KR101610970B1 - Gene involved in the biosynthesis of galbonolide - Google Patents

Abstract

The present invention relates to a composition for promoting galbanolide biosynthesis comprising a gene involved in galbanolide biosynthesis, an expression vector for the gene, a method for producing galbanolide by transfecting the expression vector, To a transformed gallonolide biosynthesis strain. More specifically, the present invention has revealed that a gene encoding an amino acid sequence represented by SEQ. ID. NO. 1 to 10 is involved in galbanolide biosynthesis, and a large amount of galbanolide, an antifungal substance, is produced using a vector expressing the gene .

Description

Gene involved in the biosynthesis of galbonolide < RTI ID = 0.0 >

The present invention relates to a composition for promoting galbanolide biosynthesis comprising a gene involved in galbanolide biosynthesis, an expression vector for the gene, a method for producing galbanolide by transfecting the expression vector, To a transformed gallonolide biosynthesis strain.

Galvo surprised Id (galbonolide) A and B are Streptomyces go booth (Streptomyces

galbus ) ssp. It was first isolated in eurythermus Tu 2253 and found to have antimicrobial activity against the gray mold (Botrytis cinerea). In addition, galblonolides A and B are micro-monosporonacinonace ( Micromonospora narashinoensis and Micromonospora chalcea . They were found to be effective against wheat stem rust fungus Puccinia graminis (wheat stem rust fungus).

A slut is amazing Eid's only neo-cryptoxanthin caucus that causes yeast increased (crptococcosis) Fortis (Cryptococcus neoformans , and the like, and its antifungal effect is presumed to be caused by inhibiting the synthesis of sphingolipid.

On the other hand, polyketide compounds such as gallonolide are biosynthesized by a megasynthase called polyketide synthase (PKS). These PKS complexes consist of several modules (a bundle of catalytic domains responsible for a single condensation reaction) involved in the acyl-CoA's claisen condensation reaction, a loading module, a plurality of extender modules, and a releasing module. Depending on the configuration of the catalyst domains constituting the module, the structure of the polyketide is correspondingly biosynthesized on a one-to-one basis. The extension module of the module is composed of acyl transferase (AT), acyl carrier protein (ACP), and beta-ketoacyl synthase, which are directly involved in the condensation reaction of each carboxylic acid. Keto reductase (KR), dehydratase (DH), and enoyl reductase (ER), which are involved in the reduction of the beta-keto group resulting from the condensation reaction with the K- ) Domains. In one module, an acyl nucleoside is added, AT transfers the acyl moiety to the corresponding ACP to produce acyl-ACP, and KS is the acyl produced in the molecule added by the previous module Increases the number of carbon atoms by allowing the acyl group of the ACP to undergo a carbon-carbon bond through claisen condensation. That is, once these sets are activated, one acyl moiety can be added to increase the carbon number of the skeleton carbon chain by two.

In the PKS catalytic domain, the acyltransferase (AT) domain within each module activates the malonyl-thioester derivative and converts it to acyl carrier protein (ACP) family To the domain of the protein. Malonyl-thioester derivatives include malonyl-coenzyme A, malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, chloroethylmalonyl- (HETWEEK, 2009) ACET (ACETP), which is a member of the ACP family, has been shown to be effective in the treatment of acute myocardial infarction (ACE) The malonyl-thioester derivative attached to the domain is inserted into the polyketide chain which is synthesized via decarboxylative Claisen condensation. This carbon-carbon bond formation reaction is the β- Ketoacyl synthase (KAS) domain. Based on the chemical structure of galbanolides A and B, it was anticipated that their biosynthesis would also be achieved by the PKS system. On the other hand, it was expected that the biosynthesis of fkbGHIJK Methoxymonyl-ACP biosynthetic The role of fkbGHIJK in the biosynthesis of methoxymonyl ACP has been elucidated through genetic and biochemical experiments (Kato et al., 2000) 2002), but the precise biosynthetic process and involved genes have not yet been identified (Chern et al., 2006; Dorrestein et al., 2006).

Accordingly, the present inventors have conducted extensive studies to isolate genes involved in the biosynthesis of galboperolide A and B, which have antimicrobial activity, and have found that a gene which is not located together with the PKS gene cluster in St. It was confirmed that the toxMalonyl -ACP biosynthesis gene ( galGHIJK ) was involved in the biosynthesis of galbanolide A, and it was confirmed that the gene including the KAS domain adjacent thereto was involved in the biosynthesis process of galbanolide A and B , And the present invention was completed by analyzing the nucleotide sequence thereof.

It is an object of the present invention to provide a gene encoding a protein necessary for galbanolide biosynthesis.

Another object of the present invention is to provide a composition for promoting galbanolide biosynthesis comprising an expression vector of the gene and a method for producing galblonolide using the same.

It is still another object of the present invention to provide a galblonide biosynthesis strain transformed with an expression vector of the gene.

The present invention relates to a gene encoding a protein necessary for galbanolide biosynthesis.

The term "galbanolide" of the present invention means a polyketide compound, which exhibits an antifungal effect by inhibiting sphingolipid biosynthesis of fungal. The gallonolide of the present invention includes both gallonololide A and B. The chemical structures of the gallonolide A and B are as shown in Formulas (1) and (2).

The gene coding for the protein necessary for galblonaldehyde biosynthesis is preferably selected from the group consisting of genes coding for the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, A methoxymalonyl-acyl carrier biosynthetic gene selected from the group consisting of a gene coding for the amino acid sequence shown in SEQ ID NOs: 6 and 7, or a gene encoding the amino acid sequence shown in SEQ ID NOs: 6 and 7 Lt; / RTI >

The present invention also relates to a gene cluster encoding a protein necessary for the biosynthesis of galbanolide having the nucleic acid sequence of SEQ ID NO: 11, which comprises a gene encoding the amino acid sequence represented by SEQ ID NO: 1 to 10.

These genes are genes isolated from streptomyces galbus (KCCM 41354), which produces galbanolide.

The protein having the amino acid sequence of SEQ ID NOS: 1 to 5 is a methoxymalonate biosynthetic protein. Among them, the galG gene encoding the amino acid sequence represented by SEQ ID NO: 1 functions as methyltransferase and the galH gene encoding the amino acid sequence represented by SEQ ID NO: 2 is acyltransferase / galI gene to phophatase) function, encoding the amino acid sequence shown in SEQ ID NO: 3 is galJ gene encoding the amino acid sequence shown the function of the dehydrogenase (degydrogenase), in SEQ ID NO: 4 is an acyl-carrier protein (acyl-carrier protein, and the galK gene encoding the amino acid sequence represented by SEQ ID NO: 5 has a dehydrogenase function. The orf2 gene coding for the amino acid sequence shown in SEQ ID NO: 7 has the function of monooxygenase, and the orf3 gene coding for the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: The orf4 gene having the function of beta-ketoacyl syntase and the orf5 gene encoding the amino acid sequence of SEQ ID NO: 10 have the function of acyl transferase.

In a specific embodiment of the present invention, the methoxymalonyl-acyl carrier biosynthesis gene galGHIJK and the genes encoding the amino acid sequences represented by SEQ ID NOS: 6 to 10, orf1 to orf5 , are located adjacent to each other , It was confirmed that these genes were not adjacent to the PKS (polyketide synthase) gene as expected (Example 1). In addition, galbanolide A was not produced when a mutant plasmid having the galI gene was transfected into the S. galbus strain (Example 2), and a mutant plasmid in which the orf4 gene was disrupted was transformed into S. galbus strain It was confirmed that neither gallonolide A nor B was produced (Example 3). Therefore, the gene encoding the amino acid sequence represented by SEQ ID NOS: 1 to 10 of the present invention is a gene involved in galbanolide biosynthesis.

In addition, the present invention provides a galblonide biosynthesis gene comprising all of the above genes.

The galblonide biosynthesis gene preferably has the nucleotide sequence shown in SEQ ID NO: 11. This is characterized in that it provides the nucleotide sequence of the galblonide biosynthesis gene group having the gene map shown in Fig.

The genes provided according to the present invention may be introduced into various host cells to be useful for producing galbanolides. These genes may be used alone, or two or more of them may be used together.

In another aspect, the present invention provides a recombinant vector comprising the gal- onolide biosynthesis gene.

The recombinant vector of the present invention is prepared by inserting a gene selected from the group consisting of genes coding for the amino acid sequence shown in SEQ ID NOS: 1 to 10 into a basic vector. The basic vector that can be used in the present invention can be used without limitation as long as it is a vector generally used for cloning or expression of a gene. In addition, the vector may vary depending on the host cell. That is, when Escherichia coli is used as the host cell, it is preferable to use a vector for Escherichia coli having a replication origin of Escherichia coli, and in the case of using a yeast, a vector for yeast having the origin of replication of the yeast is preferably used . Alternatively, a shuttle vector that has both the origin of replication of E. coli and yeast may be used.

In another aspect, the present invention relates to a composition for promoting galbanolide biosynthesis comprising an expression vector for a gene involved in galbanolide biosynthesis. More specifically, the present invention relates to a composition for promoting galbanolide biosynthesis comprising an expression vector of a gene selected from the group consisting of genes coding for an amino acid sequence represented by SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. The composition may further include an expression vector of a methoxymalonyl-acyl carrier biosynthetic gene selected from the group consisting of genes encoding the amino acid sequences represented by SEQ ID NOS: 1 to 5, A gene encoding an amino acid sequence represented by SEQ ID NO: 6 or SEQ ID NO: 7, and an expression vector for a gene selected from the group consisting of genes encoding the amino acid sequence represented by SEQ ID NO: 7.

As used herein, the term " expression vector " refers to a recombinant vector capable of expressing a desired protein in a suitable host cell, which gene construct comprises an essential regulatory element operably linked to the expression of the gene insert, Standard recombinant DNA techniques can be used to produce. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. For example, a nucleic acid sequence that encodes a promoter and a protein may affect the expression of a nucleic acid sequence that is operably linked. The recombinant vector is not particularly limited as long as it expresses a desired gene in various host cells of a prokaryotic cell and functions to produce a desired protein. However, the recombinant vector has a promoter exhibiting strong activity and a form similar to a natural state A vector capable of producing a large amount of the exogenous protein of the present invention is preferable. The recombinant vector preferably contains at least a promoter, an initiation codon, a gene encoding a desired protein, and a termination codon terminator. In addition, DNA encoding the signal peptide, an operator sequence, a non-specific region on the 5 'side and a 3' side of the desired gene, a selectable marker region, or a replicable unit may suitably be contained. The initiation codon and the termination codon can generally be regarded as part of the nucleic acid sequence encoding the immunogenic target protein and must function when the gene construct is introduced and in the coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

In another aspect, the present invention relates to a method for producing galbanolides. More specifically, the production method comprises the steps of: (a) introducing the composition for promoting galvanolide biosynthesis described above into a galblonide biosynthesis strain; And (b) culturing the transformed strain to recover galbanolides from the culture; .

The galblonide biosynthesis strain includes all strains capable of producing gallonolide, and there is no limitation on the type of galblonide biosynthesis. For example, the strain may be selected from the group consisting of Streptomyces gallus galbus , < RTI ID = 0.0 > Micromonospora & lt ; / RTI > narashinoensis ) and Micromonospora chalcea ( Micromonospora chalcea ), Micromonospora sp. Strains (strains named Micromonospora sp. MA ####), and the like. For the purpose of the present invention, the strain is preferably Streptomyces galvus. More preferably, Streptomyces galvus deposited with the number KCCM 41354 can be used.

The strain culture for producing the gallonolide of the present invention can be cultured in a culture medium containing a nutrient source to which conventional microorganisms can be used. As a nutrient source of the strain, a nutrient source commonly used in the art can be used without limitation, and a known nutrient source conventionally used for culturing Streptomyces spp. Can be used. For example, a medium containing malonic acid, ethanol, methionine, a carbon source and a nitrogen source. At this time, the carbon source is preferably at least one selected from the group consisting of starch, glucose, corn oil, glycerol, maltose, mannose and inositol, and most preferably starch, glucose and corn oil. The nitrogen source is preferably at least one selected from the group consisting of cottonseed wheat, corn steep liquor, corn steep liquor, soybean meal, peptone, and yeast extract, most preferably cotton wheat and corn steep liquor.

In addition, it is preferable that the above culture is performed by primary culturing in a viable medium, collecting the cells from the culture solution, adding an organic solvent to the cells again, and then secondary culturing at 4 ° C overnight. At this time, an inducer may be added to induce the production of galblonolide in the culture medium, and a substrate of galblonolide may be further added. As the organic solvent for eluting the gallonolide from the culture solution, methanol, acetone, ethyl ether or ethyl acetate may be used.

The seed culture can be cultured in a fermentation tank containing the culture medium and cultured. As the culture method, it is possible to cultivate shaking or stationary culture under aerobic conditions. The incubation temperature may be slightly different depending on the conditions when culturing under the above conditions, but it is usually suitable to cultivate at 20-37 ° C. The incubation period can also be cultured for a known period of time as used in the art, and the period can be adjusted as necessary. In addition, the recovery of the gallonolide from the culture can be carried out by a method known in the art using high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

In another aspect, the present invention provides a galblonide biosynthesis strain transformed by an expression vector comprising the galblonide biosynthesis gene.

Wherein said strain is a methoxymalonyl-acyl carrier biosynthetic gene selected from the group consisting of genes encoding the amino acid sequences of SEQ ID NOS: 1 to 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

The galvanolide biosynthesis strain transformed with the expression vector containing the galblonolite biosynthesis gene can increase the biosynthesis of galboperolide which is antimicrobial due to the increase of the expression and activity of the protein necessary for biosynthesis.

As the genes and genes involved in biosynthesis of galanceinol have been identified by the present invention, it has become possible to control the biosynthesis of galanceolide by using this. Also, by introducing an expression vector of a gene involved in biosynthesis into a strain, it is possible to produce a large amount of gallonolide having an antifungal activity.

Brief Description of the Drawings Figure 1 shows the organization diagram of the methoxymaronyl-ACP biosynthesis gene and its neighboring genes isolated from streptomyces galbus ( GenBank accession no. GU300145).
FIG. 2 shows the organization of the KAS- related gene domain located next to the galGHIJK gene, which was compared with homology with Burkholderia multivorans ATCC 17161 chromosome 1 (GenBank accession No. CP000868) will be. The identity of the domains was determined using pairwise alignments in BLAST-P of NCBI. The homology of orf4 and Bmul_1953 was 32%. orf3 was predicted as a KASIII (FabH) -like protein, but no catalytic residue (Cys-His-Asn). In KAS, KAS I / II (FabB) has a catalytic residue consisting of Cys-His-His, and FabB and FabH have no significant structural homology.
FIG. 3 shows the results of observing the change of antifungal activity when galI was destroyed in S. galbus . (a) shows a Kpnl -restriction map of WT and SK- galI- 5 chromosome with pD- galI . The genetic organization of galGHI was expressed as a 3.2 kb Kpnl fragment of WT. (b) shows Southern hybridization analysis results of Kpnl -digested chromosomes of WT (1) and SK- galI- 5 (2). (c) is a TLC analysis of the antifungal effect on cryptococcus neoformans. (c) is a floating extract, and (d) is a mycelium extract. Also, (e) shows the TLC analysis of the suspension extract, and (f) shows the TLC analysis result of the mycelium extract.
Figure 4 shows the HPLC-MS chromatogram results of the ethyl acetate extracts of the WT (a) and SK- gal I -5 (b) culture suspensions. The EIC of m / z 381 (1) and m / z 365 (3) in positive ionization mode, m / z 379 (2) and m / z 363 (4) in negative ionization mode, ), And the molecular ion peaks of the gallonolides A and B are represented by circles. (c) for 7.0-8.0 min (1) and 8.0-9.0 min (2) were used for the analysis of antifungal activity after silica gel TLC separation.
FIG. 5 shows HPLC-MS chromatograms of suspension extracts (A and B) and mycelium extracts (C and D).
Figure 6 shows the effect of orf4 gene disruption and its production on gallonolide production.
Figure 7 shows the antifungal effect on cryptococcus neoformans by TLC analysis. A shows the antifungal effect of the culture supernatant extract of WT, dKS-6 and dKS-7, and B shows the effect of mycelial extract of WT, dKS-6 and dKS-7.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Reference example  1: Materials and methods

Bacterial strains, media and genetic procedures

Streptomyces galbus KCCM 41354 was obtained from the Korean Microorganism Conservation Center. Escherichia coli DH5a was used as a host for general subcloning. For total DNA isolation, S. galbus was developed in tryptic soy broth with 10 mM MgCl ₂ .6H ₂ O and 0.5% w / v glycine. Glucose-yeast extract-malt extract (GYM) agar was used for S. galbus seed preparation. Escherichia ^{coli ET12567 (dam _, dcm _} , hsdS _) / pUZ8002 was non-flammable methyl plasmid provides the gene for strain cross-bonded transition to S. galbus (Flett et al., 1997). 10 mM MgCl ₂ - added GYM agar was used for the bonding experiments. Genetic procedures including genetic defect experiments were performed using standard procedures (Kieser et al., 2000). Southern hybridization was performed using a deletion kit from Roche Diagnostics (Pleasanton, Calif.) And digoxigenin DNA markers according to the procedures outlined by the manufacturer.

Cloning procedure

The 800-bp DNA fragment of the fkbI homolog from the S. galbus chromosome was amplified using PCR and primer sets of 5'-CAGGGCATGGCCGCSTGGACSGT-3 'and 5'-GATGATCTCCATSAGCTTSGCRTC-3' (Li et al., 2006) And subsequently cloned into pGEM-T Easy vector (Promega, Madison, Wis.). This DNA clone was named pHJK1001. The cosmid library of S. galbus KCCM 41354 genomic DNA was constructed according to the manufacturer's handbook (Strata gene, La Jolla, CA) using the SuperCos I and Gigapack III Gold packaging extract kits. A 3.2-kb KpnI DNA fragment was isolated from the S. galbus genome using an 800-bp fragment at pHJK1001 as a probe and the nucleotide sequence analysis confirmed the presence of the methoxymaronyl-ACP biosynthesis gene ( galGHI and truncated galJ ) Respectively. The 3.2-kb KpnI DNA fragment was then used as a probe in cosmid library screening and as a result a positive clone pHJK1011 was isolated. The 800-bp fragment internal to galI was isolated as an EcoRI fragment from pHJK1001 and ligated with pKC1139 to produce pD- gall , galI -disruption plasmids. Binding plasmid pKC1139 includes E. coli pUC replicons, Streptomyces temperature-sensitive replication origin, and the autonomic resistance cassette (Bierman et al., 1992). The 1370-bp EcoRV-BamHI fragment internal to orf4 was cloned into the same position of pKC1139 to prepare the orf4 -disruption plasmid pSK1-dKS.

Extraction of galbanolides, analysis of antifungal activity, and thin layer chromatography (TLC) separation

The fermentation of S. galbus and the extraction of gallonolide were carried out by partially modifying the procedure previously provided (Fauth et al., 1986). In summary, mycelium was collected from the culture by centrifugation and settled in a minimal volume of methanol for extraction. The culture supernatant was extracted with the same volume of ethyl acetate. The analytical microorganism C. neoformans IFO 40092 was obtained from the Culture Collection of the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chiba, Japan. Cryptococcus Neoformans were maintained in GYM agar and incubated in Bennett medium at 28 ° C in a rotary shaker. Bennett medium cultures (OD _600nm , 2.0) were added to GYM semi-solid agar (0.4% w / v agar) at 0.01% dilution and overlaid on GYM agar to prepare assay plates. For TLC analysis, a silica gel 60 F254 TLC-plate (Merck, Darmstadt, Germany) was developed with a solvent system consisting of ethyl acetate and benzene (1: 3) (Abe et al., 1985) Respectively. The assay plates were incubated at 28 [deg.] C until the assay microorganisms grew to form a confluent lawn of cells.

