CN101157929A - Safromycin Biosynthetic Gene Cluster - Google Patents

Abstract

The invention relates to the cloning, sequencing, analysis, function research and application of a biosynthetic gene cluster of safromycin, an antibiotic with anti-tumor activity produced by a streptomyces in the tetrahydroisoquinoline alkaloid family. The entire gene cluster contains a total of 30 genes: 3 non-ribosomal polypeptide synthetase genes, 4 precursor molecule 3-hydroxy-5-methyl-O-methyl-tyrosine synthesis genes, 2 peptide backbone loops Synthase genes, 7 safromycin modifier genes, 5 genes related to S-adenylated methionine synthesis, 3 regulatory genes, 2 resistance genes and 4 genes with uncertain functions. The synthesis of safromycin can be blocked by genetic manipulation of the biosynthetic gene, and its yield can be changed; the structural analog of safromycin can also be obtained. The gene cluster can be used for genetic engineering, protein expression, enzyme catalyzed reaction, etc. of tetrahydroisoquinoline alkaloid family compounds, and can also be used for finding and discovering compounds or genes used in medicine, industry or agriculture.

Description

Safromycin Biosynthetic Gene Cluster

Technical field:

The invention belongs to the field of microbial gene resources and genetic engineering, and in particular relates to the cloning, analysis, function research and application of the biosynthetic gene cluster of the antitumor antibiotic safromycin.

technical background:

Tetrahydroisoquinoline alkaloids are a class of complex and unique natural product families, and double tetrahydroisoquinoline alkaloids represented by saframycins (Saframycins, SFM) are a branch thereof, and they have good Antibacterial and antitumor activity [Chem. Rev. (2002) 102, 1669-1730]. SFM (as shown in Figure 1) was first isolated from Streptomyces lavendulae 314 [J.Antibiot. (1977) 30, 1015-1018], wherein safromycin A (SFM-A) is a relatively active component, The ID ₅₀ of its antitumor activity is about 5 nM [Gann. (1980) 71, 790-796]. SFM-A can inhibit RNA synthesis at a concentration of 0.2 μg/mL and DNA synthesis at a concentration of 2.0 μg/mL. It mainly exerts anti-tumor biological activity through the alkylation of DNA, and its C-21 cyano group helps to form an electrophilic iminium ion to carry out alkylation on the N-2 position of guanine G in the minor groove of DNA. Alkylation modification, and has a moderate degree of sequence specificity for the site of alkylation modification, such as 5'-GGG, 5'-GGC [Chem. Rev. (2002) 102, 1669-1730]. In 2004, Myers screened the target protein glyceraldehyde-3-phosphate dehydrogenase GADPH [Proc.Natl.Acad.Sci.USA.2004 (101 ), 5862-5866]. The protein generally exists in the form of tetramer, which is glyceraldehyde phosphate dehydrogenase and participates in the metabolism of glucose; when it exists in the form of monomer, it is an important part of the transcription complex in the S phase of the cell cycle, so it is speculated that GAPDH should be are proteins involved in the cell cycle. Whether the anti-tumor mechanism of SFM-A is mainly caused by the DNA breakage caused by the alkylation modification, or through the combination with the effector protein to terminate the cell cycle, so that the DNA cannot be synthesized, has not yet been determined, but SFM-A has anti-tumor activity , it is an indisputable fact that DNA can be modified by alkylation.

SFM-A has a unique chemical structure (as shown in Figure 1), and the double tetrahydroisoquinoline skeleton structure is a typical feature of this family of compounds. In 1979, Arai et al first determined the structure of SFM-C through X-ray crystal diffraction analysis [Tetrahedron Lett. (1979) 20, 2355-2358], and then determined SFM-C by comparing with the ¹³ CNMR data of SFM-C. The structure of B. In SFM-A, there is a cyano group at the C-21 position, and its structure was determined by comparing with the spectral data of SFM-C such as high-field ¹ H NMR. The structures of other members have also been confirmed by NMR, X-ray and other methods. Later, with the discovery of other compounds in this family, this unique chemical structure attracted many organic chemists to engage in their chemical synthesis research [Org.Lett.(1991)1,75-77; Org.Lett.(2000)2 , 3019-3022; J.Am.Chem.Soc.(1999) 121, 10828-10829; J.Am.Chem.Soc.(2001) 123, 5114-5115; J.Am.Chem.Soc.(2002) 124, 12969-12971]. Precursor labeling experiments show that the SFM backbone is derived from 2 tyrosines, the side chains are derived from 1 alanine and 1 glycine, and the methyl group is derived from methionine [J.Biol.Chem.(1985) 260, 344-348].

At present, a tetrahydroisoquinoline natural product Et 743 (see Figure 1) from a sea squirt (Ecteinascidia turbinate) in the Caribbean Sea has entered Phase III clinical research of Pharma Mar, and is very promising as a treatment for breast cancer. Cancer, malignant melanoma, lung and ovarian cancer new drugs. The in vitro activity of this compound is LC ₅₀ <1pM, ID ₅₀ is 0.2-0.6nM, and its activity is about 50 times higher than that of the antineoplastic drug paclitaxel [Clin.Can.Res.(2002) 8, 75-85]. As an antitumor drug with good clinical application prospect, the source of Et 743 is very limited. According to statistics, only 1g of Et 743 can be obtained from 1 ton of sea squirt, which cannot meet the needs of clinical scientific research. Some people have obtained Et 743 by culturing sea squirt cells, but its marine origin makes research very difficult. At present, Et 743 used in clinical research is mainly derived from the cyanide derivative of safracin B (safracin, SAC-B) produced by the fermentation of Pseudomonas fluorescens A2-2 through 21-step chemical semi-synthesis [Chem.Rev. (2002) 102, 1669-1730; Org. Lett. (2000) 2, 2545-2548].

SFM has a similar skeleton structure to Et 743, and its host bacterium is Streptomyces, which can obtain metabolites by fermentation. We use safromycin derived from microorganisms as the target molecule, start from cloning the biosynthetic gene cluster, use microbiology, molecular biology, biochemistry and organic chemistry to study its biosynthesis, clarify the biosynthesis mechanism, and through metabolism Engineering to produce Et 743 or generate SFM analogues combined with chemical semi-synthesis to generate Et 743 or analogues provides molecular and active diversity for the discovery and drug development of new active "non-natural" natural products.

Invention content:

The present invention relates to the cloning, sequencing, analysis and function of the biosynthetic gene cluster of saframycin (Saframycins, SFM), an antibiotic with antitumor activity produced by Streptomyces lavendulae314 in the tetrahydroisoquinoline alkaloid family research and its application. In the present invention, the entire gene cluster contains 30 gene nucleotide sequences or complementary sequences (sequence 1), of which 3 (sfmA, sfmB, sfmC) are used to encode non-ribosomal polypeptide synthetase (NRPS), including 3 modules and 12 functional domains are responsible for catalyzing the biosynthesis of SFM polypeptide chains; the proteins encoded by 4 genes (sfmO5, sfmM2, sfmM3, sfmD) are responsible for catalyzing the precursor molecule 3-hydroxy-5-methyl-O- Methyl-tyrosine biosynthesis; 2 genes (sfmCy1, sfmCy2) encode proteins responsible for catalyzing peptide backbone cyclization; 5 genes (sfmS1, sfmS2, sfmS3, sfmS4, sfmS5) encode proteins responsible for S-glandular Synthesis of glycosylated methionine (SAM), which provides cofactors for methylases; 7 genes (sfmM1, sfmO1, sfmO2, sfmO3, sfmK, sfmO4, sfmO6) encode methylases and oxidoreductases that catalyze Further modification of the molecular skeleton; 3 regulatory genes (sfmR1, sfmR2, sfmR3), 2 resistance genes (sfmG, sfmH) and 4 genes with uncertain functions (sfmE, sfmF, sfmI, sfmJ) were also included.

The present invention also provides a nucleotide sequence encoding TetR family transcription regulator, which consists of the amino acid sequence in Sequence 2 and is named sfmR1. The nucleotide sequence of its gene is located at base 786-1373 in Sequence 1.

The present invention also provides a nucleotide sequence encoding an oxidoreductase, which is composed of the amino acid sequence in sequence 3 and is named sfmO1. The nucleotide sequence of its gene is located at base 1370-2284 in sequence 1.

The present invention also provides a nucleotide sequence encoding a transcription regulator, which consists of the amino acid sequence in sequence 4 and is named sfmR2. The nucleotide sequence of its gene is located at base 2487-2996 in sequence 1.

The present invention also provides a nucleotide sequence encoding redox cyclase, composed of the amino acid sequence in sequence 5, named sfmCy1, the nucleotide sequence of its gene is located at base 3166-4704 in sequence 1.

The present invention also provides a nucleotide sequence encoding a translocase, which is composed of the amino acid sequence in sequence 6 and is named sfmG. The nucleotide sequence of its gene is located at base 4738-6177 in sequence 1.

The present invention also provides a nucleotide sequence encoding an ultraviolet repair protein, which consists of the amino acid sequence in sequence 7 and is named sfmH. The nucleotide sequence of its gene is located at base 8748-6289 in sequence 1.

The present invention also provides a nucleotide sequence encoding monooxygenase, which consists of the amino acid sequence in sequence 8 and is named sfmO2. The nucleotide sequence of its gene is located at the 10384-8819th base in sequence 1.

The present invention also provides a nucleotide sequence encoding a methylase, which is composed of the amino acid sequence in sequence 9 and is named sfmM1. The nucleotide sequence of its gene is located at base 11059-10460 in sequence 1.

The present invention also provides a nucleotide sequence encoding an unknown functional protein, which is composed of the amino acid sequence in sequence 10 and is named sfmI. The nucleotide sequence of its gene is located at the 11694-11212th base in sequence 1.

The present invention also provides a nucleotide sequence encoding pyridoxamine-5'-phosphate oxidase-related protein, which is composed of the amino acid sequence in sequence 11 and is named sfmJ, and the nucleotide sequence of its gene is located in sequence 1. 12188-11691 bases.

The present invention also provides a nucleotide sequence encoding cytochrome P-450 hydroxylase, composed of the amino acid sequence in sequence 12, named sfmO3, the nucleotide sequence of its gene is located at base 12372-13559 in sequence 1 base.

The present invention also provides a nucleotide sequence encoding ferredoxin, which consists of the amino acid sequence in sequence 13 and is named sfmK. The nucleotide sequence of its gene is located at base 13613-13798 in sequence 1.

The present invention also provides a nucleotide sequence encoding redox cyclase, which is composed of the amino acid sequence in sequence 14 and is named sfmCy2. The nucleotide sequence of its gene is located at base 15388-13874 in sequence 1.

The present invention also provides a nucleotide sequence encoding cytochrome P-450 hydroxylase, composed of the amino acid sequence in sequence 15, named sfmO4, the nucleotide sequence of its gene is located at base 16947-15520 in sequence 1 base.

The present invention also provides a nucleotide sequence encoding AL, PCP, C, A, PCP non-ribosomal polypeptide synthetase domain, composed of the amino acid sequence in sequence 16, named sfmA, the nucleoside of its gene The acid sequence is located at bases 16971-22481 in SEQ ID NO:1.

The present invention also provides a nucleotide sequence encoding a non-ribosomal polypeptide synthetase domain including C, A, PCP, composed of the amino acid sequence in sequence 17, named sfmB, and the nucleotide sequence of its gene is located in sequence 1 at bases 22618-25866.

The present invention also provides a nucleotide sequence encoding a non-ribosomal polypeptide synthetase domain including C, A, PC, RE, composed of the amino acid sequence in sequence 18, named sfmC, and the nucleotide sequence of its gene The sequence is located at bases 25863-30320 in SEQ ID NO:1.

The present invention also provides a nucleotide sequence encoding 5-methyl-O-methyl-tyrosine-3-hydroxylase, composed of the amino acid sequence in sequence 19, named sfmD, the nucleoside of its gene The acid sequence is located at bases 30317-31414 in sequence 1.

The present invention also provides a nucleotide sequence encoding peptidase, which is composed of the amino acid sequence in sequence 20 and is named sfmE. The nucleotide sequence of its gene is located at base 31411-33780 in sequence 1.

The present invention also provides a nucleotide sequence encoding an MbtH-like protein, which consists of the amino acid sequence in sequence 21 and is named sfmF. The nucleotide sequence of its gene is located at base 33777-33998 in sequence 1.

The present invention also provides a nucleotide sequence encoding a methylase, which consists of the amino acid sequence in sequence 22 and is named sfmM2. The nucleotide sequence of its gene is located at base 34031-35131 in sequence 1.

The present invention also provides a nucleotide sequence encoding a transcription regulator, which is composed of the amino acid sequence in sequence 23 and is named sfmR3. The nucleotide sequence of its gene is located at base 35223-35759 in sequence 1.

The present invention also provides a nucleotide sequence encoding prephenate dehydrogenase, which consists of the amino acid sequence in sequence 24 and is named sfmO5. The nucleotide sequence of its gene is located at base 37069-35900 in sequence 1 .

The present invention also provides a nucleotide sequence encoding S-adenosyl-L-homocysteine hydrolase, which is composed of the amino acid sequence in sequence 25, named sfmS1, and the nucleotide sequence of its gene is located in sequence 1 38550-37096 bases in the middle.

The present invention also provides a nucleotide sequence encoding methylenetetrahydrofolate reductase, which consists of the amino acid sequence in sequence 26 and is named sfmS2. The nucleotide sequence of its gene is located at base 39521-38580 in sequence 1 base.

The present invention also provides a nucleotide sequence encoding 5-methyltetrahydrofolate-homocysteine S-methyltransferase, composed of the amino acid sequence in sequence 27, named sfmS3, the nucleoside of its gene The acid sequence is located at bases 42940-39557 in sequence 1.

The present invention also provides a nucleotide sequence encoding carbohydrate kinase, which consists of the amino acid sequence in sequence 28 and is named sfmS4. The nucleotide sequence of its gene is located at base 44013-43036 in sequence 1.

The present invention also provides a nucleotide sequence encoding S-adenosylmethionine synthetase, which is composed of the amino acid sequence in sequence 29, named sfmS5, and the nucleotide sequence of its gene is located at the 45220th- 44015 bases.

The present invention also provides a nucleotide sequence encoding a methylase, which is composed of the amino acid sequence in sequence 30 and is named sfmM3. The nucleotide sequence of its gene is located at base 46245-45241 in sequence 1.

The present invention also provides a nucleotide sequence encoding an FAD-dependent oxidase, which consists of the amino acid sequence in sequence 31 and is named sfmO6. The nucleotide sequence of its gene is located at base 46553-47683 in sequence 1.

The complementary sequence of sequence 1 can be obtained at any time according to the principle of DNA base complementarity. The nucleotide sequence or part of the nucleotide sequence of Sequence 1 can be obtained by polymerase chain reaction (PCR) or digesting the corresponding DNA with a suitable restriction endonuclease or using other suitable techniques. The present invention provides a way to obtain a recombinant DNA plasmid comprising at least the DNA sequence in Partial Sequence 1.

The present invention also provides a method for producing microorganisms in which SFM biosynthetic genes are interrupted or doubled, at least one of which contains the nucleotide sequence in SEQ ID NO: 1.

The nucleotide sequence or partial nucleotide sequence provided by the present invention can be obtained from other organisms by methods such as Southern hybridization using the method of polymerase chain reaction (PCR) or DNA comprising the sequence of the present invention as a probe. Genes similar to SFM biosynthetic genes.

The cloned DNA comprising the nucleotide sequence provided by the present invention or at least a part of the nucleotide sequence can be used to locate more library plasmids from the Streptomyces lavendulae 314 (NRRL11002) genome library. These library plasmids contain at least part of the sequence of the present invention and also contain previously uncloned DNA from adjacent regions of the Streptomyces lavendulae 314 genome.

The nucleotide sequences comprising or at least part of the nucleotide sequences provided by the present invention may be modified or mutated. These pathways include insertions, substitutions, or deletions, polymerase chain reactions, error-mediated polymerase chain reactions, site-specific mutations, rejoining of different sequences, different parts of a sequence, or alignment with homologous sequences from other sources Evolution (DNA shuffling), or mutagenesis by ultraviolet light or chemical reagents, etc.

The cloned gene comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be expressed in a foreign host through a suitable expression system to obtain corresponding enzymes or other higher biological activities or yields. These exogenous hosts include Streptomyces, Pseudomonas, Escherichia coli, Bacillus, yeast, plants and animals, etc.

The amino acid sequence provided by the present invention can be used to isolate the desired protein and can be used for antibody preparation.

The polypeptide comprising the amino acid sequence or at least part of the sequence provided by the present invention may still have biological activity or even have new biological activity after removing or substituting certain amino acids, or increase the yield or optimize protein dynamics characteristics or other efforts to obtained properties.

Genes or gene clusters comprising the nucleotide sequences provided by the present invention or at least part of the nucleotide sequences can be expressed in heterologous hosts, and their functions in the host metabolic chain can be understood by DNA chip technology.

The protein comprising the nucleotide sequence code provided by the present invention can catalyze the synthesis of 3-hydroxy-5-methyl-O-methyltyrosine and 5-methyl-O-methyltyrosine and SFM polypeptide backbone , and further catalyze the synthesis of antibiotic SFM.

The gene or gene cluster comprising the nucleotide sequence provided by the present invention or at least part of the nucleotide sequence can be constructed by genetic recombination to construct a recombinant plasmid to obtain a new biosynthetic pathway, and can also be obtained by insertion, substitution, deletion or inactivation Novel biosynthetic pathways.

The cloned gene or DNA fragment comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can obtain a new structural analog or precursor of SFM by interrupting one or several steps of SFM biosynthesis. The inclusion of DNA fragments or genes can be used to increase the yield of SFM or its derivatives, and the present invention provides a way to increase yield in genetically engineered microorganisms.

The non-ribosomal polypeptide synthetase provided by the present invention may be derived from one or more non-ribosomal polypeptide synthetase domains, Modules or genes to produce new peptide compounds.

Fragments or genes comprising the nucleotide sequence or at least part of the nucleotide sequence provided by the present invention can be used to construct a non-ribosomal polypeptide synthetase library or a non-ribosomal polypeptide synthetase derived library or combined library.

The synthetic gene for catalyzing the synthesis of adenylated methionine (SAM) provided by the present invention can be used for synthesizing adenylated methionine.

The synthetic 3-hydroxy-5-methyl-O-methyltyrosine and 5-methyl-O-methyltyrosine biosynthetic genes provided by the present invention can be used to synthesize the unique synthetic 3-hydroxy- 5-Methyl-O-methyltyrosine and 5-methyl-O-methyltyrosine and other tyrosine analogs.

The post-modification gene of the SFM skeleton provided by the present invention provides a way to obtain analogues through genetic modification, and the included redox reaction can also have other applications.

In a word, the information of all genes and proteins related to SFM biosynthesis provided by the present invention can help people understand the biosynthesis mechanism of SFM family natural products, and provide materials and knowledge for further genetic modification. The gene and protein thereof provided by the present invention can also be used to find and discover compounds or genes and proteins that can be used in medicine, industry or agriculture.

Description of drawings:

Figure 1: Chemical structures of Saframycin (SFM), Et743 and Saframycin (SAC)

Figure 2: Cloning of SFM biosynthetic gene NRPS fragments

(A) Peptide chain extension catalyzed by NRPS; (B) Inferred biosynthesis of SFM peptide chain; (C) Electrophoresis of NRPS gene fragments obtained by PCR: lane 1 is the molecular weight standard, and lanes 2 and 3 are PCR products; (D) HPLC analysis of fermentation products of mutant strains with interrupted NRPS gene fragments: I is the standard product, II is the wild-type fermentation product, III is the fermentation product of NRPS gene disrupted mutants not related to SFM biosynthesis, IV is the NRPS related to SFM biosynthesis Fermentation product of mutant with gene disruption, co-injection of fermentation product of V above IV and standard.

Figure 3: Cloning of RE fragment of SFM biosynthetic gene

(A) The way of peptide chain termination in NRPS; (B) The predicted termination of SFM peptide chain; (C) Electrophoresis of RE gene fragment obtained by PCR: lane 1 is the molecular weight standard, and lanes 2 and 3 are PCR products.

Figure 4: Gene structure and restriction endonuclease map of the SFM biosynthetic gene cluster

(A) The three overlapping cosmids represent the 70kb DNA region of the Streptomyces resistant genome, B represents the restriction endonuclease BamHI, the entity represents the part that has been sequenced by DNA, and Probe-2 and Probe-3 represent the labeled probes Needle part; (B) Gene composition of the SFM biosynthetic gene cluster.

Figure 5: Biosynthetic pathway of S-adenylated methionine (SAM) contained in the SFM biosynthetic gene cluster

Figure 6: Proposed biosynthetic pathways of 3-hydroxy-5-methyl-O-methyltyrosine (A) and SFM-A (B)

Figure 7: High-performance liquid chromatography (HPLC) analysis of fermentation products of gene replacement and gene complementation mutant strains

(A) Wild-type fermentation product; (B) NRPS gene replacement mutant (ΔSfmB) fermentation product; (C) ΔSfmB gene replacement mutant fermentation product complemented by SfmB gene; (D) ΔSfmB gene replacement mutation Fermentation products of mutants complemented by SfmA-F gene; (E) SFM-A standard.

Figure 8: Synthesis of the peptide backbone catalyzed by the non-ribosomal polypeptide synthase NRPS in the biosynthesis of tetrahydroisoquinoline family compounds

(A) Biosynthetic pathway of SFM-A; (B) Biosynthetic pathway of SFM-Mx1; (C) Biosynthetic pathway of SAC-B.

Figure 9: Functional analysis of substrate recognition of three NRPS in the SFM-A biosynthetic pathway

(A) Separation and purification of recombinant proteins: lane 1, SfmA(C-A-PCP), lane 2, SfmB, lane 3, SfmC; (B) Substrate determination of three NRPS recombinant proteins.

Figure 10: Heterologous expression of SFM-A biosynthetic genes in the SAC producer Pseudomonas fluorescens

(A) Comparison of biosynthetic gene clusters of SAC and SFM-A; (B) HPLC analysis of fermentation products of mutant strains: I, wild-type Pseudomonas, II, wild-type Pseudomonas fermented with KCN treatment, III , recombinant Pseudomonas containing sfmO4 heterologous expression plasmid, IV, recombinant Pseudomonas containing sfmO4 heterologous expression plasmid were treated with KCN after fermentation.

Figure 11: Expression of SFM-A biosynthesis gene in Escherichia coli and separation and purification of recombinant protein Lane 1, protein molecular weight standard, lane 2, purified protein. (A) SfmO1, (B) SfmO3, (C) SfmO4, (D) SfmM2, (E) SfmM3, (F) SfmD.

Symbol Description:

figure 1

Saframycin: saframycin; aminated-Saframycin: aminated saframycin; Ecteinascidin: sea squirrel; Safracin: safracin; cyano-Safracin: cyanated saframycin.

figure 2

Module: module; C: condensing enzyme; A: adenylase; PCP: peptidyl carrier protein; Loading: initiation module; NRPS: non-ribosomal peptide synthetase; Wavelength: wavelength.

image 3

Acyl-S-T: acyl thioester carrier protein; TE: thioesterase; C: condensing enzyme; red or RE: reductase.

Figure 4

Probe: probe; Skeleton: skeleton; Tailoring: post-modification; Regualtion: regulation; Resistance: resistance; SAM Recycle: acylated methionine regeneration cycle; Unassigned: uncertain function;

Figure 5

S-adenosylhomocysteine: adenosylhomocysteine; S-adenosylmethionine: adenosylmethionine; L-homocysteine: homocysteine; L-methionine: methionine; 5-methyl-THF: N ⁵ -methyltetrahydrofolate; THF: tetrahydrofolate; 5,10-methylene-THF: N ⁵ , N ¹⁰ -methylenetetrahydrofolate; Adenosine: adenosine; AMP: adenosine mono Phosphoric acid; ATP: adenosine triphosphate; PPi: pyrophosphate; Pi: phosphate; MT: methylase.

Figure 6

AL: acyl-CoA ligase; PCP: peptidyl carrier protein; C: condensing enzyme; A: adenylase;

Figure 7

WT: wild type; mLLS524B: NRPS gene SfmB replacement mutant; mLLS647: ΔSfmB gene replacement mutant with SfmB gene complementation mutant; mLLS925: ΔSfmB gene replacement mutant with SfmA-F gene complementation mutant; SFM -A: safromycin A.

Figure 8

AL: acyl-CoA ligase; PCP: peptidyl carrier protein; C: condensing enzyme; A: adenylase; RE: reductase; SFM-A: safromycin A; SFM-Mx1: safromycin Mx1; SAC-B: safranin B.

