg Leu Gly Gly Ala Thr Gly Thr65 70 75 80Pro Leu Arg Ala Met Leu Ser Val Leu Leu Ala Leu Val Thr Leu Leu 85 90 95Tyr Val Ser Thr Leu Val Gly Leu Ile Thr Thr Ala Leu Thr Glu Arg 100 105 110Leu Thr Ser Leu Arg Arg Gly Arg Ser Thr Val Leu Glu Gln Gly His 115 120 125Ala Val Val Leu Gly Trp Ser Glu Gln Val Phe Thr Val Val Ser Glu 130 135 140Leu Val Ala Ala Asn Val Asn Gln Arg Gly Ala Ala Val Val Val Leu145 150 155 160Ala Asp Arg Asp Lys Thr Val Met Glu Glu Ser Leu Gly Thr Lys Val 165 170 175Gly Ser Cys Gly Gly Thr Arg Leu Ile Cys Arg Ser Gly Pro Thr Thr 180 185 190Asp Pro Ala Val Leu Pro Leu Thr Ser Pro Ala Thr Ala Gly Val Val 195 200 205Leu Val Leu Pro Pro Asp Glu Pro His Ala Asp Ala Glu Val Val Lys 210 215 220Thr Leu Leu Ala Leu Arg Ala Ala Leu Ala Gly Ala Lys Pro Arg Pro225 230 235 240Pro Val Val Ala Ala Val Arg Asp Asp Arg Tyr Arg Leu Ala Ala Cys 245 250 255Leu Ala Ala Gly Pro Asp Gly Val Val Leu Glu Ser Asp Thr Val Thr 260 265 270Ala Arg Leu Ile Val Gln Ala Ala Arg Arg Pro Gly Ile Ser Leu Val 275 280 285His Arg Glu Leu Leu Asp Phe Ala Gly Asp Glu Phe Tyr Leu Ile Ser 290 295 300Glu Pro Ala Leu Thr Gly Arg Pro Phe Gly Glu Val Leu Leu Ser Tyr305 310 315 320Ser Thr Thr Ser Val Val Gly Leu Met Arg Gly Cys Thr Pro Leu Leu 325 330 335Asn Pro Pro Pro Thr Thr Pro Val Ala Pro Asp Asp Leu Leu Val Val 340 345 350Ile Thr Gly Asp Asp Asp Thr Ala Arg Leu Asp Asp Cys Ala Glu Ser 355 360 365Val Glu Lys Ala Ala Val Ala Ser Arg Pro Pro Thr Pro Ala Pro Ala 370 375 380Glu Arg Ile Leu Leu Leu Gly Trp Asn Arg Arg Ala Pro Leu Val Val385 390 395 400Asp Gln Leu His Arg Arg Ala Arg Pro Gly Ser Ala Val Asp Val Val 405 410 415Ala Glu Pro Gly Glu Ala Thr Ile Arg Glu Ile Ser Glu Ala Glu Ala 420 425 430Asp Ser Gly Asn Gly Glu Asn Gly Gly Asn Gly Leu Ser Leu Ala Leu 435 440 445His His Gly Asp Ile Thr Arg Pro Glu Thr Leu Arg Arg Leu Asp Val 450 455 460His Ser Tyr Asp Ser Val Ile Val Leu Gly Arg Asp Pro Ala Pro Gly465 470 475 480Gln Pro Pro Asp Asp Pro Asp Asn Arg Thr Leu Val Thr Leu Leu Leu 485 490 495Leu Arg Gln Leu Glu Glu Ala Thr Gly Arg Glu Leu Pro Val Val Thr 500 505 510Glu Leu Ile Asp Asp Arg Asn Arg Ala Leu Ala Pro Ile Gly Pro Gly 515 520 525Ala Asp Val Ile Ile Ser Gly Lys Leu Ile Gly Leu Leu Met Ser Gln 530 535 540Ile Ser Gln Asn Arg His Leu Ala Ala Val Phe Glu Glu Leu Phe Ser545 550 555 560Ala Glu Gly Ala Gly Val Arg Leu Arg Pro Ala Thr Asp Tyr Leu Leu 565 570 575Pro Gly Ser Thr Thr Ser Phe Ala Thr Val Val Ala Ala Ala Arg Arg 580 585 590Arg Gly Glu Cys Ala Ile Gly Tyr Arg Asp His Ala Asp Ala Ser Thr 595 600 605Arg Pro His Tyr Gly Val Arg Ile Asn Pro Pro Lys Arg Glu Arg Arg 610 615 620Arg Trp Thr Ala Glu Asp Glu Val Val Val Ile Gly Thr Asp625 630 63536165PRTStreptomyces parvulus Tu4055 36Met Pro Ser Thr Asp Val Val Glu Leu Ile Leu Arg Asp His Arg Arg1 5 10 15Met Glu Glu Leu Phe Arg Thr Leu Arg Asn Val Glu Ala Asp Arg Ala 20 25 30Ala Ala Leu Thr Glu Phe Ala Asp Leu Leu Ile Ala His Ala Ser Ala 35 40 45Glu Glu Asp Glu Val Tyr Pro Ala Leu Arg Arg Tyr Lys Asn Val Glu 50 55 60Gly Glu Asp Val Asp His Ser Val His Glu His His Glu Ala Asn Glu65 70 75 80Ala Leu Leu Ala Leu Leu Glu Val Glu Asp Thr Ala Ser Asp Glu Trp 85 90 95Asp Asp Lys Leu Glu Glu Leu