HPLC analysis

An Agilent 1100 series LC system (Santa Clara, Calif.) Was used for HPLC-MS analysis. A Bruker HCT 3000 ion trap mass spectrometer (Billerica, Mass.) Was used in conjunction with an HPLC column and the mass scan range was m / z 100-500. The drying temperature was 350 ℃, atomizer gas was 40 psi, dry gas was 9 L min ^{_ 1.} Separation was carried out on a pair of C-18 columns (150 x 3.0 mm, 5.0 mm; Phenomenex, Torrance, Calif.) By isocratic elution. The column temperature was maintained at 25 占 폚. The flow rate was maintained at 0.5 mL min ^-1 . The mobile phase consisted of methanol and 25 mM ammonium acetate (3: 1) in water.

Varian HPLC ProStar system (Lake Forest, CA) was used for HPLC analysis using UV detection. Separation was performed on a Varian Pursuit XRs C-18 column (250 x 4.6 mm, 5.0 mm) and monitored at 230 nm. The flow rate was maintained at 0.75 mL min ^-1 . The mobile phase was composed of 25 mM ammonium acetate, pH 5.5 (A) in water, and methanol (B) was transferred using a grandient elution: 100% A for 30 minutes; 100% A to 5% A for 10 minutes; It was then maintained at 5% A for 10 min.

Example 1: The methoxymalonyl-ACP biosynthesis gene was found to be located in association with the KAS-related gene in S. galbus, while not clustering with the multi-modular PKS gene

The clone of the fkbI homologue was used to isolate the cosmid clone pHJK1011, which contains the methoxymalonyl-ACP biosynthesis gene from S. galbus . Crystallization of the complete nucleotide sequence of pHJK1011 confirmed the presence of a complete set of methoxy-malonyl-ACP biosynthesis genes, galGHIJK (Figure 1). The nucleotide sequence of the cosmid clone (41,591 bp insert) (SEQ ID NO: 11) was deposited with the GenBank database (Accession No. GU300145) at http://www.ncbi.nlm.nih.gov/Genbank. Previous reports have only reported that the methoxymonyl-ACP biosynthesis gene can be used to clone the PKS gene cluster associated with it (Li et al., 2006; Zhao et al., 2006).

We have found that no typical multicomponent PKS gene is present in the cloned 42-kb site (Figure 1 and Table 1). Instead, the galGHIJK gene was flanked by three genes coding for KAS- related proteins, orf3 , orf4 , and orf5 . product of orf3 is KAS III (FabH- type) Although protein with homology, did not have a catalyst of a cysteine residue. The orf4 protein contained the complete KAS domain, followed by the amino terminus cleaved KAS domain. In addition, orf4 contains the? -Ketoacyl-ACP reductase domain (s), but does not have the ACP domain. orf5 contained an AT domain and a thiolated motif, which are conserved in the ACP domain. orf4 and orf5 are the Burkholderia multivorans (Fig. 2), but their biological role is unclear.

The predicted functions of the methoxymalonyl-ACP biosynthesis gene and its surrounding ORFs in the present invention are shown in Table 1 below.

ORF Size (aa) Closest similarity to known proteins
(Gene Bank Accession No., size in amino acid) Similarities in%,
amino acid-size of overlap Function
-6 188 TetR family transcriptional regulator
Streptomyces hygroscopicus ATCC 53653
(ZP_05517591.1, 186)
55, 185 전류기 -5 268 Shrot chain dehydronasae
Ralstonia eutropha H16
(CAJ96572.1, 267)
55, 263 Dehydrogenase -4 353 NADH: flavin oxidoreductase / NADH oxidase
Streptomyces sviceus ATCC 29083
(ZP_05018453.1, 353)
81, 352 Oxidoreductase -3 305 Alpha / beta hydrolase fold-3 domain protein
Caulobacter sp. K31
(ABZ74245.1, 305)
52, 279 Hydrolase -2 355 Helix-turn-helix DNA binding protein
Streptomyces sp. Mg1
(ZP_05001021.1, 329)
54, 318 DNA binding protein -One 409
O- methyltransferase, family protein 2
Mycobacterium smegmatis str. MC2 155
(YP_889598.1, 345)
45, 323 O- methyltransferase galG 220 Methoxymalonate biosynthesis protein
Streptomyces neyagawaensis
(AAZ94403.1, 222)
67, 220 Methoxymalonate biosynthetic protein
(Methyltransferase) galH 384 Methoxymalonate biosynthesis protein
Streptomyces scabiei 87.22
(CBG75347.1, 385)
69, 378 Methoxymalonate biosynthetic protein
(Acyltransferase / Phosphatase) galley 369 Methoxymalonate biosynthesis protein
Streptomyces neyagawaensis
(AAZ94394.1, 371) 67, 367 Methoxymalonate biosynthetic protein
(Dehydrogenase)
galJ 100 Methoxymalonate biosynthesis protein
Streptomyces clavuligerus ATCC 27064
(ZP_05008786.1, 102)
62, 81 Methoxymalonate biosynthetic protein
(Acyl-carrier protein) galK 296 Methoxymalonate biosynthesis protein
Streptomyces scabiei 87.22
(CBG75344.1, 279)
69, 281 Methoxymalonate biosynthetic protein (Dehydrogenase) One 361 Hypothetical protein containing a Rieske [2Fe-2S] domain
Streptomyces griseus subsp. griseus NBRC 13350
(BAG 17263.1, 362)
44, 354 Unknown 2 490 Cytochrome P450 183B1 Cyp183B1
Mycobacterium marinum M
(YP_001848603.1, 462)
49, 456 Monooxygenase 3 311 Beta-ketoacyl synthase-like protein
Mycobacterium ulcerans Agy99
(YP_025567.1, 328)
33, 307 Beta-ketoacyl synthase 4 2048 Putative multi-domain beta keto-acyl synthase
Burkholderia multivorans ATCC 17616
(YP_00001945760.1, 2081)
32, 1766 Beta-ketoacyl synthase 5 501 Acyl transferase domain protein
Stigmatella aurantiaca DW4 / 3-1
(EAU66418.1, 336)
32, 235 Acyl transferase 6 505 Oxidoreductase
Streptomyces avermitilis MA-4680
(NP_822388.1, 417)
72, 417 Oxidoreductase 7 279 IclR family Transcriptional regulator
Streptomyces flavogriseus ATCC 33331
(ZP_05803308.1, 279)
80, 269 전류기 8 321 Putative 5-dehydro-4-deoxyglucarate dehydratase 2
Streptomyces scabiei 87.22
(CBG73189.1, 346)
80, 301 Dehydratase 9 445 High-affinity gluconate transport protein
Streptomyces viridochromogenes DSM 40736
(ZP_05534313.1, 445)
91, 445 Transport protein 10 444 Glucarate dehydratase
Streptomyces viridochromogenes DSM 40736
(ZP_05534314.1, 445)
91, 445 Dehydratase 11 358 Putative DNA-binding regulator
Streptomyces scabie 87.22
(CBG677401, 346)
` 58, 336 Transcription regulator 12 190 Putative sigma factor
Streptomyces scabie 87.22
(CBG677401, 346)
55, 184 Transcription regulator 13 287 Lipoprotein
Streptomyces pristinaespiralis ATCC 25486
(ZP_050121153.1, 272)
63, 271 Lipoprotein 14 199 Peptidase C60, Sortase A and B
Streptomyces ghanaensis ATCC 14672
(ZP_04687625.1, 228)
54, 198 Peptidase 15 150 Hypothetical protein SghaA1_07181
Streptomyces ghanaensis ATCC 14672
(ZP_04684942.1, 176)
39, 115 Unknown 16 315 Putative fatty acid desaturase
Streptomyces clavuligerus ATCC 27064,
(ZP_05006083.1, 381)
98, 315 Fatty acid desaturase 17 336 Putative oxidoreductase
Streptomyces viridochromogenes DSM 40736
(ZP_05529480.1, 336)
92, 336 Oxidoreductase 18 379 Conserved hypothetical protein
Streptomyces scabiei 87.22
(ZP_05001028.1, 379)
92, 378 Unknown 19 * 323 Conserved hypothetical protein
Paenibacillus sp . JDR-2
(ACT04581.1, 948) 36, 306 Unknown

* incomplete ORF

Example  2: galley - In the destruction mutation Galvalonide  A Loss of generation

The galI - break mutation was made using the gene-disrupting plasmid pD- galI . The genotype of the mutant SK- gall- 5 was confirmed by Southern analysis using an 800-bp galI fragment as a probe (Fig. 3a and b). The 3.2-kb KpnI fragment was present on the wild-type (WT) chromosome and absent from the SK- galI- 5 chromosome. Three bands with sizes of about 4.6, 5.9, and 7.4 kb were observed in the lane of the SK- galI- 5 sample. The 4.6-kb and 5.9-kb fragments tend to originate from the fusion of pD- gall to the gall position (Figure 3b), while the 7.4-kb band corresponds to pD- galI itself. Thus, we can infer that galI was successfully destroyed in SK- galI- 5. It was initially assumed that SK- galI- 5 does not synthesize gal- ololide A, but will retain its ability to produce gal- In this case, the extract of SK- galI- 5 will exhibit a low level of antifungal activity compared to the wild type. Because, gallonolide B has several hundredfold lower effects when tested against C. neoformans (Harris et al., 1998). As expected, the supernatant extract of SK- gal I- 5 showed a low level of antifungal activity compared to the wild-type level (Figure 3c). However, a similar level of activity was observed in mycelial extracts of both WT and SK- gall- 5 (Fig. 3d). It is not known whether, in addition to galbanolaide A , S. galbus has the potential to accumulate galbanolide antifungal substrates when cultured under the conditions used here. To answer these questions, silica gel-TLC separation was used with antifungal activity assays. With the TLC conditions used, the delay factor (Rf) value for galbanolide B was reported to be 0.35 (Abe et al., 1985), but there is no comparable data for galbanolide A The strong antifungal activity was eluted at a Rf value of about 0.25 in WT extract and the activity level was significantly higher in the flour extract (FIGS. 3e and f). This activity was subsequently confirmed by HPLC-MS analysis as being from gallonolide A. TLC plates were developed in triplicate for better separation. The activity of gal- ololide A in SK- gall- 5 extract was not apparent (Figs. 3e and f). In addition to SK- galI- 5, the mycelium extract of WT exhibited antifungal activity without migration under the elution conditions (Fig. 3f).

Next, the present inventors used HPLC-MS analysis to identify the gallonolide A and B from the extract. m / z 381 ([M + H] ⁺ for galbanolide A), m / z 365 ([M + H] ⁺ for galbanolide B) MH] ^-), and m / z 363 (galvo surprised [MH] for Id B ^- ion chromatogram (EICs) extraction for) it is in the rich extract from WT, 7.2 minutes and galvo in 8.7 minutes surprised Id a And B (Fig. 4A). As expected, SK- gall- 5 produced galblonolide B, but did not produce galblonolide A (Fig. 4b). In separate HPLC runs, the eluted fractions were collected at 7.0-8.0 min (fractions 1) and 8.0-9.0 min (fr. 2), concentrated and applied as an antifungal activity test after TLC separation 4c). The antifungal test showed that the WT fraction had higher activity in fraction 1 and maintained a higher antifungal activity at an Rf value of about 0.25. This activity is apparently absent in the SK- gal I -5 fraction. The eluted fraction from SK- galI- 5 had a low activity in fraction 2 at an Rf value of about 0.35. Although this activity is too low to be reproducibly observed, the Rf value is comparable to the known values for galbanolide B (Abe et al., 1985). Collectively, these experiments demonstrate that SK-gall-5 produces galblonolide B but does not synthesize galblonolide A. HPLC-MS analysis using gradient elution further supports that SK- galI- 5 has lost the ability to produce galblonolide A (FIG. 5).

Example 3: orf4  The disruptant confirmed that the production of galvalonide A and B was severely impaired.

The presence of the KAS- related genes ( orf3 , 4, and 5 ) adjacent to galGHIJK suggests that the three genes are involved in the biosynthesis of galbanolide . Thus, an orf4 -breaking mutation was generated and the genotype of the resulting mutant was confirmed by Southern analysis using a 1.4-kb EcoRV-BamHI fragment as a probe (Fig. 6a and b). -Kb PstI-NotI fragment was 3.1 was apparent in the WT chromosome, orf4 - in the two results of the fracture mutation (dKS-6 and -7), which was replaced by 2.8- and 1.7-kb fragment. The 1.4-kb fragment observed in dKS-6 and -7 will be derived from the disruption plasmid pSK1-dKS. The antifungal activity test showed that the dKS strain produced a minor amount of gallonolide A, while the production of the unknown antifungal compound (not migrated by TLC) decreased slightly (Figure 7). Although it is certain that galbanolide A biosynthesis is severely impaired in the dKS mutation, it is not clear whether the reduction of the unknown compound is associated with the destruction of orf4 . HPLC analysis, including UV detection, demonstrated that the level of mutagenesis was a low detection level for galblonolites A and B (FIG. 6C). Galboronolides A and B were collected from WT samples and their identity was confirmed by an antifungal activity test and mass spectrometry. High resolution mass spectrometry showed 381.2281 (m / e for [M + H] ⁺ , chemical ionization) and 364.2254 (for C20H33O6, calcd 381.2277) and galvanolide B (C21H32O5, calcd 364.2250) Electron impact ionization). This observation implies that orf4 plays an important role in the galbanolide biosynthesis.