Figure 9

Relative activity: relative activity; Ala: alanine; D-Ala: D-type alanine; Ala-Gly: glycerin dipeptide; Pyruvate: pyruvate; Cys: cysteine; Tyr: tyrosine; 3h5mOmTyr: 3-hydroxy-5-methyl-O-methyltyrosine; 5mOmTyr: 5-methyl-O-methyltyrosine; OmTyr: O-methyltyrosine; 3hTyr: 3-hydroxy- Tyrosine; Phe: Phenylalanine.

Figure 10

Skeleton: skeleton; Tailoring: post-modification; Regualtion: regulation; Resistance: resistance; SAMRecycle: acylation methionine regeneration cycle; Unassigned: function uncertain; Beyond boundary: outside the boundary; Wavelength: wavelength; SAC-B : safrocin B; cyano SAC-B: cyanated safromycin B; aminated SFM-S: aminated safromycin S; SFM-Y3: safromycin Y3.

Detailed ways:

The present invention will be further described in detail below with reference to FIGS. 1-11 .

1. Clone the biosynthetic gene fragment of SFM-A:

Precursor labeling experiments with SFM-A demonstrated that the dimeric tetrahydroisoquinoline ring backbone comes from two tyrosines, the side chains come from alanine, glycine, and O-, C-, N-methyl groups come from methionine [J. Biol. Chem. (1985) 260, 344-348]. The structure of SFM-Mx1, an analogue derived from Myxococcus xanthus, is very similar to that of SFM-A. Pospiech et al. cloned the 18kb part of the biosynthetic gene of SFM-Mx1 through the method of transposon insertion inactivation, indicating that it contains two non- Ribosomal Polypeptide Synthetase (NRPS) and an O-methyltransferase, prove that the biosynthesis of SFM-Mx1 adopts the NRPS mechanism [Microbiol. (1995) 141, 1793-1803; Microbiol. (1996) 142, 741- 746]. The recent cloning and analysis of the biosynthetic gene cluster of saccharin B (SAC-B) derived from Pseudomonas fluorescens A2-2 showed that the biosynthesis of SAC-B also uses the NRPS mechanism [Mol.Microbiol.( 2005) 56, 144-154]. These studies provided extremely important information for cloning the biosynthetic genes of SFM. We speculate that the biosynthesis of SFM may adopt the mechanism of NRPS, Ala-Gly-Tyr-Tyr condensation sequentially (Fig. 2A, B), combined reduction into ring, redox, dehydration, N-, O-, C-methylation, Cyanide addition, deamination and other post-modifications are completed. Therefore, the cloning strategy starts with cloning the NRPS gene.

A typical NRPS is a multifunctional enzyme in the form of modules, and each module contains a unique set of non-reusable catalytic domains, that is, the catalytic domains correspond to the sequence of product synthesis (Figure 2A). We designed NRPS universal primers based on the biosynthetic sequence A functional domain of some existing compounds, hoping to obtain related NRPS gene fragments. Using degenerate primers (5'-TAC ACG TCC GGC ACSACS GGC AAR CCN AAR GG-3' and 5'-AW CGA GKS GCC GCC SRR GSMGAA GAA-3') to PCR clone the partial sequence of the NRPS gene to obtain about 1.2kb The PCR product (Fig. 2C) was cloned into the pGEM-T Easy vector, grouped by restriction endonucleases, and DNA sequence analysis showed high homology with known NRPS genes. Further gene disruption found that some gene disruption mutant strains still produce SFM-A, indicating that they have nothing to do with the biosynthesis of SFM; while another group of gene inactivation mutant strains completely lost the ability to produce SFM-A (Figure 2, D), demonstrating that the NRPS gene fragment related to SFM-A biosynthesis has been cloned.

After the synthesis of most non-ribosomal polypeptides is completed, thioesterase (TE) dissociates the polypeptide chain from PCP and catalyzes condensation, but the biosynthesis of some natural products does not have the TE commonly used in NRPS but There is a terminal reductase called the RE domain [Proc. Natl. Acad. Sci. USA (2001) 98, 11136-11141; Gene (2004) 325, 35-42]. Such compounds generally do not form macrocyclic amides or lactones, but dissociate into aldehydes, and then form terminal alcohols, amines, amides or imines. The structure of SFM-A and SFM-Mx1 is very similar, so we guess that SFM-A also uses the reductase RE to dissociate the tetrapeptide connected to PCP. First, RE dissociates the tetrapeptide under the action of NADPH to form aldehyde, and then the molecule The internal reaction forms a ring (Fig. 3A,B). RE primers were designed according to the conserved sequences of PCP domain and RE domain in the biosynthetic sequence of some compounds, hoping to clone related genes. Using degenerate primers (5'-GAC AAC TTCTTC GAG CTG GGS GGS SAY TC-3' and 5'-GCG GAC CAA CTT CTC CGCSRC CCA YTT RCT-3') to PCR clone the partial sequence of the RE gene to obtain about 0.8kb The PCR product (Fig. 3C) was cloned into the pGEM-T Easy vector, grouped by restriction endonucleases, and DNA sequence analysis showed high homology with known RE genes.

2. Cloning, sequence analysis and functional analysis of the biosynthetic gene cluster of SFM-A:

The above-mentioned NRPS gene fragments and RE gene fragments were labeled with digoxin as probes Probe-2 and Probe-3 from about 6000 clones in the genome library of Streptomyces lavendulae 314, and the cosmids isolated covered about 70kb of the chromosome ( Figure 4A). DNA sequencing analyzed a 50kb chromosomal region with a GC content of 71.8%. Bioinformatic analysis included 32 open reading frames (Fig. 4B). It contains three NRPS modules, indicating that the biosynthesis of SFM-A adopts the NRPS mechanism. At the same time, sequence analysis also showed that the biosynthetic gene cluster contained a large number of novel NRPS post-modification genes, 2 resistance genes and 3 regulatory genes (Fig. 4B), which provided us with a basis for studying the post-modification of non-ribosomal polypeptide compounds A very good platform. The detailed analysis results are listed in Table 1.

Table 1 Functional analysis of genes and encoded proteins in the biosynthetic gene cluster of SFM-A

3. Determination of the biosynthetic gene cluster boundary of SFM-A:

According to the functional analysis of the gene-encoded protein, the biosynthetic gene cluster of SFM-A was identified as genes sfmR1 to sfmO6 (Fig. 4B), covering a region of 46.9 kb of chromosome, containing 30 open reading frames. The entire SFM-A biosynthetic gene cluster has a total of 30 genes, 3 of which (sfmA, sfmB, sfmC) are used to encode non-ribosomal polypeptide synthetase (NRPS), which contains 3 modules, responsible for catalyzing the polymerization of SFM-A. Biosynthesis of peptide chains; 5 genes (sfmS1, sfmS2, sfmS3, sfmS4, sfmS5) encode proteins responsible for the biosynthesis of S-adenylated methionine, providing cofactors for methylation; 4 genes (sfmO5 , sfmM2, sfmM3, sfmD) encode proteins responsible for catalyzing the biosynthesis of tyrosine derivative 3-hydroxy-5-methyl-O-methyltyrosine; one (sfmM1) is used to encode methylase, Responsible for catalyzing N methylation in molecules; 2 genes (sfmCy1, sfmCy2) encode proteins that catalyze redox reactions and are responsible for oxidative ring formation of peptide chain backbones; 6 genes (sfmO1, sfmO2, sfmO3, sfmK, sfmO4, sfmO6 ) encodes a protein that catalyzes redox reactions and is responsible for the modification of the molecular skeleton after synthesis; it also includes 3 regulatory genes (sfmR1, sfmR2, sfmR3), 2 resistance genes (sfmG, sfmH) and 4 genes with uncertain functions (sfmE, sfmF, sfmI, sfmJ).

4. Biosynthesis of S-adenylated methionine (SAM):

Methylation modification is a reaction that often occurs in the biosynthesis of natural products. In most cases, it is catalyzed by a methylase, which utilizes S-adenosylmethionine (S-adenosylmethionine , SAM) as a methyl donor, and the biosynthesis of SAM comes from the basic metabolism of the host in most cases. The proteins encoded by five genes (sfmS1, sfmS2, sfmS3, sfmS4, sfmS5) in the safromycin A biosynthetic gene cluster form a complete SAM synthesis cycle (Figure 5): when methylation occurs, SAM acts as The methyl donor loses the methyl group and is converted to S-adenosylhomocysteine (S-adenosylhomocysteine); at this time, the S-adenosyl-L-homocysteine hydrolase encoded by sfmS1 hydrolyzes it L-homocysteine (homocysteine) and adenosine A are produced; 5-methyltetrahydrofolate-homocysteine S-methyltransferase encoded by sfmS3 then utilizes 5-methyltetrahydrofolate ( 5-methyl-THF) acts as a methyl donor to convert homocysteine to L-methionine, while sfmS2-encoded methylenetetrahydrofolate reductase catalyzes 5,10-methylene Tetrahydrofolate (5,10-methylene-THF) is converted into 5-methyltetrahydrofolate; the carbohydrate kinase encoded by sfmS4 catalyzes the conversion of adenine nucleoside A into adenine nucleoside monophosphate AMP, and then completes A to ATP The regeneration cycle of SAM; finally, the S-adenosylmethionine synthetase encoded by sfmS5 catalyzes the conversion of L-methionine and ATP into SAM, thus completing the regeneration cycle of SAM.

5. Biosynthesis of 3-hydroxy-5-methyl-O-methyltyrosine:

The biosynthesis of 3-hydroxy-5-methyl-O-methyltyrosine is shown in Figure 6A: sfmO5-encoded prephenate dehydrogenase catalyzes the conversion of the basic metabolite prephenate to tyrosine (this reaction occurs in It also occurs in the synthesis of basic metabolic tyrosine), and then under the catalysis of methylases encoded by tyrosine sfmM2 and sfmM3, hydroxymethylation and 5-position C methylation modification of benzene ring occur, and further in the enzyme action encoded by sfMD The 3-hydroxylation of the lower benzene ring completes the biosynthesis of the 3-hydroxy-5-methyl-O-methyltyrosine unit in safrothycin.

6. The function of NRPS in SFM-A skeleton synthesis:

There are three typical NRPS genes in the SFM-A biosynthetic gene cluster: sfmA, sfmB and sfmC. In order to further confirm their correlation with SFM-A biosynthesis, a mutant mLLS524B (ΔSfmB) with NRPS gene SfmB gene replacement was constructed, and it was found that the NRPS gene inactivated mutant no longer produced SFM-A ( Fig. 7B), while placing the SfmB gene under the exogenous promoter ErmE for gene complementation can partially restore the production of SFM-A (Fig. 7C), and placing the complete operon of SfmA-F under the exogenous promoter ErmE for gene complementation Complementation can restore the production of SFM-A almost completely to the wild-type level (Fig. 7D), further confirming that the cloned biosynthetic gene cluster is indeed related to the biosynthesis of SFM-A.

Further analysis of the amino acid sequences of the proteins encoded by the three NRPS genes allowed us to infer the organizational structure of the three NRPS modules ( FIG. 8A ). SfmA includes five domains (AL-PCP ₀ -C ₁ -A ₁ -PCP ₁ ), SfmB includes three domains (C ₂ -A ₂ -PCP ₂ ), and SfmC includes four domains (C ₃ -A ₃ -PCP ₃ -RE). Sequence analysis showed that the AL domain located at the N-terminus of SfmA has high homology with many acyl-CoA ligases of bacterial origin, while AL lacks some of the adenylation domain (A) necessary Therefore, the AL domain of SfmA may not have the function of recognizing and activating amino acid substrates that the A domain should have. It is speculated that A1 of SfmA, A2 of SfmB and A3 domain of SfmC recognize and activate alanine Ala, glycine Gly and Tyr derivative (3-hydroxy-5-methyl-O-methyltyrosine), respectively. Among them, SfmC is a nonlinear NRPS, which repeatedly catalyzes the activation of Tyr derivatives twice. The C-terminal RE domain located in SfmC is highly homologous to the R domain [Proc.Natl.Acad.Sci.USA (2001) 98, 11136-11141] associated with myxochelin biosynthesis in the C-terminus of MxaA, the latter's The catalytic mechanism is to firstly catalyze the reduction of the substrate to the intermediate aldehyde, and then reduce the substrate to alcohol and dissociate. It is speculated that the RE domain of SfmC also adopts a similar catalytic mechanism. The SfmA, SfmB and SfmC responsible for skeleton synthesis in the SFM-A biosynthesis gene cluster were compared with the organization structure of NRPS responsible for skeleton synthesis in SFM-Mx1 and SAC-B (Fig. 8), which catalyzed these three substances Skeletal biosynthesized NRPS are highly similar in organization. Further SfmA-AL and SafB-A ₀ , SfmA-A ₁ , SafB-A ₁ and SacA-A ₁ , SfmB-A _{2 , SafA-A 2} and SacB-A ₂ , SfmC-A ₃ , SafA _- A ₃ and SacC-A ₃ were compared for sequence analysis. These NRPS not only had a high degree of similarity in their organizational structure, but also had a high degree of sequence homology in their corresponding tissue domains. In connection with the similarity in the molecular structure of SFM-A, SFM-Mx1 and SAC-B, it can be inferred that the biosynthesis of these three substances should use the same mechanism to synthesize a common polypeptide backbone.

A The substrate specificity of the domain can be predicted by key amino acid residues fixed at 8 positions in the protein and substrate binding region. We compared the 8 core amino acids that determine the substrate selectivity of these A domains, and the results are shown in Table 2.

Table 2. Comparison of 8 core amino acids in the NRPSA domain that determine substrate selectivity

Pospiech et al.’s speculation about the substrate activation of each module A domain of SafA and SafB is mainly based on the linear logical relationship between organizational structure and catalytic function, rather than the rules of substrate recognition [Chem.Biol.(2000) 7, 623-642]. However, many examples of biosynthesis that do not conform to the linear logic relationship have been found [ChemBioChem. (2002) 3, 490-504], indicating that it is unreliable to rely solely on this point without considering the substrate selectivity of conserved amino acids . SafB-A1 is consistent with the 8 core amino acids that determine the substrate specificity in the domain of McyA of microcystin and NRPS-7 of bleomycin that activates Ala, so Shen Ben et al. [Chem.Biol.(2000) 7,623- 642] considered that the amino acid motifs of the three proteins that determine the substrate specificity may be a new type of amino acid sequence used to recognize Ala. On the other hand, SafA-1 and Ta1 [J.Mol.Biol. (1999) 286, 465-474] and CdaPSII [Chem. Biol. (1999) 6, 385-400] have been found to activate glycine domain The homology is very high, indicating that it is very likely to activate glycine rather than tyrosine derivatives as speculated by the authors. A. Velasco et al speculate [Mol.Microbiol.(2005) 56,144-154] SacA-A directly activates an Ala-Gly dipeptide, which is obtained from a very active ribopeptidyl transferase or from proteolysis . This hypothesis lacks sufficient evidence, and the significant difference between SacB-A and SacC-A does not support the hypothesis that they also activate L-Tyr derivatives. In summary, we believe that the speculations of A. Velasco and Pospiech et al. are unreasonable, while our reasoning can explain the biosynthetic pathway of the common polypeptide backbone of these three analogs more satisfactorily.

The specificity of the A domain in the NRPS module for the substrate is the key to infer its catalytic mechanism. The polypeptide backbone of SFM-A is catalyzed and synthesized by three NRPS modules, SfmA, SfmB and SfmC. When the synthesis is initiated, the A1 domain of SfmA first recognizes and activates an alanine, and the activated alanine is captured by the sulfhydryl group of PCP ₁ to form aminoacyl-S-PCP1. The downstream SfmB-A ₂ domain recognizes a glycine for activation. The α-carbonyl carbon atom on the peptide chain coupled with alanine and PCP1 is attacked by the Gly-S-PCP2 on the downstream module through the lone pair of electrons of the amino group to form a dipeptide. SfmC-A3 recognizes and activates 3-hydroxy-5-methyl-O-methyltyrosine, and the entire SfmC is reused twice, catalyzing 3-hydroxy-5-methyl-O-methyltyrosine twice acid extension. Then tetrapeptide-S-PCP3 was reduced to aldehyde by the RE domain and dissociated, and the polypeptide backbone of SFM-A was formed after cyclization (Fig. 6B).

In order to further verify the above model, experimental verification of the selectivity of the three NRPS protein substrates of SfmA, SfmB, and SfmC is required. The genes encoding the CA-PCP three-functional domain of SfmA and the two NRPS proteins of SfmB and SfmC were cloned into the expression vector pET28a, and expressed in Escherichia coli BL21(DE3) by IPTG induction. The recombinant protein SfmA (CA-PCP) and the three NRPS recombinant proteins SfmB and SfmC were separated and purified by Ni-NTA affinity chromatography and FPLC (Figure 9A), and the activity was measured by the method of ATP- ³² PPi isotope exchange. The results showed that: SfmA (CA-PCP) specifically recognized alanine, SfmB recognized glycine, and SfmC specifically recognized L-tyrosine derivative 3-hydroxy-5-methyl-O-methyltyrosine (Fig. 9B). This is exactly in line with our speculation.

7. The complete biosynthetic pathway and post-modification of SFM-A resulted in a series of safromycin molecules:

The polypeptide chain part of the molecular backbone of SFM-A is catalyzed and synthesized by the non-ribosomal polypeptide synthase NRPS (Fig. 6B). Under the catalysis of the above-mentioned NRPS, 1 molecule of alanine, 1 molecule of glycine and 2 molecules of 3-hydroxy-5-methyl-O-methyltyrosine are condensed into tetrapeptide, and then tetrapeptide-S-PCP3 is captured by the RE domain It is reduced to aldehyde and dissociated, and the polypeptide backbone of SFM-A is formed after cyclization. Then under the action of SfmCy1 and SfmCy2, the core skeleton structure is further cyclized, and then N-methylation occurs under the catalysis of SfmM1, and the oxidation of phenol to quinone occurs under the catalysis of SfmO2 to generate SAC-B, and then the oxidation of phenol to quinone occurs under the catalysis of SfmO4. , and then transammonia, cyanide, etc. generate SFM-A. However, SfmO3 and SfmO1 may catalyze the further oxidation of the 14-position to form SFM-F, D and other analogues.

8. Application of SFM-A biosynthetic gene cluster—genes in the biosynthetic gene cluster can be heterologously expressed in the heterologous host Pseudomonas fluorescens A2-2, which can catalyze the biotransformation of SAC-B:

On the basis of cloning and analyzing the complete SFM biosynthesis gene cluster and studying the possible functions of each gene-encoded protein, the present invention has carried out a preliminary discussion on the biosynthesis mechanism of SFM-A, which helps to understand SFM-A and other The biosynthetic mechanism of tetrahydroisoquinoline family compounds also provides the possibility to further obtain SFM-A structural analogues through genetic modification of biosynthetic gene clusters. Fermentation of Pseudomonas fluorescens A2-2 produces SAC-B compounds (Figure 1), which are structurally similar to SFM but much less active. Meanwhile, SAC-B is an intermediate product in our putative SFM-A biosynthetic pathway (Fig. The relatively complex biosynthetic pathway of SFM-A was partially reorganized, thereby further converting SAC-B into other SFM-A structural analogs. When the sfmO4 gene uses pVLT33 [Gene (1993) 123,17-24] as the expression plasmid constructed by the carrier, it adopts the method of conjugative transfer to import in the host bacterium Pseudomonas fluorescens A2-2 that SAC-B produces to obtain the corresponding mutant strain, through fermentation, HPLC and LC-MS analysis (Fig. 10BIII) showed that the sfmO4-introduced mutant could convert SAC-B into a new compound-aminated SFM-S (aminated SFM-S). If potassium cyanide is added to the fermentation product, the corresponding compounds are converted into the corresponding compound SFM-Y3 containing cyano units (FIG. 10BIV). SfmO4 encodes cytochrome P450 oxidase, and there are two genes encoding cytochrome P450 oxidase (sfmO3 and sfmO4) in the SFM-A biosynthetic gene cluster, SfmO4 was successfully expressed in the heterologous host Pseudomonas fluorescens A2-2 The efficient conversion of SAC-B into aminated SFM-S also further revealed the function of the enzyme encoded by the gene in the biosynthetic pathway of SFM-A: that is, the structure that catalyzes the epoxidation of phenol to quinone.

9. Application of SFM-A biosynthetic gene cluster - the genes in the biosynthetic gene cluster can be used in heterologous hosts

Heterologous expression in Escherichia coli and recombinant protein:

The research on the catalytic mechanism of oxidoreductase is of great significance for elucidating the whole biosynthetic pathway of SFM-A. In the SFM-A biosynthetic gene cluster, the proteins encoded by 8 genes (sfmO1-sfmO6 and sfmCy1, sfmCy2) have high homology with known oxidoreductases, and it is speculated that they belong to oxidoreductases. Among them, seven oxidoreductases (sfmO1-sfmO5, sfmCy1, sfmCy2) can be well expressed in Escherichia coli BL21(DE3) using pET28a as a vector. In addition, the N-methylase gene sfmM1 and the genes sfmM2, sfmM3 and sfMD responsible for the biosynthesis of the precursor 3-hydroxy-5-methyl-O-methyltyrosine can all be well expressed in Escherichia coli. The proteins SfmO1, SfmO3, SfmO4, SfmM2, SfmM3 and SfmD can be isolated and purified to obtain soluble recombinant proteins (Figure 11) for further functional studies.

Implementation examples are further provided below, and these implementation examples are helpful for understanding the present invention, and are only used for illustration and do not limit the scope of application of the present invention.

Example 1

Extraction of SFM-A producing bacteria Streptomyces S.lavendulae 314 total DNA:

Inoculate 100 μL of 1×10 ⁸ S. lavendulae 314 spore suspension into 3 mL of ISP-2 (Yeast extract 0.4%, Malt extract 1.0%, Glucose 0.4%, pH 7.2) liquid medium, culture at 30°C, 230rpm for about After 24hrs, it reaches the late logarithmic growth phase. Take 2mL and inoculate it into 50mL ISP-2 (containing 25mM magnesium chloride), cultivate at 30°C and 250rpm for about 23hrs and reach the early stage of stable growth phase, which is milky yellow and turbid. , centrifuged for 15 minutes to collect mycelium, washed with lysate, and collected 0.5 mL of light milky yellow mycelium. Add 10 mL of lysate (containing 5 mg/mL of lysozyme) to 1 mL of mycelium, a total of four tubes, vortex until uniform, and bathe in 37 ° C for 15 min. Add 0.1mL proteinase K (10mg/mL, freshly prepared with lysate), 1mL 10% SDS, mix well and quickly put in 70°C water bath for 15mim, it becomes clear. Cool on ice, add 2.5mL 5M KAc, and cool on ice for 15min. Add 10mL saturated phenol, mix well, 10mL chloroform, mix well, centrifuge at 12000rpm, 4°C for 20min. Use a broken pipette tip to suck out the aqueous phase and place it in a new centrifuge tube, add an equal amount of CHCl ₃ -isoamyl alcohol (24:1) for extraction, and centrifuge at 12000 rpm at 4°C for 10 min. Use a broken pipette tip to suck out the aqueous phase and place it in a new centrifuge tube, add 2 times of absolute ethanol, mix well, and large groups of DNA appear. Hook it out and place it in a new centrifuge tube, add 5mL of 70% ethanol to wash, pour out the liquid, suck it up with a gun, add 5mL TE to dissolve, add RNase A to make the final concentration 50μg/mL, and incubate at 37°C for 0.5 hours . Extract twice with equal volume of saturated phenol and twice with CHCl ₃ -isoamyl alcohol, add 0.1 volume of 3M NaAc and 2 volumes of absolute ethanol to the water phase, mix gently and fully, flocculent DNA Appear. Combine four tubes of DNA into two tubes (1mL of 70% ethanol in each tube is used for washing), suck out the liquid, then wash with 1mL of absolute ethanol, suck out the ethanol, dry it in an ultra-clean bench, dissolve it in an appropriate volume of TE ( pH8.0).

Example 2

Establishment of SFM-A producing bacteria Streptomyces S.lavendulae 314 genetic transfer system:

Cultivate E.coli S17-1 containing appropriate plasmids to OD ₆₀₀ 0.3-0.4, collect the bacterial cells in 20mL LB culture medium by centrifugation, wash twice with equal volume of LB, resuspend in 2mL LB, as E. coli donor cell. Take 500 μL of 20% glycerol spore suspension of S. lavendulae 314 frozen at -80°C, wash twice with an equal volume of TES buffer (50 mM TES Na, pH 8.0), and resuspend in an equal volume of TES buffer solution, and heat shock at 50°C for 10 min to germinate the spores. Add an equal volume of TSB medium and incubate at 37°C for 2-5hr. Centrifuge and resuspend in 0.5-1 mL LB as Streptomyces recipient cells. Mix 100 μL of recipient cells with different concentrations and an equal volume of donor cells and spread them directly on a plate containing 10 mM MgCl ₂ . After incubating at 30°C for 20 hours, gently wash the surface of the plate with sterile water to wash away most E. coli , Cover the surface of each plate with 3 mL of LB soft agar containing nalidixic acid (final concentration: 50 ng/μL) and corresponding antibiotics or 1 mL of sterile water. Culture at 30°C for more than 5 days to pick zygotes.