Val Thr Ala Val Asn His His Ala Asp 100 105 110Glu Glu Glu Arg Thr Leu Leu Asn Asp Ala Arg Glu Asn Val Ala Asp 115 120 125Asp Arg Arg Arg Glu Leu Gly Gln Lys Phe Gln Glu Ala Arg Ser Arg 130 135 140Tyr Leu Glu Thr Gly Cys Gly Ser Val Glu Asn Val Arg Lys Leu Val145 150 155 160Ala Ala Ala Asp Asp 16537787PRTStreptomyces parvulus Tu4055 37Met Ala Arg Arg Leu Thr Glu Gly Arg Thr Arg Arg Glu Lys Gly Glu1 5 10 15His Met Gln Lys Pro His Gly Glu Glu Ser Glu Thr Ser Leu Ser Val 20 25 30Thr Pro Pro Lys Lys Trp Ala Ala Gly Val Pro Ala Val Val His Ala 35 40 45Leu Glu Tyr Ser Leu Glu Gln Thr Ser Pro Arg Arg Thr Gly Val Asp 50 55 60Leu Leu Thr Met Asn Gln Val Gly Gly Ile Asp Cys Pro Gly Cys Ala65 70 75 80Trp Ala Asp Pro Ala Pro Gly Arg Arg His Arg Asn Glu Tyr Cys Glu 85 90 95Asn Gly Ala Lys His Ile Asn Asp Glu Ala Thr Thr Arg Arg Val Thr 100 105 110Ala Asp Phe Phe Arg Glu His Ser Val Ala Asp Leu Ala Gly Arg Ser 115 120 125Asp Met Trp Leu Asn Gln Gln Gly Arg Leu Thr Glu Pro Met Ile Lys 130 135 140Arg Pro Gly Ser Ala His Tyr Glu Pro Ile Gly Trp Asn Asp Ala Leu145 150 155 160Gly Val Leu Ala Glu Glu Leu Lys Ser Leu Ala Ser Pro Asp Glu Ala 165 170 175Val Phe Tyr Thr Ser Gly Arg Ala Ser Asn Glu Ala Ala Phe Val Leu 180 185 190Gln Leu Phe Ala Arg Ala Phe Gly Thr Asn Asn Leu Pro Asp Cys Ser 195 200 205Asn Met Cys His Glu Ser Ser Gly Phe Ala Leu Ser Glu Thr Leu Gly 210 215 220Thr Gly Lys Gly Thr Val Gly Leu Asp Asp Leu His His Ala Asp Leu225 230 235 240Ile Phe Leu Val Gly Gln Asn Pro Gly Ser Asn His Pro Arg Gln Leu 245 250 255Ser Ala Leu Glu Glu Ala Lys Arg Asn Gly Ala Arg Ile Val Ala Val 260 265 270Asn Pro Leu Pro Glu Ala Gly Leu Arg Arg Phe Lys Asn Pro Gln Gln 275 280 285Pro Arg Gly Val Val Gly Arg Gly Thr Arg Ile Ala Asp Arg Phe Leu 290 295 300His Ile Lys Pro Gly Gly Asp Leu Ala Leu Phe Gln Ala Leu Asn Arg305 310 315 320Leu Leu Leu Glu Ala Glu Asp Ala Arg Pro Gly Thr Val Leu Asp His 325 330 335Asp Phe Ile Asp Ala His Thr Thr Gly Phe Glu Glu Phe Ala Arg His 340 345 350Ala Arg Thr Val Asp Trp Asp Asp Val Arg Ala Ala Thr Gly Leu Thr 355 360 365Arg Glu Glu Ile Glu Lys Val Arg Asp Glu Val Leu Asp Ser Glu Arg 370 375 380Val Val Val Cys Trp Ala Met Gly Ile Thr Gln His Lys His Gly Val385 390 395 400Pro Thr Val Arg Glu Ile Val Asn Phe Leu Met Leu Arg Gly Asn Leu 405 410 415Gly Arg Ala Gly Thr Gly Ala Cys Pro Val Arg Gly His Ser Asn Val 420 425 430Gln Gly Asp Arg Thr Met Gly Ile Trp Glu Gln Met Pro Asp Thr Phe 435 440 445Leu Asp Ala Leu Arg Asp Glu Phe Gly Phe Glu Pro Pro Arg Ala His 450 455 460Gly Leu Asp Ser Val Asn Ser Ile Lys Ala Met Arg Glu Gly Arg Val465 470 475 480Lys Val Phe Leu Ala Leu Ala Gly Asn Phe Val Arg Ala Ala Pro Asp 485 490 495Ser Glu Val Thr Glu Glu Ala Met Arg Ser Cys Arg Leu Thr Ala His 500 505 510Ile Ser Thr Lys Leu Asn Arg Ser His Thr Val Cys Gly Asp Thr Ala 515 520 525Leu Ile Leu Pro Thr Leu Gly Arg Thr Glu Arg Asp Val Gln Ala Asp 530 535 540Gly Glu Gln Phe Val Thr Val Glu Asn Ser Met Ser Glu Val His Thr545 550 555 560Ser Arg Gly Arg Leu Ala Pro Ala Ser Pro Met Leu Leu Ser Glu Ile 565 570 575Ala Ile Leu Cys Arg Leu Ala Arg Leu Thr Leu Asp Gly Arg Val Glu 580 585 590Ile Pro Trp Glu Thr Phe Glu Gly Asp Tyr His Thr Ile Arg