<110> Myongji University Industry and Academia Cooperation Foundation <120> Gene involved in the biosynthesis of galbonolide <130> PA100705KR <160> 11 <170> Kopatentin 1.71 <210> 1 <211> 220 <212> PRT <213> Streptomyces galbus <400> 1 Met Ala Asn Gln Ile Ala Ala Gly Pro Glu Leu Leu Gln Tyr Val Arg   1 5 10 15 Asp Phe Ser Leu Arg Glu Asp Asp Ile Leu Arg Asp Leu Arg Leu Leu              20 25 30 Thr Ser Gly Leu Pro Gly Gly Glu Ala Met Gln Val Thr Ala Glu Glu          35 40 45 Gly Gln Phe Leu Ala Phe Leu Ile Ala Ala Thr Gly Val Thr Asp Val      50 55 60 Leu Glu Val Gly Thr Phe Thr Gly Tyr Ser Thr Leu Cys Met Ala Arg  65 70 75 80 Ala Leu Pro Pro Gln Gly Arg Leu Ile Thr Cys Asp Ile Ser Glu Arg                  85 90 95 Trp Pro Glu Ile Gly Val Glu Tyr Trp Arg Arg Ala Gly Val Leu Glu             100 105 110 Arg Ile Glu Val Arg Ile Gly Glu Ala Ala Asp Thr Leu Glu Lys Met         115 120 125 Cys Ala Asp Gly Leu Ser Glu Ser Phe Gly Leu Val Phe Ile Asp Ala     130 135 140 Asp Lys Val Asn Tyr Pro Arg Tyr Tyr Glu Leu Ala Leu Arg Leu Val 145 150 155 160 Arg Pro Gly Gly Leu Leu Val Val Asp Asn Thr Leu Phe Phe Gly Arg                 165 170 175 Val Ile Asp Pro Leu Ala Asp Asp Ala Asp Thr Ala Gly Val Arg Glu             180 185 190 Leu Asn Ale Ile Ile Arg Asp Arg Arg Ile Asp Val Ser Met Leu         195 200 205 Pro Val Ala Asp Gly Ile Thr Leu Val Arg Arg Lys     210 215 220 <210> 2 <211> 384 <212> PRT <213> Streptomyces galbus <400> 2 Met Arg Gly Ala Ala Ala Thr Ala Thr Val Lys Cys Leu Val Trp Asp   1 5 10 15 Leu Asp Asn Thr Leu Trp Asn Gly Thr Leu Leu Glu Gly Asp Asp Val              20 25 30 Val Leu Ala Gln Gly Ile His Glu Val Ile Thr Arg Leu Asp Ala Arg          35 40 45 Gly Ile Leu Gln Ser Val Cys Ser Arg Asn Asp His Ala Val Ala Trp      50 55 60 Ala Arg Leu Glu Ala Leu Gly Val Ala Asp Tyr Phe Val Leu Pro Gln  65 70 75 80 Ile Gly Trp Gly Arg Lys Ser Asp Ala Val Arg Arg Ile Ala Glu Gly                  85 90 95 Leu Asn Phe Ala Pro Arg Ala Val Ala Phe Ile Asp Asp Gln Pro Thr             100 105 110 Glu Arg Ala Glu Val Ala His Ser Leu Pro Asp Val Arg Cys Tyr Arg         115 120 125 Ala Glu Asp Ala Leu Leu Pro Gly Leu Pro Glu Phe Ser Pro Gly     130 135 140 Thr Val Thr Val Asp Ala Arg Gln Arg Arg Ser Met Tyr Gln Ala Gly 145 150 155 160 Phe Arg Arg Thr Asp Glu Gln Ala Arg Phe Thr Gly Ser His Glu Asp                 165 170 175 Phe Leu Arg Ser Ale Glu Met Val Leu Arg Ile Ala Arg Ala Thr Pro             180 185 190 Glu Glu Leu Ala Arg Ile Glu Glu Leu Thr Gln Arg Thr Ser Gln Met         195 200 205 Asn Ala Thr Gly Val Tyr Pro Asp Ala Thr Leu Arg Ala Leu Leu     210 215 220 Ala Asp Pro Gly His Glu Val Leu Val Thr Thr Leu Thr Asp Arg Phe 225 230 235 240 Gly Pro His Gly Ala Val Gly Val Val Leu Leu Glu Lys His Pro Arg                 245 250 255 Ala Trp His Leu Arg Leu Leu Ala Thr Ser Cys Arg Val Val Ala Phe             260 265 270 Gly Val Gly Thr Ile Leu Leu Asn Trp Leu Ala Asp Gln Ala Ala Arg         275 280 285 Ala Gly Val His Leu Leu Ala Asp Phe Arg Ala Thr Glu Arg Asn Arg     290 295 300 Met Met Glu Ile Ala Tyr Arg Phe Ala Gly Phe Gly Asp Gln Pro Cys 305 310 315 320 Ala Cys Arg Gly Leu Val Arg Glu Gly Ser Ala His Gly Ser Ser Gly                 325 330 335 Glu Gly Gly Ser Glu Ala Gly Ala Glu Glu Ala Asn Ser Val Ala Ala             340 345 350 Glu Asp Val Gln Leu Leu His Leu Ala Pro Glu Arg Arg Gln Ala Pro         355 360 365 Thr Thr Leu Arg Leu Ile Ala Pro Asp Leu Thr Ser Gly Gly Pro Gly     370 375 380 <210> 3 <211> 369 <212> PRT <213> Streptomyces galbus <400> 3 Met Gly Asp Ala Val Ala Ala Ala Ala Ser Ala Val Thr Glu Leu Val   1 5 10 15 Gly Asp Arg Ala Ala Asp Trp Asp Arg Ala Gly Arg Leu Pro Glu Glu              20 25 30 Val Val Arg Glu Leu Gly Ala Gly Gln Leu Ala Pro Gln Ile Pro          35 40 45 Ala Arg Tyr Gly Gly Ala Gly Trp Ser Ser Ala Asp Asn Gly Glu Phe      50 55 60 Thr Ala His Val Gly Thr Leu Cys Gly Ser Leu Arg Ser Val Met Thr  65 70 75 80 Ser Gln Gly Met Ala Ala Trp Thr Val Glu Arg Leu Gly Asp Pro Ala                  85 90 95 Gln Ala Ala Glu Leu Leu Pro Arg Leu Ala Ser Gly Asp Thr Ala Ala             100 105 110 Val Ala Phe Ser Glu Pro Arg Ala Gly Ser Asp Leu Ser Ala Ile Ala         115 120 125 Thr Thr Val Thr Val Asp Gly Asp Thr Leu Val Leu Asp Gly Leu Lys     130 135 140 Thr Trp Val Thr Ala Ala Glu Gln Ala Asp Leu Leu Leu Val Val Ala 145 150 155 160 Arg Met Gly Ala Asp Gly Ala Val Val Val Val Pro Arg Asp Thr Pro                 165 170 175 Gly Val Gln Val Arg Arg Val Gly Asp Pro Met Gly Phe Arg Ala Ala             180 185 190 Gly His Ala Asp Ile Leu Leu Asp Gly Val Arg Leu Pro Leu Ser Ser         195 200 205 Leu Leu Gly Gly Gly Arg Gln Pro Leu Ser Leu Leu Val Thr Thr Ala     210 215 220 Leu Ala Phe Gly Arg Leu Ser Val Ala Trp Gly Cys Val Gly Ile Leu 225 230 235 240 Arg Ala Cys Val Ala Ala Val Gly His Ala Ala Ser Arg His Gln                 245 250 255 Phe Gly Arg Pro Leu Gly Arg His Gln Leu Val Ala Arg His Leu Ala             260 265 270 Asp Leu Trp Thr Gly Glu Gln Ile Ala Thr Arg Ala Cys Glu His Ala         275 280 285 Ser Arg Cys Trp Asp Glu Asn Ala Ala Asp Leu Ser Ala Ala Ile Val     290 295 300 Leu Ala Lys Tyr Val Gly Ala Glu Ala Ala Ala Arg Gly Ala Ala Thr 305 310 315 320 Ala Val Gln Val Leu Ala Ser Ala Gly Ala Arg Asp Gly His Val Val                 325 330 335 Ala Arg Ala Tyr Arg Asp Ala Lys Leu Thr Glu Ile Ile Glu Gly Thr             340 345 350 Asp Glu Val Cys Gln Leu Ile Leu Ala Asp His Val Leu Gly Ser Arg         355 360 365 Gly     <210> 4 <211> 100 <212> PRT <213> Streptomyces galbus <400> 4 Met Thr Met Pro Ser Asn Arg Ala Val Pro Glu Pro Leu Gly Asp Pro   1 5 10 15 Gly Asp Ile Glu Arg Glu Leu Leu Arg Phe Leu Glu Leu Arg Thr Gly              20 25 30 Lys Thr Trp Ser Ala Asp Met Asp Val Phe Gly Ser Gly Gly Leu Ser          35 40 45 Ser Leu Phe Ala Met Glu Leu Val Val His Leu Glu Arg Thr Tyr Gly      50 55 60 Ile Ala Val Arg Gly Ala Asp Leu Gln Met Asp Asn Phe Arg Thr Ala  65 70 75 80 Arg Arg Met Ala Ala Leu Val Gly Arg Leu Arg Arg Pro Ala Val Gly                  85 90 95 Gly Ser His Gly             100 <210> 5 <211> 296 <212> PRT <213> Streptomyces galbus <400> 5 Met Ala Ala Gln Arg Gly Thr Pro Gly Thr Gly Arg Arg Leu Ala Val   1 5 10 15 Leu Gly Ala Gly Val Gly Gly Ser Gly Ile Ala Ala Leu Ala Ile Gly              20 25 30 His Gly Met Ala Val Ser Leu Val Asp Leu Asp Arg Arg Arg Leu Asp          35 40 45 Glu Ala Pro Gly Arg Ile Asp His Gln Leu Arg Thr Ala Arg Leu Met      50 55 60 Gly Ala Leu Pro Glu Glu Ala Val Pro Gly Ala Leu Val Thr Gly Arg  65 70 75 80 Ser Leu Ala Asp Leu Met Thr Asp Gly Pro Ala Phe Thr Ala Val Val                  85 90 95 Glu Ala Val Thr Glu Asp Pro Asp Ala Lys Ala Lys Val Leu Ala Asp             100 105 110 Val Ser Ala Leu Ala Ala Pro Gly Thr Pro Ile Ile Ser Asn Thr Ser         115 120 125 Ala Ile Pro Ile Asp Glu Leu Ala Arg Ala Thr Ala Arg Pro Val Asp     130 135 140 Leu Val Gly Thr His Phe Met Asn Pro Pro Cys Leu Ile Arg Thr Val 145 150 155 160 Glu Val Ile Arg Gly Thr Gln Thr Ser Glu Ala Thr Leu Asp Ala Leu                 165 170 175 Arg Ala Leu Leu Ala Leu Gly Arg Glu Ser Val Val Val Asp Asp             180 185 190 Ala Pro Gly Phe Val Thr Ser Arg Ile Leu His Pro Met Ile Asn Asp         195 200 205 Ala Val Val Ala Ala Gly Thr Ala Ser Ala Glu Ser Val Asp     210 215 220 Arg Leu Met Gln Gly Cys Leu Gly His Pro Thr Gly Pro Leu Arg Thr 225 230 235 240 Ala Asp Leu Ile Gly Leu Asp Asn Leu Val Asp Ser Leu Trp Val Leu                 245 250 255 His Gly Arg Thr Gly Asp Glu Gly Cys Arg Pro Cys Asp Leu Leu Leu             260 265 270 Glu Met Val Arg Ala Gly His Leu Gly Arg Lys Ser Gly Arg Gly Phe         275 280 285 Tyr Glu Tyr Glu Gly Glu Trp Ala     290 295 <210> 6 <211> 361 <212> PRT <213> Streptomyces galbus <400> 6 Met Lys His Leu Leu Asp Ser Arg Ala Ile Ser Leu His Gly Ala Glu   1 5 10 15 Pro Ala Asp Thr Leu His Ala Pro Ser Arg Pro Tyr Pro Asn Gly Trp              20 25 30 Phe Cys Val Ala Phe Ser Asp Glu Leu Arg Ala Gly Ser Leu Thr Thr          35 40 45 Arg Arg Leu Ala Gly Ala Glu Val Val Leu Tyr Arg Thr Ala Lys Gly      50 55 60 Val Leu Arg Ala Val Arg Pro Gln Cys Pro His Leu Gly Ala His Leu  65 70 75 80 Gly Val Gly Gly Ser Val Glu Gly Glu Asn Ile Val Cys Pro Phe His                  85 90 95 Arg Phe Ala Phe Asp Pro Ser Gly Thr Cys Val Arg Thr Gly Tyr Asp             100 105 110 Gln Gln Pro Pro Lys Ala Ser Leu Thr Gln Tyr Pro Val Cys Glu Ala         115 120 125 Asn Gly Ser Val Tyr Val Trp Ser His Ala Leu Gly Met Pro Pro Gln     130 135 140 Trp Asp Val Pro Leu Phe Pro Met Asp Asp Arg Gln Pro Phe Ser His 145 150 155 160 Arg Thr Phe Asp Ile Ala Gly His Pro Gln Asp Val Ile Glu Asn Ala                 165 170 175 Phe Asp Trp Gly His Leu Pro Ala Leu His Gly Leu Glu Glu Val Asp             180 185 190 Ile Gly Gly Leu Pro Val Thr Gly Glu Pro Ile Ser Thr Val Thr Ala         195 200 205 Thr Ala Arg Asn Thr Met Met Arg Gly Phe His Gln Ser Tyr Thr Leu     210 215 220 Thr Val Ile Gly Leu Ala Thr Ile 225 230 235 240 Gly Ala Gly Asn Leu Tyr Val Met Leu His Ala Thr Pro Thr Gly Pro                 245 250 255 Gly Arg Met Gln Val Arg Phe Gly Thr Lys Leu Glu Leu Asn Gly Phe             260 265 270 Pro Gly Val Pro Asp Trp Leu Gly Arg Pro Ala Val Thr Pro Leu Ala         275 280 285 Arg Leu Leu Ser Glu Val Leu Gln Arg Val Gly Ser Val Asp Thr Gly     290 295 300 Ala Asp Leu Leu Met Trp His His Gln Glu His Val Ala Arg Pro Arg 305 310 315 320 Leu Ala Lys Gly Asp Gly Pro Ile Gly Arg Tyr Arg Gln Trp Ala Gln                 325 330 335 Gln Phe Tyr Thr Glu Pro Thr Gly Asp Leu Val Pro Pro Arg Pro Glu             340 345 350 Arg Ser Glu Ala Ser Arg Ser Glu Glu         355 360 <210> 7 <211> 490 <212> PRT <213> Streptomyces galbus <400> 7 Met Arg Phe Ser Met Thr Asp Thr Ser Ala Val Ser Ala Glu Ser Val   1 5 10 15 Pro Leu Ala Pro Arg Ala Trp Pro Leu Leu Gly His Ala Leu Pro Leu              20 25 30 Leu Arg Asp Pro Leu Ala Phe Ile Thr Ser Leu Ser Gly Tyr Arg Glu          35 40 45 Met Val Arg Val Val Arg Leu Gly Pro Ser Ser Ala Val Met Ile Cys Asp      50 55 60 Pro Asp Leu Thr Arg Gln Val Phe Leu Asn Asp Arg Thr Phe Asp Lys  65 70 75 80 Gly Gly Pro Ile Tyr Asp Arg Ile Arg Glu Val Ile Gly Asp Gly Leu                  85 90 95 Ser Thr Cys Ala Tyr Pro Leu His Arg Arg Gln Arg Arg Leu Cys Gln             100 105 110 Pro Ser Phe His Pro Ala Arg Leu Ala Gly Tyr Gly Ala Val Phe Ala         115 120 125 Arg Ala Gla Ala Lys Ala Gly Ser Trp Arg Asp Gly Asp Val Ile     130 135 140 Asp Val Thr Gln Glu Met Met Thr Leu Thr Thr Arg Ala Thr Met Glu 145 150 155 160 Thr Met Phe Ser Gly Ala Leu Pro Glu Glu Thr Met Arg Arg Ser Leu                 165 170 175 Ala Asp Thr Ala Val Val Ser Ala Phe Phe Arg Arg Met Met Thr             180 185 190 Pro Ala Leu Leu Arg Arg Leu Pro Thr Arg Arg Lys Arg Arg Tyr Asp         195 200 205 Asp Ala Arg Thr Arg Leu Ser Ala Thr Ile Ala Glu Ile Ile Ala Glu     210 215 220 Arg Arg Ala Asp Pro Ala Asp His Ala Asp Leu Leu Ser Thr Leu Ile 225 230 235 240 Ala Ala Val Asp Glu Glu Ser Glu Asp Gly Arg Arg Gln Leu Asn Asp                 245 250 255 Ala Glu Leu Ala Asp Glu Ala Leu Thr Phe Leu Gly Gly Met Glu             260 265 270 Thr Ala Ile Thr Leu Ala Trp Ala Leu His Leu Leu Ser Thr His         275 280 285 Pro Asp Val Gln Arg Arg Leu Lys Ala Glu Thr Asp Arg Val Leu Glu     290 295 300 Gly Gly Lys Leu Asp Pro Ala Arg Leu Pro Ser Leu Gly Leu Ala Ser 305 310 315 320 Arg Val Val Thr Glu Thr Leu Arg Leu Tyr Pro Pro Ala Trp Met Met                 325 330 335 Thr Arg Val Val Arg Glu Asp Ala Glu Leu Gly Gly Val Arg Phe Lys             340 345 350 Gly Gly Ser Thr Ile Val Leu Ser Pro Tyr Leu Leu His Arg Arg Pro         355 360 365 Asp Leu Tyr Glu Glu Pro Asp Arg Phe Asp Pro Asp Arg Trp Leu His     370 375 380 Ala Gln Pro Asp Arg Ala Thr Tyr Ile Pro Phe Gly Ala Gly Ala Arg 385 390 395 400 Lys Cys Ile Gly Asp Gln Phe Ala Leu Asn Glu Ala Ile Leu Ala Leu                 405 410 415 Thr Ala Ile Val Ala Arg Trp Glu Leu His Pro Val Gly Asp Arg Pro             420 425 430 Phe Leu Pro Lys Val Glu Thr Ser Leu Ser Ser Gly Leu Arg Leu         435 440 445 Arg Leu Thr Glu Arg Arg Ser Ala Asp Ala Asp Gly Ala Glu His Gly     450 455 460 Ala Ala Ala Pro Leu Ser Ala Ser Gly Arg Pro Ser Thr Gln Pro Gly 465 470 475 480 Val Ala Cys Pro Val Lys His Gly Glu                 485 490 <210> 8 <211> 311 <212> PRT <213> Streptomyces galbus <400> 8 Met Asp Asn Gly Pro Pro Ser Leu Thr Ala Leu Asn Gly Phe Thr Ser   1 5 10 15 Ala Ala Val Ser Thr Asp Ser Ser Pro Ser Gln Met Ala Ala Arg Ala              20 25 30 Gly Leu Lys Ala Leu Arg His Ala Asp Val Thr Gly Ala Asp Leu Ser          35 40 45 Leu Leu Val His Ala Asn Phe Gln Asp Glu Asp His Tyr Ala Pro Ser      50 55 60 Ala Tyr Leu Leu Arg Val Leu Gly Gly Pro Asp Thr Asn Ala Ile Glu  65 70 75 80 Leu Gly Ala Ala Ser Asp Gly Gly Gly Ala Ala Leu Val Thr Ala Ala                  85 90 95 Glu His Leu Thr Ala Arg Pro Ala Ser Lys Ala Ala Leu Val Thr Ala             100 105 110 Gly Val Arg Phe Arg Pro Glu Arg Trp Gly His Val Ala Glu Met Gly         115 120 125 His Leu Ala Gly Asp Ala Gly Ala Ala Ala Val Leu Thr Arg Gly Thr     130 135 140 Gly Phe Ala Arg Leu Met Ala Thr Ala Gln Ala Gln Pro Gln Leu 145 150 155 160 Glu Val Leu Thr Arg Ala Arg Thr Ala Ala Asp Gly Ser Gly Arg Pro                 165 170 175 Leu Leu Val Gln Glu Thr Gly Leu Met Pro His Val Asp Ala Leu Arg             180 185 190 Gln Ser Thr Arg Arg Cys Val Glu Thr Val Leu Asp Glu Thr Gly Val         195 200 205 Arg Pro Glu Gly Ile Ala Arg Val Val Pro Ile Ala Ile Gly Ala Glu     210 215 220 Val Leu Asp Leu Leu Leu Gly Gly Ala Pro Leu Gly Leu Arg Ala Ala 225 230 235 240 Asp Thr Ser Trp Thr Phe Gly Arg His Leu Gly His Val Gly Pro Cys                 245 250 255 Asp Val Leu Leu Ala Leu Asp Arg Glu Phe Arg Ser Gly Thr Leu Arg             260 265 270 Ala Gly Glu Arg Val Leu Val Val Ser Phe Gly Leu Gly Phe Arg Trp         275 280 285 Thr Ala Leu Leu Glu Ile Thr Arg Asp Pro Ala Thr Gly Gly Pro     290 295 300 Ala Gly Arg Ser Thr Lys Lys 305 310 <210> 9 <211> 2048 <212> PRT <213> Streptomyces galbus <400> 9 Met Arg Arg Leu Ala Gln Gly Gly Ala His Val Ile Val Asn Tyr Phe   1 5 10 15 His Ser Val Asp Gln Ala Lys Leu Leu Gln Glu Glu Leu Glu Gln Ala              20 25 30 Gly His Ser Cys Glu Phe Ile Arg Ala Ser Val Ala Lys Thr Ser Glu          35 40 45 Ile Asp Arg Met Phe Asp Leu Val Gln Glu Arg His Gly Gly Leu Asp      50 55 60 Ile Leu Ile Asn Asp Ala Aspen Gly Asp  65 70 75 80 Val Asp Asp Thr Tyr Trp Gln Arg Ala Trp Ser Thr Asn Val Met Ala                  85 90 95 Ala Tyr His Cys Ser Arg Arg Ala Ala Glu Leu Met Ala Gly Arg Glu             100 105 110 Gly Ala Ser Ile Leu Cys Leu Ser Ser Val Gly Ala His Gln Pro Val         115 120 125 Pro Gly Tyr Gly Pro Gly Gly Val Thr Lys Ala Ala Leu Glu Ser Leu     130 135 140 Val Arg Tyr Leu Ala Leu Glu Leu Val Gly Arg Gly Ile Arg Val Asn 145 150 155 160 Thr Val Leu Leu Gly Ser Val Ala Ser Glu Ile Val Val Asn Leu Asp                 165 170 175 Gly Pro Ala Ala Arg Gly Gly Pro Ala Ala Ala Asp Glu Leu Leu Asn             180 185 190 Arg Thr Leu Ser Thr Pro Glu Ala Ala Arg Leu Ile Val His Phe Leu         195 200 205 His Glu Asp Ala Gly Phe Ile Thr Gly Gln Thr Leu Val Ala Asp Gly     210 215 220 Gly Ile Ser Ile Gly Gly Met Arg Gly Leu Arg Leu His Ser Arg Leu 225 230 235 240 Ala Asp Gln Val Ser Lys Pro Ala Arg Arg Pro Val Ala Ser Pro Pro                 245 250 255 Ala Pro Ser Pro Pro Val Pro Thr Ala Pro Val Pro Thr Ala Pro Val             260 265 270 Ala Gly Leu Pro Ala Pro Gly Arg Pro Gln Pro Ala Arg Pro Ala Pro         275 280 285 Pro Ser Thr Ser Ser