Since S.lavendulae 314 is sensitive to thiostrepton, apramycin and erythromycin, finally determine the concentration of antibiotics used for genetic transfer: thiostrepton 25 μg/mL, apramycin 50 μg/mL , erythromycin 50 μg/mL. IWL-4 (soluble starch 1.0%, K ₂ HPO ₄ 0.1%, MgSO ₄ 0.1%, NaCl 0.1%, (NH ₄ ) ₂ SO ₄ 0.2%, CaCO ₃ 0.2%, FeSO ₄ 0.0001%, MnCl ₂ .6H ₂ O 0.0001%, ZnSO ₄ 0.0001%, yeast extract 0.05%, tryptone 0.1%, agar 2.0%, pH7.2) medium is the best, and the conjugative transfer efficiency is the highest. On IWL-4 medium, whether it is the plasmid pKC1139, pHZ1358 that can replicate autonomously in Streptomyces, or the plasmid pSET152 that integrates site-specifically, the efficiency of conjugative transfer is higher. The transfer efficiency is about 10 ^-4 .

Example 3

Construction of SFM-A producing bacteria Streptomyces S.lavendulae 314 genome library:

First, through a series of dilution experiments to determine the amount of Sau 3AI, on this basis, a large number of enzyme-digested DNA fragments slightly larger than 40kb, dephosphorylated. SuperCos1 was first cut and dephosphorylated from the middle of the two cos sequences with Xba I, and then cut with Bam HI from the multiple cloning site to obtain two arms, which were ligated with the prepared 40kb DNA fragment overnight. Take out the packaging mixture from the -80°C refrigerator and place it in a dry ice bucket. Melt the packaging mixture quickly between your fingers. Add 4 μL of the ligation product when it just starts to melt, mix well with a gun, and place in a 22°C water bath for 2 hours. Add 500 μL SM buffer [(L ⁻¹ ): 5.8 g NaCl, 2.0 g MgSO ₄ , 50.0 mL 1MTris.HCl (pH=7.5), 5.0 mL 2% (w/v) gelatin] and 50 μL chloroform, mix gently Evenly, centrifuge for 4 seconds, and the upper and lower interfaces appear precipitated. Transfer the supernatant to a new tube and store at 4°C. The strain E.coli VCS 257 frozen at -80°C was spread on LB medium for recovery. Pick a single colony and inoculate it in 50mL LB medium (peptone 1.0%, yeast extract 0.5%, NaCl 1.0%, pH=7.0; add 0.2% maltose and 10mM MgSO ₄ ), shake at 220rpm for 6.5 hours at 37°C, and measure its When OD ₆₀₀ = 0.83, take 100 μL of packaging supernatant and 100 OD ₆₀₀ = 0.83 bacterial solution, flick the tube wall to mix well, and perform 5 parallel operations. Water bath at 22°C for 30 minutes, add 800 μL of LB medium, bathe in water at 37°C for 1.5 hours, invert once every 15 minutes. Spread a large plate with LB agar (Amp: 100 μg/mL) (15 mm×15 mm), wash each portion with 200 μL of LB medium, and incubate at 37° C. for 18 hours. Plaque forming units (pfu) were measured to estimate library potency. Then scrape and wash with LB medium, add ampicillin and glycerol, so that the final concentration of ampicillin is 50 μg/mL, and the final concentration of glycerol is 18%. Dispense into 1mL×12, 0.5mL×48, 025mL×24. Store at -80°C. A total of about 10,000 clones were obtained, with a titer of 4,000 cfu/μg DNA. For Streptomyces, the size of its chromosomal DNA is about 8Mb, and if the titer of the library with an insert fragment of 20kb is 2000-5000cfu, it is enough to represent its entire genome. The titer of our library reaches 4000cfu/μg DNA, which has good quality and meets the needs of library screening.

Example 4

Fermentation, product isolation, purification and identification of SFM-A producing bacteria Streptomyces S.lavendulae 314:

Take 100 μL of 1×10 ⁸ S.lavendulae 314 spores and spread them on YSA (0.1% yeast extract, 0.5% soluble starch, 1.5% agar, tap water, pH7.5) plate, culture at 30°C for seven days, and use a puncher to Make a spore-filled agar block and inoculate it into 50mL (500mL Erlenmeyer flask) fermentation medium (glucose 0.1%, soluble starch 1.0%, NaCl 0.5%, K ₂ HPO ₄ 0.1%, Casein acid hydrolysate 0.5%, Meat extract 0.5%, tap water, pH7.0), cultured at 27°C, 250rpm. After about 20 hours, a dark blue pigment was produced, and the culture medium was turbid with many foams. The culture was stopped after 6 hours, the supernatant was collected by centrifugation, the pH was adjusted to 6.8, KCN was added to make the final concentration 1 mM, and shaken at 35°C for 30 minutes. The fermentation broth was extracted with ethyl acetate, the organic phase was washed with deionized water, the organic phase was concentrated and dried and dissolved in 100 μL ethyl acetate. Take 20 μL and detect it by HPLC.

HPLC analysis:

UV=270nm; Column: EC 150/4.6 NUCLEOSIL 100-5 C18 Ser.No.2085011 Batch21302092; Mobile phase conditions: V=1mL/min; A=H ₂ O (0.05% TFA) B=CH ₃ CN (0.05% TFA)

time/min 0 5 25 27 29 30 B/% 10 10 85 85 10 10

Example 5

PCR cloning of SFM-A biosynthesis gene:

PCR system includes: DMSO (8%, v/v), MgCl ₂ (25mM), dNTP (2.5mM), degenerate primer (40mM), Taq DNA polymerase (2.5u) and an appropriate amount of template S.lavendulae 314 total DNA . First 95°C, 3min, 1 round; then 94°C, 1min, 68°C, 1min, 72°C, 2min, 5 rounds; 94°C, 1min, 65°C, 1min, 72°C, 2min, 30 rounds; finally 72°C, 10 minutes, 1 round. After PCR, the results were checked by 1% agarose electrophoresis. A DNA fragment of the expected size was recovered by low melting point gel, connected with pGEM T Easyvector, transformed into Escherichia coli DH5α competent cells, and coated on a medium containing ampicillin, IPTG (isopropylthio-β-D-galactoside) and X- Blue-white screening was performed on LB plates of gal (5-bromo-4-chloro-3-indole-β-D-galactoside). Pick the white colony and culture it overnight, extract the plasmid, and digest it with EcoR I to determine whether it contains a DNA insert of the expected size. Sequencing of plasmids inserted with DNA fragments of expected size.

Example 6

Nucleic acid molecular hybridization:

1) DIG DNA labeling: Dilute the DNA to be labeled with sterile water to a total volume of 15 μL, heat and denature in a boiling water bath for 10 minutes, and immediately place it in an ice-salt bath to cool. Then add 2 μL of primer mixture, 2 μL of dNTP mixture, and 1 μL of enzyme, mix well, and place in a 37° C. water bath for about 16 hours. Add 0.8 μL 0.8 MEDTA (pH 8.0) to terminate the reaction, add 2.5 μL 4M LiCl and mix well, then add 75 μL pre-cooled absolute ethanol to precipitate the labeled DNA, and place it at -80°C for 40 minutes. The DNA was collected by centrifugation at 12000 rpm for 20 minutes at 4° C., and the DNA precipitate was washed with pre-cooled 70% ethanol, dried in vacuo and redissolved in 50 μLTE ((pH 8.0).

2) Quality detection after DIG DNA probe labeling: Dilute the labeled DNA probe to the following six gradients, 1, 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 . Dilute the labeled control DNA to concentrations of 1 μg/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, respectively. Take 1 μL of the above-mentioned gradient DNA samples and spot them on the nylon membrane for hybridization, carry out the color reaction according to the steps described in 7), and compare the color intensity of the labeled DNA probe and the DIG-labeled control DNA to determine the color of the labeled DNA probe. needle concentration.

3) Membrane transfer of colony hybridization (library screening): thaw the gene library stored at -80°C for a while, take 50 μL, dilute with 450 μL LB to obtain a dilution factor of 10 ^-1 , and double dilution to obtain 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 . 300μL was spread on a plate (15cm×15cm, the plate was LB/50μg/mL kanamycin). Choose an appropriate ratio to make about 1200-1500 clones per plate. Spread four plates evenly according to the selected ratio, and incubate overnight at 37°C. Cut the nylon membrane according to the size of the plate, carefully cover the surface of the plate to avoid air bubbles, and mark the position. After 1 minute, remove the nylon membrane and place it on dry filter paper, and dry it for 10 minutes until the colonies are combined on the nylon membrane. The original plate was placed in the incubator for 4-5 hr to allow the clones to re-grow as the original plate. Place the nylon membrane on filter paper saturated with denaturing solution (0.25M NaOH, 1.5M NaCl) for 15 minutes (do not soak the membrane), transfer to neutralizing solution (1.0M Tris.HCl, 1.5M NaCl, pH7.5) saturated on filter paper for 5 minutes. Transfer to filter paper saturated with 2×SSC (20×SSC stock solution (L ⁻¹ ): NaCl, 175.3 g, sodium citrate, 88.2 g, pH=7.0) and air-dry naturally. Remove the nylon membrane and place it in an oven, and fix it at 120°C for 45 minutes. Shake and wash in 3×SSC/0.1% SDS solution at room temperature for 3 hours to remove cell debris.

4) Membrane transfer of Southern hybridization: DNA samples are electrophoresed to an appropriate distance on an agarose gel of appropriate concentration, and marked. Soak in 400mL 0.25M HCl for 20 minutes to depurinate the bromophenol blue, and wash it several times with deionized water. Immerse in alkaline buffer (0.5M NaOH, 1M NaCl) for 15 minutes at room temperature with gentle shaking. Change the medium once and continue to soak the gel for 20 minutes, shake gently, and wash with deionized water three times. Take a nylon membrane that is 1 mm larger than the gel on each side, soak it completely with deionized water, and mark it. Using the upward capillary transfer method, transfer with 10×SSC transfer buffer for 8–24 hr. Wash the membrane slightly with 2×SSC and bake at 120°C for 30 minutes.

5) Pre-hybridization and hybridization: preheat the hybridization solution (20mL/100cm ² ) to a hybridization temperature of 68°C, put it into a hybridized nylon membrane, shake gently and incubate for 30 minutes. Denature the DIG-labeled DNA probes in a boiling water bath for 5 minutes, and immediately place them in an ice-salt bath to cool. After cooling, the DNA probe was mixed evenly with an appropriate volume of DIG hybridization solution (2.5 mL/100 cm ² ). Remove the pre-hybridization solution and immediately add the DNA probe/DIG hybridization solution, shake gently and keep the hybridization temperature at 64°C or 68°C for about 16 hours.

6) Stringent elution after hybridization: wash twice with 2×SSC/0.1% SDS at room temperature, 5 minutes each time. At 68°C, shake and rinse twice with 0.1×SSC/0.1% SDS, each time for 15 minutes.

7) Color reaction and detection: the nylon membrane after stringent elution is equilibrated in washing buffer (0.1M maleic acid, 0.15M NaCl, pH=7.5, 0.3% (v/v) Tween 20) for 1-5 minutes, Then block for 30 minutes in blocking buffer (blocking reagent dissolved in 0.1M maleic acid at a concentration of 10%, 0.15M NaCl, pH=7.5), and then soak in the antibody for 30 minutes. Rinse the nylon membrane twice with washing buffer, equilibrate with detection buffer (0.1M Tris-HCl, 0.1M NaCl, pH=9.5) for 2-5 minutes, and finally place the nylon membrane in 10mL of newly prepared chromogenic solution [NBT (nitroblue tetrazolium chloride) was dissolved in 70% DMF with a concentration of 70mg/mL, and BCIP (5-bromo-4-chloro-3-indolyl-phosphate) was dissolved in water with a concentration of 50mg/mL. Add 45μL NBT, 35μL BCIP] to 10mL chromogenic solution, and place in the dark for color development. After proper color development, rinse with deionized water to terminate the reaction.

Example 7

Obtaining of gene disruption mutant strains:

The obtained transformants were inoculated into liquid medium ISP-2 (Am 25 μg/ml), and shaken at 30° C. for about 28 hours. Take out 200μl and spread it on the ISP-2 (Am 50μg/ml) plate, culture at 30°C for 6-8 days, collect the spores and store at -80°C; take out 10μl and draw a line on the ISP-2 (Am 50μg/ml) plate, 37 Cultivate at ℃ and place for 2-3 days. Pick a single colony integrated and grown at 37°C, inoculate it into liquid medium ISP-2 (Am 25 μg/ml), shake at 37°C for 2-3 days. Take out the plate coated on ISP-2 (Am 50μg/ml), integrate at 37°C for 2-3 days, collect spores, and store at -80°C. Take out 200μL and spread it on the ISP-2 plate (Am 50μg/ml), and place it at 30°C for 2-5 days to identify its biological activity.

The vector used for gene interruption or gene replacement is pOJ260 or pKC1139, and the gene replacement uses the erythromycin resistance gene to replace the DNA fragment in the middle of the target gene. The constructed plasmid was introduced into S. lavendulae 314 by intergenus conjugal transfer as described in Example 2 to obtain double crossover mutants, and the obtained mutants were genotyped by Southern hybridization.

Example 8

Gene expression in Pseudomonas and analysis of fermentation products:

First, the target protein gene in pET28a was excised with XbaI/HindIII, cloned into the corresponding site of vector plasmid pVLT33, transformed into E.coli CC118 (Kana 50 μg/mL), and identified by enzyme digestion. The correct plasmid was introduced into Pseudomonas fluorescensA2-2 by conjoint transfer. The method of conjugative transfer is as follows: transform the derived plasmid of the constructed pVLT33 plasmid into E. coli S17-1, and culture it in LB (Kana 50 μg/mL) at 37°C. Take the strain of P.fluorescens A2-2 and streak culture on the LB plate, and culture at 30°C. A single colony of S17-1 (derived plasmid containing pVLT33) was cultured in LB (Kana 50 μg/mL) overnight at 37°C. Pick a single colony of P.fluorescens A2-2 and culture it overnight in LB at 30°C. Take the overnight culture of the above bacteria, transfer it to the corresponding LB culture medium in an amount of 1%, and continue to cultivate at the corresponding temperature until the OD ₆₀₀ reaches about 0.8-1.0. Take 1mL and 4mL of the above two bacterial solutions, mix them evenly at a ratio of 1:4, filter through a 0.22μm membrane, remove the filter membrane, and place the bacteria side up on an LB plate (without antibiotics). Incubate at 37°C for 8hr. Remove the filter membrane and wash the membrane with 3 mL of 10 mM MgSO ₄ . 1ul and 0.1ul were diluted and spread on LB plates (Kana 50μg/mL and Ap 100μg/mL or Tc 12.5μg/mL were added to inhibit the growth of E.coli), and cultured overnight at 30°C. Pick 3 to 5 single colonies to LB medium (Kana 50 μg/mL and add Ap 100 μg/mL or Tc 12.5 μg/mL) and culture overnight at 27-30 °C to preserve the strains. Further culture and expression are the same as Escherichia coli, using LB medium (Kana 50μg/mL), culture temperature is 25°C-30°C; add IPTG induction at OD ₆₀₀ at 0.6-1.0, after induction, the culture time can be extended appropriately (24hr) .

Fermentation of Pseudomonas fluorescens A2-2 and mutants: Inoculate 0.1% of the bacteria into 50mLYMP3 (glucose 1%, beef extract 0.25%, tryptone 0.5%, CaCO ₃ 0.8%, pH6.5) medium (250mL shake flask), 27°C, 250rpm for 30hr, transfer 2% to 50mL (500mL shake flask) M-16 medium (D-mannitol 15.2%, dried yeast 3.5%, (NH4)2SO4 1.4%, FeCl3 0.018%, CaCO3 2.6%, pH6.5), 27°C, 220rpm for four days. The fermentation broth was centrifuged, the supernatant was adjusted to pH=9, extracted and concentrated with ethyl acetate. Or first adjust the pH to 3, extract with ethyl acetate to remove impurities, then adjust the water phase to pH = 9, extract and concentrate with ethyl acetate, the concentrated solution contains SAC-B. If the fermentation broth is adjusted to pH=6.8, add KCN to a final concentration of 1mM, react at 35°C and 250rpm for half an hour, then extract and concentrate with ethyl acetate, the concentrate contains cyano-SAC-B. The fermentation of the mutant was similar to this. After 40 hours of transfer, IPTG was added to a final concentration of 0.2 mM, and cultured at 24° C. and 220 rpm for 96 hours (or 72 hours). The supernatant of the fermentation broth was centrifuged to adjust the pH to 9, extracted and concentrated with ethyl acetate. Or first adjust the pH to 3, extract with ethyl acetate to remove impurities, then adjust the water phase to pH = 9, extract and concentrate with ethyl acetate, the concentrated solution contains the compound aminated SFM-S. If the pH of the fermentation broth is adjusted to 6.8, add KCN to a final concentration of 1 mM, react at 35° C. and 250 rpm for half an hour. Extraction and concentration with ethyl acetate, the concentrate contains SFM-Y3.

Example 9

Gene expression in Escherichia coli and recombinant protein isolation and purification:

The target gene to be expressed is mainly obtained by PCR method (when expressing NRPS gene, the gene encoding N-terminal and C-terminal is obtained by PCR, and the middle part is obtained by cosmid subcloning), cloned into a common vector such as pSP72 with EcoRI/HindIII, and sequenced by DNA After the analysis was confirmed, the insert fragment was cut out by NdeI/HindIII, and connected into the corresponding enzyme cutting site of the expression vector pET28a to obtain the expression plasmid. Transform Escherichia coli BL21(DE3), pick a single colony into 3 mL LB (Kana, 50 μg/mL), and culture overnight at 37°C. Take 30 μL of the overnight culture and insert it into 3 mL of LB (Kana 50 μg/mL) culture solution, incubate at 37°C for two hours, add IPTG to a final concentration of 1 mM/L, continue to cultivate at 37°C for four hours, collect 1 mL of bacteria by centrifugation, and add 50 μL of protein for electrophoresis buffer solution, vortexed and boiled in a boiling water bath for 3 minutes, took out the sample, centrifuged at 12,000 rpm for 5 minutes, took 10 μL of the supernatant to load the sample, and checked the expression by SDS-PAGE protein electrophoresis. For massive expression, inoculate 2ml of well-expressed overnight culture into 500ml LB medium (kanamycin 50μg/ml), culture at 37°C for 2.5 hours (A _600nm is about 0.5), transfer to 25°C for 0.5 hours, add IPTG to The final concentration is 0.1mM, expressed at 25°C for 6 hours or at 15°C for more than 24 hours. Cool in ice, centrifuge at 5000rpm at 4°C for 5 minutes to collect the bacteria (take 1ml of the bacteria liquid to collect the bacteria as an SDS-PAGE sample to analyze the whole bacterial protein), STE buffer (10mM Tris-HCl, 10mM NaCl, 1mM EDTA, pH8.0 ), wash once with bacteriostasis buffer (50mM NaH ₂ PO ₄ , 300mM NaCl, 10mM imidazole, adjust pH to 8.0 with NaOH), add 10ml (2ml/g wet cells) bacteriostasis buffer (add Resuspend the enzyme to a final concentration of 1mg/ml) and place it at 4°C (or in ice) for 30 minutes. Freeze at -80°C for 30 minutes, then thaw at room temperature, and sonicate for 10 minutes in an ice bath (200-300W, sonicate for 10 seconds with an interval of 50 seconds). The supernatant was transferred to a 50ml conical centrifuge tube. Add 1-2ml of Ni-NTA affinity chromatography column filler (depending on the expression amount and size of soluble protein), and shake in ice bath for 1-2 hours (if the protein is greater than 150KD, the time can be extended, and if the protein is more than 200KD, it can be overnight). Centrifuge at 2,000rpm at 4°C for 5 minutes, pour off the supernatant, spin the column packing gently with 10-20ml of washing buffer (50mM NaH ₂ PO ₄ , 300mM NaCl, 25mM imidazole, adjust the pH to 8.0 with NaOH), and place in an ice bath. Shake the column for 10 minutes, centrifuge at 2,000 rpm at 4°C for 5 minutes, pour off the supernatant, add 10ml of washing buffer to spin up the column packing, pack the column, and rinse with 10-20ml of washing buffer for 2-3 times. The target protein was eluted with 0.5ml×6 times of elution buffer (50mM NaH ₂ PO ₄ , 300mM NaCl, 250mM imidazole, adjusted to pH 8.0 with NaOH). Analyze the results of SDS-PAGE. If the purity of the target protein reaches more than 90%, combine the target protein and dialyze against the dialysate (50mM Tris-HCl, 25mM NaCl, 5mM β-mercaptoethanol, 10% glycerol, 0.02% NaN ₃ ) overnight at 4°C After quantification, aliquots are stored at -80°C for activity testing; if the purity is poor, the target protein is combined, and further separation and purification are combined with other methods (such as gel filtration) according to the properties of the target protein.