Asp Arg 595 600 605Ile Ala Arg Ile Val Pro Gly Phe His Asp Phe Asn Ala Arg Val Thr 610 615 620Arg Pro Gly Gly Phe Gln Leu Pro Asn Pro Val Asn Glu Gly Val Phe625 630 635 640Asn Thr Glu Val Gly Lys Ala Leu Phe Thr Arg Asn Glu Ser Val Val 645 650 655Pro Arg Ala Pro Glu Gly His Leu Leu Leu Gln Thr Leu Arg Ser His 660 665 670Asp Gln Trp Asn Thr Val Pro Tyr Thr Asp Asn Asp Arg Tyr Arg Gly 675 680 685Ile His Gly Ser Arg His Val Val Leu Val Asn Pro Ala Asp Leu Ser 690 695 700Glu Leu Gly Leu Ala Gln Gly Asp Arg Val Asp Leu Val Ser Val Trp705 710 715 720Ala Asp Gly Thr Glu Arg Arg Ala Glu Asn Phe Gln Val Val Pro Tyr 725 730 735Pro Ala Ala Lys Gly Ser Ala Ala Ala Tyr Tyr Pro Glu Thr Asn Val 740 745 750Leu Val Pro Leu Asp Ser Val Ala Asp Ile Ser Asn Gln Pro Thr Ser 755 760 765Lys Gly Ile Val Val Arg Leu Glu Pro Val Pro Asp Arg Thr Gln Pro 770 775 780Ser Pro Ala78538206PRTStreptomyces parvulus Tu4055 38Met Ala Glu Gln His Glu Gly Pro Arg Ala Val Pro Asp Thr Pro Gly1 5 10 15Ala Arg Thr Ser Gly Asp Arg Ser Thr Gly Arg Arg Pro Leu Arg Glu 20 25 30Arg His Val Asp Gln Thr Val Glu Val Ala Val Pro Val Arg Thr Ala 35 40 45Tyr Asn Gln Trp Thr Gln Phe Lys Ser Phe Pro Arg Phe Ser Ala Val 50 55 60Val Arg Asp Val Glu Gln Val Arg Pro Thr Val Thr Ala Trp Thr Leu65 70 75 80Gly Tyr Gly Pro Leu Arg Arg Arg Phe Ala Val Glu Ile Leu Glu Gln 85 90 95Asp Pro Asp Ala Tyr Leu Ala Trp Arg Gly Leu Glu Gln Arg Pro Trp 100 105 110His Arg Gly Glu Val Glu Phe Arg Pro Thr Glu Ser Gly Gly Thr Ala 115 120 125Ile Thr Val Arg Val Leu Leu Glu Pro Arg Gly Ala Ala Arg Ile Leu 130 135 140Thr Arg Ser Ser Arg Ala Val Arg Leu Thr Thr Arg Leu Val His Gly145 150 155 160Glu Leu Thr Arg Phe Lys Arg Phe Met Glu Gly Leu Gly Gln Glu Gly 165 170 175Gly Ala Trp Arg Gly Thr Ile Arg Asn Gly Arg Val Gln His Asp Arg 180 185 190Pro Glu Pro Pro Arg Ser Arg Val Ala Arg Trp Pro Val Gly 195 200 20539251PRTStreptomyces parvulus Tu4055 39Met Leu Leu Leu Ile Ser Pro Asp Gly Val Glu Glu Ala Leu Asp Cys1 5 10 15Ala Lys Ala Ala Glu His Leu Asp Ile Val Asp Val Lys Lys Pro Asp 20 25 30Glu Gly Ser Leu Gly Ala Asn Phe Pro Trp Val Ile Arg Glu Ile Arg 35 40 45Asp Ala Val Pro Ala Asp Lys Pro Val Ser Ala Thr Val Gly Asp Val 50 55 60Pro Tyr Lys Pro Gly Thr Val Ala Gln Ala Ala Leu Gly Ala Val Val65 70 75 80Ser Gly Ala Thr Tyr Ile Lys Val Gly Leu Tyr Gly Cys Thr Thr Pro 85 90 95Glu Gln Gly Ile Ala Val Met Arg Ala Val Val Arg Ala Val Lys Asp 100 105 110His Arg Pro Glu Ala Leu Val Val Ala Ser Gly Tyr Ala Asp Ala His 115 120 125Arg Ile Gly Cys Val Asn Pro Leu Ala Leu Pro Asp Ile Ala Ala Arg 130 135 140Ser Gly Ala Asp Ala Ala Met Leu Asp Thr Ala Val Lys Asp Gly Thr145 150 155 160Arg Leu Phe Asp His Val Pro Pro Asp Thr Cys Ala Glu Phe Val Arg 165 170 175Arg Ala His Ala Ala Gly Leu Leu Ala Ala Leu Ala Gly Ser Val Arg 180 185 190Gln Thr Asp Leu Gly Arg Leu Thr Arg Ile Gly Thr Asp Ile Val Gly 195 200 205Val Arg Gly Ala Val Cys Glu Gly Gly Asp Arg Asn Ala Gly Arg Ile 210 215 220Arg Pro His Leu Val Ala Ala Phe Arg Ser Glu Met Asp Arg His Ala225 230 235 240Arg Glu His Arg Ala Gly Val Thr Thr Ala Ser 245 25040467PRTStreptomyces parvulus Tu4055 40Met Pro Thr Pro Ala Pro Asp His Ala Pro Ala Gln Arg Ala Ala Pro1 5