Ala Ser Ser Ala Ser Ala Ser Ser Ala Ser Ala     290 295 300 Pro Ser Gly Ser Ala His Val Ser Pro Pro Ala Pro Pro Thr Val Ala 305 310 315 320 Ala Pro Ala Val Ala Ala Ala Glu Ala                 325 330 335 Asp Pro Gly Ala Val Ala Val Val Gly Leu Gly Leu Ala Leu Pro Gly             340 345 350 Ala Asn Ala Ala Glu Phe Trp Asp Arg Leu Arg Glu Gly Val Leu         355 360 365 Leu Ser Ser Glu Pro Ser Ala Phe Asp Leu Lys His Phe Trp Ala Pro     370 375 380 Thr Arg Glu Glu Thr Asp Ala Phe Tyr Val Arg Glu Ala Gly Tyr Leu 385 390 395 400 His Asp Phe Val Pro Asp Pro Ala Ser Val Ala Glu Pro Asp Gly Asn                 405 410 415 Val Gly His Pro Gly Trp Pro Arg Thr Thr Arg Trp Leu Arg His Cys             420 425 430 Ala Val Gln Ala Leu Ala Gly Val Arg Arg Arg Pro Thr Asp Arg Trp         435 440 445 Leu Thr Ala Thr Thr Gly Ile His Asp Gln Thr Gly Leu Gly Pro Gln     450 455 460 Gly Val Val Leu Gly Asp Glu Tyr Arg Arg Met Val Arg Glu Ala Leu 465 470 475 480 Pro Asp Asp Pro Asp Gly Asp Trp Leu Ala Glu Leu Ala Asp His Ala                 485 490 495 Ile Ser Ala His Tyr Gly Gly Asp Gly Gly Asp Pro Gln Ala Tyr Leu             500 505 510 Pro Ala Ser Val Val Arg Asn Ala Leu Arg Gly Leu Ile Pro Gly Asp         515 520 525 Thr Gln His Leu Thr Leu Asp Ala Ala Cys Ala Ser Gly Leu Phe Ala     530 535 540 Leu Asp Ala Ala Val Lys Ala Leu Arg Glu Gly Ser Cys Asp Ile Ala 545 550 555 560 Leu Ala Gly Gly Thr Ser Val Ile Glu Pro Ile Gly Phe Thr Leu Phe                 565 570 575 Cys Arg Ala Gln Gly Ile Ser Met Ser Gly Lys Val Arg Pro Phe Asp             580 585 590 Arg Ala Ala Asp Gly Thr Leu Ile Gly Glu Gly Ala Val Ala Leu Val         595 600 605 Leu Lys Pro Tyr Ala Arg Ala Val Ala Asp Gly Asp Arg Val Leu Gly     610 615 620 Val Ile Arg Gly Thr Gly Leu Ala Ala Asp Gly Arg Gly Lys Gly Ile 625 630 635 640 His Ala Pro Ala Thr Arg Gly Gln Glu Leu Ala Ile Asp Arg Ala Trp                 645 650 655 Ala Asp Ala Gly Val Glu Ala Gly Asp Val Asp Trp Ile Val Ala His             660 665 670 Gly Thr Gly Thr Pro Val Gly Asp Glu Ile Glu Leu Arg Ser Leu Leu         675 680 685 Ser Arg Leu Gly Pro Arg Glu Arg Ala Cys Leu Leu Thr Ser Asn Lys     690 695 700 Gln Val Phe Gly His Thr Gly Val Leu Ala Gly Leu Val Ser Val Ala 705 710 715 720 His Ala Leu Val Ala Leu Glu Arg Gly Ala Val Pro Gly Gln Pro Val                 725 730 735 Val Thr Asp Pro His Pro Leu Leu Gly Asp Gly Ala Arg Leu Thr Val             740 745 750 Pro Val Gly Asp Ala Pro Trp Pro Ala Asp Gly Ala Arg Pro Arg Val         755 760 765 Val Gly Val Ser Ser Phe Gly Leu Gly Gly Ala Asp Ala His Val Val     770 775 780 Leu Ser Asp His Ala Pro Arg Ser Val Pro Val Arg Arg Arg Gly Ile 785 790 795 800 Asp Val Arg Asp Asp Arg Glu Asp Leu Val Val Val Gly Trp Asn Thr                 805 810 815 His Leu Pro Gly Met Glu Pro Asp Gln Val Pro Ala Trp Leu Arg Gly             820 825 830 Asp Gly Pro Leu Pro Glu Pro Gly Phe Gly Leu Pro Tyr Pro Leu Pro         835 840 845 Ser Pro Arg Asp Val Arg Ile Pro Pro Leu Thr Met Arg His Met Asp     850 855 860 Ala Ala His Leu Met Val Leu Gln Ala Leu Gly Pro Leu Leu Glu Gln 865 870 875 880 Leu Gly Glu Pro Gly Thr Ser Leu Arg Pro Asp Thr Ala Ile Val Val                 885 890 895 Gly Ser Thr Leu Pro Thr Thr His Asn Thr Gln Ala Ala Leu Arg Val             900 905 910 His Ala Ala Glu Cys Ala Thr Ala Phe Asp Leu Leu Pro Asp Pro Ala         915 920 925 Gln Ala Arg Leu Leu Lys Glu Tyr Leu Ala Lys Gly Met Ala Glu Ala     930 935 940 Glu Gly Val Ile Pro Gly Asp Leu Asn Glu Asp Asp Phe Thr Gly Ala 945 950 955 960 Val Ser Cys Ile Leu Ser Gly Arg Ala Ala Asn Tyr Tyr Asp Phe Arg                 965 970 975 Gly Leu Gly Ser Ser Val Tyr Ser Gln Arg Asp Ser Ala His Thr Ala             980 985 990 Val Asp Leu Ala Leu Arg Gln Leu Arg His Arg Ala Cys Asp Leu Ala         995 1000 1005 Leu Ile Gly Ala Val Cys Leu Arg Pro Ile Ser Gly Trp Asp Arg His    1010 1015 1020 Leu Asp Gln Leu Val Pro Glu Gly Arg Ser Ile Ala Glu Gly Ala Ala 1025 1030 1035 1040 Phe Leu Ala Val Thr Arg Arg Ser Thr Ala Leu Glu His Gly Phe Pro                1045 1050 1055 Val Leu Gly Thr Leu Ser Thr Glu Val Ala Val Ala Asp Ser Arg His            1060 1065 1070 Pro Ala Val Pro Ser His Ala Leu Pro Val Leu Ala Arg Thr Gly His        1075 1080 1085 Thr Tyr Leu Ser Leu Asp Ala Leu Leu Thr Val Leu Glu Ala Val Val    1090 1095 1100 Thr Arg Thr Asp Thr Ala Val Arg Pro Ala Ala Ala Gly Ser Pro Leu 1105 1110 1115 1120 Ile Arg Phe Thr Pro Pro Ala Asp Pro Trp Asp Ala Pro Gly Ala Asp                1125 1130 1135 Arg Gly Glu Thr Gly Asn Gln Ala Glu Leu Thr Gly Pro Gln Trp Ala            1140 1145 1150 Asp Ala Val Arg Asp His Pro Glu Arg Ala Gly Thr Val Ala Asp Gly        1155 1160 1165 Ser Gly His Thr Asp Ser Ala His Thr Asn Pro Glu Pro Ala Gly Ser    1170 1175 1180 Asp Arg Pro Gly Pro Asp Arg Thr Glu Ser Glu Gln Ala Gly Ser Gly 1185 1190 1195 1200 Pro Ala Gly Thr Glu Arg Thr Glu Asp Phe Thr Gly Arg Arg Gln Thr                1205 1210 1215 Phe Arg Leu Val Pro Thr Asp Arg Pro Pro Thr Glu Ser Ala Val Pro            1220 1225 1230 Ser Ile Pro Pro Gly Thr Val Val Ile Thr Asp Ala Pro Glu Leu Ala        1235 1240 1245 Glu Ala Val Thr Gly Pro Asp Thr Ala Val Trp Ser Pro Arg Pro Gly    1250 1255 1260 Val Ala Gly Thr Ala His Val Pro Glu Glu Ala Pro Gly Ala Leu 1265 1270 1275 1280 Ser Gly Leu Pro Phe Val Pro Arg His Ile Arg Val Leu Cys Arg Leu                1285 1290 1295 Pro Val Asp Asp Asp Ala Tyr Gly Glu Ala Glu Ala Ala Arg Ala Glu            1300 1305 1310 Asp Leu Gln Asp Leu Ala Phe Thr Thr Leu Gln Ala Ala Leu Pro Ala        1315 1320 1325 Leu Arg Ser Gly Gly Ser Leu Gly Val Leu Leu Leu Gly Glu Leu Pro    1330 1335 1340 Asp Asp Leu Pro Pro Leu Ser Gly Leu Phe Thr Gly Leu Val Arg 1345 1350 1355 1360 Ser Val Arg Ala Glu Val Pro Gln Cys Gly Gly Val Thr Leu Ile Ser                1365 1370 1375 Asp Ala Ala Asp Val Ala Thr Ala Leu Asp Gln Phe Ala Arg Ala Gly            1380 1385 1390 Ala Val Arg Val Pro Thr His Thr Leu Ala Cys Arg Gly Asp Asp Trp        1395 1400 1405 Phe Ala Leu Ala Val Ala Asp Asp Pro Val Ser Pro Ala Asp Gly Val    1410 1415 1420 Pro Leu Pro Ser Gly Ala Val Val Ala Phe Gly Gly Ala Arg Gly 1425 1430 1435 1440 Ile Thr Pro Glu Leu Leu His Glu Val Ala Leu Gly Thr Asp Arg Pro                1445 1450 1455 His Val Tyr Val Ile Gly Arg Thr Pro Leu Pro Glu Ser Asp Ala Pro            1460 1465 1470 Leu Pro Pro Gln Thr Glu Phe Ile Ala Ala Glu Arg Arg Arg Arg Pro        1475 1480 1485 Asp Ala Ser Leu Arg Glu Leu Arg Val Ala Tyr Glu Lys Ala Gln Ala    1490 1495 1500 Arg Leu Glu Val Arg Arg Thr Val Arg Arg Leu Thr Glu Leu Cys Gly 1505 1510 1515 1520 Pro Asp Arg Val Val His His Arg Val Val Cys Asp Val Leu Asp Ala Glu Gly                1525 1530 1535 Thr Ala Val Leu Gly Glu Ile Leu Asp Arg His Gly Arg Ile Asp            1540 1545 1550 Leu Leu Ile Asn Thr Val Leu Asp Leu Arg Ser Ser Ala Leu His Ala        1555 1560 1565 Lys Thr Leu Pro Asp Phe Arg Ala Val Arg Ala Thr Lys Ala Thr Gly    1570 1575 1580 Tyr Arg Asn Leu Lys Arg Ala Leu Ala Gly Arg Met Pro Arg Ile Trp 1585 1590 1595 1600 Cys Asn Phe Ser Thr Leu Ala Thr Leu Ala Pro Ala Pro Gly Asp Ile                1605 1610 1615 Asp Tyr Cys Ala Val Asn Glu Tyr Leu Ala Tyr Ala Ser Ala Arg Ala            1620 1625 1630 Gln Arg Arg Ala Pro Ala Gly Arg His Glu Val Ala Val Leu Trp Ser        1635 1640 1645 Gly Trp Arg Glu Val Gly Val Ala Ser Ser Val Thr Met Arg Glu Thr    1650 1655 1660 Leu Lys Arg Asn Lys Met Asp Ala Tyr Ile Ser Thr Ala Gln Gly Arg 1665 1670 1675 1680 Ala Gln Phe Leu Ser Ala Val Thr Val Pro Ala Asn Gly Val Ala                1685 1690 1695 Phe Phe Ile Arg Glu Glu Glu Arg Val Leu Leu Ala Arg Arg Gly Ile            1700 1705 1710 Ser Thr His Gly Ser Arg Ala Glu Pro Ala Val Pro Ala Pro Asp Ala        1715 1720 1725 Pro Pro Pro Ala Pro Pro Asp Pro Glu Leu Thr Thr Pro Leu Leu Asp    1730 1735 1740 Gly Val Leu Tyr Arg Gly Asp Asp Trp Ala Val Phe Thr Lys Thr Trp 1745 1750 1755 1760 Asp Pro Arg Thr Leu Ala Glu Arg Asp Gly Arg Trp Met Arg His His                1765 1770 1775 Gln Val Asn Gly Glu Tyr Thr Leu Ala Gly Thr Phe Thr Leu Glu Ala            1780 1785 1790 Ala Ala Gly Ala Ala Ala Leu Cys Pro Gly Leu Arg Val Thr Gly        1795 1800 1805 Phe Arg Gly Leu Val Cys Arg Ser Ser Ile Thr Val Arg Leu Ala Gly    1810 1815 1820 Pro Pro Arg Thr Val Ala Val Glu Ala Arg Val Val Arg Arg Glu Gly 1825 1830 1835 1840 Ala Arg Ala Glu Val Ala Val Arg Ile Thr Ala His Arg Ile Gly Lys                1845 1850 1855 Asn Gly Lys Val Leu Arg Phe Asn Asp Leu Leu Cys Glu Thr Arg Val            1860 1865 1870 Leu Leu Ala Asp Arg Phe Pro Ala Leu Thr Gly Pro Pro Asp Phe Ser        1875 1880 1885 Ala His Glu Pro Glu Pro Asp Phe Ala Met Pro Val Tyr Ser Pro Asp    1890 1895 1900 Pro Pro Ile Ser Leu Thr Gly Pro Phe Ala Gly Thr Gly Asp Tyr Ala 1905 1910 1915 1920 Arg Gly Pro Asp Gly Asn Ser Ala Arg Phe Arg Leu Asp His Asp Thr                1925 1930 1935 Trp Gly Pro Ala Leu Ala Gly Met Thr Val Pro Ala Ile Leu Leu Asp            1940 1945 1950 Ala Met Val His Leu Leu Leu Leu Pro Pro Arg Gly Asp Val Pro Pro        1955 1960 1965 Arg Val Gly Pro Met Ala Gly Leu Asp Glu Val Asp Leu Gly Gly Pro    1970 1975 1980 Gly Asp Asp Cys Arg Leu Ser Ala Leu His Pro Thr Ile Arg Leu His 1985 1990 1995 2000 Cys Asp Phe Ala Thr Gly Glu Leu Thr Ala Val Ala Gly Asp Gly Gln                2005 2010 2015 Val Leu Ala Arg Val Ser Gly Val Ser Ala Tyr Ala Leu Asp His Ser            2020 2025 2030 Gly Glu Leu Val Arg Pro Gly Asp Ala Pro Arg Pro Val Arg Ser        2035 2040 2045 <210> 10 <211> 501 <212> PRT <213> Streptomyces galbus <400> 10 Met Ala Ala Pro Arg Gly Gly Leu Leu Arg Asp Leu His Arg Arg Tyr   1 5 10 15 Glu Thr Val Arg Asp Ala Leu Ser Arg Ile Asp Lys Ala Ala Glu Gly              20 25 30 Leu Gly Leu Pro Asp Ile Ser Ser Leu Ile Glu Gly Asn Gly Thr          35 40 45 Ser Asp Gln Arg Gly Pro Glu Val Gln Tyr Leu Glu Ile Phe Ala Val      50 55 60 Ser Leu Ala Thr His Gln Met Leu Ile Ala Glu Gly Val Glu Pro Ile  65 70 75 80 Ala Ile Val Gly Gln Ser Ile Gly Glu Leu Trp Ala Leu Ala Ala Ala                  85 90 95 Gly His Leu Ser Val Glu Asp Ala Ala Arg Leu Ala Val Ala Arg Ser             100 105 110 Gln Ala Leu Thr Arg His Ser Trp Lys Gly Lys Met Leu Ala Val Gly         115 120 125 Val Asp Gly Arg Arg Ala Glu Ser Leu Ala Gly Leu Ile Asp His Pro     130 135 140 Asp Leu Val Leu Ala Cys Glu Asn Ala Pro Arg Gln Ser Val Ile Ser 145 150 155 160 Gly Pro Glu Glu Leu Ile Arg His Val Glu Arg Val Ala Asp Thr Leu                 165 170 175 Gly Trp Pro Ser Leu Pro Leu Asp Val Pro His Pro Thr His Thr Pro             180 185 190 Ala Met Ala Gla Ala Ala Arg Glu Ala Arg Ala Thr Ala Pro Arg Val         195 200 205 Ala Tyr Gly Ser Gly Arg Trp Arg Val Val Arg Ser Pro Trp Leu Gly Arg     210 215 220 Asp Val Ala Asp Asp Asp Pro Val Asp Leu Val Ala Gly Ala Leu Thr 225 230 235 240 Ala Arg Val Ala Met Leu His Thr Val Arg Glu Leu His Ala Ala Gly                 245 250 255 Ala Asp Ala Phe Val Glu Cys Gly Glu Trp Pro Val Val Thr Lys Phe             260 265 270 Val Glu Ser Ser Ile Pro Gly Val Arg Cys Val Val Pro Leu Ser Asp         275 280 285 Ser Asp Pro Val Gly Ala Val Arg Ala Leu Ala Glu Asp Ser Thr Ala     290 295 300 Ile Gly Ala Phe Arg Ser Ser Leu Pro Ala Arg Pro Ser Glu Arg Leu 305 310 315 320 Leu Pro Ser Ala Arg Val Ser Ala Pro Glu Pro Asp Gly Pro Ala Ser                 325 330 335 Pro Leu Thr Val Ala Val Thr Val Ala Glu Ala Pro Thr Pro Ala Thr             340 345 350 Ala Val Ala Thr Pro Ala Pro Leu Glu Ser Tyr Ala Glu Pro Thr Ala         355 360 365 Ala Ala Thr Ala Ala Thr Thr Ala Ala Ala Thr Ala Ala Val Ala Ser     370 375 380 Ile Ala Pro Ser Thr Val Thr Ser Val Ala Gln Ala Ala Thr Ala 385 390 395 400 Thr Pro Ala Pro Ala Ala Val Ala Pro Pro Ser Ala Gly Leu                 405 410 415 Asp Tyr Glu Thr Val Leu Thr Glu Leu Arg Thr Leu Tyr Gly Asp Phe             420 425 430 Leu Gly Tyr Pro Pro Asp Leu Leu Gly Glu Asp Asp Gly Leu Glu Ser         435 440 445 Glu Leu Gly Val Glu Ser Leu Lys Gln Val Thr Leu Leu Gly Arg Val     450 455 460 Ser Asp Arg Tyr Asp Leu Pro Asp Leu Arg Ser Ser Ser Ser Leu Leu 465 470 475 480 Thr Val Gly Thr Leu Arg Arg Ile Ala Glu Ser Val Val Gln Gly Arg                 485 490 495 Ala Glu Ala Ala Gly             500 <210> 11 <211> 41591 <212> DNA <213> Streptomyces galbus <400> 11 tcgaccgagt agggcgctgg atcggcgttc ttgagcgccg cctcgacttc gaacgcgccc 60 ttggaccctg gtgtggccga tgctcccgtc ttcgacgtgg atgcgggagc cttcccggca 120 tctccggcgc cacaaccggc aagcaggacg gcggccgcga ttccggccgt cacggtggcc 180 aagtggggca tggcggggga cctcctcagc gtggttcaca gaccgctgag tctgtcatgg 240 gcacagccgc gagccctcca ccccttctca cggcgccacc atcccttgct cgcccgcggt 300 gtccagcaat ccaggtcatg acccgggcgc cgcagtgagg gccttgtggg tcgactccag 360 gatcttctcc gacagttctg tgcccgccgt ggcccgtgcg agcaggatcg cgccgaccag 420 agtggccacg acgggaaggc cgtcggcggc gtcggtggcc atccacgcgg cgaattcctc 480 gactccggtc gcgtacgttg cgcgcacctc ctcagccagg ggctcgcgag ccatgtcccc 540 tgcgaatccc gcggtggggc aaccggtgcc gggctggtct cggtgctcgg ccgacaggta 600 gaaatcgagc agggcgccgc gcgcggtgtc gtgatcgccg tgggccgtgt cgaacgactc 660 caggagcagg tcgaggtccc cgaaagcggc atgggccgcc tcggccacca gcgcctcctt 720 ggacgagaac tgcttgtaga agccgccggt ggtcagcccg atggacttca tcagatcggc 780 aacgctgatg ccgttcacgc cgcgctcccg gaagagctgg gaggctgcgg cgaccgcgcg 840 cctgcggttc tcgagcgcct gagcctgcga aacgcgactc atcagtcacc tgcccacgtt 900 agatagtaaa tcgcatctat catatgcgaa gcgccgggca ggcggctgcg gcgccctctc 960 tctgggctct ttctctcggt ccatgcgcgg ccggccgagg cgggcccgcg cttattccga 1020 ggggtcgggg ggatctggag ccgggtcgag ggcggctcac gcggcgcacg gcgagtgtgc 1080 ggcgtgcggg tctccgccgg cgtgccgttc gccgtcccgc gcaatcgtcg cgagggcggc 1140 gatgcccgca atcgtccacg gcgaccatac gccggaaggg atctcccttc gcgcgcaccg 1200 gatcgggggc acctgatcga ctgtcgacgc cgtagtcccg tatgcgcgtc gaggtgctcc 1260 agccgcggac ggtgactgac ggcatcgggg ccgttgactg gcacgcttta gataactaac 1320 gtaatctaaa gcgaggtcga tggttccggc ccgctactca ttccgcgaga ccagaaatga 1380 gatccacctc catgaccacg cagaacaaga ccgcggtcgt caccggcgcg tccgccgggc 1440 tgggtgccgc ttacgcgcag cggctcgcag accggggcta cgacctggtg ctggtggccc 1500 ggaacaccgc gcggctggaa acgctggcgg cggacatccg cagccgtacg ggccgctcgg 1560 tggacgtcgt gaccgccgac ctcaccgacg cggcgcagat ctccgtggtc gaggagcgtc 1620 tgcggaccga cgagagcatc gaggtgctga tcaacaacgc cggcgggtcg ctgttcaccc 1680 cgctggcgac ctccgacgcc gcggacgccg aggcattgat caacctcaac gtgacctcgc 1740 tgaccaggct gaccatcgcg gtcctgccgg gcctgacagc ccgggggcac ggcaccgtgg 1800 tgaatgtgtc ctcggcgctt gctctcaaca tcctgcccgt cagtgccgtc tacagcggca 1860 ccaagagtta cgtgctgacc ttcactcagg cgctgcagca ggagctcgcc gagagccccg 1920 tcgtggtgca ggccgtgctg ccgggggccg tccgcacgga gttctgggac ggctccggcc 1980 tcgatctcgc ggcgtttccc gacgagtgga tcatgagcgc ggacgacgct gtggacgcgg 2040 cactcgccgg gctcgacgcg ggggagcccg tcaccatccc gtcactgcct cagatcagtg 2100 actgggaagc gttcgagaag gcccgtcaga cgctcgtccc gaacctgtcg cagcgggtcc 2160 cggctgatcg ttaccgcggc tgaacaccgc tcagactcgg gtgcggaagt cctgattgac 2220 cccgtgccaa cggcacgcgc gacggcggcg gtcatgtgcg ccccatgccc gaccacaccg 2280 tcgattaccc ctccacgcag tccggtgccg cctcggtgca gtcaccggtc cccttcggcc 2340 ccaccggtcc catccgaccc accggtccca tccgacccac cggtcgcata gagcccgccg 2400 gtcccatccg gccggtccgc gcgtaggtgc ccgccgcggg gctgcctgcc accccccccc 2460 ggttctctca cccgtttact taaggaacat ccatgttgaa cgccctgtgg aacccgatcg 2520 tcgccgggga gatctccctg ccgcaccgcc tggcgatggc ccccctgacg cggaaccggt 2580 ccacaccgga cggcgtgccg accgagctga acgccgagta ctacgcccag cgcgcctcgc 2640 acgccctcat catcaccgag ggcacccagc cctccgccga cggccagggt tacccggtga 2700 cgccaggcat ctacaccgat gagcacatcg ccggctggcg caaggtcacc gacgccgtgc 2760 