The following gene and protein sequences are provided according to the contents of the present invention:

Amino Acid/Nucleotide Sequence Listing:

SEQUENCE LISTING

<110>Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences

<120>Saframycin biosynthesis gene cluster

<130> specification, claims

<160>1

<170>PatentIn version 3.3

<210>1

<211>49362

<212>DNA

<213> Streptomyces lavendulae 314 (NRRL11002)

<400>1

tcatggcgga ggcggcgaag gccggcgcca aggtcctcaa gcccgccgac gccctcccct 60

ggggcggtta cggcggtacc ttcgccgacc ccgagggcta catctggagc ctcggctaca 120

gcgcccaggg aaccgaccag ccctacgcgg aatagccgaa cgccagagca gaccggagag 180

aacgatgaaa ttccgcaccc acgtcgagcc ccccgagccc atgcggggcc tggaagtccc 240

gcccgaggtg gtggccgcgc tcggcggggg cgcgcggcca ccggtgacga tcactctggg 300

cggccactcc tggaagagcc ggatcgccct cctccgcggc cgccacctgc tcggcctcag 360

caacgccaac cgccaggccg ccggcgtcgc cgtcggcgac gaggtcgagg tcgagctgga 420

actcgacacc gaaccccgcg tcgtcgtcga gcctgcggac ttcgcccagg ccctggacgc 480

cgacccggca gcccgcgccg cctacgacaa cctggcgtac agccgcaagc gcgagcacgt 540

acgcgccatc gagagcgcga agaagcccga aacccgccga agccgcatcg aaaaggccct 600

caccaccctg cggggctgac aaacaccccc accgcaccga tgaaggccag aacgtgcccc 660

cgggtggccg cagccgcgac attttgcatg gtggacgccc gccctgcttt aataaatgcg 720

cagtcattta ttaactcgcc aagctggagg gcactgtggt gggacgtccc cgaggggtcg 780

acgacgtgga catcctgcgt gcggcggcag acgtgatcgg ccgggtgggg cccgcgggac 840

tcacgctcgc cgccgtggcc ggcgaggtgg gcctcgtacc gggcaccctc ctgcagcggt 900

tcggctccaa gcgcgggctc atgctggcca tcgcccggga gtcggccaag gaggccgaat 960

ccgtgtacga gagcatgcgt gacgagcacg ccactgccct catggccgtg acggacctgg 1020

ccgtgacctc aatgggcgtc atgaccaagc cggaggtcta cgcgaaccat ctggccttcc 1080

tgtgcatcga cctcacggac ccggagttcc acgcgcacgc cctcaccatc caccaggccc 1140

agcggcgggt atacgaggcc ctgctcaccg aggcggtgga caacggggag ctgttggccg 1200

gcaccaatgt cgcggcgctg gccaccacccc tccaggctgt cgtcgcgggc gcgggcctca 1260

cctgggccct ggaccgcgag ggcaccctgc cccaccggct cgaacgcgag gtcatcacgg 1320

ccctagcccc ccacatcacg ccgcagggtg actccgcagg ggagggatcg tgaccgacgg 1380

cgtccgcaca ctggccggca aggtcgccct cgtcgccgga ggcacgcgcg gaggcgggcg 1440

gggcatcgcc gtcgaactcg gtgccgccgg cgcgacggtg tacgtcagcg gccgcagcag 1500

ctcggccaac ggaagctccg acatgggccg cccggagacc atcgaggaga cggcgcgggc 1560

ggtcaccgcc gccggaggcg tgggcatccc ggtgcgcacc gaccacagca gccccgaaga 1620

ggtccgcgcg ctggtcgacc ggatcgacgc cgagcagcag gggcgcctgg acgtcctggt 1680

caactgcgtc tggggcgggg accggctgac cgactggaac cgtccgctgt ggcagcagga 1740

cctcgacaag gggttgaggc tgctgcgcca ggccgtcgag actcacgtga tcaccagccg 1800

gtacgccgtg cggctgatgg ccgcccgccg cagcggcctc gtcgtggagg tcaccgacgg 1860

caacaccgcc cgctaccgcg gatccttctt ctacgacgtg gcgaagtcca cggtgatccg 1920

cctggccttc gcacaggccg ccgacctgaa ggaacacggc gtcgcggccg tcgccatcac 1980

gcccggcttc ctgcgctcgg aggccatgct ggagcacttc ggcgtcaccg aggacaactg 2040

gcgcgacggc gtggccaggg acccggactt cgcgcattcc gagaccccgg catacctggg 2100

cagggcggtg gccgccctgg cggccgatcc cgacatcatg gcgaagaccg ggcgggcgct 2160

ggccacctgg ggcctgtaca aggaatacgg gttcaccgac atcgacggca gccagccgga 2220

cttcgccgcc cactgggcca ggaccctgga gccgcggctc ggaccgctgg gtaccccctt 2280

gtagggcgct tcccggaaag agccacgcta atacctgcgc acagccgttg tccgcggcgc 2340

cccaggggtt ctccaccact gcgcggaaag acgcttgaga ccaactggag gtggctgcca 2400

ggacgggatc tcccttggct actgtgcaca caggtacgtg ccaatccgga atccaccgtg 2460

cccatggcac aggggaggtct gcttcggtgc ttaacgcaat tgaatcgaaa gtggaattga 2520

tcaggtggcc ggtggacgca gcccgtcggg cggcatgcag ggaccagggg gtcatgcggc 2580

tcctggtgct ggaagcaggg gtcgaagccc cggtgtgtac ggacgtccgg gaggactggg 2640

tccgtacccc ggtcagtagg agcgaccttc gcgcgcggat cgacgcgctg cgcgtgaagg 2700

gcagcgcgga cttcctgccg acggtggatc cgaacggcgt cctgcgctat cgcaggatgt 2760

ccgtggtgct ttccccgacc gagacggatt tggtgaatcg cctcgcggaa tcgttttccc 2820

atgtcgtccc gcgggaagtc ctgctggaag cgctgtcccg gacttcgcag cccagccgaa 2880

atgccctgga cttgcacatc atgcggatcc gccgccgggt gaaacccctc gacctcgccc 2940

tgcgtacggt gtgggggcgc ggctacgtca tggagtccgc ggacgccgcc tcgtagcggg 3000

ccgcaccagg gcctccccgg cgggacaccc gcggattcgt ctttcaggga ctcgaaggat 3060

tcgacccggc atcgaccgct gcgcgtcgac aatcgctggc gacgcgcatg tggatccgac 3120

cgcgcgcgtc cccgtacccg cgcagagtcc accaggggga gaaccatgct tgagcgccgt 3180

gccgtgttgc ggatgagtgc aggagcggcg gccgtcgccg tcggaggctc catggtcgcg 3240

ggcgtggcgt ccgccgccgc cgacaccggc gtccgccgct ccgcgggcgt ggactggagc 3300

aggctgagca gccgcctctc cggcgatctg gtgcttcccg ccgacgcccg ttacgagcag 3360

gccagccgtc tggcgatcgg acagttcgac gcgatccgcc cgcaggccgt cgcctactgc 3420

cagagcgagc aggacgtgcg cacggccctc gccttcgcac aggacaacag cctgcgcctg 3480

gtgccgcgca gcggtggcca cagcttcggc ggctactcca cgacgactgg tctggtactc 3540

gacgtctcgc gcctcaactc ggtccgcctg acggccgaca cggtggtggt gggtcccggc 3600

ctccagcagg tcgacgcgct gacccagctg gcgccgcgcg gcgtccaggt cgtgagcgga 3660

ctgtgcccgg gcgtctgccc gggcgggttc atccagggcg gcggcctggg ctggcagacc 3720

cgcaagttcg gcatggcgct cgaccgtctc gtgtcggcca gggtcgtgct tgccgacggc 3780

cgtgccgtca ccgcctccgc caaggagaac tgcgacctgt tctgggcgct gcgcggcggt 3840

ggcggcggca acttcggcgt cgtcaccagt ttcgaggtcg agcccacgca cgtcccgtcc 3900

atcgtgaact tcaacctgac ctggccctgg gaagccgccc agaaggtcat cgcggcgtgg 3960

cagcgctggg tcatcggcgg ctctcgcgac ctgggcgccg gcctcggcgt gcagtggctg 4020

gacgccggta ccggccggcc cgaactgctc gtctacggcg gctggctcgg ccgccccgac 4080

gccttcaccg ccgtgctcga cgccttcgtc cgcgatgccg gcagcgcccc cctcacccgc 4140

tcggtcgagg agcagccgta ccagaaggcg atgatgtcct actacggctg cgccgacctg 4200

acgccggacc agtgccaacac ggtcgggtac tcgcccgagg ccgcggtacc gcgcaccaac 4260

ttcgcgatcg accgcagcag gctcttttcg aaggccatcc cggactccgg gatcgagaag 4320

gccctggagg ccttcgtcgc caacccgcgc gccggccagt tccgcttcct cagcttcttc 4380

gcgctgggcg gcgcggccaa ggagcccggc cgcaccgaca ccgccttcgt gcaccgcgac 4440

accgagttct acgccggcta ctcgctgggc ctcaacgaag cgaagtacac ggccgaggac 4500

gaggcggcgg gacgggcctg ggccgaggcc ggcttccgcg ccatcgaccc gttctccaac 4560

cgggagagct accagaactt catcgacccc tccctgacgg actggaagac ggcctactac 4620

ggcgagaact acgcgcggct tctcacggtc aagcggaagt acgaccggca ccaggtcttc 4680

tccttcgcgc agggaatcgc ctaattcccg aaagatccca accgagaggt gaaacacatg 4740

gtcgccgagc agggcgcgcc gagcgccaag gcgggcaaga ccctcctcac cctgttgtgc 4800

ctggcccagt tcatgctgat cctggacgtc gccgtggtcg cggtcgccgt cccgtcgatg 4860

cagtcgcagc tgaacatctc cgcggccaac atccaatggg tcagtaccgc gtacggcctg 4920

gcgttcggtg gcttcctggt cgcttccggc cgagcggccg acctgttcgg ccccaagcgg 4980

atcctgctca tcgggctctc gctgttcaca ctcgcctcgg ggctgtgcgg catcgcgccc 5040

aacgagctga cgctgttcgt ggcccgcgcg gtgcagggct tcggcgccgc tctggtgtcg 5100

ccggcggccc tgtcgctggt cacgaccagc ttcgggggagg gcgaggagcg caacaaggcg 5160

ctcggggctgt ggggagcggt ctcgtccggc ggtgccatcg ccggccagct gctcggcggc 5220

gtgctcgcgg acctcgcggg ctggcggtcc atcttcctca tcaacgtccc gctcggcctg 5280

atcgtgatcg tggccgtcgc ccgcatggtc cgcaaggacc ggtcgaacga caccggccgc 5340

atcgacatgg cgggcgccgg cctgctcacc gccggagtgg tgctcctcgt gacggccgtg 5400

tcgcaggccg ccgagcgcgg tgtgggcctg cccgtcctga tcagcggtgt gctcggagcg 5460

gtcctgctgg tcttcttcgt ggtgcacgaa ggccgcgtcc agaaccccgt cctgcgcctg 5520

tcgatgttcc gcaaccccaa tgtccgttac ggcaacatga tctgcatgct gacggccgga 5580

ggcgcgacgg tctcggtgtt cctcggcact ctctacatgc agcaggtcat cgggatgagc 5640

ccgatgatgg caggcctggg cttcgctccg gtcaccctgc tgatcctggg cgtgtcgatg 5700

cgcatgggcc cgctggtgca gaagttcggc gtgaagaacc tgctgctggc ctcggccgcg 5760

ctgttcgcgg tgggttcgct gctgctgagc ttcgtcacgg tcgacggctc gtactgggtg 5820

caggtgttcc ccggcctcgc catcggaggc gtcggcgcgg ccctgagctt cgccccctcg 5880

atgatcctct gcaccaccgg ggtccccgac gaggagcagg gcctggcttc ggggctgctc 5940

ggcacctcgc agcagatcgg tgcggcgctc ttcctggcca tcctgagcgg ggtcaccgcc 6000

gcggtggccg cctcgtccgg cggcgacacc cccgaggcgc tgaccgaggg cttccaggcg 6060

gggttcctgt ggtcgctggc gatcccggca ctgatcgtcc tgtgcctcct ggccctgccc 6120

aggaagaagg acgaaccgca ggtggagccg gcggcggagc agaccgaaac ggtctgaccc 6180

ctcatacggt ccgacacgtc gaacggcccg acccgtcata cggcgcctcc gggccacaag 6240

cgaaagcgct cggccggtac ctcaccggcc gagcgcttcc gttcggcgtc agccgcgtgg 6300

gacgagccgg cgcaggtggc cggcggtggc gctgtcggcc gcccggatct gctcgggggt 6360

gccctcggcg acgacgcgcc cgccgtgctc gccggctccg ggccccatgt cgaccagcca 6420

gtcggcgctg tccgccaggt gcaggtcgtg ctcggcgacc agcagggtgt ggcccgcggc 6480

cagcagggcg tcgaaggcgc tcaccatccg ctgcacatcg gccgggtgca gcccggtcag 6540

cggctcgtcc agcaggacca cgccgccgcg gccaccacg gcgccccggc gtatcgcctc 6600

cgccagcttg aggcgctgcg cctcgccccc ggacaggtcg gtggcgctct ggccgagctg 6660

gaggtagccg aggccgacct gctggagggc gtgcaggatg gccgtcaggg tgcgcggttc 6720

gcggaagaac tccgccgcct cgtcgacggt cagcccgagc acctggtcga tggccaggcc 6780

ctggtaggtg acggacagga cctcgggcgt gaacctgcgt ccggcgcaca cgtcacaggt 6840

cgcccacacg tcgggcagga agtgcatgtc caccagcttc tggccgtagc ccgtgcacgc 6900

ctcgcagcgt cccgcggccg cgttgaagga gaaggcggag gcgcccaggc ccagcgcgcg 6960

ggcgcggtcc gtggccgcgt acagcttgcg gaccgcgtcg aacgccttgg tgtacgtggc 7020

cggattggac cgcggggtgc ggccgatcgg actctggtcg acgcagtcca cccacgccac 7080

ggactcgatg ccggtgaccg cccccaccgc aggatggccc gaaccggcga gggcggcggt 7140

gagcgcgggg gcgacggcat ggcccaggag gctgctcttg ccgcttccgc tgacgccggt 7200

gaaacaggtc agggtgttca gcgggacgcg gagctcgtcc acccgtacgg tgtgcgcccg 7260

gacgtccttg aacgtcagcc acctgccgct gccgccgccg gaccgggcgc ggttgatgcg 7320

cggccggctg cggtccaggt agcggccggt caccgagtcg ggatgggccg cgagctcctg 7380

cgggggaccg gagaacatca cccggccgcc accgctgccg gcgccggggc cgaggtcgat 7440

gacccagtcc gcctgggcca cgacgtccgg gtcgtgctcg accagcagaa cggtgttgcc 7500

cgagtcccgc aggtcttcca ggatgtcgcg cagcttctgc ttgtccgcgg gatgcagtcc 7560

ggaccccagc tcgtcgagca cgaagacgat gcccgtcagc gaggtgctga gctgagcggc 7620

cgtacgggcg cgctggagct ccccgccgga gagggtcggg gccgtgcggc cgagttcgag 7680

atgggccagg cccaaccggt cgagcagccg cagccggttc gtcacctcgg gcagcagcgc 7740

ctcgccgatc tcgcgctgga cgccgctgag gacgccgccg agctcggcgg cccacttctg 7800

cacctcgtgc acccgcaggg cggacagctg ctggtacgtg acgcccgcca gggccaccgt 7860

acgggcggcc tcgccgtagc cgctgccacc gcactgcgcg caggggatct ggcgcatgta 7920

cggcaggtag cgctgcttgg cgttctcggt accggccgcg gcgaacagcc gctcgacctc 7980

ggccagcgca ccgcgcagcg gctggttgag ctggtagagg acctcggcgt cggagttctt 8040

gttggggacc tgcacgctga cgctcacggt ttccgtgccc gttccgtaca gcaaggcgtc 8100

gcggaaggac tggggcagct cccgccacgg cagggagagg tccgcgccgt ggcgctccgc 8160

caggaagggc accgccacct gctccggcga gcgcagtttg tcgaaccacg gcgatccgcc 8220

ctcctggagg gggaggccgg ggtgggtgat gatcaggtcg gcgtccgcac ggggagtgcc 8280

gccggcgccg tggcagacgg ggcagccgcc ctccgggctg tagcggctga agtgcccgct 8340

ggacagctcc ggtccctccc cggccaccgc gggcagccgg gagtagagca gcccgaggta 8400

ggcgtcgacg ccggtcatcg tggcgaccgt ggaccgcgaa tggcggttga ggcggcgctg 8460

gtcaacggcg agggtggcgc cgaggccgtc gatgcggtcc acctgcgggc ggtccttggg 8520

ggtgatgtac tggcggacga agggcgaaag gccctccagg tagcgcagtt gggcttccgc 8580

gtggaccgtg tcgatcgcca gtgaggtctt gccgctcccg ctgacccccg tgaaggcgac 8640

gatcccgccc cgtggaatgt ccacggtgac gtccttcagg ttgtgcgtcc gggcgcgtac 8700

caccctgatc cattccgtgc tgctgtgggg gaaggggctt tccggcatgc tgtttccgct 8760

cgttcggggg cgtacgggaa caggcccccg ggggaccggg ggcctgctga aaatcagatc 8820

acaccgccgc gggggccgcg gccggtgcgg ccggccggcg ccgcgccgcg ccaggggcct 8880

ccggctcgcc gcaccagtgg tgcagagcgg cccgcaggcc gggggtgtct ccggcgtggc 8940

ccgcccaggc gacgtagccg tccgggcgta ccagcaacgc ctcgggcagg tccttcgggt 9000

tcttgcggcc tacgctcacc gaccgcaccg cggtgacgcg gtccgcccag cccgcttcgg 9060

ccacctcggc gcagaagccg ccgtgggact ggtcgagcag cacgaaacca cccgtgcgga 9120

agtgggtgta gaggcggctg tccacgccgt cacgtgagcg cagcgcggtg tcgtgcaccc 9180

gtcggccgac cagccggtgc ggtacccgtc ccgtcgtgcc ggccagggggg cggtagcgga 9240

gcttcagccc ggacagctcc tccaggacgg agttctgtac gggcgccagc cccatcaggc 9300

gggtgctgag cccgcgcagc agccgggccc ccgcggagtc ggagacctcg aacttgtaga 9360

ccaggtccgt gcggcgcagg gtcgccgcag cgatggggcg gcgctcgcgc tggtaggtgt 9420

cgaggaggtg ggccggtgcc cgccccgaca ggtgggcggc gagcttccac ccgaggttca 9480

tggcgtcctg gatccctgtc tgcaggccct ggccgccgga cgggatgtgg gtgtgcgcgg 9540

cgtcgcccgc aagcaggacc cggccgagcc ggtagcggtc gctgagccgc tggtcgctgc 9600

ggaagcggga catccacagc ggatcgtgga gcccgaagtc ggtgcccagt accgcccggc 9660

agctctccct cagctcgtcg accgtgacgg gctcgtccac cggcgcgccc atccgcgcgt 9720

ggtcgagcgc gatgagccgg aacgtgccgt cgccgaacgg gaagagcacg accatcccgc 9780

gcttgccggt ccgggcgtac acctgcgggt ccggcggggt ggccagccgg acgtcggcca 9840

cgatcagcga actgtcgtag gaggatccgc tgaaggagat accggcgagt tcgcgcaccg 9900

tgctgtgcac cccgtcgcag cccaccacga agtccgcgtg ctcggtgcgc ggacccccgg 9960

gcccctccac ctccacctcg acgccgccgt cgtgctggcg cagtccggtc acggtcgccc 10020

cgcgctcgat gacggcgccc gagtccaggg cccaggaggt cagctgctcc tcggtgcggc 10080

tctgcgggat gacgagcatg tacgggaagg ggctgtcgac gtcgcggtag tccagccagc 10140

gcttgccgtc gccgagcgcg gcatgcgccc agggcaggcc ctgcgccacg aacgactcgg 10200

cgcgcccgcg catgtcgagc agttcgaggg tgcggggaag cagtccgaac gcccgcgagt 10260

ggggcgaggg ggtgctccgc ttgtcgagta cacggcaggc gatcccggcc gcggagagct 10320

cggcggcgag ggcgagaccg gtcggtcccg cgccgacgat cagcacggac gggaactgg 10380

ccatgaaatg ctccttggtc tgactcggac tgactcggtc cgacccgtcg cgtcgcggat 10440

ccgggcgacg gcgggcggat cagccgttgc gggtcgcgtg ggtgacgacg aaggtgccgg 10500

ggacggtctc cagacgctcg ggggtgagac cggcgtccgc gaccagcgtg cgcatcccgt 10560

cgacgtcgta cagccggggc agccgcccca tggccttgag cagcagccgg agcagcactc 10620

cgggcggggc gtgctcggcg atcatcacgg aaccgcccgg gcgcaggatc cggcgtatct 10680

cccgcagccc cgccggggcg tccgaccagt ggccgaacgc ggtggtgctc accacgaggt 10740

cgacgctcgc gtcggccggc ggcagccgct cggcgcggcc gaccgtcagc tcggcgccgg 10800

gcagccgccc ccgggcgatc tccaccatgc ggtcggcggg gtcgacgccg tacaggcggg 10860

cctgcggcca gcggcggccc gcctcctcca gcagctttcc ggtgccgcag ccgacgtcga 10920

gtacggaccg gggctccagg cccgcctggg cggcccagtc gagtacgacg gtgtgggcgg 10980

tctcgcagtc cttgccgaac ttggcgtcgt accgcggtgc gatccggttg aaggtgtcga 11040

cgtgcccggg atccgccatg gggttccgct ccaggtctcc gtgccgatgg gatttccacg 11100

ctaggaaggc ggccgggcgc gcgggcgaac ggcgacaggt cgctgaaagg atcccgcccg 11160

cgccgccgcc ccgccgcgcg gtcctggcgg tgcgggtgga acccgcaggc gtcagggcag 11220

ctcggcccag gcgcgggcgc gctcctcgtt gtccttgcgg gcgctctcgg cgatccgctc 11280

cagggccgcc cagtcgatgt cctggggggc ggtcaggttc cgctcctcca ggaaccgcgc 11340

ggtgtcccgt tcgatcttct ccacgacacc cgcccagccc ccgggtccaa gaccggggccc 11400

cggcagcacc cgccggggcc ggaaggacca gccgtcgccg tgggcggcga ggtcaccgga 11460

gacggcgagg gcctgcaacg agccgaactc ctcgtcgtgg ctgtgccaca gcagccacgc 11520

gggtccctcc gcggccggca cgccctccgg cagccggacc accatgagac ccgtgtcccg 11580

gtcctgccgg ggacggcagc gcaggctgaa ggggaagccg tcggggtccc ggaaggagag 11640

caccgggctg ctgaagaggt gcaggtagtc gtcgatcttg tgccagacgc tcatgccaca 11700

cgctccagcg cggtgccgcc catcgcgtcc ttgcggaagg cccagacctt ctccggcgtc 11760

accgtgatgc gcaggcgcca gtagaaactc ggatggaact gcgcgcggat ccgcgggtcg 11820

agcgcctcga gcgcgccggc gggacgcttg cggaacagct ccgcccagaa gtcctccagg 11880

ccgtccacgg tcatgatctc ctctggggcg gtggcccgcc cctgcacgag tacggcggga 11940

gcgctgccgg gtagggcgct gcccgtgaaa tcggacatga ggagggagac ccggtcgtcg 12000

cggttgatgt gctccgcctt ccgggccacc gagatgctgg aactgaggac gaactcgttc 12060

tcctccggct tccagaggaa ggccatcggc caggtcaccg ggcggccggt tctggcaaga 12120

gtggagaact cacaggcacg tacacggcgc agaacacgct tcacttcgcc ggaaagggtg 12180

gctggcatgg ttcctcttga tcgtcgggtg attcctgtca cgatgaggcc gggacatggg 12240

taccacccgg ttttcttccg gtcaaggctg gaattcgagc ctcagtcgac ggtctcggac 12300

cgtccgaata cggctgacgg attcctgaaa gaaacagggt ccggtcgaat tccgaatgct 12360

agagaaaggg catgacaacg acgcccgcac ccagctaccc cttccccgcg aaatacctcg 12420

acgaggccgc cgaactggcc aggctgcgcc aggagagcc catctcacgg gtcaccatgc 12480

cctacggcgg cgaggcctgg ctggtgaccc gtatggcgga cgtcaaggag gtgctcgccg 12540

accccaggtt cagccgtcag ctgcagaccg aggcggaccg cccgcgcttc ttccccgagc 12600

ccgtggtcga gggcataggc atcatggacc cgccggagca gacgaggctg cgccggctgg 12660

tcgccaaggc gttcacggcg cgccgggtcc aggagttcgg gccgcgcgtg cagacgatcg 12720