10 15Leu Ala Val Val Asp Pro Ala Thr Gly Thr Val Phe Asp Glu Ala Pro 20 25 30Asp Gln Gly Pro Asp Val Leu Asp Ala Val Val Asp Arg Ala Arg Arg 35 40 45Ala Trp His Gly Trp Arg Ala Asp Pro Asp Ala Arg Thr Thr Ala Leu 50 55 60Arg Ser Ala Ala Asp Ala Val Glu Ala Ala Gly Asp Asp Leu Ala Arg65 70 75 80Leu Leu Thr Arg Glu Gln Gly Lys Pro Leu Ala Glu Ser His Ala Glu 85 90 95Val Ala Arg Thr Ala Ala Arg Leu Arg Tyr Phe Ala Gly Leu Ala Pro 100 105 110Arg Thr Arg Arg Ile Thr Asp Gly Arg Pro Val Arg Ser Glu Val Arg 115 120 125Trp Arg Pro Leu Gly Pro Val Ala Ala Ile Val Pro Trp Asn Phe Pro 130 135 140Leu Gln Leu Ala Ser Ala Lys Phe Ala Pro Ala Leu Ala Ala Gly Asn145 150 155 160Thr Met Val Leu Lys Pro Ser Pro Phe Thr Pro Leu Ala Thr Arg Leu 165 170 175Leu Gly Ser Val Leu Ala Thr Ala Leu Pro Glu Asp Val Leu Thr Val 180 185 190Val Thr Gly Arg Glu Pro Leu Gly Ala Arg Leu Ala Ala His Pro Gly 195 200 205Ile Arg His Val Thr Phe Thr Gly Ser Val Pro Thr Gly Arg Ala Val 210 215 220Ala Arg Ala Ala Ala Ala Ser Leu Ala Arg Val Thr Leu Glu Leu Gly225 230 235 240Gly Asn Asp Ala Ala Val Leu Leu Asp Asp Val Glu Val Asp Arg Ile 245 250 255Ala Asp Arg Leu Phe Trp Ala Ala Phe Arg Asn Cys Gly Gln Val Cys 260 265 270Met Ala Val Lys Arg Val Tyr Ala Pro Ala Arg Leu His Ala Gln Val 275 280 285Val Glu Ala Leu Thr Glu Arg Ala Lys Ala Val Ala Val Gly Pro Gly 290 295 300Leu Asp Pro Arg Thr Arg Leu Gly Pro Val Ala Asn Ala Pro Gln Leu305 310 315 320Ala Arg Val Glu Gln Ile Thr Arg Arg Ala Leu Ala Asp Gly Ala Arg 325 330 335Ala Ala Ala Gly Gly His Arg Leu Asp Gly Pro Gly Cys Phe Phe Ala 340 345 350Pro Thr Ile Leu Thr Asp Val Pro Pro Asp Ser Pro Val Val Thr Glu 355 360 365Glu Gln Phe Gly Pro Val Leu Pro Val Leu Pro Tyr Arg Ser Leu Asp 370 375 380Glu Ala Val Asp Ala Ala Asn Gly Thr Gly Phe Gly Leu Gly Gly Ser385 390 395 400Val Trp Gly Thr Asp Leu Asp Arg Ala Glu Ala Val Ala Asp Arg Leu 405 410 415Glu Cys Gly Thr Ala Trp Val Asn His His Ala Glu Leu Ser Leu Ala 420 425 430Gln Pro Phe Ala Gly Asp Lys Asp Ser Gly Val Gly Val Ala Gly Gly 435 440 445Pro Trp Gly Leu Tyr Gly Asn Leu Arg Pro Phe Val Val His Arg Pro 450 455 460Arg Gly Glu46541368PRTStreptomyces parvulus Tu4055 41Met Ser Phe Arg Ala Ala Val Leu Arg Gly Tyr Glu Asp Pro Phe Thr1 5 10 15Val Glu Glu Val Thr Leu Gly Thr Glu Pro Gly Ala Gly Glu Ile Leu 20 25 30Val Glu Ile Ala Gly Cys Gly Met Cys Arg Thr Asp Leu Ala Val Arg 35 40 45Arg Ser Ala Gly Arg Ser Pro Leu Pro Ala Val Leu Gly His Glu Gly 50 55 60Ser Gly Val Val Val Arg Thr Gly Gly Gly Pro Asp Thr Ala Ile Gly65 70 75 80Val Gly Asp His Val Val Leu Ser Phe Asp Ser Cys Gly His Cys Arg 85 90 95Asn Cys Arg Ala Ala Ala Pro Ala Tyr Cys Asp Ser Phe Ala Ser Leu 100 105 110Asn Leu Phe Gly Gly Arg Ala Glu Asp Pro Pro Arg Leu Thr Asp Gly 115 120 125Ser Gly Ala Ala Leu Ala Pro Arg Trp Phe Gly Gln Ser Ala Phe Ala 130 135 140Glu Tyr Ala Leu Val Ser Ala Arg Asn Ala Val Arg Val Asp Pro Ala145 150 155 160Leu Pro Val Glu Leu Leu Gly Pro Leu Gly Cys Gly Phe Leu Thr Gly 165 170 175Ala Gly Ala Val Leu Asn Thr Phe Ala Ala Gly Pro Gly Asp Thr Leu 180 185 190Val Val Leu Gly Ala Gly Ala Val Gly Leu Ala Ala Val Met Ala Ala 195 200 205Thr Ala Ala Gly Ala Pro Ser Val Ala Val Asp Arg Asn Pro Arg Arg 210 215 220Leu Glu Leu Ala Glu Arg Phe Gly Ala Val Pro Leu Pro Ala Ala Thr225 230 235 240Ala Gly Leu Ala Glu Arg Ile Arg Arg Leu Thr Asp Gly Gly Ala Arg 245 250 255Tyr Ala Leu Asp Thr Thr Ala Ser Val Pro Leu Ile Asn Glu Ala Leu 260 265 270Arg Ala Leu Arg Pro Thr Gly Ala Leu Gly Leu Val Ala Arg Leu His 275 280 285Thr Ala Leu Pro Leu Glu Pro Gly Thr Leu Asp Arg Gly Arg Ser Ile 290 295 300Arg His Val Cys Glu Gly Asp Ala Val Pro Gly Leu Leu Ile Pro Gln305 310 315 320Leu Thr Arg Leu Trp Gln Ala Gly Arg Phe Pro Phe Asp Gln Leu Val 325 330 335Arg Thr Tyr Pro Leu Ala Asp Ile Asn Glu Ala Glu Arg Asp Cys Asp 340 345 350Ala Gly Leu Val Val Lys Pro Val Leu Leu Pro Pro Ala Arg Ser Arg 355 360 36542301PRTStreptomyces parvulus Tu4055 42Met Thr Gly Thr Ala Pro Gln Tyr Thr Asp Val Glu Gly Val Asn Gly1 5 10 15Gly Val Gly Leu Thr Ala Phe Leu Val Ala Ala Ala Arg Ala Ile Glu 20 25 30Thr His Arg Asp Asp Ser Leu Ala Gln Asp