acaaggccga cggacgcatc gtcatccagc tcatgcacgc cggacggatg tcccaccccg 2820 acaacacccc ccaccaccgg cagccggtgg ccccctccgc cgtccggccg gcgggcgaga 2880 tgttcaccgc gtccgggctc caggagatgc cggtgccgcg tgagttgtcc acggaggagg 2940 tctccgcgac ggtcgacgac ttccggcgcg ccgccgcggc cgccgtcgcg gccggcgccg 3000 acggcgtcga gatccacggg gccaacggct acctggtgca ccagttcctg gccaccaaca 3060 ccaaccagcg caccgaccag tacggcggct ccgtcgacaa ccgcatccgt ttcgccgtcg 3120 aggtcgccac cgccgtggcc gacgagatcg gcgccggccg caccggtatc cggatctctc 3180 ccgggaaccc cttcaacgac atcgtggaga gcgacaccca cgagctgtac ccggcactcg 3240 tgagcgccct cgccccgctc gacctcgcct acctgcacat cgcccacggc ggcgacgacg 3300 aacttctgca caccctgcgc aaggtgtggc cgaccgccct cgtcctcaac cgggccggca 3360 ccgacatcgc cacccgcgcc aaggacgtcg aagacggcct ggccgacatc gtcaccgtcg 3420 gcaccatggc gctcgccaac cccgacctgg tcgagcgcgt acgcgccggc gcaccgctga 3480 acactcccga ccccgccacc ttctacggcg gcgacgcaac cggctacacc gactacccca 3540 ccctcactgc ctgacaaacg ccggcgacac cggtcctggc gggccggtat gcccggatgg 3600 ccccatggag gtacacgtgt ctgagcacac tgcagttgaa ctgagcccgg aagcgatcca 3660 gttcgcggaa tggttggccg agggggcgaa gcccccgtcc gagctggatg cggcgcgcat 3720 ccagggggag caggtccatc tcgcggcccg ggagccggaa ggcgtcacct atcgggaggt 3780 ggacgcgggt ggcgtgctgg gaatctggtg cgaacccgtc gacgccaaca ccgaccacgt 3840 cctgctgcac agtcacgccg gcggctccgt tctggcgtcg gcacacgctg accggaagct 3900 cgcaggccat atcgccaagg ccgcaggggc ccccgtcctg gtcctggact tccggcgggc 3960 gccggaacac aagtacccgg ctcaggtgga cgacgtggag gcggccttca actggctgct 4020 ctccgaaggg tatgagccgg ggaacatcat cacgatcggc cactcgatcg gcgggttcct 4080 cgccgtcgcc gtggcgcttc gcctccgcga caagaagcaa ccgctgcccg gtgccatcgt 4140 gtcgatctcc ccgtggtgcg acctcgagat cgccaacgag accatcgcgt ccaacgccgg 4200 gacggacaag atactcagca aggatctgct ggagttcttc cgcgagtcct ggatcggcgg 4260 cacgggcatc gagttcacgg acaccaggat caatctgaac cgtgcggacc tgagcggtct 4320 ccccccgacc ctcgtgtcct ggggaacgta cgaggtgctg gccggcgagg acgaggagtt 4380 cgccgcccgc gtcaaggacg ccggcatcga caccacgacc gtggtggtct ccggaggcca 4440 gcactcgtac gtctacggtg ccggccgggt tccggagacc gacgccgcca tcgcgcggat 4500 cggcgcgtgg gtccgggaga agacgaagat ctgaccgagc gctcggcagg tgcaggatcc 4560 gcgcctgccg gagcgcgact tcgtcctggt ccagcagacc acgactacgg cctcagctcg 4620 gtcgcgcgca ccggcgtgcg cgaccgagct cgcactggcg ccgaagccga cgactggctc 4680 accgccacac tgcgtccgct ggatgtgccg gccgatcagc gctcgcggat gagcgactcg 4740 ggcaggtagg cggaccggat gcccagctcc cagtcatggg ccagagaagc gagcgcggtc 4800 agtgttgatg gcctccaccg gcagcagact ccgggggcgg ggtcggtgcc ggccatcgat 4860 ctcaccctgg accggctcag ctccgttcgg cgtcaggggc tgtcggccga cgaccggggg 4920 cggtgccgct gcccgtgggg cggggtccgg gcaccgtgct cctcgaccgg acgaactggc 4980 tgggcgtttg gccgtaccgc ttcttgaagg cacgcacgaa gtgactgctg tcggcgaatc 5040 gccagcgggc ggcgagttcc gagatgctcg gtcggccaag cggtgacgcg agctcaaggc 5100 gtgcttgctc cagccgcctg cgccggatgt acgcgctcac cgactcctcg gccgcggcga 5160 acgcccggtg cagcgtcctg acggagacgt tgagttcgcg ggccagggcc gagggcgaga 5220 gctcaggatc tgtgaggcgg ctgtccacga tgcccttcgc cgcttcggcc agcgcggggg 5280 ccagccaggg ctcgtggtcg tcgaatccct gcgtcagcac gcccttggtc agttcgatca 5340 gagagttgcg ggcggcttgt agaccggcgg gcgtgaggtc gttcaggctc gcatccacca 5400 acccggcgta gcccatgagc agacgtactt cggtcgagtc ggccggtccg acgacaggcc 5460 ggtcgccgat cagcggcctg agctgtgcgg cgggcaggat cagcaccttc gccgtcgtgc 5520 gcggctccat ctcgaaccgc accggcggac cgttgtgccg cgcgatgaac tgcccggccg 5580 ggacggcgaa ctcaccgcgc tcgcgcggtc ggacgaagcg ccactcgccg cgccgcacca 5640 catgcacgag gacccggtcg tcgaggtggt gaccggcgcc ccgggtgccg ccggccaacg 5700 actcaccttg caggtccgcg atcaccgcgt cgtgcatctt ggaggcgcgg acggtgaccc 5760 ggaagtcacc ggaaccgagc ggttcgaggg gtggcagggg gagcgcctct ccgacctgtc 5820 cctcccactc gcgtcggaag acgtcgaagc cgtcccgcgc ggtgctcggg gcggtgacgt 5880 ccacgaagaa attcctgccc ggcccgccgg atctcggtgc ggtgcttcct gtgttgctca 5940 tggccgacgc cccaactcga caacggatcg caggacttac gcggcccgct tcacctttca 6000 ccttgccaca ccgcccttgg cactgacgtt ccatcggagt ggcgccgcag tacaagcgtg 6060 cgccgcaacg atctccctaa ggtgagcggt ggagcctgaa ccgcagagca cggggcggcg 6120 tcggaagccg aagccgacgc ggcgctactc cggcgtcgtg ccgctcaagg agccgccgcg 6180 tcgcctgaca ccggacctcc cgcccctcgt cccccacggg ctccgaccca tcctgaatcc 6240 tgagaggtta ctggctgtca tgactgacac gaccgacact acgcgtggca tcgaagtcga 6300 gccgagcgat ccagcagcgg acactgctgc cgttttcgac ctcctgggcg gccttgtcgt 6360 cgcccagatc ctgcgggccc tggccgccct gcgcatagcc gatcatctcg ccgacggacc 6420 cctgacggcc gaggagttgg ccgagcgcga gggcagccat ccgcaggcca cgtaccggct 6480 gatgcgcgcg gccgcgtcct cgggcctgct gagctacgaa gggcgccgtc gcttcgccct 6540 caccgggcga gggcggctgc tgcgttcagg tgtgcccggc tccctgcgct cactggtact 6600 gcccagacg ggacacgcgc actggcaggc ctgggcccac ttccccgaag cggtgcggca 6660 gggagccact cagaccagga aggcgctggg agcggacatc ttcgagtact tcgcgcgacc 6720 ggagaacgct gacgaggccg cgctcttcgc tcaggcgatg ggagacctgt ccggcctggt 6780 gacgcggggc gccgtgagcg cggtctccac cgtcggtgta tccaccgtgg tcgacgtcgg 6840 cggttcgaac ggtgacttcg tcctggcgct gatggaggcc gacccccagc tgagcggaca 6900 ggtactcgac ctgcctcacg ccgtcgaggg cgcacgcggc gaggccgcga aacgtggtct 6960 gtccgaccgc ttccacgcgg tcgccggcga cttcttcgcc gaggtccctc cggcggacct 7020 ctacctcctg aagacgatcc tgcacgactg ggacgacacc cagtgcgccg tgatccttcg 7080 gaactgccgt tcagccgtga acgagggggg acgagtcctg gtcgtcgaga cggtcatcgg 7140 tgagatcggt gagcccgact tcgccacccg ggccgacatg accatgctcg ccatgacaaa 7200 cggcatggaa cgcgatctgg acgagttcga cgccctcttc gcggcctcgg gatggcgccg 7260 ctcgcggacg tatcccgtgg gtggcggata cttcggcatg gagctcctgg ccgtctgacg 7320 gttgcgcggc gcgcgctgcc attggctcca cgccgcgcag gactggtgat gagccccacc 7380 acggccgggt cggccgtcga cgcggaactc gtcggtagcc ggtacctcac tgcggccgag 7440 cgtgcgaaaa tgcgctgttg tatttcacct gacgctcttc tagcctgtta ttcatggcga 7500 atcagatagc agccgggccg gaactcttac aatacgtcag ggacttctcg ctcagagaag 7560 acgacattct gcgtgacttg cgcctgctga cctcgggact gcccggcgga gaggccatgc 7620 aggtcacggc ggaggagggc cagttcctcg ctttcctgat cgccgcgacc ggtgtcacgg 7680 acgtgctgga ggtcggtacg ttcaccggat acagcacgct ctgtatggcg cgtgccctac 7740 cgccgcaggg gcgactgatc acatgtgaca tcagtgagcg gtggcccgag atcggtgtcg 7800 agtactggcg gcgcgccggt gttcttgagc gaatcgaagt gcgtatcggc gaggcggcgg 7860 acacattgga gaaaatgtgc gccgacggat tgtccgaaag cttcggtctt gttttcatcg 7920 acgccgacaa ggtgaattat ccgcgctatt acgaactggc attgcggctg gtccggcccg 7980 gcgggctgct ggtggtggac aacacgctct tcttcggccg ggtcatcgac ccgctcgccg 8040 acgacgcgga caccgccggg gtacgtgagc tgaacgcgat catccgggac gaccgacgga 8100 tcgatgtctc catgctcccg gtggccgacg gcatcaccct ggtccgccgg aagtgagatc 8160 gggagcgatc agccggagcg tcgtgggcgc ctggcgccgc tcgggggcga ggtgcagcag 8220 ttggacgtcc tcggcggcga cggagttcgc ttcttcggct ccggcctcgc tcccgccctc 8280 gcccgagctc ccgtgggccg agccctcgcg gaccaggccc cggcaggcgc agggctggtc 8340 tccgaacccg gcgaaacggt aggcgatctc catcatgcga ttgcgctcgg tggcccggaa 8400 gtcggccagc aggtggacac cggcccgcgc cgcctggtcg gccagccagt tcaggaggat 8460 ggtgccgacg ccgaacgcca ccacacggca cgaggtggcg agcagcctga gatgccaggc 8520 ccgtgggtgc ttctccagca gcaccacgcc cacggcgccg tgcggtccga aacggtcggt 8580 gagcgtggtc accagcacct cgtggcccgg gtcggcgagc agggcgcgca gtgtcgcgtc 8640 cgggtagtgc accccggtcg cgttcatctg gctggtgcgc tgggtcagtt cctcgatccg 8700 cgccagctcc tccggcgtgg cgcgcgcgat gcgcagcacc atctccagcg accgcaggaa 8760 gtcctcgtgc gagccggtga accgcgcctg ttcgtccgtg cgccggaatc cggcctggta 8820 catggagcgg cgctgtcgcg cgtcgacggt cacggtcccg gggctgaact cgggcaggcc 8880 gggcaggagc gcggcgtcct cggcccggta gcagcgcacg tccgggaggc tgtgggccac 8940 ctcggcccgc tcggtcggct ggtcgtcgat gaaggcgacg gcgcggggag cgaagttcag 9000 gccttcggcg atccggcgta ccgcgtccga cttgcgcccc cagccgatct gcggaaggac 9060 gaagtagtcg gcgacgccca gcgcctccag ccgcgcccag gcgacggcgt ggtcgttgcg 9120 gctgcacacc gactggagga tgcctcgcgc gtccaggcgg gtgatcacct cgtggatccc 9180 ttgggccagg acgacgtcgt cgccttccag caacgtgccg ttccagagcg tgttgtcgag 9240 gtcccagacc aggcacttca cggtcgcggt cgccgccgcc cccctcacgc tcatccccgg 9300 ctcccgagca catggtcggc caggatcagc tggcacacct cgtcggtgcc ctcgatgatc 9360 tccgtgagct tggcgtcgcg ataggcccgc gcgacgacgt ggccgtccct cgcccccgcc 9420 gacgcgagca cctggaccgc ggtcgccgcg cccctggccg cgtgctcggc gccgacgtac 9480 ttggccagca cgatggcggc gctcagatcg gcggcgttct cgtcccagca gcggctggcg 9540 tgctcgcagg cccgggtggc gatctgctcg ccggtccaca gatcggccag atgccgggcg 9600 acgagctgat gccgccccag cggccggccg aactggtgcc gggacgcggc gtggccgacg 9660 gccgccgcga cgcaggcccg caggattccg acgcaccccc aggcgacgga gagccgcccg 9720 aaggccaggg cggtcgtcac cagcagggac agcggttgcc gcccgccgcc cagcaggctg 9780 ctcaggggca gccgcacccc gtcgagcagg atgtccgcgt gcccggccgc cctgaagccc 9840 atcggatcgc ccacccgccg cacctggacc ccgggggtgt ccctcggcac cacgaccacg 9900 gcgccgtccg cgcccatccg cgcgacgacc aggaggagat cggcctgctc ggcggcagtc 9960 acccacgtct tgaggccgtc gagcacgagg gtgtcgccgt cgacggtgac ggtggtcgcg 10020 atggccgaca ggtcgctgcc cgcccgcggt tcactgaagg ccacggccgc cgtgtcgccc 10080 gggccagcc gggggagcag ctccgccgcc tgcgccgggt cgccgagccg ttcgacggtc 10140 cacgcggcca tgccctggga ggtcatgacg ctgcgcagcg agccgcacag ggtgccgaca 10200 tgagcggtga actcaccgtt gtccgcgctg ctccaccccg ctccgccgta tcgcgccggg 10260 atctgcgggg cgagctgccc tgcggcaccg agttcgcgca ccacctcctc gggcagccgc 10320 cccgcgcggt cccagtccgc ggcccggtcc ccgaccagct cggtgaccgc ggaggccgcc 10380 gcggcgacgg cgtcacccat gggagccgcc cacggccggt cgccggagcc ggccgaccag 10440 cgcggccatc ctccgcgcgg tccggaagtt gtccatctgg agatccgcgc ctcgcacggc 10500 gatgccgtac gtccgctcca gatgcaccac cagctccatg gcgaagagcg acgacagccc 10560 gccggagccg aagacgtcca tgtcggccga ccaggttttc ccggtacgca gttcgaggaa 10620 ccgcagcagt tccctctcga tgtcgccggg gtcgcccagg ggctccggta ccgcgcggtt 10680 ggatggcatg gtcatgccca ttctccctcg tactcgtaga agccgcgccc gctcttgcgg 10740 ccgaggtgcc ccgcccgcac catctccagc agcaggtcgc aggggcggca gccttcgtcg 10800 ccggtccggc cgtggagcac ccacagcgag tcgacgaggt tgtccagccc gatcaggtcg 10860 gcggtgcgca acggacccgt cggatgcccc aggcacccct gcatgagccg gtcgaccgac 10920 tcggcgctcg cggtgcccgc cgccaccacc ctcaccgcgt cgttgatcat cgggtgcagg 10980 atccggctgg tgacgaagcc gggcgcgtca tcgaccacga cggactcccg gccgagcgcc 11040 gcgagcagcg cccgcagcgc gtccagcgtg gcctcgctcg tctgggtgcc ccggatgacc 11100 tcgaccgtcc gtatcagaca gggcggattc atgaagtggg tgccgaccag atccaccgga 11160 cgggccgtgg cccgggccag ctcatcgatg gggatcgccg acgtgttcga gatgatcggg 11220 gtgcccggcg cggccagcgc ggacacgtcc gccagaacct tcgccttggc gtcggggtcc 11280 tcggtgacgg cctcgacgac cgccgtgaac gccgggccgt cggtcatcag gtcggcgagt 11340 gccggccgg tgacgagggc gccgggcacg gcctcctccg gcagcgcgcc catcagccgc 11400 gccgtcctca gctggtggtc gatccgcccc ggagcctcgt ccaggcggcg ccggtccagg 11460 tcgaccaacg acaccgccat cccgtgcccg atcgccagcg cggcgatgcc cgaccccatc 11520 actcccgctc cgagcacggc caatcgccgc ccggtgccgg gcgtacccct ctgcgctgcc 11580 atatcctccc tcttccggtg ctggaccccg acgcctgtgc tcactcctcc gagcggctcg 11640 cctcggagcg ctcgggccga ggcgggacca ggtcgcccgt gggttccgtg tagaactgct 11700 gcgcccactg ccggtaccgg ccgatcggac cgtcgccctt ggcgagccgg ggacgcgcga 11760 cgtgttcctg gtggtgccac atcagcaggt ccgcgccggt gtcgacgctg cccacccgtt 11820 gcaggacctc gctgagcagc cgggccaggg gcgtgaccgc cgggcggccc agccagtccg 11880 gcacacccgg gaatccgttc agctccagct tcgtgccgaa gcggacctgc atccggcccg 11940 ggccggtcgg ggtcgcgtgg agcatcacgt agaggttccc cgcgcccgcg ggcaggaccg 12000 tccgcgccgc gatggtggcc agtccgatca ccgtgagcgt gtacgactgg tggaatccgc 12060 gcatcatcgt gttgcgcgcg gtcgcggtga ccgtgctgat cggttccccg gtcaccggca 12120 ggcccccgat gtcgacctcc tcgagaccgt gcagggccgg cagatgcccc cagtcgaacg 12180 cgttctcgat gacatcctgt gggtggccgg cgatgtcgaa cgtgcgatga ctgaacggtt 12240 ggcggtcgtc catcgggaag agcgggacgt cccactgcgg gggcatcccc agggcgtgcg 12300 accacacata gaccgagccg ttggcctcgc acaccgggta ctgcgtcagc gacgccttcg 12360 gcggctgctg gtcgtaaccc gtgcgcacac aggtaccgga cgggtcgaac gcgaaccggt 12420 ggaaggggca gacgatgttc tcgccctcga ccgatccacc gacgccgagg tgcgctccga 12480 ggtgcgggca ctggggcctg accgcccgca gcacgccctt ggccgtccgg tagaggacga 12540 cctcggcgcc cgcgagccgt cgggtggtga gggatccggc ccgcagctcg tcgctgaagg 12600 ccacacagaa ccagccgttc gggtaagggc gcgagggagc gtgcagggtg tcggccggct 12660 ccgcaccgtg caggctgatc gccctggagt cgagcagatg cttcaccagg ggcttcaggt 12720 gaggtctcat gggctcactc cccgtgcttc accgggcagg cggcgacgcc cggctgtgtg 12780 gacggccggc cggaggcgga cagcggtgcg gcggcgccgt gctccgcgcc gtcggcgtcg 12840 gcggacctcc gctcggtgag ccgcaggcgc agcccccgtg aactcaggct ggtctccacc 12900 ttcggcagga acggccggtc acccaccgga tgcagctccc agcgggcgac gatggcggtc 12960 agcgccaaga tggcctcgtt gagggcgaac tggtccccga tgcacttgcg ggcaccggcg 13020 ccgaacggga tgtaggtggc ccggtcgggc tgggcgtgga gccagcggtc cgggtcgaac 13080 cggtcgggct cctcgtagag gtcgggcctg cggtgcagca ggtaggggct gagcacgatg 13140 gtgctgccgc ccttgaaccg cacacccccc agttcggcgt cctcccgcac gacgcgggtc 13200 atcatccagg ccggcgggta caaccgcagc gtctcggtca cgacccggga ggccaggccg 13260 agcgagggaa gacgcgcggg gtcgagcttc ccgccctcca gaacccggtc ggtctcggcc 13320 ttcagccgcc gctggacgtc cggatgcgtg gagagcaggt ggagcgccca ggccagggtg 13380 atggccgtgg tctccatgcc accgaggaag aacgtgaggg cctcgtcggc gagttcggcg 13440 tcgttcagct gccgtcggcc gtcctcgctc tcctcgtcga ccgccgcgat cagtgtggag 13500 agcaggtcgg cgtggtcggc cgggtcggcc cgccgctcgg cgatgatctc cgcgatggtg 13560 gcggacagcc gggtgcgggc gtcgtcgtaa cgccgcttcc gccgggtcgg cagccgccgc 13620 agcagggccg gcgtcatcat ccggcggaag aacgcgctga cgacgaccgc ggtgtccgcc 13680 agggaccgcc gcatggtctc ctccggcagc gcgccgctga acatcgtctc catggtggcc 13740 cgcgtggtga gcgtcatcat ctcctgggtg acgtcgatga cgtcgccgtc acgccaggag 13800 cccgctttgg cctcggccgc ccgcgcgaac accgcgccgt acccggcgag ccgggccggg 13860 tggaacgacg gctggcacag ccggcgctgc cgccggtgca gcggataggc gcaggtggac 13920 aggccgtcgc cgatcacctc gcggatccgg tcgtagatcg ggccgccctt gtcgaaggtc 13980 cggtcgttga ggaacacctg ccgggtcagg tcgggatcgc agatcatcac ggcggaactc 14040 ggaccgagcc ggacgcgaac catctcgcgg tacccggaca gcgacgtgat gaacgccagc 14100 ggatcacgca gcagaggcaa cgcgtgaccc agcagcggcc acgcccgcgg agcgagcgga 14160 accgactcgg cggagaccgc actcgtgtct gtcatggaaa acctcacttc ttcgtcgagc 14220 ggccggccgg ccccccggtg gcgggatcgc gggtgatctc cagcagcgcg gtggtccagc 14280 ggaaaccgag cccgaaactc acgaccagga cccgttcccc ggcccgcagc gtgccggagc 14340 ggaactcccg gtcgagggcc agcagcacgt cgcacggtcc gacgtgaccg aggtgccggc 14400 cgaaggtcca gctggtgtcg gcggcgcgca ggcccagggg cgcaccgccg agcagcaggt 14460 ccagcacctc cgccccgatg gcgatgggca ccacgcgcgc gatgccctcc ggccgtacgc 14520 cggtctcgtc gaggacggtc tccacacagc ggcgcgtgga ctgccgcagc gcgtcgacgt 14580 gcggcatcag tccggtctcc tggacgagca gcggccggcc gctgccgtcg gccgccgtcc 14640 gggcccgggt cagcacctcc agctgtggct gagcggcctg cgcggtcgcc atcagccgcg 14700 cgaagcccgt gccccgggtg aggacagcgg cggcaccggc gtcaccggcc aggtgtccca 14760 tctccgcgac atggccccac cgttcgggcc ggaaccggac accggccgtc accagcgcgg 14820 ccttcgaggc cggtctggcg gtgaggtgtt cggccgcggt gaccagcgcg gcgccgccgc 14880 cgtcgctggc ggcgcccagc tcgatcgcgt tggtgtccgg gcctccgagc acccgcagca 14940 gataggccga gggcgcgtag tggtcctcgt cctggaagtt ggcgtgcacg agcaggctca 15000 ggtcggcgcc cgtgacgtcc gcgtggcgca gtgccttgag gccggcgcgt gcggccatct 15060 gggacggcga gctgtccgtg gagacggcgg ccgaggtgaa tccgttgagc gcggtcaagg 15120 acggcggccc gttgtccacg ggcttgtccc acggctcgtg tttctgcctg atcggcggta 15180 tccacgcacc ggtaccggcg atcgtgatgt cctgccacaa cactgtgctg cctcctcggc 15240 tgtcacgaac ggaccggtcg tggcgcgtcg ccgcggggcc ggacgagttc tccgctgtgg 15300 tccagggcat acgcgctgac cccggacacc cgggccagca cctgcccgtc accggcgacc 15360 gcggtcagct ccccggtggc gaagtcgcag tgcaaccgga tcgtcggatg cagcgcgctg 15420 agccggcagt cgtcgcccgg cccgccgaga tcgacctcgt cgaggccggc catcggcccg 15480 acccgcggcg gaacgtcacc gcgcggcggc agcagcaaca ggtgcaccat cgcgtccagc 15540 aggatcgccg gtacggtcat cccggccagg gccggacccc aggtgtcgtg gtccaggcgg 15600 aacctggcgc tgttgccgtc cggtccgcgg gcgtagtcac cggtgccggc gaacggtccg 15660 gtgagggaga tcggcgggtc cggggagtac acaggcatcg cgaagtcggg ttcgggttcg 