tcgacgaact gctcgacgcg gtcgaggcca agggcgcccc cgccgacctc tacgccgact 12780

tctcctggca gctgccgggc atctccatct gcgagttcat gggcgtcccc tacgaggacc 12840

gggaccggtt cgtgccgttc ttcgactcgg tcgtctcgac cacgtccaag accccggacg 12900

agatccgcca ggccatcggc gacctccacg cgtacttcgg cgagctcatc gagaggcgtc 12960

gcgccacccc cggggacgac ctgttcaccg ctctgatccg ggcgcgcgac gaggacgacc 13020

ggctctcgga gaaggagctc atcgagatga gcttcggcct cctcgtcgcc ggcatggaga 13080

ccaccgcctc ccagctcgcc aacttcctct acctgctgtt ccgcaaccccc gaccagctgg 13140

agctgctgcg cgccaagccc gaactggtgc cgaacgccgt cgaggagctg ctgcgcttcg 13200

tgccgctgct cgccgtcgac cagccgtcgg tggcgcgcga ggacgtcacc ctgggcggcg 13260

tgctgatccg ggccggcgag accgtggtcc cgtcgatgaa ctccgcgaac cgcgacgccg 13320

acgtcttcga gaacccgcag cgcatcgacg tcacccggga gaacaacccg cacctcgcct 13380

tcagccacgg agcgcaccac tgcgtgggcg cccagctggc gcggatggag atggtcacgg 13440

cgctgcgctc cctgctggtg cgcttccccg gccttcacca ggcggtgccg gacgaggagc 13500

tccgctggaa ggccggggtc gtcctgcggg gcgtcgaggc gctgccggtg gcgtggtagc 13560

cgtggccgac gagaacgcat ggagcgtacg ggtcgaccgg caggcctgcc tgatgtcggg 13620

gctgtgcatg tcgctggccc ccaacgtctt cggggagggc gacgaccacc gggtcgtggc 13680

gctgaccgac cggtccgcgc ccgacgggcg gctgctggag gccgcggagt tctgcccggc 13740

cgaggcgatc gcgatcagca gcctcgcgac cggggagacg gtcgagggca ccggctgagg 13800

cgggccactc tcccgcggaa ccacccgagg gcggcggacc ggtgtgtccg ccgccctcgg 13860

gcacgtttcc ggatcaggtg atcgactggg cgaagcggaa gaagcccttg gggtcgtagg 13920

cgcgcttcac cgccgacagc cgctcgtagt tgaccccgta gtaggcctcc cgccagtccg 13980

cgaggtccgg gtcgatgtag ttctggtacg cctggcccga ggaccagggg tgcacccgtg 14040

cgtaggcccg gtcgacccac tcctggcacg cgcgcctgcg gtcgggcgcc agcacctcgc 14100

cctccggcac gtcgatgccc accgaccagc cggcgtagaa cagggcgttg cggtggacgt 14160

acgcggtggc ggtcgcgggc acgcggttgt gggcgccgcc cagcccctgg aggtcgaagc 14220

tgcgggcctc gccctcgctg cgggcggccg tgaaggcctt caggaccgcg tcgatgcccg 14280

cactgtcgag cggcctgtcg aagaagttgc cgcgcgccag cgcgaatccg taacgggcca 14340

gctgggcctg ggggttgtgt ccgacacggt ggcacgcggc cggttcgagg gtgtcgcatc 14400

cgaaccagtg gcgcatgccg aagcggtagg agtcggtgcg ccgctcgctc gtcgcggggg 14460

cggtgcccac ggcggcggtc agccggtcca gcagcgggcc gagcccgtcc ggcgagccga 14520

gccaacacgcc ggagaccgtc acacccggct cgacaccggc gtcggcgccg tacgtcgaga 14580

tgttgagcag cggcgtcagc gggtcggggg cctcggccgt ccaccgctgc caggcggaca 14640

gcaccgcccg gacgggaggcc cacgtccagg tgagggtgaa gctggtcatg tcgggggcgg 14700

gcaccggctc gaagtcgtac tcggtgacga tcccgaagtt gccgccgcca ccgccgcgca 14760

gggcccagaa caggtccgcg tgctggtggg cgctgctctc gaccacgcgc ccgtccgcga 14820

ggaccacctg ggccctgcgc atgcggtcgc ttgcgacgcc gtacatccgg gtcaggaagc 14880

ccagcccgcc gccgagggcg aggccgccga tggcgaccgt gggacaccag ccggtcggca 14940

ccgcgacccc gtgcacggcg agcgcctcct ccaggtcgac gagctggcag ccgggctgga 15000

tccgggcgac cgaaccggcc agccgcaccg cgttcatgag agagaggtcg atgaccatgc 15060

cggtcgtcgt ggagtatccg gcgaaggagt ggccgccgct gcgcgccacg gccggcacgc 15120

gatggcgcgc ggcgaaccgt acggcctcgc agacgtcctc gaccgtggtg cagcgcacca 15180

cggcctgcgg cctgaccgag tcgaactggc gcagggccag ggagcggacg gtgtcgtact 15240

gcgggtcgcc gggcagcacc accgtgccgt cgaggctccg ctggagggcg tcccacggcg 15300

ggcctcccgc cgaggcggtg gcggccggca ggagcaggcc gcccgcagct gaggccgccg 15360

cggtccccag gaaagtgcgg cgtttcatac aggtctcccc ttttccgggc gcggtctgtc 15420

ggttccgacg gccccagagtg tggtgactgt gttcgcgcca cacggcggag atggcaggaa 15480

ttcgacagtt ctcggccaca caagggggcg gctcccgggt caggggtgct ccggcagctc 15540

gatgatctcg ttcaccgatc cggtgacgcg gtacgcccgc ggagccggcc cgtccggccc 15600

ggccaccagg gcccggacgt gcccgtcggg gaccgtggag atccgcacga cgggatccac 15660

cacagtcccg gcgggcagca cgacaggtgc gcgctgcatg atgcgggtga gcaccagctt 15720

gctctccagc agggcgaggt tgcgcccgat gcagttgtgc tggcccagac cgaaggggaa 15780

gtactcgtgc tgcgagggct gggcggtctc ccagcgctcc gggcggaagc gcaggggctc 15840

ggggaacgtc ccggcgtcgc ggtgggtgac gaaggagctg aagatcgtca tggcgccctt 15900

ggggatcggg tggccgtcga tcacggcgcc ctcctccgcg tagcgcacgc cgaatccggc 15960

cggcggcatc agccgcacgc tctccttgac gatccggtcc agcacgggca tccgtacgag 16020

cacgtccggt cccggagccg cgtcacccgc gacctccgcc acctcggcgc gcagcttctc 16080

ccaccactcc cggtgctggt cgagcaggaa gagggtccag aacagggacg aggcgacgct 16140

gtcgtggcac agcgcggtgt acgcctcgcc cagcagctcc tcgtcgctca actcctcggc 16200

gtcggcctcc gcgtcgggca cggtcagctt gctcagcagg tcgggcgcgg ccgctccgga 16260

gcgccgtgcg gcgatcagcg cgcgcaggat gtcctcgatc tcgcccgcga tccgggtcat 16320

gcggcggaag ggcgtaccgg gcagcggtgc ctgtatcacc gccgtgagcg ggtgccccgc 16380

cgcggcagcc agctcgacca tcttgtcgtt gaggtggcgt gccgccgcct cgtcctccag 16440

accggccatg gtggccagcg agatccgcgt cacgaggcgg gcgagctcct cgtgcaggtc 16500

cacggaacgg cccggcgtcc acccgtccag catgtcgtcg gcgaagcgca cgatggtgtc 16560

ggtgtagacg gtgacgtggc gcggggagaa ggcctgctgc atggcctgac ggtgcttgcg 16620

gtggatggtg ccgttcagcc gcagcagccc actggtcagc agcatcatcg gcgagccggg 16680

cggcaggcgc aggtggcgga aggagtccga gacgaaggac ttcccggtca gcacctggcg 16740

tgcctggtcg ggcccgaacg cgaagaccgt gcgcgggttg cggccgggca tcgcgcagac 16800

gtcgccgtaa ccgctcgcga gctcctgcag gaaaggcagc gggcggcgca ggagattcag 16860

tccggccgag agcctgcgca acccgagagg atctgccggt ccggccagtc ggtccgccgg 16920

attctgcaca acagggggttc caggcacggt gattctcctc cgggaacggc atggatgaag 16980

actcttcctt gtaccccacc gccgcccgcc cgagccgccg gccgaaagaa ccccgcaaga 17040

tggtcctcgg cgacttcttt cgggaatccc tcatggttct gaaagaaatt ctccggaggg 17100

tccgggcccc ctttagcttc gggaaggcgg gcttcgggag cggactttcc ccccaccccg 17160

ctgaacgcgg cgtatgtcat cggcagacgg gtggggacac aatgcaggcg agcacgggcc 17220

gggagactgt tctcggggcc attcgggccg tgtgcgagac cgacccgggt gcgacggcgg 17280

ccgttttccc cgcccagccg gcgatcggtc tcaccgaggc ggtcaccttc agccgcgagg 17340

gactggaccg cgaggcccgg gccctcgcgg tcaagctgcg cgaggtggcc gagggccccg 17400

tactgctgct gatgcagccc ggcccggact acctcaaggg gttcctggcc acggtctacg 17460

cgggactgcc agccgcaccg gtctacccgc ccaacccgc cgacgtgcgc cgcgacttca 17520

tccggctcgg cgcgatcctc gccaagctgc cggacgccac cctgctgacc gagccggggc 17580

tgctggagcc gctgcgcgag ctcttcgcgg agcggctgcc cgacgtacgt cccgaacggc 17640

tggtggacac cagcgtcccg gccggcgccg aggacgactg gcgggcgccc gcggtccggc 17700

ccgaggaacc gctgttcatc cagttcacct ccggttcgac cggcacccccc cgcggcgtgc 17760

tcgtctcgca ccgcaacctg ctcgccaacg tccgcgccat caccgaccgg ttcggcctgg 17820

acacgtcgag caccggtgcc ctgtggctgc cgccgtacca cgacatggga ctggtcggcg 17880

gagtactcac tccgctcgtc agcggcttcc cgatccacct gctctcgccg ctctccttcg 17940

tctccgaccc gatgggctgg ctccggctgg tctccgagac cggagccacc cacaccgggg 18000

cgcccaactt cggctacgcg ctggccaccc gccgcgcccg ggacgaggac gtggccgcgc 18060

tcgacctcag ccgactccag gtggccttct ccggtgccga gccggtggac gcctcgaccc 18120

tgcgcgcttt cgccgagcgc ttcgcccccg ccggcctgag cccggacgtc ttcctgccct 18180

gttacgggct cgccgagtcc accctgatcg tcagcggcgg ccgccccggc gccggactgc 18240

gtacgcaccg gctcgacccc gaagccctgg cactgggcag ggccgaaccg gcccggcccg 18300

gcgccccggc caccgaactc gtctccagcg gcccggtggt ggccggcacc gaggtacgga 18360

tcgccgaccc cgtcaccggg gcggcggccg gacccggcga gatcggcgag gtcttcgtcg 18420

cctcggacag cgtcgccgag gggtacttcg aggacccccga ggagacggcg cgcaccttcg 18480

gcgcgaccct ggcgggctcg agtcgcagct ggatgcgcac cggcgacctc ggcttcctcg 18540

gcgccgacgg cgacctcgtg ccggtcgccc ggatcaagga cgtcatcgtc gtccgcggcc 18600

gcaacctgca cccgcaggac atcgagcgga cggtgcagga caccgacccc gggatccgca 18660

agggctgtgt cgcggccgcg ggcgtacccg gcccggacgg cggggaggag atcctggtcg 18720

tggcggagct gcggcccgag gccgcggacg acgaagccca ggcccgcagg atcgcccggg 18780

aagtccgcgc agccgtcgca cgcgagcact ccgccccggt gcgcggcatc cacctgatcc 18840

gtccctcgac cctgcccaag acctccagcg gcaaggtgca gcgctccgcc gcccgcgccg 18900

cccacctcga cggctccctg gccaccgtcc tgtgcgacac cgccgacact gcggcgccga 18960

cgggcgcgcc cctccccgcc gggcccgcag cgggcggctc cgacgacctc gtcgtcaacc 19020

tcgcagcgac ggtcatcagg cacttcgtcg gccacggcgg caccaccggc gcactggact 19080

cgctcggcgc ggcccggctc gccggggagg cgtcgcagat cttcaaggtc gcccttcccg 19140

tcaccgacat cctggccggt gtcgacgccg aggcgctcgc cgcgctcatc cggaccgccg 19200

cccccctcgg cggccccgcc gccgcaaccg gctccgccgg gctggccgaa ctccccgcga 19260

cggcacgcca ggaaaccctg tgcctgctcc aggaactcga ccccgccgcc cccggcctcg 19320

ccctcggcat cgccttcgcc ctgcccgcca cggccgaccc gcagcgcgtc gccctggccc 19380

tgaccgctct cgtggcccgc cacccggcac tgcgcacccg cctggtgcgc cgcggcgaac 19440

actggtcgcg cctcgtcgac ccggccgaca ccgccgccgg tgcgccctgg ggccggtact 19500

gcaccctcac ccggctcggc gggatcgggg agcgggacct cgaagaggcc ctcgccgagc 19560

gctgccgacg cgtacccgat ctcgccgaag gcccgctgtt ccaggccgag gtcgtcctcg 19620

cggacgggca cggcgcccac ctggtgatca ccgtccacca cgccgtcgcc gacctgtggt 19680

cgatcggtgt cctggcggcc gagctcgccc acctgctcgc cgccggtgat ccggaggccg 19740

gggccctgac cctcccgccg gccccggagg tctcctgcga cgtccccgac gaccggcgcc 19800

tggaacgggc gtggaacttc tggcagggac tgctcaccga cgggtgcggc cccctccagc 19860

tgccgtccgc cgacggcccg gcgcagaacg tcccggccgg ccgcgcgcgg caggccagcc 19920

acgccccgct caccctggac gcccggcgca ccgccgcgct caccgccctc gccaaggagt 19980

gcggcgtcac cctgtacgcg gtcctgctcg ccgtgcagtc gctcaccctg tcccggctca 20040

ccggaaccga ccgcgtcccg gtcgccgtac cgctgcacgg ccggggaagc gccacgcacc 20100

gggccgtcga ctacctcgtg tccacggtca ccctgccgat ggagaccgcc accggcaccg 20160

tccgcgacct ggtcggccgt gcggccgacg ccctgcgcgg cgcgctcgcc caccgcaccg 20220

tcggctaccc cgagctggtg gccctgtccg cggcccgcgg cggccccgac gtacccgccc 20280

ccgacgcggc gctgctgctc cagcaggaca cccccggcgc accgcgcggg gtcggcgccg 20340

gcctcctcgg cggcggagtg cggctcgggg acgtggaact gggcgtcgcc gtggtcccgc 20400

ccagcatcgg ccccttcggc ctgaccaccc tgctcacgga gacggacggc accctcaccg 20460

ggcgcgtcga ggtcgacccc agccgccatg ccgactggct ggcaggccgg ttcgccgacg 20520

ccttcctcgc catcgccgac tcggtggccg ccggcgcagg actccccctc gcggaggcgt 20580

ccgcggtggg ggcggaacag gccgaacggc tgcgcggctg gtcgcgctcg cccgtgcccg 20640

aggacggcga gggcaccctg cacgacctcg tcctgtcagc ggcggcccgc cacccgcggc 20700

gcaccgcggt ggtcgccggc gacgggagcc tcagctacgg cgaactcgca caccgctcgg 20760

ccgtcgtggc ggccgcgctc gccgcggaag gcgccggccc cggctgcacc gtcggcgtgc 20820

tgatgcagcg cggccgcgac ctgcctgcgg tcctgctcgg agtactgcgc tccggcgccg 20880

cctacctgcc cctggacgcg gccactccgc ccgcccggct ggccgccgtg gtcgaggacg 20940

ccggctgccg ccacgtcctg gtgggcgacg tcccggtgga acgggggcag ttcttccccg 21000

tccgcaccct ggacgtggac gcggtcctcg cggcgggccc ggccgagccg gtgccgccgc 21060

ggcccctgac caccccccgac gaccccgcct acctgctctt cacctccggc tccaccggcc 21120

ggcccaaggg cgtggtgatc ccgcaccgcg gaccggtcaa cctgatccgc tgggcgggcc 21180

gggagttcgg cacggacgcc ctcgcccgca ccctcgccgt cacgcccacc accttcgacc 21240

tgtccgtctt cgaactcttc acgcccctgg cgcacgggtg cgaggtgcgg ctgctcgacg 21300

gcgtcctgga cctggtggac agccccgcgc acgccgccga cgccaccctg ctcaacaccg 21360

tcccctcggc ggtggcgagc ctgctggaac aggacgcgct tcccgccggc ctcagcgtgg 21420

tcaacgtggc gggcgaaccg ctcaccgccg aactcgtcca ctccgtgcac cggcgccttc 21480

ccggtgtccg catggtcaac ctctacggcc ccagcgagac caccacctac tccacgtacg 21540

ccgaactggg cccggacacc agcggcgccg tccccatcgg ccgacccgtc ggcggcacca 21600

ccctgagcgt cgtcgacgcc tccctgcggc cgctgccgca gggagccacc ggcgaactgc 21660

tcatcggcgg cgcgggcgtc gccgtcggat acgcggggcg cccgggcatg accgcggccc 21720

ggttcctgcc cgaccccgaa cacccgggcc gcaggctcta ccgcaccggc gacctcgtcc 21780

gctggcgggc cgacggcctg ctggagttcc tcgggcgctc cgaccaccag gtcaaggtcc 21840

gcggcttccg catcgaactc ggcgacgtcg agcgcgcgct gaccggcctc gacgcggtcc 21900

gggaggccgt cgtcgtcgcc ctgggccagg gcaccgaccg ccgcctggcc gcctacctcg 21960

tgcccgagcg gcccctggaa ggcgaccccg gcagctggct gcgcggcgtc cgccggcgcc 22020

tcggccacga actgcccggg tacatggtgc cgggcgagtt cgcggtactc gacgaactgc 22080

cgcgcaaccg gcacggcaag ctcgaccgcg gccggctcgc ctccaccgcc accgttccgc 22140

tcgtcacggg cagccggatc gccccgcgca acgacagcga gcgccgtgtc gcggcctgct 22200

gggcgcaggc actgccggtt cccgagatcg gcgtgaccga cgagttcctc gacgtcggcg 22260

gacactccct gatgctcacc acgatcgcgc acctgatcgc cagggagttc tcggtccagg 22320

tcccgctgac cgccctgcgc acgcacacga cggtggccga acaggccagg tacctcgacg 22380

agctcacctc ggccgcgccc ccgcacgccg caggcccgcg gtccgtgcga cgcctggacc 22440

gcagcaggta caccgcggcc gggaacggcc ggaccacctg atgggccgcc cccctcagga 22500

ccgcccgacc ccagcccgac gaaacgtttt ggagcactcg tgagcgaccg gatcccaccc 22560

gacgcccatc tcttccccgc ctccccggga caggaacggc tctggttcct ggaccggatg 22620

aacgagatgg ccggcaaggc gtacggcctc gccgtccgcc tggacctgcg cggcgccctg 22680

gagcgcaccg ccctccagtc cgccctcagc gccgtcgtcg aacgccacga ggccctgcgc 22740

accggcctgc gccagatcga cggcacgctg acgcaggtcg tcgtccccgg cgtcaccgtc 22800

tcgctgcccg tcgtcgacct cggcggccgc ggaccggacc ccgcgcagct cgaccgcgag 22860

gtgcggcgcc tggcacgcca ggaggcccag cgcggctgga acctggccca gccgcccctg 22920

ctgcgcgggc tgctggcccg gctcgccgac gaccaccacg tcctgctgct gtgcgtgcac 22980

cacgccgtgt gcgacggcct gtccctccag atcgtgctgc gcgaactgct ggagaactac 23040

accggcggcg gtcccgccgg tgccgacgag cccctccagt tcgcggacta cgtggtgtgg 23100

aacaacggcg gtgaggagtt cccggaccccc gagtggtccg cgcgccgcca ggccgcccgg 23160

gaacactgga gcagcaccct cgcgggcgcc ccccaggttc tcgacctgcc caccgaccgg 23220

cgccggccgc ccctgcagtc ctacgccgga gcccgcgttc ccgtccggct ggacaccgcg 23280

ttcgccgacc gggtgcgcga ctggtccgcc cagcgcgggg tgaccccctt caccacgctg 23340

ctggccgcct aacccgtcgt cctcgcccgc aacggcggcg ccgacgacct gctggtcggc 23400

ctgcccgtcg ccaaccgctc ccacgcggac ctcgccggga cggtcggcta cctggccaac 23460

acctgcccgc tccgcgccga cctgcgcgcc gaccccaccc tcggcgaact ggtccgagac 23520

ctgtacgacc ggctcacggg cgtcctcgaa cacgccgacc tccccttcgg cgagctggtg 23580

gaactgctgg caccgccgcg catgcccgag cgcaaccccg tcttccaggt catgttcggc 23640

ctccagcagg acgtccggcg cggctgggac ctgccaggcc tgcgcgtgga cgtcgaggac 23700

gtggactgcg gcaacgcccg cgtcgacctg tccctgttcc tcttcgagga ggctgacggg 23760

gcgatcgacg gattcctgga gtacgcctcg gcgctcttcg accgcgccac cgccgagcgc 23820

ttcgccgacc agctccacac cgtgctccgg cagatcctgc gggacgcgcg gatccccgtc 23880

tccgccgtcg acctcgtcgg caacggctcc gccaccacca tcgactcctt cctcgacggc 23940

gggccgctgg aacagccctg gccgctggtg tggccgcgga tccgcgaact cgcggcgcgc 24000

cgtccgtcgg ccgaggccgt ccgcgacgac gcggaagccc tggactacgc ctcgctcgtc 24060

gaccgcgtcg acgccgccgc cgcgcggctc accgctgccg gcgcgggccc cggcgaccgc 24120

gtcgccgtcc tcgccgaacg cggcgtccgc gccgtggtcg cgatgctcgc ctgctggcgt 24180

gccggcggcg tgtacgtgcc cgtcgacccg gcggccccgc tgccgcgccg ggagctgatc 24240

ctcgaacagg ccgctcccgc cgtcctcgtc tgcgaggacc ccgacgagca gccgccgcac 24300

caccggagcc gagcggtggc gatcggcgac ctcaccgccg aggcggacgc cggtgcgggc 24360

acaccggcgg agccggcccc gcggccgcac gatcccgcgt acctgatgtt cacctccggc 24420

tccaccgggc gccccaaggg cgtggccgtc agccacgcca acctctcctc gttcctgcac 24480

gcgctgaccg gccgcctggc actggggccg gccgaccggc tgctggcgct gacgacgacg 24540

gcgttcgaca tctcgctgct cgaactcctc ggcccgctcg tcaccggcgg caccgtcgtc 24600

gtggcaccct cctccgccca gcgcggcgcc gccgacctcg ccgcccggct cagctccccc 24660

ggaatcacca ccgcccaggc cacaccggcc gtctggcgcc tggccctgtc cgccggatgg 24720

cgcccccggg agggcttcac actcctgtgc ggaggcgagg cactgccccc ggacctcgcc 24780

gacctgctgg ccgccacccc cgccgaggcc cacaacctct acggtccgac cgagaccacc 24840

atctggtcct gcgccgcccg gatccgcccg ggtgagcccg tcaccatcgg ccgtcccatt 24900

cccggcaccc gcgtcctggt ggccgacgcc gcgctgcggc ccgtaccgcc cggcgtctgc 24960

ggcgaactcc tcgtcggcgg ccccggagtg gccctcggat acctggacga ccccgcccgc 25020

accgcggcgc ggttcgtgcc cgacccctac caccccggcg aacggctcta ccgcaccggc 25080

gacgtcgtac gcctgcgctc cgacgggttg atcgagttcg tgggccgcgt cgacgaacag 25140

gtcaaggtac gcggccaccg catcgaactc ggcgagatcg agagcgcgct gcgcgccctg 25200

cccggagtgc gcgacgccgc cgcgaccgtg ctcgacccgc gcggcaacgc ccgtatcgcc 25260

ggctacctcg tcgccgacga cggcgccctc gacacggcag gacgggcggc ccggctgcgt 25320

caggacctgt ccgaggcact gcccgcgtcc atggtgccct ccgagctgta cgccgtgccc 25380

gccatccccc tcaaccccaa cggcaaggtc gaccgccggg ccctgccggg caccggtcgg 25440

cgcctggagg gcggcagcga gcgcgtcgcc ccctcgaccg atgccgagca cgccgtcgcc 25500

gcgctgtggt gcgaactcct cagccttccg gaagtcggcg tccgagagga cttcttcgga 25560

ctcggcggcc actcgctgct cgccgccgac ctcctgcagc gcctcgaacg cgacctgggc 25620

gcccgcgtcc ccgtcgccga gttcttcatg gaaccgacgg tcgcccgcct cgcggcgacc 25680