Val Tyr Ala Glu His Phe 35 40 45Val Arg Ala Ala Pro Ala Cys Ala Asp Trp Pro Val Arg Ile Glu Gln 50 55 60Val Pro Asp Gly Asp Gly Asn Pro Leu Trp Gly Arg Phe Ala Arg Tyr65 70 75 80Phe Gly Leu Arg Thr Arg Ala Leu Asp Asp Phe Leu Leu Arg Ser Val 85 90 95Arg Thr Gly Pro Arg Gln Val Val Leu Leu Gly Ala Gly Leu Asp Thr 100 105 110Arg Ala Phe Arg Leu Asp Trp Pro Ser Gln Cys Ala Val Phe Glu Ile 115 120 125Asp Arg Thr Gly Val Leu Ala Phe Lys Gln Gln Val Leu Thr Asp Leu 130 135 140Ala Ala Thr Pro Arg Val Glu Arg Val Pro Val Pro Val Asp Leu Arg145 150 155 160Ala Asp Trp Ala Gly Ala Leu Thr Ala Ala Gly Phe Asp Pro Ala Ala 165 170 175Pro Ser Val Trp Leu Ala Glu Gly Leu Leu Phe Tyr Leu Pro Gly Pro 180 185 190Ala Glu Ser Leu Leu Val Asp Thr Val Asp Arg Leu Thr Thr Asp Gly 195 200 205Ser Ala Leu Ala Phe Glu Ala Lys Leu Glu Lys Asp Leu Leu Ala Tyr 210 215 220Arg Asp Ser Ala Ile Tyr Thr Ala Thr Arg Glu Gln Ile Gly Ile Asp225 230 235 240Leu Leu Arg Leu Phe Asp Lys Gly Pro Arg Pro Asp Ser Ala Gly Glu 245 250 255Leu Ala Ala Arg Gly Trp Ser Thr Ser Met His Thr Pro Phe Val Phe 260 265 270Thr His Arg Tyr Gly Arg Gly Pro Leu Pro Glu Pro Asn Asp Ala Leu 275 280 285Glu Gly Asn Arg Trp Val Phe Ala Arg Lys Pro Gly Pro 290 295 30043179PRTStreptomyces parvulus Tu4055 43Met Cys Met Arg Asp Glu Ala Ala Lys Arg Val Glu Leu Val Phe Ser1 5 10 15Leu Phe Asp Ala Asn Gly Asn Gly Val Ile Asp Ser Asp Asp Phe Asp 20 25 30Leu Met Thr Asp Arg Val Val Ala Ala Ala Ala Gly Ser Asp Asp Ser 35 40 45Ala Lys Ala Ala Val Arg Ala Ala Phe Arg Arg Tyr Trp Thr Thr Leu 50 55 60Ala Thr Glu Leu Asp Ala Asp Gly Asp Gly Val Ile Thr Val Glu Glu65 70 75 80Phe Arg Pro Phe Val Leu Asp Pro Glu Arg Phe Gly Pro Thr Ile Ala 85 90 95Glu Phe Ala Arg Ala Leu Ser Ala Leu Gly Asp Pro Asp Gly Asp Gly 100 105 110Leu Ile Glu Arg Pro Leu Phe Val Ala Leu Met Lys Ala Ile Gly Phe 115 120 125Glu Glu Ala Asn Ile His Ala Leu Phe Asp Ala Phe Ala Pro Asp Ala 130 135 140Ala Asp Arg Ile Thr Val Ala Ala Trp Ala Ser Gly Ile Glu Asp Tyr145 150 155 160Tyr Ala Pro Asp Leu Ala Gly Ile Pro Gly Asp Arg Leu Val Ala Ala 165 170 175Arg Thr Val4433DNAArtificial SequenceDescription of Artificial Sequence oligo CM410 44aaaatgcatt cggcctgaac ggccccgctg tca 334533DNAArtificial SequenceDescription of Artificial Sequence oligo CM411 45aaatggccag cgaacaccaa caccacacca cca 334632DNAArtificial SequenceDescription of Artificial Sequence oligo CM412 46aaagtcctag gcggcggccg gcgggtcgac ct 324735DNAArtificial SequenceDescription of Artificial Sequence oligo CM413 47tttagatctc gcgacgtcgc acgcgccgaa cgtca 354834DNAArtificial SequenceDescription of Artificial Sequence oligo CM414 48aaactgcaga gtcgaacatc ggtcacacgc aggc 344935DNAArtificial SequenceDescription of Artificial Sequence oligo CM415 49aaaatgcatg atccacatcg atacgacgcg cccga 355036DNAArtificial SequenceDescription of Artificial Sequence oligo CM416 50taaatgcatt ccattcggtg caggtggagt tgatcc 365136DNAArtificial SequenceDescription of Artificial Sequence oligo CM417 51ataggatccc ctccgggtgc tccagaccgg ccaccc 365235DNAArtificial SequenceDescription of Artificial Sequence oligo CM368 52tttcctgcag gccatcccca cgatcgcgat cggct 355335DNAArtificial SequenceDescription of Artificial Sequence oligo CM369 53tttcatatga caggcagtgc tgtttcggcc ccatt 355436DNAArtificial SequenceDescription of Artificial Sequence oligo CM370 54tttcatatgg cggatgccgt acgtgccgcc ggcgct 365532DNAArtificial SequenceDescription of Artificial Sequence oligo CM371 55tttcatatgc cccaggcgat cgtccgcacc ac 325641DNAArtificial SequenceDescription of Artificial Sequence oligo CM372 56tttcatatgg tctcggcccc ccacacaaga gccctccggg c 415720DNAArtificial SequenceDescription of Artificial Sequence oligo B1819A 57gtcatgcatg cggcgggctc 205820DNAArtificial SequenceDescription of Artificial Sequence oligo B1819B 58ggtctagaac ggccgaactt 205920DNAArtificial SequenceDescription of Artificial Sequence oligo B1819C 59gttctagaac ctcggtcggc 206020DNAArtificial SequenceDescription of Artificial Sequence oligo B1819D 60ctggatccca cgctgctgcg 206119DNAArtificial SequenceDescription of Artificial Sequence oligo BLDA 61ggagacttac gggggatgc 196219DNAArtificial