15720 tgtgccgaga agtcaggtgg ccccgtgagc gccggaaaac ggtccgccag cagcacccgc 15780 gtctcgcaca gcaggtcgtt gaagcggagc accttgccgt tcttgccgat ccggtgtgcc 15840 gtgatcctga cggccacctc ggcgcgggca ccttcccggc gcacgacccg ggcctccacc 15900 gcgacggtgc gcggcgggcc ggcgagcctc accgtgatcg acgaccggca gacgagaccg 15960 cggaacccgg tgacccgcag cccgggacag agggccgccg ccgcgccggc cgccgcctcc 16020 agggtgaacg tccccgccag cgtgtactca ccgttgacct ggtgatgacg catccaccgc 16080 ccgtcccgct cggcgagggt gcgcggatcc caggtcttgg tgaacacggc ccagtcgtcg 16140 ccgcggtaca gcactccgtc gagcagcggt gtggtcagct ccgggtccgg cggcgccgga 16200 ggtggcgcgt ccggggccgg gaccgcaggc tccgctcgcg agccgtgggt gctgataccg 16260 cgccgggcca gcaggacacg ttcctcctcc cggatgaaga acgccactcc gttggccggc 16320 ggcacggtca ccgcggacag gaactgcgcg cggccctgcg ccgtgctgat gtaggcgtcc 16380 atcttgttgc gtttgagggt ctcccgcatg gtgacgctgc tggcgacacc gacctcgcgc 16440 cagccgctcc acaggacggc gacctcgtga cggcccgccg gcgcgcgccg ctgcgcccgg 16500 gcgctcgcgt acgcgaggta ctcgttgacg gcgcagtagt cgatgtcgcc gggtgcgggg 16560 gcgagcgtcg ccagggtgct gaagttgcac cagatccggg gcatgcgtcc ggccagggcc 16620 cgcttcaggt tgcggtagcc ggtcgccttg gtggcccgca cggcccggaa gtccgggagg 16680 gtctggcgt gcagcgcacg tgagcgcagg tcgaggacgg tgttgatgag caggtcgatc 16740 cgtccgtgcc ggtcgaggat ctcgccgagg acggcggtgg tgccttcggc gtcgaggacg 16800 tcgcagacgc ggtggtggac ccggtccggg ccgcacagct ccgtcagccg tcgcaccgta 16860 cggcgcacct ccaggcgtgc ctgggccttc tcgtaggcga cgcgtaactc gcgcagggag 16920 gcgtcgggac gccgtcggcg ttcggcggcg atgaactcgg tctgcggtgg cagcggggcg 16980 tcgctctccg gcagcggggt gcggccgatg acgtagacat ggggccggtc ggtgccgagg 17040 gccacctcgt gcaggagttc gggagtgatc ccccgggcac cgccgaaggc gaccacgacc 17100 gcgccggagg gcagcgggac gccgtcggcc ggggacacgg ggtcgtcggc cacggccagg 17160 gcgaaccagt cgtccccgcg gcaggccagc gtgtgcgtcg gcacccggac ggcgcccgcc 17220 cgtgcgaact ggtccagggc ggtcgccaca tccgccgcgt cgctgatcag ggtcaccccg 17280 ccgcactgcg gcacctcggc gcgtacggag cggacgagtc cggtgaacag cccactcagc 17340 gggggtggca gatcgtccgg cagctctccg agcagcagta cgccgagcga gccgccggaa 17400 cgcagcgcgg gcagcgccgc ctgcagcgtg gtgaacgcga ggtcctggag gtcctccgcc 17460 cgggcggcct cggcctcccc gtacgcgtcg tcgtcgacgg gcagccggca caggacccgg 17520 atgtgccgcg ggacgaacgg caggccgctc agcgcgccgg gcgcttcctc gggcgggaca 17580 tgggcggtcc ccgcgactcc gggtcgcggt gaccagacgg cggtgtccgg gccggtgacc 17640 gcctcggcga gttccggcgc gtcggtgatg acgacggtgc ccggcgggat cgacggcacg 17700 gccgactccg tgggcggccg gtcggtcggc acgagccgga aggtctgacg gcggccggtg 17760 aagtcctcgg tccgttccgt gccggccggt cccgacccgg cctgctcgga ctcggtgcgg 17820 tccggcccgg gacggtccga cccggcaggt tcggggttgg tgtgcgccga gtcggtgtgc 17880 ccggagccgt cggctactgt gcccgcgcgt tctgggtggt cgcgtaccgc gtcggcccac 17940 tgcgggccgg tgagctcggc ctggttcccc gtctcccccc ggtccgctcc gggagcgtcc 18000 cacgggtcgg cgggcggggt gaaccggatc agcggggagc cggcggcggc cggccggacc 18060 gcggtgtccg tcctggtcac gacggcttcg agaaccgtca gcagggcgtc cagggacaga 18120 taggtgtgtc cggtgcgggc cagcacggga agggcgtgcg agggcacggc cggatggcgg 18180 gagtcggcga cggcgacctc ggtggagagc gtgcccagga ccgggaatcc atgctcgagc 18240 gcggtcgagc gccgggtgac cgccaggaac gcggcacctt ccgcgatgga ccggccctcc 18300 ggcaccaact ggtcgaggtg tcggtcccag ccgctgatcg gccgcagaca cacggcgccg 18360 atcagggcga ggtcgcaggc ccggtgccgg agttggcgca gtgccagatc gacggctgtg 18420 tgcgcggagt cccgctggga gtagacgctg gaccccaggc cccggaagtc gtagtagttg 18480 gcggcccggc ccgacaggat gcaggagacg gccccggtga agtcgtcctc gttcaggtcg 18540 cccgggatca ctccctcggc ctccgccatg cccttcgcga ggtactcctt gagcagcctg 18600 gcctgcgccg ggtcgggcag cagatcgaac gcggtggcgc actcggcggc gtgcacccgc 18660 agcgccgcct gggtgttgtg cgtggtcggc agggtcgagc ccaccacgat cgccgtgtcc 18720 ggccgcaggc tcgtgcccgg ctcgccgagc tgttccagca gcgggccgag cgcctggagc 18780 accatcaggt gggcggcgtc catgtgccgc atcgtcagcg gcgggatccg tacgtcacgc 18840 ggggagggca gggggtacgg caggccgaaa ccgggctcgg ggagcggccc gtcgccacgt 18900 aaccaggcgg gcacctggtc gggctccatg ccgggcaggt gggtgttcca gcccacgacg 18960 accaggtcct ccctgtcgtc gcgcacgtcg atgccccgcc tgcgcacggg gacggaccgt 19020 ggcgcgtggt cggacaggac cacgtgcgcg tccgccccac cgaggccgaa cgacgacacc 19080 ccgacgacgc gcggtcgcgc cccgtcggcc ggccaggggg cgtccccgac cggcacggtc 19140 agccgcgcgc cgtcgcccag cagcggatgc ggatcggtga ccacgggctg gccgggcacc 19200 gctccgcgtt cgagggcgac cagggcgtgt gcgaccgaga ccaggcccgc cagcaccccg 19260 gtgtgcccga agacctgctt gttcgacgtc agcagacagg cccgctcgcg cggcccgagc 19320 cgggacagca gcgagcggag ttcgatctcg tcgcccaccg gcgtgccggt gccgtgcgcc 19380 acgatccagt ccacgtcgcc ggcttccaca cccgcgtcgg cccaggcccg gtcgatcgcc 19440 agttcctgcc cgcgcgtggc cggggcgtgg atgcccttgc cccggccgtc ggcggcgaga 19500 ccggtgcccc ggatgacacc gagcacccgg tcgccgtccg ccacggcccg cgcgtacggc 19560 ttgaggacga gcgcgacggc gccctcaccg atcagggtgc cgtcggccgc ccggtcgaag 19620 ggccgcacct ttccgctcat cgagatgccc tgggcccggc agaacagcgt gaacccgatc 19680 ggctcgatca ccgaggtgcc accggccagc gcgatgtcgc aagaaccctc acggagcgcc 19740 ttgaccgccg cgtccagggc gaacagccca ctggcacagg ccgcgtcgag agtcaggtgc 19800 tgtgtgtcgc cggggatcag cccgcgcaac gcgttgcgga cgacggacgc gggcaggtat 19860 gcctgcgggt cgccgccgtc accgccgtag tgcgcgctga tcgcgtggtc ggccagctcc 19920 gccagccagt cgccgtccgg gtcgtcgggg agcgcctcgc gcaccatgcg ccggtactca 19980 tcgccgagca cgacgccctg ggggccgagc cccgtctggt cgtggatgcc cgtggtcgcg 20040 gtgagccagc ggtccgtcgg acgtcggcgc actcccgcca gggcttggac ggcacagtgc 20100 cgcagccagc gagtggtccg cggccagccg gggtgtccga cgttcccgtc cggttcggcg 20160 accgaggcgg ggtcggggac gaagtcgtgg agatatccgg cctcgcggac gtagaaggcg 20220 tcggtctcct cgcgggtcgg agcccagaag tgcttcaggt cgaaggcgga cggttcgctg 20280 gagagcagga cgccctcgcg cagccggtcc cagaactccg ccgcgttgtt cgccccgggc 20340 agtgccaggc ccagcccgac aaccgcgacg gcgccggggt ccgcctccgc cgcggccgac 20400 cggtcgggtg cctgagccac cgccggggcc gccactgtcg ggggtgccgg gggagatacg 20460 tgtgcggacc cggatggtgc ggacgcggat gatgcggacg cggatggtgc ggatgacgtg 20520 ggggcggcg cggggcgggc gggctgcggc cgcccgggtg cgggcagacc cgcgacggga 20580 gctgtcggca ccggcgctgt cggcaccggc ggcgacgggg ccggcggcga cgcgaccggg 20640 cggcgggcgg gcttgctcac ctggtcggcc agcctgctgt gcagtcgcag acctcgcatc 20700 ccaccgatgc tgatgccgcc gtcggcgacg agggtctgcc cggtgatgaa accggcgtcc 20760 tcgtggagga agtgcacgat caacctggcc gcctccggcg tcgagagggt gcggttcaat 20820 aactcgtcgg ccgcggccgg gccgccgcgc gccgccggtc cgtcgaggtt cacgacgatc 20880 tcgctggcga cggagcccaa cagcacggtg ttgacccgga tgccccggcc gaccagttcc 20940 agggccagat agcggaccag tgactccagg gcggccttgg tgacaccacc ggggccgtag 21000 cccgggaccg gctgatgtgc cccgacgctc gacaggcaga ggatgctcgc gccctcccgt 21060 cccgccatca gctcggccgc gcggcgagaa cagtggtagg ccgccatcac attggtggac 21120 ccgcccgtt gccagtaggt gtcgtcgacg tcgaacaggg gcaggaacgc cccgcccgcg 21180 gcgttgttga tcaggatgtc caggccgccg tggcgctcct gaaccaggtc gaacatccgg 21240 tcgatctcgc tggtcttggc cacggaggcg cggatgaact cgcagctgtg gccggcctgc 21300 tccagttcct cctggagcag cttcgcctgg tcgaccgagt ggaagtagtt cacgatcaca 21360 tgtgcgccgc cctgcgccag gcggcgcacg atgtccgcgc ccaggctctt cgcggcgccc 21420 gtgacgaggg cgaccttgcc cctcaggctg tcgtccgtca cccggcagcc tccgccctgc 21480 cctggacgac gctctccgcg atccggcgca gtgtgccgac ggtcagcagc gaggagttcg 21540 acctcagatc gggcaggtcg taccggtcgg agacccggcc gagcagggtg acctgcttga 21600 gcgactccac gcccaactcg gactccaggc cgtcgtcctc gccgagcagg tccggcgggt 21660 atcccaggaa gtcgccgtag agcgtgcgca gttcggtgag caccgtttcg tagtccagcc 21720 cggcggacgg tggggcgacc gcggccggcg gagcgaccgg cgtggcggta gcggcctgag 21780 cgacggaggt gaccgtaggg gacggcgcga tggaggcgac cgcggcggtg gccgcagccg 21840 tggtcgcagc ggtggccgca gcggtcggct ccgcgtagga ctccagggga gcgggggtgg 21900 ctacggcggt ggccggggtc ggtgcctcgg cgacggtgac ggcgacggtg aggggggagg 21960 ccgggccgtc cggctcgggc gcgctcaccc gcgcggaggg caggagcctc tcgctcggcc 22020 gcgccggcag acgggagcgg aacgcgccga tcgcggtgga gtcctccgcc aacgcccgta 22080 cggcgccgac cggatcgctg tcgctgagcg gtacgaca ccgcacccca ggtatcgacg 22140 actcgacgaa cttggtgaca accggccact ccccgcactc cacgaaggcg tcggcgcccg 22200 ccgcgtgcag ttcgcggacc gtgtgcagca tggcgacgcg ggccgtgagc gcgccggcca 22260 ccaggtcgac cgggtcgtcg tcggcgacgt cccgtcccag ccagggagag cggacccgcc 22320 agcgcccgga accgtacgcg accctcggtg cggtcgcccg cagctcccgc gctgcccccg 22380 ccatcgcggg agtgtgcgtc ggatgcggga catccagcgg gaggctcggc cagccgagcg 22440 tgtcggccac ccgctccaca tgccggatca gttcctcggg cccgctgatg acactctgtc 22500 gaggggcgtt ctcgcaggcc agtaccaggt cgggatggtc gatcagcccg gcgagagact 22560 ccgcccgacg gccgtccacg ccgaccgcga gcatcttccc tttccagctg tgccgggtca 22620 gtgcctggga gcgtgcgacg gccaggcgcg cggcgtcctc cacggacagg tggccggcgg 22680 cggcgagggc ccacagctcc ccgatgctct gcccgacgat cgcgatgggc tcgacgccct 22740 cggcgatcag catctggtgg gtcgcgaggc tgacggcgaa gatctccagg tactggacct 22800 cgggaccgcg ctggtcactc gtaccgttgc cctcgatcag ccgactgctg atgtcgggca 22860 gccccaggcc ctcggctgcc ttgtcgatgc gggacagcgc gtcacggacg gtctcgtaac 22920 ggcggtggag gtcgcgaagg agtcccccgc gtggagccgc cacggttccg ggcatgacaa 22980 agacagcatc catcatatac atcaccctca ttccgtcccg gcggcctccg cggcgatgtc 23040 acgtggggcc agccgggctc tgaaacgcca tggcggcgca ccaaaccgac gggatacgtt 23100 cacttgttgc gctatacgag agtggcgggt ccggtgaagt ggaccccggc ggcatgttgt 23160 ttcctgtttg tttcttgtcg aaagggaacc gcattgacgg tatctggggc tcttccgaga 23220 gggcaatgat attcaggaaa ctattttcag cctacatcga tcaggaactc gaatcggcca 23280 atcacatgca ggcgcccttg tggctgcgcg aggctgcatg tcagtcaggt gggccagtcg 23340 ccggatcggt cactcaaagg accgttttga tgtgttttat tttgtgacgc taacgcccca 23400 ttcgcccgcc cggccggctt ggctgcgacg atccgacggg gccgagaacg tcgcaggggc 23460 ggcgccggtc gcctaccgtc gcccgccccg gtccgggcct ccacccggtg caccaattcg 23520 gcaatcggcc aatcggcggg gacgcgaggc ggagtcgggt tttagcggca ccaccgctga 23580 acggccaacg gaacgttcac ttctcggcgg cagctgcggt ccgtgacctc gcgccgccgc 23640 ggtgacctga tttcggcggc ggagacaggc cgatggccgg aaagccgaac tggagatctt 23700 cgatggctga gatggcgacg gccggaattc ggcgagggac tactccaaag tcgcgagtaa 23760 gggggcggtg ggccctcgcc cgcggtcctg ggtcgtactt cgccacgacg tcggtctgcg 23820 gggagcggca gccgtaccgg gccggtggtg catccgtggg ccggtcgcgc gcggaacact 23880 gcggtgtagg ggcggtgtgg gccgcgccgg caggcgggag aggtggtcgt gtgtccgcaa 23940 ccgaggacac gagggacgtg ctggtggtcg gggccggcgt ggccgggctc gcgtgcgcgc 24000 gggatctgct ggcggccggt gtcggggtac gggtgctgga ggccggtgac gaggtcggcg 24060 ggcggatgcg ttcggaccgg gtcgaggggt tcgtggtgga ccgcgggttc caggtggtca 24120 acacgtcgta cccacagctc cggcgccgcg tcacactgaa ggacctgaaa ctgcggccct 24180 tcacccccgg cgtcctcatc cagggcccct cgggccggct ccgcttcagc gaccccaccc 24240 gccggccccg tacgttgccc gacctgctgc cgggccgtct cgccgggcct cgtgacctgg 24300 ccgccctcgg cctgttgtcc gcccgcgaca tgctctcgtc cccccgtcgg ctgaaacggc 24360 tcgcggacac caccacccgc accgcgctcg cggacgccgg gttctccgag agcttcgtcg 24420 aaggcttctt ccgccccttc ctgtcgggag tcttcctgga ggacgacctg gagacctcag 24480 cccggatgtt ccacctggtg tggcggagca tgctgcgcgg caccctgtgc ctgcccgccg 24540 agggcgtcgg agcggttccc cgggccctcg cggccgccct gccccagggt gccgtgcggc 24600 tggagagccc cgtggcgggg ctcacggacg acggggtgct gacggccgag ggtcgggaga 24660 tccccgcacg cgcggtcgtg gtcgccacgg gccccggctc cgtcggcgtg ctgctgcccg 24720 aggtcgccct tcccggctac cggaccgtga cgacgtacta ccacgtcact tcgcgttccc 24780 cgctcgggga gccgaccctg ctcgtggaca cccggcgccg gttcctgaac acctgtgtcc 24840 tcagcgacgt cgtgccctcc tgcgccccgc cgggccacgc cctcgtagcc acgtcggtcc 24900 tcggccgagg ggaggaggac ggggagcccc gggaggcggg ggagcggcgc atccgggagg 24960 ccctcggcga ggcgtacggc accgggacgg gcgactggag cctgctgacc gtccgccgca 25020 tcggggacgc gctgcccgcc atggcgccgc cgcagccgct gacccgcacc acacgcgtgg 25080 caccgggccg ctacgtgtgc ggcgaccacc gcgccaccgg ctccgtccag ggcgcgctgg 25140 cctccggggc acgggcggcg cgcgaggtac tgcgggacct acgacgctga ggcgcgtggc 25200 cgtcgtcccg tcggggtggg caatcgctgg gagccgatgt cgtcggggtg ggcggtcgtc 25260 gggagccgct gtcgtcagcg cggaccgtcg ccgcgccggc tctcctggcg ccgcagctcg 25320 tccacaaccg cgagacgatc cagctcgcgg accggctgag ccatccggag atggcgctcc 25380 aggcgggctc ggtgccggtg ctggaccaat gccctcatca gagcctcagc gccagtcagg 25440 cacgcccgag accggcggta gctgggattc gatcttcgct cgcatctccc gcatcacgga 25500 gacgatgcgg gactcgtgct cgggggtcag acgggccacc gggaccgagc agctgatggc 25560 gtcggtggcc ggggtcgtgt agcggagagc gaacccgaag ccggcgatgc cgatgacggt 25620 ttcctcgcgg tctatggagt agccgcgttc gcggatccgg gccaggtcgg cgtggaggga 25680 agcgcgggtg gtgtgtgtgt tctccgtgag ggcggtcaac gggccattcg gaagagggag 25740 gtcgtcgtcg cggagctcgg cgaggagggc tttgccgagt ccgccggcat gggccgggac 25800 gcgacggccg acgcggctga tggtgcgcag gtactcgtgg gactcgcgag tggcgaggta 25860 gacgacgtcc gggccgtcga gccgggccat gtggatcgtc tcaccgaggg cgtccgacgc 25920 ctcatcgagg taggggcgga cgaccctgat gcgccggtcg ccgtcgagat agctggtgcc 25980 ggtgagcagt gcgcggatgc cgatgccgta gagcgagccg gtgacgtcgg tgcggaccca 26040 gccgcagtcg acgagggtct gcagcagttg gtacatgctg ctgcgcggca cgtccagttc 26100 ctccgcgagc tggtccaggc gtgagggctg ctcgccccgc ccggccagga gttccagcag 26160 cgcgacggtt cgggccgccg acttcacccc acggacaccc gtcttctcga ccatgaactc 26220 atcgtaggcg cgcgcgaatt cgtgtcggca tacccattga cccactccgt tcacagatct 26280 agcctccatc tgcatacgca gacagcatct gtatacaggg acggctttca tggcagggat 26340 cagcatcgag gtggcacagg tggcacaggt ggcacaggtg gcacaggtcg ctcagcggct 26400 ccggggcggc atggagagtg gggttctgtc cttccccctc accagctttc gggacgacgg 26460 aggcctcgac ctggacgcat accgggtgta cctggccgac cggctggcca ccgcccccgg 26520 cgcggtgttc cccgcctgcg gcaccggcga gttcttctcg ctggacgagg acgaataccg 26580 ggcggtcgtg caggcaacgg tcgaagtcgc tgacgggcgc ctcccggtgg tcgcgggcat 26640 cggctacggc tgggcccagg ccatccggtt cgctcgcatc gccgaggacg ccggcgccga 26700 cgccgccctg gtactgcccc actacctggt cagcgccccg caggacggcc tcgtggagca 26760 actgcgccgg atcgccgggg gcacccggct gccgctcatc gcctatcagc gcggacaggt 26820 cgccttcacg gcagcctctc tgcggcgtgt cgcggagatt ccgaacgtca tcgggctcta 26880 ggacggccac agcgacctcg accggctcca gcgcctcacg ttcgccgcgc ccgaggactt 26940 cctcttcttc aacggggccg ccaccgcgga gatccaggcc cgcgcgtacg ccaccgtcgg 27000 cgtcccggcc tactcctctg ccgtccacgc cttcgcccct gagatcgcga acgccttctt 27060 cgtcgccctc gggcaaggcg accacggcac ggtcggcaag ctgctgcgcg atttctacgt 27120 cccgctggtc gaactccgag accgggtgcc gggctacgcc gtgtccctgg tgaaggccgc 27180 cgcccggctg cgcggcctcc cggtcggccc ggtgcgtgcc ccgctcgtcg accccggccc 27240 ggccgacctg tccgacctgg agaaggtgct ggaccacggg ctgagcctgg tcggcgcgga 27300 acgacgacct gcctgacccg cctgactcgc ctcacccgcc tgacgaatcc cgcacggcga 27360 gtcgtccgac gcggccgcag gcacctcgcc gtgcctgccg ggctcctccg cgcctcactg 27420 cccctctccg ccctgcggcc tcctctcgct cgacagttca aaggagaact gccatgcctc 27480 ttctcgtagt cgggatcagt gtcctggtcc tgctgctcct catgaccagg ctgaaactca 27540 acggcttcgc ggcactgctg ctcgtggcgg tcggcgtcgc gctggtccag gggattccgg 27600 tggcgaccat cccggacgtc ctctccgagg gcatcggggg gcagatcggc gacaccatgc 27660 tcaccatcgg gctcggtgcc atgctcggcc gggtcatggg tgactccggc gccgcccagc 27720 gcatcgcagg caagctcctc gacgccttcg gcccgcgctg ggtgcaggtg gcgatggtgg 27780 tgaccgcgat gctgatcggc gtgaccatgt tctacgaggt cgccttcatc atcatcgtgc 27840 cgatcgcgtt cacactggtc agggtcacgg gggcaccgct gctgtgggtc ggtctgccga 27900 tgtccatcac gctgtcgacg atgcacagtt tcctgccgcc gcaccccggt cccaccgccg 27960 tcgccgcgag cttcgacgcc tccgtcgggc tgaccctgtt ctacggcctg ttcatcgccg 28020 tccccgccgg cgcgctcatc gccctggtct ggccccgcct gccgttcatc aaggccatga 28080 cgccctccat tccccagggt ctggtcagcg aacgcgagtt caccgacgag gagatgcccg 28140 gcctcggctg gtcgctgttc gtggcgctgt tccccgtggc gctgatcgtg gcggctgccg 28200 tgacggacat ggccacttcc gccgagacgg ggttcctgca cttcgtcgcc ttcatcggtt 28260 cggcaccgat cgcgctgctg ctgacgctgt gcctggcgat ctgggcgtgc gggccgcgga 28320 tcggccggag cctggaggag gtcggcgcct cctgctcctc cgcggccaag gcgatggcga 28380 tgatcctgct ggtgatcggc gcgggcgggg cgttcaagaa cgtcctcgtc gagggcggga 28440 tctccgacta catcaaggac gccaccgacg gctggtccat ctcaccgatc attctggcct 28500 ggctcgtcgc cgtcatcctc cgcatagccc ttggctcggc gaccgtggcc gtcgtcacgg 28560 cctccggcgt ggtgctgccg