gtcagccagc tcagcggtgc cgtgcaccag acaccgaccc aagaccaga cgggcgtgcg 25740

gagcagaccg cagagccctc gcccgaggac cgcggggcag acggtgacgg atgggacttc 25800

cccaccgtgc gccggtccgt cacggtcccc ggctccgacc ccgttcccct ggaggtttcg 25860

ccgtgacccg gcacgagccc accgagacgt tcaccgacac cctgcaccgc gtactcgaag 25920

aacgcggcgc ggaatcggcc accccgctgt ccgtggaaca gcgccgcctg tggctgctcg 25980

gcgggatcgc cgacggctcc tgggccgtcg tcaccggccg ctaccggctc cagggcgtcc 26040

cggacgccgc gcggctccaa ttgcggctgg cctccctggt ctcccgccac gaggccctgc 26100

gcagcgtgtt cgtgcaggtc gccgagcgtc ccgtccgcct ggtcctgccg ttcgccgagg 26160

tcgccctgcg cacggtgaac gcacccgact ccaccgaccc cgcgcgggcc gacgagcacg 26220

tccgccacct cgtcgaggag gagttctccc tcggccacgg accgctgctg cgcgccctgc 26280

tgctgcgctc cgccgacggc ggcgccgccg aactcgtcct cgtcgggcac cgcctcgtcc 26340

tggacgccac ctcactggac ctgctcgtcg ccgaactgct cggcgaggac gccccccacg 26400

gccgcgaggg cgccgcggac accgccggcg ctctggagac caccctcgcg gcggaacgcg 26460

agcgcctcgc cgaccccgcc ctcacgcaag cggtgaccgg ccgggccgcc gaactggccc 26520

tccccgccgc caccgagatc cccggctacg gccggcggcc cgagatcaag ggcacctcct 26580

acgcctccgt ggcgctgccc ctgccgctcg ccctcgcccc gcgcgccgcc gaggccgccg 26640

actgggaggc cgcggtcgcc gccgcctggc tcgtggtcct catgcgctcc caggccaccg 26700

gctccgccgt ctgcggtgtc cgcaccgccc gcggcgcgga gcaggccggg atcgtcggcc 26760

cgctcgacgg cctgcgcctg gtccgcgtcg acgacgcgga cgacgcgccg ctgtcggacc 26820

tgctcgccgc ggtcgggcga cagctgcgcg cccccgccgt ggacgtgccg ctcgcacacc 26880

tcctcgaagt ggccccgccc cgccgcgaca tcagccgcac cccgtacgcc cagaccgtcg 26940

tgcgggccgt ggacgcccgc gaaccgctcg gcggggcgtc gggcaccggc cggcagatcg 27000

gcggagccgc ctcgggcacc gagtacgaca tcgaggtgac ggtgcgcctc cagcccggcc 27060

tcgccgtcgc acagatcgac tacgacgtcc agctgtactc cgaggagcgg gtccgcgccc 27120

tcggcaacca gctcgccgcc gtcctcgacg ccatcctgcc cggcggcacc ccggaggtca 27180

ccgccaccgt cgtcccgctg ctcgacgccg agggcgcccg gctcgccctg gaggcgggcc 27240

gcggcgagcg gaccgcgccc gacacccgat cgctcgtcga cctcgtcgag gcccaggtgg 27300

ccgccgcacc ggacgccgtc gccctgtggc agggagacac ccgagtcacg tacgcccagc 27360

tgtgggccga cgccactcgc ctggccgacg agctggccgc ccgcggggtg cgccccggcg 27420

accgcgtcgc ggtctggctg cgccgcggcc cctcgacggt caccgccctg ctcgccgtgc 27480

tcgccgccgg cgccgccttc gtgcccgtcg acgccgccta ccccgaggag cgggtgcgct 27540

acctgctctc cgactcgcgg ccctcgctcg tggtcaccga gagctccgtg cacctgctcg 27600

gcgaactcgg ccttcccacg ctgctgctgg acgagctgtc cggtgcgccg gccgccgtgg 27660

acggcgcgcg ccgccccgac cgggtcgccg ccgacactcc cgcctacctc atctacacct 27720

ccggcaccac gggccgcccc aagggcgtcg tcgtgcgcca ctccagcgtg gtcaacaaca 27780

ttgcctggcg ccaggcgaac tggcagctca ccgaagacga ccgggtgctg cacaaccaca 27840

gcttctgctt cgacccctcc gtctgggcgg cgttctggcc gctcgccacg ggcgccgcca 27900

tcgtcctcgc caccgaggag cagatgaagg accccggcga gatgatcacg acgctccgcg 27960

accaccaggt caccgtgctc ggcggcgtgc cctccctgct gtccctgctg ctcgaccacc 28020

gcgatgcggg cacctgcacc cgcgtccgcc tggtgctcag cggtggcgaa cccctcaccg 28080

acaccctgct ggagtccgtc gagtcgacct ggtccgccga ggtcgccaac ctctacggcc 28140

ccaccgaggc caccatcgac gccaccggcc accgcgtacc gcgcggcgac cgcacggtcc 28200

cggttccgat cggccgcgcg gtgtccaaca ccgccgtcca cgtcgtcgac gccgaactgc 28260

ggcccgtacc cgaaggcgtc cccggtgaga tcgtggtcac cggcgcggga gtcgccgtcg 28320

gctaccacga ccggccggcg ctgaccgccg cgcgcttcct gcccgcgccc ttcgccgacg 28380

cgtccgacga cccgggtgcc accctctacc gaaccggcga cctgggccgc aggctccccg 28440

acggctccgt ccagttcttc ggccgggtcg acgaccaggt caagatccgc ggccaccgcg 28500

tcgaggtctc cgaggtcgag tcggtgctca aggccctggc cggggtccag gacgcggccg 28560

tggtcgccct ggacgcgggt accgagaacg cccggctggc cgccgccctg gtccttccgg 28620

ccggctccga cgccccgtcc ctcgaagacg tccgctcggc actcgccggc gaactccccg 28680

actacctcgt gccggaccgc ttcgccgtcg tggacgagct gccgctcacc gccaacggca 28740

agacggaccg ccgcggagtc gccgaactgc tctcccggca ggcagccgca ccggtcgccg 28800

gccaggacgg gcccaccgag ccccgcaacg ccctggagcg ctccgtcgcc gaggccttcg 28860

cctccgtgct gcgcctcccc gcgatcgaca tccacgccga cttcttcgac gtgggcggca 28920

cctcgctcat cctcgcgaag ctggcctccc tgctcggccg caagcacgat gtcgagatcc 28980

cgctgcacga gttcttccgt acgccgaccg tcgccggcgt ctccgagacg atcgaggtct 29040

accgccgcga gggcctcgcc ggcgtcctcg gccgcaagca cgccgcgacg ctggagggcg 29100

acggcaccct cgacccctcc atcagccccg agggccttcc cgaggcggaa tgggagaacc 29160

cgcggcgcgt cttcctcacc ggcgccaccg gctacctggg cctccacctc gtcgagcagc 29220

tcctgcgccg caccgacgcg gaggtcgtga ccctgtgccg ggcccgcgac gaacagcacg 29280

cgctcgaacg cctcaaggag ggcttcgccc tctacgagat cgacgtcgag gaccagctgc 29340

accggatctc ggccgtcatc ggcgacctgg ccgagccgcg cctgggcctc acccaggagc 29400

agtgggacga cctggccgcc accgtggacg tgatctacca caacggcgcg ctcgtcaact 29460

tcgtctaccc gtactcggcg ctcaaggccg ccaacgtcgg cggcactcag cgcgtactgg 29520

aactggcctg caccacccgc ctcaaggccg tccaccacgt gtccaccatc gacaccctgc 29580

tcgccaccca catgccgcgc cccttcctgg agaacgacgc gccgctgcac tcggccgtcg 29640

gcgtcccggc cggctacacc ggcagcaagt gggtggcgga gaaggtcgtc gacgaggccc 29700

gccgccgcgg catcccggtc accgtcttcc gccccggcct catcctcggc cacacgaaga 29760

acggcgccac gcagaccatc gactacctgc tggtcgcgct ccgcggcttc ctgcccatgc 29820

gcatcctgcc cgactacccc cgcatcttcg acgtcatccc ggtcgactac gtggcctccg 29880

ccatcgtgca catatcccgg aagcgggaag ccatcgacgg cttctaccac ctgttcaacc 29940

ccgcgccggt gccgctgctc accttctgcg actggatcaa gagctacggc tacgagttcg 30000

acatcgtgcc cttcgaggag ggcaggcgcc gggccctcgg agtcggcccc agccacctcc 30060

tgtacccgct cgtcccgctc atcaaggacg cggaggccga gccgcaccgg gccctcgacc 30120

ccaagtacat ggacgaggtc cagcccgccc tcgaatgcgc ggagacgctg cgcatgctgg 30180

ccggcagcga catcgcctgc ccgccgacca ccgaggccga cgcccacgcc gtcatggact 30240

acctggtgcg caccggcttc atgccggcgc ccgcggacgt cgtccacgac gagccgagca 30300

gcacgctgga ggagcgatga cggcaccggc cgacaccgtc caccccgccg gccagcccga 30360

ctacgtggcc caggtggcca ctgtgccctt ccggctggga cggcccgagg aactgcccgg 30420

cacgctcgac gaactccggg ccgccgtctc ggcccgggcg ggtgaggccg tgcgcggcct 30480

caaccgcccg ggagcccgga ccgacctggc cgcactgctg gcggcgaccg agcggacccg 30540

agcggccctc gccccggtcg gcgccggccc ggtgggggac gacccctcgg agtccgaggc 30600

caaccgcgac aacgacctcg ccttcggcat cgtgcgcacc cgcggcccgg tggccgaact 30660

cctcgtggac gccgccctcg ccgcgctcgc cggaatcctg gaggtcgccg tcgaccgcgg 30720

ctcggacctg gaggacgcgg cctggcagcg cttcatcgga ggattcgacg cgctcctcgg 30780

ctggctcgcc gacccgcaca gcgcgccccg gccggcgacc gtaccgggcg caggcccggc 30840

cgggccgccc gtccaccagg acgccctgcg ccgctgggtg cgcggacacc acgtcttcat 30900

ggtcctcgcc cagggctgcg cgctcgcgac ggcctgcctg cgcgacagcg cggcccgcgg 30960

cgacctgccc ggggcggagg cctcggcagc cgcggccgaa gcgctgatgc gcggctgcca 31020

gggagcgctg ctctacgccg gcgacgccaa ccgggagcag tacaacgagc agatccgccc 31080

caccctcatg cccccggtcg caccgcccaa gatgagcggc ctgcactggc gcgaccacga 31140

ggtgctgatc aaggagctcg ccggctcccg cgacgcctgg gagtggctct ccgcccaggg 31200

ctcggagcgg cccgccacct tccgggcggc cctcgccgag acgtacgact cccacatcgg 31260

ggtgtgcggc cacttcgtcg gcgaccagtc accccagcctg ctggccgcgc agggctcgac 31320

gcgttccgcc gtcggcgtca tcggacagtt ccgcaagatc aggctgagcg cgctccccga 31380

acagcccgcc acccagcagg gagaaccctc gtgaaccgcg gctcccgctc cctgcgcgaa 31440

agcctggccc gctacggccc ggccgccgcg gcctgcgccg cggcggccta cgccggcacc 31500

tcgctcacct caccgcgccc ccgcccggca agcgctcccc aggagcagtt ctccgccgag 31560

cgcgccatgc gccacgtccg ggccgtggcc gccgaaccccc acccggtcgg ctcgcgggcc 31620

gcggcccgcg tccgggacta cctgctcgcc gaactgaagg acctgggctt cgagacggag 31680

gtccaggagg ccgtcgcctc ccacgacctg gggccaccc cctacggacc ccggtacctc 31740

accgggggag tggtgcgcaa cgtcatcggc cggctgccgg gctccatccc cggacatgcc 31800

gtggcgctga tgacgcacta cgactcggtg tcccagggcc cgggggccag cgacgcgggc 31860

gtaccggtgg cagcgctgct ggaagcggcc cgcgcactgc ggaccgacgg cgtgcagccg 31920

gtcaacgacc tcctggtggt gttcaccgac ggcgaggaag cgggactgct cggcgcccgg 31980

gccttcttcg accggcaccc gctggcgaag acggtgggcg cggccttcaa cttcgaggcc 32040

cgcggcaccg agggccccgt cctgatgttc gaggccggcc cgggcaacgg ccccatgctg 32100

gaggaactgg cccgcaccgg cgtacccgtg ttcgccagct ccctcttcga cgccatctac 32160

cggcgcatgc ccaacgccac cgacttcgcc ctcgtcaagg aggggggat cccgggtctc 32220

aacttcgccc acatcggagg cttcgccgcc taccatggcc ccctcgacga catcgaccat 32280

gtcgaaccat cggccctcca gcaccagggc gaactcgcac tggcgctggc ccgccgcctc 32340

ggctccgccg acctggccga actcaccggc gaggacgcgg tcttcttccc cgccggacgc 32400

ggaaagctga tcagggtccc cggcagagcc gtcgtccccc tggccgtcgc ggcaggcctg 32460

ctgggcgcgg gcggcctcgg acacgccctg cgcaagggcg gcctgaccgg ccgccggctg 32520

gccggcacca gcgccctgct gctcggccgg atcgccgccg gcgcggccgc gggtacggcg 32580

ttcaccgccg ccatgggcaa actggcgccc gagttccggc ggcacggcga cttccacggc 32640

tccggcgaca tctacgccgc cgtcgccggg ctcgcctgcg ccgcggtcct cgccggtccc 32700

ggcgacgccc cggcggcggc cgcccggacc gccgcggcca cactcccgct ggccgccgcc 32760

tctgccgcgc tggcctcggg actgcccggt ggcagctacc tgacgacctg gccgctcgcc 32820

ggcgcggggc taggactgcg gctgctcgac tccgcgcagc cgacgggcac cggacgggtt 32880

ccgtccgtac gccacgcggt gggcgcggcc gccgctgccg ccccggccgc cgccctcctc 32940

gtcccgctgt cccgactcct cttcaagggg ctcaccccgc gcctggccgg caccgccgtc 33000

gtcgccctcc agctctgcgg cgaactggcc gcccccgcac tggccggact gccccggcgc 33060

acccgcaggg ccgccgccgc gggcggcctg ctgctcgccg ggggcgtggc cgcgcggcgc 33120

ttcctgcggc gcgggaaggg tgctcccccg ccggagacca tcagctacct cctggacccc 33180

gtgcactcca cggcactgtg gatcagcagc gacgaatcgg cctcggagcg gacccgggcc 33240

ttcctcgggg aacagccgga gaagggccgc ctgccggagc acttccccgg ctggcagcgg 33300

gacttcctgc acgcccccgc ccccaggctc gacctccccg caccggaagt gacggtgctg 33360

gaggacgggg ccggcgaggc cggccgacgc cggctgctgc tccacctgcg ttcaccgcgc 33420

ggcgcacgcc agctgtccct cgccgtcgtc ggcgcaggag tgcggcggtg gcgcgtcgag 33480

gacggcccgt ggtccgagcc gtccccggcc gaggacgaca gctggggagct gtggctccac 33540

gccgtgccgg acccgggcat ccgggtcgaa ctcgaacttc cgtcgtccgg aacccggctc 33600

cgggtcctcg accggatcga cggactgccg caggagctcg cggcggctcc gggcacagat 33660

cccgccaccg ggatcccgga gcacatcagc ccggcagccg ccctcgacgt ggacacctgg 33720

ggcaacgcct cactcgtcgt acggacggtg caccttccgg cccaggaagg gccggcatga 33780

caacgaaagg agtgacaggg ttggccactt tcctgggcga tgacgacctc gtcccctgcg 33840

cggtcgtcgt caacgacgcc gcccagtact cgatctggcc gcaggaccgt cccgtccccg 33900

ccggctggcg gccggccggg ttcgaaggcc cgcgcagcgc atgcctggaa cacatcgaga 33960

cggtatgggc cggtcccacc cccgcccccg ccgcctgacc gcacaccacc acacaaggag 34020

aaccagcacc atgaccatca gcctcgagaa caccaccgtc ggccagaacc ccgccggtgg 34080

ccccccgacc ggcaaggccc ccctggacat ggagggtctc gcctggatcc tcttcggcgc 34140

ctccgccttc cagtacctca acgccgcgtg cgagctcaac ctcttcgagc tgctggagaa 34200

caagcccggt ctgaccaagc cgcagatcgg cgccgaactc ggcctggccg accgcgccaa 34260

cgacatcctg ctgctgggcg ccaccgcgac cggcatgctc accgtcgagg acggccgcta 34320

ccagctcgcc acggtcctgg ccgagctcct caagacggac gactggcagc gcttcaagga 34380

cacggtcggc ttcgagcagt acgtctgcta cgagggccag atcgacttca ccgagtcgct 34440

gcgctccaac agcaacgtcg gcctgcgccg ggtccgcggc tcgggccgtg acctgtacca 34500

ccgcctgcac gagaacccgc agatggagca ggccttctac aagtacatgc ggtcctggtc 34560

ggagctggcc aaccagcacc tggtcgaggt cctggacctg tccggcacct ccaagctcct 34620

cgactgcggc ggcggcgacg ccgtcaactc gatcgcgctg gcccaggcca acccgcacat 34680

cgaggccggc atcctggaga tcccgccgac tgccccgctg accgagaaga agatcgccga 34740

ggccggcctc agcgaccgca tcacggtcaa gccgggcgac atgcacacgg acgagttccc 34800

caccggctac gacacggtga tgttcgccca ccagctcgtc atctggaccc cggaggagaa 34860

cacggcgctg ctgcgcaagg cgtacaacgc cctgcccgag ggcggccggg tgatcatctt 34920

caactcgatg tccaacgacg agggtgacgg cccggtcgtg gcggcgctcg acagcgtcta 34980

cttcgccgcg ctgcccgccg agggcggcat gatctactcc tgggccacct acgaggagtc 35040

cctgaccaag gccggcttca accccgagac cttccagcgg atcgacttcc cgggctggac 35100

cccccacggc gtcatcatcg ccaccaagta atccacggcc cgtgcagcgg cccggtcccg 35160

ccctctgggc ggaggccggg ccgctgcacg ggcttccgcc atgccgcacc gcggaggacc 35220

tgatgctgac cactccggac gaagacaagg tgaggatcgt ccgctggccg ctggaaacgg 35280

ccaagcggga gcgatgcagg gaagccggag ccctgcggat cctcctcgtc gagaacggcg 35340

cgaagccacc gctgacggcc gacctccggg aggactgggt cagggccccc gtcagcaggg 35400

aagacctcgc ggcccgggtc acggccctgc gcgccaaagc cggtgcctat cgggtcccca 35460

ccctcgacgt ggacggcgag ctgcgcttcg acggccgctc ggtcgtgctc tcccgcgcgg 35520

aggtcgccat cgtctccgta ctcgtgcgca attacggctc cctggtcgcc agggagtcgc 35580

tcgcgcacct gccggacgct ccccgagcgg gcagccgcaa cgcgctcgac ctgcacgtga 35640

tgcgcatccg ccggcgcatc gccccgctgg gcctgggcat cagcacggcc tggggccgcg 35700

gctacctgct ccaggagctg acgcaggaag cgcagggaca ggacgcggga ggaacctagg 35760

gccgccccct ccgtatccgg cccggcccgc gaccaggcgg aagcggcggg agcggaaacg 35820

gcggacgggg cgctcaccgt cggctgacgg ctcgcgcccc gctccgctcc gcggcccgct 35880

cttcgcgggc cggccctctt cacaccctcc cggccaccgg atccgctgac atggactccc 35940

ccagcaccgg ccagccgcgc tccgccagcc agtccgacag catggacgcc atgccggcgt 36000

cgaccgagag gacggcgagg ccgacggggg aatccgcgga gtcctcgatc cacagatcct 36060

cgacgttcac gttcaactcg ctcacatccg ccaacagccg ggccagttcc ccctcgcggt 36120

ccgtgaggag cacctggagc ttgtcgtacg cgcgtcctgc cgccccgtac ttctgcggga 36180

tcagcagctg tcccgcccgg ccctcctcca ggagccggcg gagcagctcg cggtaggccg 36240

gggcctcggc ggcctcggcg gcccccgggg aagcggcgag cgcgtgcagg atacgacggg 36300

tcatctccag gtcgtgctgc aggttttcca gggcgccgag caccgcggtg gcgttggatc 36360

cgaggatgtc ggtccacagg tcggggtcgc cggccgcgat cctggtgaag tcacgcagcc 36420

cggagccgga cagctccagc gcccggtggt cgtaggaccg cagcaggcgg gccatcagac 36480

tgctgacgag gtgcggcagg tgggaggtca tggcgacggc ccggtcgtgt gcctccggct 36540

gcatcgtcac cacgtccgcc ccgcacaggc cgatcagccg gcgggccgtg gcgatcgtct 36600

cctgcgaggt gtcagcgctg ggggtgagga tccacggctt ggccctgaac atgtcgctgc 36660

gggccgcgcc gggccccggg tgttcccggc cggcgatggg atgtcccccg atgaaccggg 36720

aggcgtccca gccgaggtcc gcccccgccg cggggagcgc cgccttgacg ctggccacgt 36780

cggtgtagtg gcgggcgatc cgccgtcgct gggcgtcagc gagagccgtt cgcacgctct 36840

gcggaggcac ggcgatgacc gccaggtccg tctggcgctc gggggcgagc gcggtgcccg 36900

cccccctggc ctcggcgatc cgcaccgatt ccgcatggac gtcctggagg tggacccgga 36960

caccgtactc ggtgagtgcc aggccgatgg aggtccgat caggccggtg ccgatcacgg 37020

tgacgctgat cagcgaaccg gcgtcttcgg tgtttccgat ggaggtcacc tggtggtccg 37080

tccgttccac ggccgtcagt agcggtagtg gtcgggcttg tagggccct cgacctggac 37140

gccgatgtag gcggcctgct ccgggcgcag ggtggtgagc ttgacgccga gcgcgtcgag 37200

gtggaggcgg gcgaccttct cgtcgaggtg cttggggagc acgtagacgt cggtcgggta 37260

ctcggacggc ttggtgaaga gctcgatctg ggccagggtc tggtccgcga aggagttcga 37320

catcacgaac gaggggtggc cggtcgcgtt gcccaggttg agcaggcggc cctcggagag 37380

cacgatcagg accttgccgt cggggaactt ccaggtgtgg acctgcggct tgacctcgtc 37440

cttgacgatg ccctcgatct ttgccaggcc ggccatgtcg atctcgttgt cgaagtggcc 37500

gatgttgccc acgatcgcct ggtgcttcat cttggccatg tccgaggcca tgatgatgtc 37560

cttgttgccg gtcgtggtga tgaagatgtc cgcgatctcc accacgtcgt ccagcgtggc 37620

gacctggtag ccgtccatcg ccgcctgcag cgcgcagatc gggtcgatct ccgtgacgat 37680

gacccgcgcg ccctgcccgc gcagcgactc cgcgcagccc ttgccgacgt cgccgtaacc 37740

gcacacgacc gcgaccttgc cgccgatcag cacgtcggtg gcgcggttga tgccgtcgat 37800

cagggagtgg cggcagccgt acttgttgtc gaacttcgac ttcgtcacgg cgtcgttcac 37860

gttgatcgcc gggaacagca gggtgccctc ggccatcatc tcgtagagac ggtggacacc 37920

ggtggtggtc tcctccgtca caccgcggat ctcggacgcg agctgcgtcc acttctgcgg 37980

ggtctcgccg agggtgcggt tgagcagcgt gaggatgtgg ccgtactcct cggagtccgc 38040

ggtggacggg tccggggccg cgcccgcctt ctcgaactcg acgcccttgt ggacgaggag 38100

ggtggcgtca ccaccgtcgt cgaggatcat gttcgggccg ccggtggggg tgttcggcca 38160

ggtcagcgcc tgctccgtgc accaccagta ctcctccagc gtctccccct tccaggcgaa 38220

gacggggacg ccctgcgggt tctccggggt gccgtccggg ccgaccgcga tggcggccgc 38280

ggcgtggtcc tgggtggaga agatgttgca ggaggcccag cggacatccg cgccgagcgc 38340

gacgagggtc tcgatgagga cggccgtctg gaccgtcatg tgcaggggtc cggtgatacg 38400

ggcgccggcc agcggctgcg ccgcggcgta ctccttgcgg atcgacatca ggcccggcat 38460

ctcgtgctcg gccagggtga tctccttgcg gccgaaggcg gcaagggaaa ggtcggcgac 38520

cttgaaatcg gcgggggaca ggttcgtcat cgttgctccg tgaggcggtg agggacggtt 38580

cagtggcggg ctgcggcggg ctgccgggca ggcgacagac cgaggttccg atggatctcg 38640