SequenceDescription of Artificial Sequence oligo BLDB 62ctccagcagc gaccagaac 196320DNAArtificial SequenceDescription of Artificial Sequence oligo B19A 63cccatgcatc accgacatac 206420DNAArtificial SequenceDescription of Artificial Sequence oligo B19B 64gcgatatccc gaagaacgcg 206520DNAArtificial SequenceDescription of Artificial Sequence oligo B1920A 65gccaagcttc ctcgacgcgc 206620DNAArtificial SequenceDescription of Artificial Sequence oligo B1920B 66cactagtgcc tcacccagtt 206720DNAArtificial SequenceDescription of Artificial Sequence oligo B1920C 67cactagtgac ggccgaagcg 206820DNAArtificial SequenceDescription of Artificial Sequence oligo B1920D 68tcggatccgt cagaccgttc 206936DNAArtificial SequenceDescription of Artificial Sequence oligo CM384 69aacctgcagg taccccggtg gggtgcggtc gcccga 367024DNAArtificial SequenceDescription of Artificial Sequence oligo CM385 70cgccgcacgc gtcgaagcca acga 247124DNAArtificial SequenceDescription of Artificial Sequence oligo CM386 71tgtgggctgg tcgttggctt cgac 247234DNAArtificial SequenceDescription of Artificial Sequence oligo CM387 72ggtgcctgca gcgtgagttc ctcgacggat ccga 347326DNAArtificial SequenceDescription of Artificial Sequence oligo CM388 73gaggaactca ccctgcaggc accgct 267426DNAArtificial SequenceDescription of Artificial Sequence oligo CM395 74cgaacgtcca gccctcgggc atgcgt 267528DNAArtificial SequenceDescription of Artificial Sequence oligo CM396 75tggcacgcat gcccgagggc tggacgtt 287635DNAArtificial SequenceDescription of Artificial Sequence oligo CM397 76tttcctgcag gccatgccga cgatcgcgac aggct 357736DNAArtificial SequenceDescription of Artificial Sequence oligo CM398 77aaacatatgg tcctggcgct gcgcaacggg gaactg 367835DNAArtificial SequenceDescription of Artificial Sequence oligo CM399 78tttcctgcag gcgatgccga cgatggcgat gggct 357943DNAArtificial SequenceDescription of Artificial Sequence oligo CM400 79aaacctgcag gttccccggc gacgtggact cgccggagtc gtt 438041DNAArtificial SequenceDescription of Artificial Sequence oligo CM401 80ttttctagag cgacgtcgca ggcggcgatg gtcacgcccg t 418120DNAArtificial SequenceDescription of Artificial Sequence oligo B25A 81ttctgcagcc gcggccttcg 208220DNAArtificial SequenceDescription of Artificial Sequence oligo B25B 82agaattcgcc ggcgccgctg 208320DNAArtificial SequenceDescription of Artificial Sequence oligo B7T1 83ggctgcagac gcggctgaag 208420DNAArtificial SequenceDescription of Artificial Sequence oligo B7T2 84ccggatccca gagccacgtc 208520DNAArtificial SequenceDescription of Artificial Sequence oligo BP4501 85cgtatgcatg gcgccatgga 208620DNAArtificial SequenceDescription of Artificial Sequence oligo BP4502 86agccaattgg tgcactccag 208720DNAArtificial SequenceDescription of Artificial Sequence oligo BNHT1 87gtcatgcatc agcgcacccg 208820DNAArtificial SequenceDescription of Artificial Sequence oligo BNHT2 88gtgcaattgc cctggtagtc 208920DNAArtificial SequenceDescription of Artificial Sequence oligo BTRNAS1 89tgtctagact cgcgcgaaca 209020DNAArtificial SequenceDescription of Artificial Sequence oligo BTRNAS2 90tgaattccga agggggtggt 209120DNAArtificial SequenceDescription of Artificial Sequence oligo B5B 91aactagtccg cagtggaccg 209220DNAArtificial SequenceDescription of Artificial Sequence oligo B5A 92tcgatatcct caccgcccgt 209320DNAArtificial SequenceDescription of Artificial Sequence oligo B6B 93aactagtgtg gcagacggtc 209420DNAArtificial SequenceDescription of Artificial Sequence oligo B5A 94tcgatatcct caccgcccgt 209520DNAArtificial SequenceDescription of Artificial Sequence oligo B6T1 95cggatgcatc accggcacgg 209620DNAArtificial SequenceDescription of Artificial Sequence oligo B6T2 96tgggatccgc ggggcggtac 209720DNAArtificial SequenceDescription of Artificial Sequence oligo BBB 97aactagtgcg atcccgggga 209820DNAArtificial SequenceDescription of Artificial Sequence oligo BBA 98cgtcgatatc ctccaggggc 209920DNAArtificial SequenceDescription of Artificial Sequence oligo BBT1 99tactgcagca cacccggtgc 2010020DNAArtificial SequenceDescription of Artificial Sequence oligo BBT2 100tgggatccgc tgtgtcatat 2010120DNAArtificial SequenceDescription of Artificial Sequence oligo BCB 101cactagtcct cgccgggcac