ctcctggcgg ggagcggggt ccacccggag gtgatggtcc 28620 ttgccgtcgc ctgtggctcc atcgcgttct cccacgtcaa cgaccccggt ttctggctct 28680 tcaaggagta cttcaacctc tccgtcatcg aagcgatcaa ggtccgcacc agctacacga 28740 ccgtgctcgc catcctcggc ctgggcggcg tcctggtcgc cgagcaggta ctcgacgttc 28800 tcaacctctg aaccacacgc acgacccctt ggctcggaac actccgcgcg ccccgtacca 28860 cctcacccca gaagggcacc cgatgagcat cggacagccg accgtcacgg cgttcgccgt 28920 ctaccccgtc gccggacggg acagcatgga gctgaacctc tccggcgcgc acggccccta 28980 cttcacccgc aacgtcgtcg tcctcaccga ctccgagggg cgcaccggac tgggggaggt 29040 gcccggaggg gagaacatca cgcggacact gcgggacgcc gagtcactga tcgtcggggc 29100 caaggtgggc gactacaagc gggtgctgcg cgccataggt gaccggttcg ccgaccgcga 29160 cgccggcggg cggggcaccc agaccttcga cctgcgcacc accgtccacg cggtcaccgc 29220 cgtcgagtcg gcactgctcg acctcctggg acagcacctc gatgtaccgg tcgcggcgct 29280 gctcggcgac ggccaacagc gggattccgt ccgggtactg ggctacctct tctacgtcgg 29340 cgacccggac cgcaccgacc tggagtacgt ccgtgaaccc gactccccgg tggagtggta 29400 ccgcgtccgg cacgaggagg ccctcacctc ggaggcgatc gtccggcagg ccgaggcggc 29460 ctacgacctc tacgggttca gggacttcaa gctcaagggc ggtgtcctgg agggcgccga 29520 ggaggtcaag gccgtacgtg ccctcaagaa ccgcttcccc gaggcgcgga tcactctcga 29580 ccccaatggc gcctggtcac tgcgcgaggc ggtcgagctg tgccgtccgc tcaccgacac 29640 gctcgcctac gccgaggacc cctgtggagc tgagggaggc tactccggac gggagatcct 29700 cgccgagttc cgtcgcgcca ccggcctgcc caccgcgacc aacatgatcg ccaccgactg 29760 gcgccagctg acccatgccc tggccctcca gtcggtctcc attccgctgg ccgacccgca 29820 cttctggacc atgcagggct cggtccgcgt agcacagttg tgcaacgcga tgggcctgac 29880 ctggggttgt cactccaaca accacttcga catctccctg gccatggtga cccactgcgg 29940 cgccgcggcc cccggtgagt acaacgccct ggacacgcac tggatctggc aggagggact 30000 ggaacggctc accaccacac cgccccgtat cagcggcggc gagatcgccg ttccggacgc 30060 cccgggcctg ggggtccacc tcgacatgga ccgactgctc gcggctcacg agctctacca 30120 gcagaaggcg ttgggagccc gcgacgacgc agtcaccatg caatacctca tccctgcctg 30180 ggcattcgac aacaagcggc cctgcctggt gcggtaggcg acgttcgcag gtcgccaaga 30240 gctcctggtg gaccgccggc atcgccgccg tggcaacctt gctcgcctcg gaccagcacg 30300 gttcggcgat gcgcatgggg cgcaaccgcc gatggaccga tagcgccgat gcctgcgggg 30360 ctgggcgtgg ccgccgctgt cgggcaccgg gactccgacg tggtggtctt cgcccatccg 30420 acgggccgca ctcagccgcg tacggcggtc tccagcggaa accgtgtccc cgtcgtgccc 30480 caggtggttc gtgtcgaact gcgtctcgaa gccgtcccgc gtgcgctgct acggccacac 30540 tgccccacga cgacccgtac cggcggcgcc gccacccccg agaggcggtc cgggaagccc 30600 tcgccgcacg acgccgggca cgccgggcgg cgctcggccc cgcaccggat tccaccccgg 30660 cgcccgcgct cacgcagccc ccggaccgaa tcggggaccc gtccctgagc gtgaaggagt 30720 cgcctgccca cccccgtgga accaccccga gggcccgccc cgcgatcagg gcaggccctc 30780 gggggacgtg gcggggtcac agggtgacca tgcgtaccag gaagaaggcg gcgacgagca 30840 gaccgccgac cgcgagcagg aacagccaca ggtgcggatg ctcccagagg cctccgtccg 30900 gtcgttcgcg gtgcgcctcg gccgtcgacc cttcggcggc gggggtctca ccgggcgggg 30960 aaagctgctc gctcccggca cctgcctcgt gcggcctctt ggggttcatc agcactcctc 31020 cgggctcggg acgatcgtcg tccgctacgg aacagaagcc gtatgcgggg acgcatcaca 31080 gctgctcttc cgcttggagg gcgagtccgg atgcgtgtgc ggagctaggg cgcttctgat 31140 ggatctccgt gggagaagga gcggcgtcgt gggggcccct cccggtcgag cgaagccgag 31200 actgggggag cgtgctctcg gcgtgccgcg cggaagcccg cgtactggac gtactggggt 31260 tttcgcgcgg tgcggcgaga gggcgtgccg ggcgtcgcga cgccgcggag atccatcaga 31320 agcgccctag ccgccgggcg actcggtggt ccggcctccc ggttcactca cggcgcagcg 31380 gatcacacgg actccggtgc gtgctcctca ctcgccgtgg acgcgtcgtc gccggggcga 31440 ccggcggaga ccgtgatcct cacggcgtcg cccagaccga aggggcgcag gagctccgga 31500 tccgaacgca gcccccatcc cgggccgcac agcagaacat cactgcgggt ccgggcaccg 31560 tggatccccc agcgtatggc cgcgaggtgc cgggcgagcg ggaggttcgc ggtactccgg 31620 gactgcgacc acaggaccac ggcggccggg gcgaccctgc ggaccgtggc ctccagcgct 31680 tcgaccggca ccgaaccgcc cagcatcagg acgcgcagcc cccgctcggc cagtacggcg 31740 gtgagcacct ccagcgggag ggtgtgctgt tcgcccggca ggcacgccag gaggacctgg 31800 cgcgtgcctg cggctcggcc ttgggccgtg gcgcgtgcgt aggcgtaccg gaggaccgtg 31860 gagatgtgcc aggacagcag gtgctccact tccacgtacc ggtcgtcgga ggactcccac 31920 ctgcgtccga cgtaccgcag cgtcggaacg aggatctcct cccacgcggc caccaggccg 31980 tacgcgtcca ccgtcgccgt gatctgctcc tgtacggcct cggcgtccat acggaccgcc 32040 gcctgcgcga gcccccggca cagtcggcgc gagtcggcga tcggcgggac atccgccggg 32100 gccgcgtcca cgggtgagaa cggctcgtcg accggtgcga gagggcgggg aggcgggtgg 32160 cctgccaggg gtgtctcggc ggccggggag cgcacggtgc cggttctgag cgcctgggcc 32220 ttggcggccc gggccgcctc cccgggaggg aggccctccg cggtcatgcg gcacatctcc 32280 cgcaccatgg cgacgtcctc ggccgtccat cgccggtgac gcccctcgac cctggtggcc 32340 ggcccgagac cgtagcggcg gtcccaggaa cgcagtgtgg tgggagagat gccgagcctg 32400 cgcgcgagca tcccggtcgt catgccggcc ccggcgccgg gttcctcgtg cgcgtccatc 32460 ctgtcgcctc cgccgtagcc gatgtccctg gcctcgatgt cccgggcctc ggggcacggg 32520 aggtattcac cgtccggcgt gcggacggat gcgatgcggg cgatgtgggc aaggcgccga 32580 gggatgcgc cgagacgatg tctgggggca ggcgcggtga gcgcgcccgc cgtccagggc 32640 gggcgccccg ggaacgcggc gggcgacgag cgccgggacg cggtcagaca gcgggttcca 32700 tcacccggcg cagcgagtgc agggccaggc ggatatggct cttcaccgtg cccaggggca 32760 gccccgtgag ctcggcgatc tgcgcctggg tcatgtcgcc gtacacggcc atgcccagca 32820 cacgccgctg caccggcgtc agacgctgga gcttctgcag gacgaggacg cggtcgacgg 32880 cggcctggct ctcgtcgacg ccccagtacc gcaccgcctc ctcctgctgg gccgcggaca 32940 tgctctcgac gtcggccctg cgcgcccgcg ccgcatgcgc gtccgctatc gcatggcgcg 33000 tgatgccgac gagccatccg cccagcgcgc cgcgctccgg gcggtaccgc tcacgtcccc 33060 gccaggcggc cagaaagacc tgctgggtga cgtcctcggc gtcccgctca tcgttcagcg 33120 agcgccgggc gagcgtgtgg acgagggaac cccaccgccg gtacgccagg gcgatcgcct 33180 gctcgtcccc ggcgtggaac cgccgggcga gttcggtgtc atcggtttcc tcgggcgccg 33240 ggagcggacc ggtcaccctg cggggatcgg ggtggcgggg atgggtgcgc tccgcgtcgg 33300 cgctcaggtg tgtgtcgggc ggtgcagtgg tcatcgtgag ctccgaggtc gaaggagtac 33360 gacacgacga ccgtccgtcg aaacgatgcg gcgggcaacg tgcagcgtta gcgcatcgaa 33420 atgctccact caagcgccca tgcggccggc ccgcttctcc ggagcccacg cacagggcct 33480 tcgagcgacg caagcccttc aagcacacgt ttcgtccacc cgcaaagggc gccaggaaaa 33540 gacgccccgg gtaagacgcc ccgaaaaaga cgcccggaaa gaatctgcat cccccgccgc 33600 ggcacgcacc gaagaccagg tgcactcact cacagggagt gggcagttcg aagggacagc 33660 cagccatgat ctcccggacc cccgttgccg cagcagcctc gctcggcgtg tgcgcgctcg 33720 tgctgtgcgg cgccggaaca gcgaacgccg ccgccgacga cgacaaggcc atggtctccg 33780 tcttccacgc cgttcccggc ctgaacgtcg acgtgtacgc caacggcgac gaactgattc 33840 ccgacttcga accgggcacc ctcaccgacc cgcagcccct tcccgccggc tcctacgacc 33900 tgaaggtctt cgaggcaggg gcggacccga agggcgagcc cgccatccag aagactgtcg 33960 aggttcccgc gggggccgac gcgaccgtcg tggcccacct gaacgccgac ggtgcgccac 34020 agttggacgc ctacgtcaac gacacctccg aagtggctgc cggccaggcc cgactgacgg 34080 tccgtcatgt cgcggcggct ccggccgtcg atgtcagggc tggcggcaca ccggtcttca 34140 aggatctcgt caacccgaag gaggacaccg ccaccgtgga cgccggcacg gtctcggcgg 34200 acgtcgtgct ggccggcacc gacgacgtcg ccatcggtcc cgccgacctc gccctcaagg 34260 agggcacctc caacatcgtc tacgcctggg gaagcgccga tgacaagaac ctcgctctga 34320 aggtgcagac cctgaccggc atgcactcct cgccctccgg tgtgaacgcc ggttcgacca 34380 acgacaccga cgcatggctg gcctacgccg ccggagccgg cctcctcggg cctcgccggc 34440 gcgctgaccg cccgccgtgt ggtgtcccga ggccgtgcgt gacaccgggc cggtcccgtg 34500 gttccggcgg cgtctccaga cagcggtgac agccctgctg gcggtgtccg gagccacggt 34560 gctggcactg gcaccgtccg gcggtgagag ccttccgccg accgcggact tcgacgcgag 34620 ggccgccgac tcgtcgccca tggccccgcc cgaccgcgcg gagagcacca gcactccggc 34680 cacggcggcg aacctgggcc ccgcctccga cccgaggcgg ctcgacatcg agcgcgtcgg 34740 tctcagcgcc gcggtcgagc ccggcggagt ggccccggac ggcaccgccg agattcccga 34800 ggacccggag aagatcggct ggtaccgctt cggtcccgct ccgggcgaac cgtccggttc 34860 cgccgtcatg gtgggacacg tggactcccg cacgggcgac ctcggggcct tcgccgccct 34920 cttcgacgta cgcgcgggcg acgaggtgac cgttcggcgg gccgccgcac cgcccgtcat 34980 gtaccaggtc gtcgcgcgca tacaggtgga caaggaccgg ctgcccgcct cgaccttcgc 35040 caggaccggc gaaccggtcc tcacgatgat cacctgcgcg ccccccttcg acggggatcg 35100 aggcggctac cagcggaacc tggtggtgac ggcggtcccg atcacctcca cggaatgagc 35160 ccgtggcccg gtgcgcccgc ttacgctgcc gtcgattgcc gtcgattgcc gtcaattgct 35220 gtccatgccc ggggcggggg aggggaccat gcctgagggg cgaccggctg cacgagggga 35280 ggcggagcat ggcgcagcac ctggaccccg acgagctgac cgacctcgca ctcggacccg 35340 ttgcccgccc gaccccgggc cagcacgctc atctggcacg gtgtccggcg tgccgcgatg 35400 aactcgacca gctgcgacgc gtggtccgtg ccgcccggac cgtctcgacc gacgacctcc 35460 tgaccgcacc gcccgacgag gtgtggcgct cgatctcggc ggaactggag tcggacacgg 35520 ccccgcaccg gcaggccggg agccgcgaca ccccaccggt gccgggtcga agcgacggac 35580 gtgacggaac cgacgaaacc gacggaaccg actgaagcgg cggacgcggt ccagcgtggc 35640 tgcgccgccc cgcccgccct ggcggtcgcc tccgcgagtc tcgtcgtggg ggttgctatg 35700 cgtgccgtc accgcatggc ggctcgccga cgacacaccg agggccgagg ggagtgcgcc 35760 tgatccgcgg ctccggggca gaacgggagg tcacggtgac cgtcaaccgg tctgccccgc 35820 acggacggct actgggcgac ctcggccggg accccctgat cgacgtgtcg gcgcaggagg 35880 acgacggcaa cccggcccat tccaaccgga gcgtcgttcg cggccccctg ccggctgacc 35940 cggtaccgcc gaagcgagcg ggaacccggc ggcagcggct ccctctctga accgtcggaa 36000 caggcctcgc ttgcgatgtc gttcacggcc ggagccgatc ccagaatcag gccgcgacga 36060 gttcctgctc gcggtccggg gttttgacct tgggcttctt gttcggcagg gagagccgga 36120 agaccttgtg ccacgcggag aacacctgct tgggcagcgg cccggtgacg tactccagct 36180 cgtacttctc gaacaacgcg cgcaccttca ccgcgacctc ggcgtaccgg ttgctcggca 36240 ggtccgggaa caggtggtgc tcgatctggt gcgacaggtt gccggtcatg aagtgcatgg 36300 ccttgctgcc gctgatgttc gccgagccca tcatctggcg caggtaccac tggccgcgcg 36360 tctcgccctt gatcgaccgg cgctcgaaga cctgcacgcc ctcggggaag tgcccgcaca 36420 tgatcaccga gtgggtccag aggttgcgga ccaggttcgc ggtgaacgtg gcggcgagcg 36480 tggtgaggaa cgacgggccc gacagcagcg ggtggatcac gtagtccttg agcacctgct 36540 tgcggatctt gcggcccacg gccctggccc gcgcgcggaa ctccgggttc ttgcggcggc 36600 gcttgtgcag gttcttgccg agttccaggt cgtacgcggc gatgccgtac tcgaagaagc 36660 aggcgttgat gaagttccac agcggctggc cgaggtggaa cgggtgccac cgctggtcct 36720 cgtcgacgcg catgatgccg tagccgaggt cgttgtcctt gccgatcacg ttggtgtacg 36780 tgtggtgcag ctcgttgtgc gagtgcttcc actggtcggc cggcgagacg tgatcccatt 36840 cccaggtggt ggagtggatc ttcgggtccc gcatccagtc ccactggccg tgcaggatgt 36900 tgtggccgat ctccatgttg tccatgatct tcgccacgga cagaccggcg gtgccgatca 36960 gccacgcggg cgggaagaac gagaacagca gcacgcccct gctgaccagc tcgagcttgc 37020 gctgcgccga gatgaccttg cggatgtagg cggcgtcctt ctcgccgcgg tcggcgagaa 37080 cctcgtcgcg gatcgcgtcc agctcgcggc cgagctcctc gatctgctcc gcggtcaggt 37140 gggcggtggg gtcgatggcg gtcaaggtgc tcctaccgtt cgatgtcaca ggggcccgcc 37200 gcggcggaca cgcaggtctg gatgaggacg cccggctcgg cctcggtgat ctcgccggtg 37260 cgcaggtcgc ggacggcgcc cgccttgagc ggcgtgacgc agccgaagca gatgcccatg 37320 cggcacccgg acggcatgag cacgccggcc tcctcgccga catccagcaa cggcgtggcg 37380 ccgtccgcgt cgacggtctt gccggtggtg ctgaacgtga cctcgccacc gtcgccgacg 37440 gcgacgacgc tggggcggaa gcgctcggtg tgcagacgct ctcggacgcc gtgctcggtc 37500 cagtgctcct cggcggcgtc gagcaggccc gtgggcccgc aggcccaggt ctctcgctcg 37560 gcccagtcgg gcacgagttc gtcgagacgg gagatgtcga gcatgccgtg cgtgtcggtg 37620 tgcagctcgg tgagacgcag cttcttgtcc gcgaccaggc cgtgcagatc gtcgcggaag 37680 atcacgtctt gcgggcgtgg cgcggagtgg accatgacga cgtcgtcgaa ctcggtgtcg 37740 cgcagcatgc ccatcacagg cgtgatgccg ctgcccgctg tcagatagag caccttggcg 37800 ggcttggcct ccggcagcac gaagtcaccg gtcgcctggt cgagctggat cagcgtgccc 37860 gt; atcgagacgc ggccgtcctg gcggtctgtc ggtgaggtga gcgagtaggc gcgccacagg 37980 cgcaccccgt cgacgtcgac cccgatccgc acgtactgac cggctgtgtg gccgcgccag 38040 ccccgtcccg gcctgatcac gatggtcgcg gcgtcacgtg tctcggggtg cacggcctcg 38100 atacgacccc gcaggtcggc gcccgcacgc agcgggctga ccaggtcgag gtagtccgac 38160 ggcagcagcg gcgtcgtgac catctccagc attttccacg ccctgctgcg gagggctgca 38220 ctcgtcatga gtccagcttg atgcgccgca gggcgtaaag tcctgtccgc aggacgtgaa 38280 tctgatcggc tgaattgttc acagggaaca aaatatgagc catgtaatcc ggagggccac 38340 cgaactggcc ctggatgaga cgacggtcgc cgcgctccgg gccgcgctga gaaccaccgc 38400 cgacgaggtc gtccaggcga tcatcgacga ggtccctcct tacgccaacg ccctgtcggg 38460 ccgcatgggc ggcaccatcc gacgagccgt ccgcaccgcc ctgggcaact acctggacgt 38520 ggcgagcggg aacgccacgg gcggcgacgc cggtgacgcg gcttacgagc tgggccgcgg 38580 cgaggtgcgc gcggccgtt cgatggacgc cctgctcagc gcctaccgcg tcggcgcccg 38640 cgtggcctgg cgatgcctgg ccgcgggcgc cgtacccgca ggtctgcccg ccgccgaggt 38700 cgccaagttc gccgagctga ccttcgccta catcgacgag ctctccgccg cgagcgccgc 38760 gggccacgcc gacgaactgg ccgcccgggg ccgggtgcat gagcgtcacc tggaacaact 38820 ggtccgggac ctcctcgccg gcgcgagccc ggacgtgctg caggcatctg tccaacgggc 38880 cgcccggcc tgcctaccgg gtgctcgacc cgagcaccct cgtcctcgac gatctgccgg actcctcggg 39000 cgtgctgctc gtccccgatg ccgaccgacc acatctcttg cgccagctgg ccgaccgcac 39060 cgccgtggtc ggcccggccc ggccatggac tcgtgcgtcc gcctcgtacg cacgagccgt 39120 gcgcgcgcgt tcactctcct gcgacattcg cgacaccgag gagcacctgc ccgagctggt 39180 actgagcgcc gacggggacg cactcgcgga cctgcgcgcc cgagccctcg caccgctgcg 39240 gccctgcct gtcgcgaccg cgcggcggct ggaggagacc ttgcgggcgt ggctgctgca 39300 ccaggggagg cgggacgagg tggcggcggc gttgttcgtc catccccaga ccgtccggta 39360 ccggatgtcg cagctgcggg agctgtttcc cgacctcggg tcgccgcacc gggtccttga 39420 actgacgctg gcggtcggtc ttcgggacag ctgacacgtt cttcagacct tctgcggctc 39480 gccgcgggcc aggagtccct gccctcatgc ccctacggcc ggaagatcac ccgacccggc 39540 ggttgcccgc cgttccccgc cacatgaggc attcgaggag catcacccca gctgatgtgg 39600 gccgtgcgga cccgccgtgg ggggactcgt cgtcctctcg aagccagacg acggcaggcg 39660 cgacacctgg cgtgcgcgcc tgccgcattg cggcctttca gtcccgcggt gcgacgcgga 39720 tacggttccc gtcaggatcg gctgcgagga aggtcagccc gaaccccgcg tcgtgaggct 39780 cgcgcacgat cgtgacgccc ttggactgcc actgcttgaa gatcgcgttg agctcgtcgg 39840 gaccaccgtc gatggccagg cagacctcac tggtacgcgg gacgtccggt gacagatgct 39900 cgaactggcc agaccacaga ccgaggtcag cgcccggccc gaggtcgaag gtgatgtatc 39960 ccggagtctc gaacgagggg ctcatgtcga ggaggtcgct cgtgggagcg cacctgcatt 40020 gcgggtctgc actgcgtgtc ttcactgcgc tcatcgcgca ggccgacgcc cgaagaggac 40080 ctcgattggg ccgtctcggt tgattccacc ctcgtgggcg ctcaccagca cgctgccgga 40140 gctcacggcc tcgatcacat tggccgcagc acgagcggga acgccgcagg gcgatcggga 40200 tcgtccagaa cggcggcttc ctggcgtgcg gtcattccga tggagacgga gtcgtcccat 40260 ggccatccgc gagaacttgc ccgaagctcg tgtcaggagc gagtcttccg atgggcgagg 40320 aaagcgaaga ccgccagaag cgctccgcct gctgctgcga tcactccgag cacaatgggc 40380 acagtgtcgc ccgtgactgc tcccgcgagg cacgcacata tgccgccgat gatcgcaatc 40440 acggaaaagg ttcgcactcc cctgatcgca tccctcatgg acgcgtccct ccctcgcaac 40500 cagcgctcct tccgaatccg caggaaaatg ttgggtcgtt gagcctgaca gtgcacgagg 40560 acgccgcagc cggacgtggg ccgtcgcggc gtccgccgtg aggggtggtg acgcctgcct 40620 accggaccgg tgtgtccggc agcccccacg gctcctccgg cacgggatcg gggttgtagg 40680 agtggtggta gctctccggc ggcgcgaggt aggtgacggg cagcccgccg gtgtcgatga 40740 cgatctggtc cacggcgatc gcgggatcga ccatgaagag ccgtaggacg tgttcgccgg 40800 gttcggtgac ggtcacggtg gtggtcagtt tctcgatgcc gtcctcgacg ttgcgggccc 40860 aggcgtcgcc gcggttgccc gtggcgacgg cctgtcccgc caggacggcg acaggctggt 40920 cgtcgagggc cagggcgacc cggcgacggc cgcgttcgtc gagggagggg agcctgaagg 40980 cagtgacccg gaagtccccg gtgccggcga aacggacccg gtagcgcaac tccggtgccc 41040 gagtggtgag ttcgtcggtg atcggcgccg ccgtcgaagg caccgcctcc atggcggccg 41100 tacggcggcc gagcccgcgt accgtccgcc agcgggcccc gccgcgcgcc acccggtggt 41160 cgtagtgcgc ggcgtccatc gagacatacc catgggcctc gacgaagccg cgcgcccgcc 41220 ggcgcgcccg cctcccgtcg ttgagcaccc gtaggggtac ggcgaggctg ttgcctgcac 41280 cggtgacggt caccgtggct tcgtgtgcgc ccttgggcac ccgctcccag tcgatctcca 41340 cggacacccg ggtctgttcg gtcatcgtgc cgccggaggt gctcagccgt acccacgggt 41400 gt; gtcggtcacg ggtgtaggag gagaaggaca gaggccgtcc ggtgccggtc tcgttgccct 41520 cggccgccac gcccagaccc gaggtctcct tccgggcgac cctggtgacg ctcggacgtc 41580 ctggcgcctt g 41591