acggtcgcgg tggaccggtt gagggtgatg tagtgcaggc cgggcgcacc ttcggccaag 38700

aggcggtcgg ccatggcggt ggcgtggtcg acgccgatgc ggtggccctc ggaggggtcg 38760

tcgcgggcgg cgtcgagccg tcgggcgagt tcgtcgggga acgacgcgtt gctgagttcc 38820

gcgaagcggc ggatctggcg tacgtcggtg gcgggcatga tcgcggggat gatcggcgtg 38880

ttgcagccga cgaccgcgac cctgtcgcgc agccggaggt agtcgtcgac gcggaagaac 38940

atctgggtga tggcgtagtc ggcgccggcc cggcacttgg cgacgaagtg acggatgtcg 39000

ctgtcccagt ccggcgagcg tggatgccgc tcgggaaagg ccgccacgcc aaccgtgaaa 39060

tctcccagtt ccctcgtgag ggaaacgagt tcagcggcat gacggagccc gtcgggatgg 39120

gggacccact cgcccttggg atcgcccggc gggtcgcccc gcagagccag gacatcccgg 39180

atgccggcgt cggcgtactg gccgatgatg tggcgtagtt gggcgatcga gtggccgacg 39240

gcggtcaggt gcgcgaccgg gcgcagcgtg gtgtccgccg cgatccgctt ggtcacctcg 39300

accgtccggt cccgggagga acctccggcc ccgtacgtga cggagacgaa cgacggtgag 39360

aaggcctcga tgcggcggat cgccctccac agcgtccgct cgccggctgc cgtcttcgga 39420

gggaagaact cgaacgaata cgacttctgc cccgtgcgga gcaggtcccc caggcggccc 39480

ggacgggcgc ggtacgggaa ctccgcggtc tctgctgcca tggtggtcct tcgggattcg 39540

gtacggggga gccggatcag cgcgcgttga agtacgtggc gtccgggtgg tggatgacga 39600

tggcgtcggt ggactgctcg gggtggagct ggaactcctc cgagaggtgg acgccgatcc 39660

gctccggctg gaggaggtcg gcgatcttcg cgcggtcctc caggtcgggg caggcgccgt 39720

agccgagcga gaagcgggcg ccgcggtact tgaggtcgaa catgtcctcg accttctcgg 39780

ggtcctcgcc gccgaagccg agctcggcgc ggacgcgggc gtgccagtac tcggcgaggg 39840

cctcggcgag ctggacggag agcccgtgca gctccaggta ctcgcggtag gagtcggcct 39900

cgaacagctt ggccgtggcc tcgccgatcc tcgaaccgac ggtgaccacc tggaggccga 39960

tgacgtcggt ctcgccggac tcctccgggc ggaagaagtc ggcgaggcac agacgccggc 40020

cccggcgctg gcgggggaag gtgaagcggg tccgctcgtt gccctgctcg tcgaggatga 40080

tcaggtcctc gcccttggag acgcacggga agtagccgta gaccacggcc gcttccagca 40140

ggttctccgt gtggagcttc tccaggaggc cgcgcaggcg cggacggccc tcgctctcga 40200

cgagctcctc gtaggtggcc ccgccggcac gggcctgctt gaggccccac tggcccttga 40260

acagggcgcc ctcgtccagc caggaggcgt agtccttgag cgggatgccc ttgatcacgc 40320

gggtccccca gaagggcggg gtgggcaccg ggttgtcgac ggccacgtcg gagcggccgc 40380

cggtctcctc cggctcctcc acctggaggg ccggggtgtc ccgcttggcg acgcggcgct 40440

gcttgagggg cgggagttcg gcgccgggga ttccgcgctt gacggcgatg agcgcgtcca 40500

tcagccgcag accctcgaac gcgtcccggg cgtagcggac ctcgccttcg tagatctcgt 40560

ggaggtcctg ctccacgtag gcgcgggtga gggcggcgcc gccgaggatg accgggtagt 40620

cggctgccag cttgcgctgg ttgagctcct ccaggttctc cttcatgatc accgtcgact 40680

tgacgaggag tccggacatg ccgatcacgt cggccttgtg ttcctgcgcg gcctccagga 40740

tcgcggagac cggctgcttg atgccgatgt tcacgacgtt gtagccgttg ttggtgagga 40800

tgatgtcgac gaggttcttg ccgatgtcgt ggacgtcacc gcggacggtg gccaggacga 40860

tggtgccctt gccgtcggcg tcggtcttct ccatgtgcgg ttcgagatag gccaccgccg 40920

tcttcatcac ctcggccgac tgcaggacga acggcagctg catctgcccg gaaccgaaca 40980

gctcaccgac gaccttcatg ccctccagga gggtgtcgtt cacgatgtcc agcgccggac 41040

gggatgcgag ggcctcgtcc aggtcggcct ccaggccgtt cttctccccg tcgatgatcc 41100

gccgctggag ccgctcgtcc agcggcagcg cgaggagctc ctcggcacgg cccgccttca 41160

acgacttcgt gttgacgccc tcgaagaggg ccatcagctt ctgcaggggg tcgtagtcgc 41220

cctcacgccg gtcgtagatc agatcgagtg cggtgctgac ctgctcctcg tcgaaccggg 41280

cgatcggcag gatcttcgac gcgtgcacga tcgccgaatc cagacccgcc ttcacacact 41340

cgtccaggaa aaccgagttc agcagcacac gggcggccgg gttcaacccg aaggagatgt 41400

tcgacagacc cagcgtcgtc tgcacctccg ggtgacggcg cttgagctcc cggatcgact 41460

cgatcgtcgc gatcccgtcc ttgcgggact cctcctgacc ggtgcagatc gtgaacgtca 41520

gcgtgtcgat caggatgtcc gactcccgga tcccccagtt actggtgagg tcctcgatca 41580

gccgctcagc gatggccacc ttgtgctcga ccgagcgggc ctggccctcc tcgtcgatcg 41640

tcagcgcgat cagcgcagca ccatgctcct gcgccagccg ggtcaccttc gcgaaacggg 41700

actccggccc gtcaccgtcc tcgtagttga cggagttgat gaccgcacgc ccgcccagct 41760

tctccagacc cgcctggatc accgggacct cggtcgagtc cagcacgatg ggaagggtgg 41820

aggcggtggc gaagcggccg gcgagttcct gcatgtccgc gacaccgtca cggccacgt 41880

agtccacgca caaatccagc atgtgcgcgc cctcacggat ctggtcccgg gccatctcca 41940

cacagtcgtc ccagcgggcc tccagcatcg cctcacggaa cttcttcgac ccgttcgcgt 42000

tcgtccgctc accgatcgcc atgtacgagg tgtcctgccg gaacggcacc gtctgataca 42060

gcgacgccgc acccggctcc ggctgcggcg accgctcggt gaccgccgtg ccccggaccc 42120

gctccacgac ctgccgcagg tgctccggcg tcgtaccgca gcagccacccc accagcgaca 42180

gcccatactc ccggacgaac gtctcctgcg cgtccgccag ctcggccgcc gacagcggat 42240

agtgcgcccc ctgcttcgtc agcaccggca gacccgcgtt cggcatgcac gacagcggaa 42300

tccgcgcatt gcgggccaga tagcggaggt gctcgctcat ctccgccgga cccgtcgcac 42360

agttcagacc gatcatgtcg atccccagcg gctccaacgc cgtcagcgcc gcaccgatct 42420

ccgaaccgag cagcatcgtg ccggtcgcct ccaccgtcac cgagcagatg accggcatgt 42480

cttccccgca gttgctcaac gcccggcggg cgccgatgat cgcggccttc gtctgcagga 42540

ggtcctgggt cgtctccacc agcagggcgt cggcgccgcc ggagatcagg ccctcggcgt 42600

tgacctggta ggcgtcgcgg atctgggcgt agtcgatgtg gcccagcgtc ggcagcttcg 42660

tgcctgggcc catcgacccc agcacccagc gctgccgccc ggtcgaggcc gtgaactcgt 42720

cggcgacctg gcgggcgatg cgggcgccgg cctcggacag ctcgaagttg cgttcgggga 42780

tgtcgtactc ggccagcgcc gcgaagttcg cgccgaacgt gttggtctcc acgcagtcga 42840

cgcccacggc gaagtactcg cggtgcacgt tggccacgat gtcgggccgc gtcacgttca 42900

agacctcgtt gcagccctcc agctgctgga agtcctccat cgacgggtcc tgcgcctgca 42960

gcatcgtccc catcgccccg tcggcgacga ccacgcgggt cgcgagcgcc tctcgcaggg 43020

cgtcacttcg tgcgctcacg gcttggcctg gaggtggggc gtgatctcgg cggccggtcc 43080

gtcaccgtac gcgtccgcga tccgggccgt gagggacgac cggtccagcc ggtattcctg 43140

cgtgcctacc gactccagga cggttgtggc cagcgcgcag cccagctggg ctgcgcgctc 43200

ctgcgacagc ccctggccga tccccgccag gtacccggcg cggaacccgt cgccgacgcc 43260

cgtcgggtcg gcgacgttct tggcggggac ggccggcacg ctgacggacg gggtgtcggt 43320

gttgtggatc gtgacgccgt caccgccgca ggtgatgatc caggtgccca cccgctggag 43380

gacctggtgt gcggtccatc cggtgcactc ctggagcagg gcggcctcgt actcattggt 43440

gaacagcaac tcggcgccgt ccaccagacg ccgggtctcg tcgccattca gccgcgcgag 43500

ctgctgcgac ggatccgcag cgaggggaat gcccagttca cgggtcttcg ccgagtgccg 43560

cagcatcgcc tcggggtcgt ccggcgcgac gacgacccgg tcctgcggtc ccgaacggcg 43620

caccacatcg gccaggtcga tctcgcgggc ctcgaccatg gcaccggtgt agaaggaggc 43680

gatctggttc tggtccgcgt ccgtggtgca catgaaacgg gccgtgtgcc gtgtggcgct 43740

caccctcacg gagccggtgt cgtgttcgcg aagccaggag tcgtactcgg ggaagtcggt 43800

gccggcggcg ccgaccagca acggggagaa tcccagacgt cccaggccga atgcgatgtt 43860

cgccgcgacg ccgccgcggc gcacctccag gtcgtccacc aggaaggaca acgagatcgt 43920

gtccagccgg tcgggtatca gctgctcggc gaaacggccg gggaaggtcg tcagatggtc 43980

ggtggcgatg gatccggtga tcgcgatgcg catgtcagac ccccgcgacc ctgcgcaggg 44040

cgtcggcgcg gtcggtgcgt tcccacgtga agtcggtcag ctcgcggccg aagtggccgt 44100

acgcggcggt ctgcgcgtag atgggccgca gcaggtccag gtcgcggatg atggcggcgg 44160

ggcgcaggtc gaagacctga ccgatggcct cttcgatctt cgaggtgtcg accttggcgg 44220

tgccgaaggt ctccacgaac aggccgacgg gctcggcctt gccgatcgcg tacgcgacct 44280

gcacctcgca gcgcgaggcg agcccggcgg cgaccacgtt cttcgcgacc cagcgcatcg 44340

cgtacgcggc cgaacggtcg accttggacg ggtccttgcc ggagaacgcg ccaccaccgt 44400

gacgggccat cccgccgtac gtgtcgatga tgatcttgcg gccggtcaga cccgcgtcgc 44460

ccatcgggcc gccgatctcg aatcggccgg tcgggttcac cagcagccgg tagccgtcgg 44520

tgtcgagctt gatcccctcc tggacgagct gcttcaggac gtgctcgacc acgtgctcgc 44580

ggacgtcgag ggtgaggagg gcgtccaggt cgacgtccgc ggcgtgctgg gtggagacga 44640

cgacggtgtc gaggcgcaca gccctgtcac cgtcgtactc gatcgtgacc tgcgtcttgc 44700

cgtcgggacg caggtagggg atggtgccgt ccttgcggac ctcggacagc cggcgggaga 44760

gccggtgggc gaggtggatc ggcagcggca tcagctcggg cgtctcgtcg caggcgtacc 44820

cgaacatcag gccctggtcg ccggcgccct gcttgtccag ctcgtcgccc tcgccctcga 44880

cgcgctgctc gtaggcggtg tccacgccct gggcgatgtc gggcgactgc gcaccgatcg 44940

agatcgacac gccacaggaa gcgccgtcga agcccttggc ggaggagtcg tagccgatgt 45000

cgagaatcgc gtcgcggacg agctgcgcta tcggcgcgta cgccttcgtc gtcacctcgc 45060

cggcgacatg cacctggccg gtggtgatca gggtctcgac cgcgaccctg ctgctcgggt 45120

cctcgcgcag cagggcgtcg aggatggtgt cgctgatccg gtcggcgatc ttgtcggggt 45180

gaccttcggt caccgattca gacgtgaaga gacggcccat gacgagttcc tccaggattc 45240

tcaggcggcg cggaccgcac gaatgacgtt catgccactg ggcagggagt ccaacacgggt 45300

aatgcggaac ccggccttgc tcagcaggac ttcgacctgc gccgcagtcc gttcctggcc 45360

gccgttgccg ccgaacaggg agagcatgta gaggtccatc agggccgcgg tgtgcagccg 45420

ctcgtcccgc tgtccgcggt tcgcggcgat caggtcgacg accatcagcg tcgcgtcgtc 45480

ggacatggcg gcgcgacagg tgcgcaggat ctgcaccgct cgatcgtcgt cccagtcatg 45540

caggatgtgc gagagcagat agaggtcgcc ccccctcggg gaggctgtcg aagaagtccc 45600

ccgttgtcac gggagtagcg gccctccagc ggacccgcgg tggggcgctc cgcctggagg 45660

gacgtcgagc cgggcagatc gaagacgatg ccgctgatgt cgggatgtgc tgacaggatc 45720

ctgcctgcag gtcgactcta gaggatccgg cgaagaaacg ttccgtcgct gccgcctacg 45780

tccacgaccg tgggataagc ggaaaagtcg atcgcgcgca ggatctcgtc cagctccaga 45840

gccagtccgc ggccctggga gtcgtcgaag accgcgcgaa gctcctcgtc ctgctccatg 45900

tgggtgaaga gcgagacgcc tcggctgcgc tcgaacggcg actgtcccgt ccgcacggtc 45960

tccagcaagt cgccccaggc ggcaccgaac tgccctgcca ccagcgtggc cgacggaagt 46020

gcggaagccg ggtgcccgga ggtgagggcc tcaccgagcg gagtcagcga gtagacctgt 46080

tcggcgccct cctcgaacac gccgaacacg gcgagaccac gcagcaggcg ggcgagtgcc 46140

acggcgtccg tgctggtccg ctccgcgagg gtcgagatgt ccgcaggccc ctccgcgagg 46200

atgtccgcga tccccaactg cgcgacgacg accagcgcac gagaaggaag ctgggcatac 46260

agcatccgtc tcatgacggc agccgcctcc cgctgttccg cgctcaactc gtacacggtc 46320

gcctcctgac tggtctcctc gcgccaccgg gatggagacg ccccacgatg gtgaccgtcg 46380

gcggaccggc ccttccgcca cgggccagat cccgaacaga tttccccgga ccccgctcgc 46440

gccggtccga gaagccccca ccaccccggtt ccggctccac cgcgcttccg tcaggactcg 46500

gtcatccgtt gagcggccct accacactc atggcggccc ggcaccctgt cggccgctga 46560

accgagagggg gggaggaacc ggtgacggag aacccgcgac gcccgcccct ggtggtgggg 46620

gcgagcgccg cgggcctgac ggtcgcccac gaactggccc ggcgtggact gcccgtgcgc 46680

gtcatcgaag cctcagccgg accggccacg acagggcccg cggtgacact ccagccgcgt 46740

accctggaag tgctcgacca gatggcggtc atcgacggct tcctccgcca cggacgcaag 46800

ggcgagaccc tcgccgacca cacgacgctg cccacccttc acccgtacac gctcgtcatc 46860

gaccgggccc gggccgcggc cctgctccgc gaagccgtcg tcgacctcgg cgtaagcgtc 46920

gaggggggcg cgcgggaccg gcaccctgcc gccccggctt gtctgatcac ctgcgacggc 46980

ttcccgcagc gctcggatcc ggtcggccgc cgcctcaccg tcgtggcgga ccacgacccc 47040

gacaccggga tccggcaggc ttacaacctc gcctggaaac tggcgatggt cgagcagggc 47100

ctcgtgggtc cgcgcttgct ggacacctac gacgaggagc atcctgcgga gcgaggccac 47160

cggccgggga tccgtgcact cctcaacgct gtccccgctc tccggcgcac tcatccaagc 47220

caccgctcag ggctggggct cacctaccgg aacagctccc tgaccacgcg gaacaccctc 47280

gacttcctct ccggttacca cgtcggcgcg gtgggattcg aaccgactcc cgtccccgcg 47340

cccggttccc gcgtcactga ggccgggccg gcctgcaacc tgctgcgctc cgagctgagg 47400

gacccacgat ggtccctgct gttcgcgacg gacccagccg gcacccaggg ctcaccggac 47460

tccgtggccg ccaccgccgc tgcccagtac agcgcctact tgtcggtccg catcctggga 47520

gccgcttccc tccccgaccc cctctgccga ctgcgcgccg cgctgggcac gcctccgggc 47580

ggctggctcc tgatccggcc ggacggctac gtggctgcac gcggcagcct cctgaccagc 47640

caagccctcg accgggcact gtccccgctc tccgtcgccc ctctaagaat cggagccaac 47700

ccatgtggct gatgtggttc ttccgccgtc tcgtgacaac ctccgcggac gtacgcccct 47760

accatcacga gcagccccgg caccgccgcc gccacctgcg gcgggccact gccggccgac 47820

gcgactgacg gcctgcccgg tcatgtttca acgcacacga gccgagtgag ccatgaccgg 47880

ccggaagaac gcccccgaac cggcgcggtt gtcgggacga cccgcagacg ccgctgcggc 47940

tgcggcgact gacgggctgc gcagagacgg cggtcgaagg cgttgaggga agtctccgcg 48000

gcagccacat gccggccgga cacggggtcg catcggcacg cccttgagcc ggattccgct 48060

cgttctcacc tcagaccatg cacagtcgat gccctgccac cagactcagc gccacggtca 48120

caccgatcgg caccacgacg tcgagcgtgc tggacgccca tgcccaagcc gcccgcgacg 48180

gcctgcggga gaagcagtcc ccgcactatg tgattctgac gctctccggt gacgggtcgt 48240

cgatctcagt ggagcaacag ggaccggggag accgctacga gttcctcgac aaactggccg 48300

ggaggagctg ccgctacggc ctgtgtaccg tgccctccaa gatgggcgaa acacaagtcg 48360

tcttcgtccg ctggacccct gtggacgcgc ccgccgaaga aaagacgctc tatgactcct 48420

acgaggagag cgcccggcaa gcgctcactg ctccccagga cgtggctccg cgcaccgacc 48480

gctccgaacc gcaggacctt cgcgggcgcc gaacacctga ggcaacccga gggcggtgaa 48540

aggcccagga ccctgaccgc cacacggccc gtctgagcgt gaggtctcgc ccggccaccc 48600

gaccacctgg accatgcctt cgccccgaac cacaagggga tgccggtgcg cgacggagcc 48660

cccgagcccg accgggcacc gcccactggg tgatgagcgg cgtgtcggtt catgctgccg 48720

ccaggctttg gtgaggcgga cgacgcgagc gggggtggcg atgccccgca cggtcgcgag 48780

cttgccgtcc gagatgtcga acgtcacggc gccgacgacc ctgccgtcgg gcacgaaaag 48840

aatggcgagt gcgtagtgga cagcggagac gccgccgagc cgctgcttcg cgggagttga 48900

cctgaagccg gcccgtgcga tggcggcgat gcgctgagga gtgtcgcacc gcagcagctt 48960

ctcggtcagg ccggctccgt cggagatcgc ggtcgcgtcg tcggtgagca gtgccaccag 49020

gtgttcggtc ttccccgctg ccgccgcgcc gcgccccgac gatgcgctgc cgggcccggt 49080

ggaggtgctg ctggcttgcg gactcggtga tagcgaggat ctcgacgatc tcggcgcggg 49140

tgcaggagaa cgcttcgcgc aggacggtgc gttcgcggcg ggcctgtgcc gagcggagct 49200

tgacgaggca cagtgaccag cgtcgaagac cactgttctc ccgacccgcg aggccgggcc 49260

actggccgcc tcgccgtcga gacctgcgtt gccgcacctc gcgactgcct gcgcaaccag 49320

tcgcccaatg gcgcgctggc ggagcccacc cgactcggat cc 49362

<210>2

<211>195

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Asp Ile Leu Arg Ala Ala Ala Asp Val Ile Gly Arg Val Gly Pro

1 5 10 15

Ala Gly Leu Thr Leu Ala Ala Val Ala Gly Glu Val Gly Leu Val Pro

20 25 30

Gly Thr Leu Leu Gln Arg Phe Gly Ser Lys Arg Gly Leu Met Leu Ala

35 40 45

Ile Ala Arg Glu Ser Ala Lys Glu Ala Glu Ser Val Tyr Glu Ser Met

50 55 60

Arg Asp Glu His Ala Thr Ala Leu Met Ala Val Thr Asp Leu Ala Val

65 70 75 80

Thr Ser Met Gly Val Met Thr Lys Pro Glu Val Tyr Ala Asn His Leu

85 90 95

Ala Phe Leu Cys Ile Asp Leu Thr Asp Pro Glu Phe His Ala His Ala

100 105 110

Leu Thr Ile His Gln Ala Gln Arg Arg Val Tyr Glu Ala Leu Leu Thr

115 120 125

Glu Ala Val Asp Asn Gly Glu Leu Leu Ala Gly Thr Asn Val Ala Ala

130 135 140

Leu Ala Thr Thr Leu Gln Ala Val Val Ala Gly Ala Gly Leu Thr Trp

145 150 155 160

Ala Leu Asp Arg Glu Gly Thr Leu Pro His Arg Leu Glu Arg Glu Val

165 170 175

Ile Thr Ala Leu Ala Pro His Ile Thr Pro Gln Gly Asp Ser Ala Gly

180 185 190

Glu Gly Ser

195

<210>3

<211>304

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Thr Asp Gly Val Arg Thr Leu Ala Gly Lys Val Ala Leu Val Ala

1 5 10 15

Gly Gly Thr Arg Gly Gly Gly Arg Gly Ile Ala Val Glu Leu Gly Ala

20 25 30

Ala Gly Ala Thr Val Tyr Val Ser Gly Arg Ser Ser Ser Ala Asn Gly

35 40 45

Ser Ser Asp Met Gly Arg Pro Glu Thr Ile Glu Glu Thr Ala Arg Ala

50 55 60

Val Thr Ala Ala Gly Gly Val Gly Ile Pro Val Arg Thr Asp His Ser

65 70 75 80

Ser Pro Glu Glu Val Arg Ala Leu Val Asp Arg Ile Asp Ala Glu Gln

85 90 95

Gln Gly Arg Leu Asp Val Leu Val Asn Cys Val Trp Gly Gly Asp Arg

100 105 110

Leu Thr Asp Trp Asn Arg Pro Leu Trp Gln Gln Asp Leu Asp Lys Gly

115 120 125

Leu Arg Leu Leu Arg Gln Ala Val Glu Thr His Val Ile Thr Ser Arg

130 135 140

Tyr Ala Val Arg Leu Met Ala Ala Arg Arg Ser Gly Leu Val Val Glu

145 150 155 160

Val Thr Asp Gly Asn Thr Ala Arg Tyr Arg Gly Ser Phe Phe Tyr Asp

165 170 175

Val Ala Lys Ser Thr Val Ile Arg Leu Ala Phe Ala Gln Ala Ala Asp

180 185 190

Leu Lys Glu His Gly Val Ala Ala Val Ala Ile Thr Pro Gly Phe Leu

195 200 205

Arg Ser Glu Ala Met Leu Glu His Phe Gly Val Thr Glu Asp Asn Trp

210 215 220

Arg Asp Gly Val Ala Arg Asp Pro Asp Phe Ala His Ser Glu Thr Pro

225 230 235 240

Ala Tyr Leu Gly Arg Ala Val Ala Ala Leu Ala Ala Asp Pro Asp Ile

245 250 255

Met Ala Lys Thr Gly Arg Ala Leu Ala Thr Trp Gly Leu Tyr Lys Glu

260 265 270

Tyr Gly Phe Thr Asp Ile Asp Gly Ser Gln Pro Asp Phe Ala Ala His

275 280 285

Trp Ala Arg Thr Leu Glu Pro Arg Leu Gly Pro Leu Gly Thr Pro Leu

290 295 300

<210>4

<211>169

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Leu Asn Ala Ile Glu Ser Lys Val Glu Leu Ile Arg Trp Pro Val