2010220DNAArtificial SequenceDescription of Artificial Sequence oligo BCA 102gaggatcccg gtcagcggca 2010320DNAArtificial SequenceDescription of Artificial Sequence oligo BCT1 103gcctgcagcg acctcgccgg 2010420DNAArtificial SequenceDescription of Artificial Sequence oligo BCT2 104cgggatcccg tggcgtggtc 2010520DNAArtificial SequenceDescription of Artificial Sequence oligo B23A 105atctgcagcg gcatcggtgt 2010620DNAArtificial SequenceDescription of Artificial Sequence oligo B23B 106agaattctcc actgcggtcg 2010720DNAArtificial SequenceDescription of Artificial Sequence oligo B9A 107acctgcaggc cgggctcatc 2010820DNAArtificial SequenceDescription of Artificial Sequence oligo B9B 108agaattcggg cgagccgccg 2010920DNAArtificial SequenceDescription of Artificial Sequence oligo B231 109atcaagcttc gtgtccatgg 2011020DNAArtificial SequenceDescription of Artificial Sequence oligo B232 110gtcatgcatc aggcgttcgg 2011120DNAArtificial SequenceDescription of Artificial Sequence oligo B251 111cttctagatg aacccctcca 2011220DNAArtificial SequenceDescription of Artificial Sequence oligo B252 112gggcaattgc gcggcagctt 2011390PRTStreptomyces parvulus Tu4055 113Met Leu Gly Phe Tyr Ala Leu Leu Leu Ala Pro Ala Glu Leu Asp Leu1 5 10 15Leu Phe Val Gln Asp Gly Thr Gln Gly Arg Gly Ile Gly Arg Leu Leu 20 25 30Val Asp His Met Lys Arg Arg Ala Arg Ala Ala Gly Leu Asp Arg Val 35 40 45Arg Val Val Ser His Pro Pro Ala Glu Gly Phe Tyr Arg Ala Val Gly 50 55 60Ala Leu Pro Thr Gly Thr Ala Arg Ala Asn Pro Pro Ala Val Ala Trp65 70 75 80Asp Arg Pro Val Leu Glu Phe Leu Ile Pro 85 90