Claims (15)

A gene encoding the amino acid sequence of SEQ ID NO: 9, which is a protein required for galbanolide biosynthesis.
The gene according to claim 1, wherein the gene encoding the amino acid sequence represented by SEQ ID NO: 9 is a β-ketoacyl synthase.
11. A gene cluster encoding a protein necessary for biosynthesis of galblonide having the nucleic acid sequence of SEQ ID NO: 11 and comprising the gene of claim 1.
A composition for promoting galbanolide biosynthesis comprising an expression vector of the gene of claim 1.
5. The method according to claim 4, further comprising an expression vector of a methoxymalonyl-acyl carrier biosynthesis gene selected from the group consisting of genes encoding the amino acid sequences represented by SEQ ID NOS: 1 to 5 Wherein the biosynthesis enhancing composition is a biosynthetic composition.
5. The composition for promoting biosynthesis according to claim 4, which further comprises an expression vector of a gene selected from the group consisting of genes coding for the amino acid sequence shown in SEQ ID NO: 6 and SEQ ID NO: 7.
(a) introducing the composition of any one of claims 4 to 6 into a galblonide biosynthesis strain to transform; And
(b) culturing the transformed strain and recovering galbanolid from the culture; &Lt; / RTI &gt;
The method according to claim 7, wherein the strain is selected from the group consisting of streptomyces galbus , Micromonospora narashinoensis and Micromonospora chalcea . Production method.
The production process according to claim 7, wherein the galbanolid comprises galalonoids A and B.
A methoxymalonyl-acyl carrier biosyntation gene selected from the group consisting of genes encoding the amino acid sequences represented by SEQ ID NOS: 1 to 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, And a gene coding for the amino acid sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
11. The method of claim 10, wherein the strain is selected from the group consisting of streptomyces galbus , Micromonospora narashinoensis , and Micromonospora chalcea . A galbanolide biosynthetic strain.
A gene encoding the amino acid sequence represented by SEQ ID NO: 8, which is a protein required for galbanolide biosynthesis.
13. The gene according to claim 12, wherein the gene encoding the amino acid sequence of SEQ ID NO: 8 encodes beta-ketoacyl synthase.
A gene encoding the amino acid sequence shown in SEQ ID NO: 10, which is a protein required for galbanolide biosynthesis.
15. The gene according to claim 14, wherein the gene encoding the amino acid sequence represented by SEQ ID NO: 10 encodes acyl transferase.

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