Claims

1-73. (canceled)

74. A host cell capable of expressing a polyketide synthase for borrelidin or a borrelidin derivative or analogue, in which a borrelidin biosynthetic gene involved in production of the borrelidin starter unit in said cell, has been deleted, disrupted, or otherwise inactivated wherein said gene is selected from the list consisting of borC, borD, borE, borF, borG, borH, borK, borL, borM, and borN.

75. The host cell according to claim 74 wherein the gene is borG.

76. The host cell according to claim 74 wherein the gene is borE.

77. The host cell according to claim 74 in which one or more borrelidin biosynthesis genes or borrelidin polyketide synthase domains or modules are additionally deleted, modified or replaced.

78. The host cell according to claim 74 which is an Actinomycete.

79. The host cell according to claim 74 which is a Streptomycete.

80. The host cell according to claim 74 wherein the host cell is selected from the group consisting of Saccharopolyspora erythraea, Streptomyces coelicolor, Streptomyces avermitilis, Streptomyces griseofuscus, Streptomyces cinnamonensis, Micromonospora griseorubida, Streptomyces hygroscopicus, Streptomyces fradiae, Streptomyces longisporoflavus, Streptomyces lasaliensis, Streptomyces tsukubaensis, Streptomyces griseus, Streptomyces venezuelae, Streptomyces antibioticus, Streptomyces lividans, Streptomyces rimosus, Streptomyces albus, Streptomyces rochei ATCC23956, Streptomyces parvulus Tu113, and Streptomyces parvulus Tu4055.

81. A method for modifying a host cell to increase its capacity for the production of borrelidin, or a borrelidin derivative or analogue, the host cell being capable of expressing a polyketide synthase for borrelidin or said derivative or analogue, the method comprising deleting, disrupting, or otherwise inactivating a borrelidin biosynthetic gene involved in production of the borrelidin starter unit in said cell, wherein the gene is selected from the group consisting of borC, borD, borE, borF, borG, borH, borK, borL, borM and borN.

82. The method according to claim 81 wherein the gene is borG.

83. The method according to claim 81 wherein the gene is borE.

84. The method of claim 81, wherein the gene is borG and the method additionally comprises deleting, modifying or replacing one or more borrelidin biosynthesis genes or borrelidin polyketide synthase domains or modules.

85. A method for producing borrelidin, or a borrelidin derivative or analogue, said method comprising fermenting a host cell according to claim 74 and feeding an exogenous carboxylic acid.

86. The method of claim 85 wherein the gene is borG and wherein the exogenous carboxylic acid is selected from the group consisting of trans-cyclobutane-1,2-dicarboxylic acid, 2,3-dimethylsuccinic acid, 2-methylsuccinic acid, and trans-cyclopentane-1,2-dicarboxylic acid.

87. The method according to claim 85, further comprising the step of isolating the borrelidin, borrelidin derivative or borrelidin analogue.