1 5 10 15

Asp Ala Ala Arg Arg Ala Ala Cys Arg Asp Gln Gly Val Met Arg Leu

20 25 30

Leu Val Leu Glu Ala Gly Val Glu Ala Pro Val Cys Thr Asp Val Arg

35 40 45

Glu Asp Trp Val Arg Thr Pro Val Ser Arg Ser Asp Leu Arg Ala Arg

50 55 60

Ile Asp Ala Leu Arg Val Lys Gly Ser Ala Asp Phe Leu Pro Thr Val

65 70 75 80

Asp Pro Asn Gly Val Leu Arg Tyr Arg Arg Met Ser Val Val Leu Ser

85 90 95

Pro Thr Glu Thr Asp Leu Val Asn Arg Leu Ala Glu Ser Phe Ser His

100 105 110

Val Val Pro Arg Glu Val Leu Leu Glu Ala Leu Ser Arg Thr Ser Gln

115 120 125

Pro Ser Arg Asn Ala Leu Asp Leu His Ile Met Arg Ile Arg Arg Arg

130 135 140

Val Lys Pro Leu Asp Leu Ala Leu Arg Thr Val Trp Gly Arg Gly Tyr

145 150 155 160

Val Met Glu Ser Ala Asp Ala Ala Ser

165

<210>5

<211>512

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400> 1

Met Leu Glu Arg Arg Ala Val Leu Arg Met Ser Ala Gly Ala Ala Ala

1 5 10 15

Val Ala Val Gly Gly Ser Met Val Ala Gly Val Ala Ser Ala Ala Ala

20 25 30

Asp Thr Gly Val Arg Arg Ser Ala Gly Val Asp Trp Ser Arg Leu Ser

35 40 45

Ser Arg Leu Ser Gly Asp Leu Val Leu Pro Ala Asp Ala Arg Tyr Glu

50 55 60

Gln Ala Ser Arg Leu Ala Ile Gly Gln Phe Asp Ala Ile Arg Pro Gln

65 70 75 80

Ala Val Ala Tyr Cys Gln Ser Glu Gln Asp Val Arg Thr Ala Leu Ala

85 90 95

Phe Ala Gln Asp Asn Ser Leu Arg Leu Val Pro Arg Ser Gly Gly His

100 105 110

Ser Phe Gly Gly Tyr Ser Thr Thr Thr Gly Leu Val Leu Asp Val Ser

115 120 125

Arg Leu Asn Ser Val Arg Leu Thr Ala Asp Thr Val Val Val Gly Pro

130 135 140

Gly Leu Gln Gln Val Asp Ala Leu Thr Gln Leu Ala Pro Arg Gly Val

145 150 155 160

Gln Val Val Ser Gly Leu Cys Pro Gly Val Cys Pro Gly Gly Phe Ile

165 170 175

Gln Gly Gly Gly Leu Gly Trp Gln Thr Arg Lys Phe Gly Met Ala Leu

180 185 190

Asp Arg Leu Val Ser Ala Arg Val Val Val Leu Ala Asp Gly Arg Ala Val

195 200 205

Thr Ala Ser Ala Lys Glu Asn Cys Asp Leu Phe Trp Ala Leu Arg Gly

210 215 220

Gly Gly Gly Gly Asn Phe Gly Val Val Thr Ser Phe Glu Val Glu Pro

225 230 235 240

Thr His Val Pro Ser Ile Val Asn Phe Asn Leu Thr Trp Pro Trp Glu

245 250 255

Ala Ala Gln Lys Val Ile Ala Ala Trp Gln Arg Trp Val Ile Gly Gly

260 265 270

Ser Arg Asp Leu Gly Ala Gly Leu Gly Val Gln Trp Leu Asp Ala Gly

275 280 285

Thr Gly Arg Pro Glu Leu Leu Val Tyr Gly Gly Trp Leu Gly Arg Pro

290 295 300

Asp Ala Phe Thr Ala Val Leu Asp Ala Phe Val Arg Asp Ala Gly Ser

305 310 315 320

Ala Pro Leu Thr Arg Ser Val Glu Glu Gln Pro Tyr Gln Lys Ala Met

325 330 335

Met Ser Tyr Tyr Gly Cys Ala Asp Leu Thr Pro Asp Gln Cys His Thr

340 345 350

Val Gly Tyr Ser Pro Glu Ala Ala Val Pro Arg Thr Asn Phe Ala Ile

355 360 365

Asp Arg Ser Arg Leu Phe Ser Lys Ala Ile Pro Asp Ser Gly Ile Glu

370 375 380

Lys Ala Leu Glu Ala Phe Val Ala Asn Pro Arg Ala Gly Gln Phe Arg

385 390 395 400

Phe Leu Ser Phe Phe Ala Leu Gly Gly Ala Ala Lys Glu Pro Gly Arg

405 410 415

Thr Asp Thr Ala Phe Val His Arg Asp Thr Glu Phe Tyr Ala Gly Tyr

420 425 430

Ser Leu Gly Leu Asn Glu Ala Lys Tyr Thr Ala Glu Asp Glu Ala Ala

435 440 445

Gly Arg Ala Trp Ala Glu Ala Gly Phe Arg Ala Ile Asp Pro Phe Ser

450 455 460

Asn Arg Glu Ser Tyr Gln Asn Phe Ile Asp Pro Ser Leu Thr Asp Trp

465 470 475 480

Lys Thr Ala Tyr Tyr Gly Glu Asn Tyr Ala Arg Leu Leu Thr Val Lys

485 490 495

Arg Lys Tyr Asp Arg His Gln Val Phe Ser Phe Ala Gln Gly Ile Ala

500 505 510

<210>6

<211>479

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Val Ala Glu Gln Gly Ala Pro Ser Ala Lys Ala Gly Lys Thr Leu

1 5 10 15

Leu Thr Leu Leu Cys Leu Ala Gln Phe Met Leu Ile Leu Asp Val Ala

20 25 30

Val Val Ala Val Ala Val Pro Ser Met Gln Ser Gln Leu Asn Ile Ser

35 40 45

Ala Ala Asn Ile Gln Trp Val Ser Thr Ala Tyr Gly Leu Ala Phe Gly

50 55 60

Gly Phe Leu Val Ala Ser Gly Arg Ala Ala Asp Leu Phe Gly Pro Lys

65 70 75 80

Arg Ile Leu Leu Ile Gly Leu Ser Leu Phe Thr Leu Ala Ser Gly Leu

85 90 95

Cys Gly Ile Ala Pro Asn Glu Leu Thr Leu Phe Val Ala Arg Ala Val

100 105 110

Gln Gly Phe Gly Ala Ala Leu Val Ser Pro Ala Ala Leu Ser Leu Val

115 120 125

Thr Thr Ser Phe Gly Glu Gly Glu Glu Arg Asn Lys Ala Leu Gly Leu

130 135 140

Trp Gly Ala Val Ser Ser Gly Gly Ala Ile Ala Gly Gln Leu Leu Gly

145 150 155 160

Gly Val Leu Ala Asp Leu Ala Gly Trp Arg Ser Ile Phe Leu Ile Asn

165 170 175

Val Pro Leu Gly Leu Ile Val Ile Val Ala Val Ala Arg Met Val Arg

180 185 190

Lys Asp Arg Ser Asn Asp Thr Gly Arg Ile Asp Met Ala Gly Ala Gly

195 200 205

Leu Leu Thr Ala Gly Val Val Leu Leu Val Thr Ala Val Ser Gln Ala

210 215 220

Ala Glu Arg Gly Val Gly Leu Pro Val Leu Ile Ser Gly Val Leu Gly

225 230 235 240

Ala Val Leu Leu Val Phe Phe Val Val His Glu Gly Arg Val Gln Asn

245 250 255

Pro Val Leu Arg Leu Ser Met Phe Arg Asn Pro Asn Val Arg Tyr Gly

260 265 270

Asn Met Ile Cys Met Leu Thr Ala Gly Gly Ala Thr Val Ser Val Phe

275 280 285

Leu Gly Thr Leu Tyr Met Gln Gln Val Ile Gly Met Ser Pro Met Met

290 295 300

Ala Gly Leu Gly Phe Ala Pro Val Thr Leu Leu Ile Leu Gly Val Ser

305 310 315 320

Met Arg Met Gly Pro Leu Val Gln Lys Phe Gly Val Lys Asn Leu Leu

325 330 335

Leu Ala Ser Ala Ala Leu Phe Ala Val Gly Ser Leu Leu Leu Ser Phe

340 345 350

Val Thr Val Asp Gly Ser Tyr Trp Val Gln Val Phe Pro Gly Leu Ala

355 360 365

Ile Gly Gly Val Gly Ala Ala Leu Ser Phe Ala Pro Ser Met Ile Leu

370 375 380

Cys Thr Thr Gly Val Pro Asp Glu Glu Gln Gly Leu Ala Ser Gly Leu

385 390 395 400

Leu Gly Thr Ser Gln Gln Ile Gly Ala Ala Leu Phe Leu Ala Ile Leu

405 410 415

Ser Gly Val Thr Ala Ala Val Ala Ala Ser Ser Gly Gly Asp Thr Pro

420 425 430

Glu Ala Leu Thr Glu Gly Phe Gln Ala Gly Phe Leu Trp Ser Leu Ala

435 440 445

Ile Pro Ala Leu Ile Val Leu Cys Leu Leu Ala Leu Pro Arg Lys Lys

450 455 460

Asp Glu Pro Gln Val Glu Pro Ala Ala Glu Gln Thr Glu Thr Val

465 470 475

<210>7

<211>819

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Pro Glu Ser Pro Phe Pro His Ser Ser Thr Glu Trp Ile Arg Val

1 5 10 15

Val Arg Ala Arg Thr His Asn Leu Lys Asp Val Thr Val Asp Ile Pro

20 25 30

Arg Gly Gly Ile Val Ala Phe Thr Gly Val Ser Gly Ser Gly Lys Thr

35 40 45

Ser Leu Ala Ile Asp Thr Val His Ala Glu Ala Gln Leu Arg Tyr Leu

50 55 60

Glu Gly Leu Ser Pro Phe Val Arg Gln Tyr Ile Thr Pro Lys Asp Arg

65 70 75 80

Pro Gln Val Asp Arg Ile Asp Gly Leu Gly Ala Thr Leu Ala Val Asp

85 90 95

Gln Arg Arg Leu Asn Arg His Ser Arg Ser Thr Val Ala Thr Met Thr

100 105 110

Gly Val Asp Ala Tyr Leu Gly Leu Leu Tyr Ser Arg Leu Pro Ala Val

115 120 125

Ala Gly Glu Gly Pro Glu Leu Ser Ser Gly His Phe Ser Arg Tyr Ser

130 135 140

Pro Glu Gly Gly Cys Pro Val Cys His Gly Ala Gly Gly Thr Pro Arg

145 150 155 160

Ala Asp Ala Asp Leu Ile Ile Thr His Pro Gly Leu Pro Leu Gln Glu

165 170 175

Gly Gly Ser Pro Trp Phe Asp Lys Leu Arg Ser Pro Glu Gln Val Ala

180 185 190

Val Pro Phe Leu Ala Glu Arg His Gly Ala Asp Leu Ser Leu Pro Trp

195 200 205

Arg Glu Leu Pro Gln Ser Phe Arg Asp Ala Leu Leu Tyr Gly Thr Gly

210 215 220

Thr Glu Thr Val Ser Val Ser Val Gln Val Pro Asn Lys Asn Ser Asp

225 230 235 240

Ala Glu Val Leu Tyr Gln Leu Asn Gln Pro Leu Arg Gly Ala Leu Ala

245 250 255

Glu Val Glu Arg Leu Phe Ala Ala Ala Gly Thr Glu Asn Ala Lys Gln

260 265 270

Arg Tyr Leu Pro Tyr Met Arg Gln Ile Pro Cys Ala Gln Cys Gly Gly

275 280 285

Ser Gly Tyr Gly Glu Ala Ala Arg Thr Val Ala Leu Ala Gly Val Thr

290 295 300

Tyr Gln Gln Leu Ser Ala Leu Arg Val His Glu Val Gln Lys Trp Ala

305 310 315 320

Ala Glu Leu Gly Gly Val Leu Ser Gly Val Gln Arg Glu Ile Gly Glu

325 330 335

Ala Leu Leu Pro Glu Val Thr Asn Arg Leu Arg Leu Leu Asp Arg Leu

340 345 350

Gly Leu Ala His Leu Glu Leu Gly Arg Thr Ala Pro Thr Leu Ser Gly

355 360 365

Gly Glu Leu Gln Arg Ala Arg Thr Ala Ala Gln Leu Ser Thr Ser Leu

370 375 380

Thr Gly Ile Val Phe Val Leu Asp Glu Leu Gly Ser Gly Leu His Pro

385 390 395 400

Ala Asp Lys Gln Lys Leu Arg Asp Ile Leu Glu Asp Leu Arg Asp Ser

405 410 415

Gly Asn Thr Val Leu Leu Val Glu His Asp Pro Asp Val Val Ala Gln

420 425 430

Ala Asp Trp Val Ile Asp Leu Gly Pro Gly Ala Gly Ser Gly Gly Gly

435 440 445

Arg Val Met Phe Ser Gly Pro Pro Gln Glu Leu Ala Ala His Pro Asp

450 455 460

Ser Val Thr Gly Arg Tyr Leu Asp Arg Ser Arg Pro Arg Ile Asn Arg

465 470 475 480

Ala Arg Ser Gly Gly Gly Ser Gly Arg Trp Leu Thr Phe Lys Asp Val

485 490 495

Arg Ala His Thr Val Arg Val Asp Glu Leu Arg Val Pro Leu Asn Thr

500 505 510

Leu Thr Cys Phe Thr Gly Val Ser Gly Ser Gly Lys Ser Ser Leu Leu

515 520 525

Gly His Ala Val Ala Pro Ala Leu Thr Ala Ala Leu Ala Gly Ser Gly

530 535 540

His Pro Ala Val Gly Ala Val Thr Gly Ile Glu Ser Val Ala Trp Val

545 550 555 560

Asp Cys Val Asp Gln Ser Pro Ile Gly Arg Thr Pro Arg Ser Asn Pro

565 570 575

Ala Thr Tyr Thr Lys Ala Phe Asp Ala Val Arg Lys Leu Tyr Ala Ala

580 585 590

Thr Asp Arg Ala Arg Ala Leu Gly Leu Gly Ala Ser Ala Phe Ser Phe

595 600 605

Asn Ala Ala Ala Gly Arg Cys Glu Ala Cys Thr Gly Tyr Gly Gln Lys

610 615 620

Leu Val Asp Met His Phe Leu Pro Asp Val Trp Ala Thr Cys Asp Val

625 630 635 640

Cys Ala Gly Arg Arg Phe Thr Pro Glu Val Leu Ser Val Thr Tyr Gln

645 650 655

Gly Leu Ala Ile Asp Gln Val Leu Gly Leu Thr Val Asp Glu Ala Ala

660 665 670

Glu Phe Phe Arg Glu Pro Arg Thr Leu Thr Ala Ile Leu His Ala Leu

675 680 685

Gln Gln Val Gly Leu Gly Tyr Leu Gln Leu Gly Gln Ser Ala Thr Asp

690 695 700

Leu Ser Gly Gly Glu Ala Gln Arg Leu Lys Leu Ala Glu Ala Ile Arg

705 710 715 720

Arg Gly Ala Val Gly Gly Arg Gly Gly Val Val Leu Leu Asp Glu Pro

725 730 735

Leu Thr Gly Leu His Pro Ala Asp Val Gln Arg Met Val Ser Ala Phe

740 745 750

Asp Ala Leu Leu Ala Ala Gly His Thr Leu Leu Val Ala Glu His Asp

755 760 765

Leu His Leu Ala Asp Ser Ala Asp Trp Leu Val Asp Met Gly Pro Gly

770 775 780

Ala Gly Glu His Gly Gly Arg Val Val Ala Glu Gly Thr Pro Glu Gln

785 790 795 800

Ile Arg Ala Ala Asp Ser Ala Thr Ala Gly His Leu Arg Arg Leu Val

805 810 815

Pro Arg Gly

<210>8

<211>521

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Ala Ser Ser Pro Ser Val Leu Ile Val Gly Ala Gly Pro Thr Gly

1 5 10 15

Leu Ala Leu Ala Ala Glu Leu Ser Ala Ala Gly Ile Ala Cys Arg Val

20 25 30

Leu Asp Lys Arg Ser Thr Pro Ser Pro His Ser Arg Ala Phe Gly Leu

35 40 45

Leu Pro Arg Thr Leu Glu Leu Leu Asp Met Arg Gly Arg Ala Glu Ser

50 55 60

Phe Val Ala Gln Gly Leu Pro Trp Ala His Ala Ala Leu Gly Asp Gly

65 70 75 80

Lys Arg Trp Leu Asp Tyr Arg Asp Val Asp Ser Pro Phe Pro Tyr Met

85 90 95

Leu Val Ile Pro Gln Ser Arg Thr Glu Glu Gln Leu Thr Ser Trp Ala

100 105 110

Leu Asp Ser Gly Ala Val Ile Glu Arg Gly Ala Thr Val Thr Gly Leu

115 120 125

Arg Gln His Asp Gly Gly Val Glu Val Glu Val Glu Gly Pro Gly Gly

130 135 140

Pro Arg Thr Glu His Ala Asp Phe Val Val Gly Cys Asp Gly Val His

145 150 155 160

Ser Thr Val Arg Glu Leu Ala Gly Ile Ser Phe Ser Gly Ser Ser Tyr

165 170 175

Asp Ser Ser Leu Ile Val Ala Asp Val Arg Leu Ala Thr Pro Pro Asp

180 185 190

Pro Gln Val Tyr Ala Arg Thr Gly Lys Arg Gly Met Val Val Leu Phe

195 200 205

Pro Phe Gly Asp Gly Thr Phe Arg Leu Ile Ala Leu Asp His Ala Arg

210 215 220

Met Gly Ala Pro Val Asp Glu Pro Val Thr Val Asp Glu Leu Arg Glu

225 230 235 240

Ser Cys Arg Ala Val Leu Gly Thr Asp Phe Gly Leu His Asp Pro Leu

245 250 255

Trp Met Ser Arg Phe Arg Ser Asp Gln Arg Leu Ser Asp Arg Tyr Arg

260 265 270

Leu Gly Arg Val Leu Leu Ala Gly Asp Ala Ala His Thr His Ile Pro

275 280 285

Ser Gly Gly Gln Gly Leu Gln Thr Gly Ile Gln Asp Ala Met Asn Leu

290 295 300

Gly Trp Lys Leu Ala Ala His Leu Ser Gly Arg Ala Pro Ala His Leu

305 310 315 320

Leu Asp Thr Tyr Gln Arg Glu Arg Arg Pro Ile Ala Ala Ala Thr Leu

325 330 335

Arg Arg Thr Asp Leu Val Tyr Lys Phe Glu Val Ser Asp Ser Ala Gly

340 345 350

Ala Arg Leu Leu Arg Gly Leu Ser Thr Arg Leu Met Gly Leu Ala Pro

355 360 365

Val Gln Asn Ser Val Leu Glu Glu Leu Ser Gly Leu Lys Leu Arg Tyr

370 375 380

Arg Pro Leu Ala Gly Thr Thr Gly Arg Val Pro His Arg Leu Val Gly

385 390 395 400

Arg Arg Val His Asp Thr Ala Leu Arg Ser Arg Asp Gly Val Asp Ser

405 410 415

Arg Leu Tyr Thr His Phe Arg Thr Gly Gly Phe Val Leu Leu Asp Gln

420 425 430

Ser His Gly Gly Phe Cys Ala Glu Val Ala Glu Ala Gly Trp Ala Asp

435 440 445

Arg Val Thr Ala Val Arg Ser Val Ser Val Gly Arg Lys Asn Pro Lys

450 455 460

Asp Leu Pro Glu Ala Leu Leu Val Arg Pro Asp Gly Tyr Val Ala Trp

465 470 475 480

Ala Gly His Ala Gly Asp Thr Pro Gly Leu Arg Ala Ala Leu His His

485 490 495

Trp Cys Gly Glu Pro Glu Ala Pro Gly Ala Ala Arg Arg Arg Pro Ala

500 505 510

Ala Pro Ala Ala Ala Pro Ala Ala Val

515 520

<210>9

<211>199

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Ala Asp Pro Gly His Val Asp Thr Phe Asn Arg Ile Ala Pro Arg

1 5 10 15

Tyr Asp Ala Lys Phe Gly Lys Asp Cys Glu Thr Ala His Thr Val Val

20 25 30

Leu Asp Trp Ala Ala Gln Ala Gly Leu Glu Pro Arg Ser Val Leu Asp

35 40 45

Val Gly Cys Gly Thr Gly Lys Leu Leu Glu Glu Ala Gly Arg Arg Trp

50 55 60

Pro Gln Ala Arg Leu Tyr Gly Val Asp Pro Ala Asp Arg Met Val Glu

65 70 75 80

Ile Ala Arg Gly Arg Leu Pro Gly Ala Glu Leu Thr Val Gly Arg Ala

85 90 95

Glu Arg Leu Pro Pro Ala Asp Ala Ser Val Asp Leu Val Val Ser Thr

100 105 110

Thr Ala Phe Gly His Trp Ser Asp Ala Pro Ala Gly Leu Arg Glu Ile

115 120 125

Arg Arg Ile Leu Arg Pro Gly Gly Ser Val Met Ile Ala Glu His Ala

130 135 140

Pro Pro Gly Val Leu Leu Arg Leu Leu Leu Lys Ala Met Gly Arg Leu

145 150 155 160

Pro Arg Leu Tyr Asp Val Asp Gly Met Arg Thr Leu Val Ala Asp Ala

165 170 175

Gly Leu Thr Pro Glu Arg Leu Glu Thr Val Pro Gly Thr Phe Val Val

180 185 190

Thr His Ala Thr Arg Asn Gly

195

<210>10

<211>160

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Ser Val Trp His Lys Ile Asp Asp Tyr Leu His Leu Phe Ser Ser

1 5 10 15

Pro Val Leu Ser Phe Arg Asp Pro Asp Gly Phe Pro Phe Ser Leu Arg

20 25 30

Cys Arg Pro Arg Gln Asp Arg Asp Thr Gly Leu Met Val Val Arg Leu

35 40 45

Pro Glu Gly Val Pro Ala Ala Glu Gly Pro Ala Trp Leu Leu Trp His

50 55 60

Ser His Asp Glu Glu Phe Gly Ser Leu Gln Ala Leu Ala Val Ser Gly

65 70 75 80

Asp Leu Ala Ala His Gly Asp Gly Trp Ser Phe Arg Pro Arg Arg Val

85 90 95

Leu Pro Gly Pro Gly Leu Gly Pro Gly Gly Trp Ala Gly Val Val Glu

100 105 110

Lys Ile Glu Arg Asp Thr Ala Arg Phe Leu Glu Glu Arg Asn Leu Thr

115 120 125

Ala Pro Gln Asp Ile Asp Trp Ala Ala Leu Glu Arg Ile Ala Glu Ser

130 135 140

Ala Arg Lys Asp Asn Glu Glu Arg Ala Arg Ala Trp Ala Glu Leu Pro

145 150 155 160

<210>11

<211>165

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Pro Ala Thr Leu Ser Gly Glu Val Lys Arg Val Leu Arg Arg Val

1 5 10 15

Arg Ala Cys Glu Phe Ser Thr Leu Ala Arg Thr Gly Arg Pro Val Thr

20 25 30

Trp Pro Met Ala Phe Leu Trp Lys Pro Glu Glu Asn Glu Phe Val Leu

35 40 45

Ser Ser Ser Ser Ile Ser Val Ala Arg Lys Ala Glu His Ile Asn Arg Asp

50 55 60

Asp Arg Val Ser Leu Leu Met Ser Asp Phe Thr Gly Ser Ala Leu Pro

65 70 75 80

Gly Ser Ala Pro Ala Val Leu Val Gln Gly Arg Ala Thr Ala Pro Glu

85 90 95

Glu Ile Met Thr Val Asp Gly Leu Glu Asp Phe Trp Ala Glu Leu Phe

100 105 110

Arg Lys Arg Pro Ala Gly Ala Leu Glu Ala Leu Asp Pro Arg Ile Arg

115 120 125

Ala Gln Phe His Pro Ser Phe Tyr Trp Arg Leu Arg Ile Thr Val Thr

130 135 140

Pro Glu Lys Val Trp Ala Phe Arg Lys Asp Ala Met Gly Gly Thr Ala

145 150 155 160

Leu Glu Arg Val Ala

165

<210>12

<211>395

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Thr Thr Thr Pro Ala Pro Ser Tyr Pro Phe Pro Ala Lys Tyr Leu

1 5 10 15

Asp Glu Ala Ala Glu Leu Ala Arg Leu Arg Gln Glu Glu Pro Ile Ser

20 25 30

Arg Val Thr Met Pro Tyr Gly Gly Glu Ala Trp Leu Val Thr Arg Met

35 40 45

Ala Asp Val Lys Glu Val Leu Ala Asp Pro Arg Phe Ser Arg Gln Leu

50 55 60

Gln Thr Glu Ala Asp Arg Pro Arg Phe Phe Pro Glu Pro Val Val Glu

65 70 75 80

Gly Ile Gly Ile Met Asp Pro Pro Glu Gln Thr Arg Leu Arg Arg Leu

85 90 95

Val Ala Lys Ala Phe Thr Ala Arg Arg Val Gln Glu Phe Gly Pro Arg

100 105 110

Val Gln Thr Ile Val Asp Glu Leu Leu Asp Ala Val Glu Ala Lys Gly

115 120 125

Ala Pro Ala Asp Leu Tyr Ala Asp Phe Ser Trp Gln Leu Pro Gly Ile

130 135 140

Ser Ile Cys Glu Phe Met Gly Val Pro Tyr Glu Asp Arg Asp Arg Phe

145 150 155 160

Val Pro Phe Phe Asp Ser Val Val Ser Thr Thr Ser Lys Thr Pro Asp

165 170 175

Glu Ile Arg Gln Ala Ile Gly Asp Leu His Ala Tyr Phe Gly Glu Leu

180 185 190

Ile Glu Arg Arg Arg Ala Thr Pro Gly Asp Asp Leu Phe Thr Ala Leu

195 200 205

Ile Arg Ala Arg Asp Glu Asp Asp Arg Leu Ser Glu Lys Glu Leu Ile

210 215 220

Glu Met Ser Phe Gly Leu Leu Val Ala Gly Met Glu Thr Thr Ala Ser

225 230 235 240

Gln Leu Ala Asn Phe Leu Tyr Leu Leu Phe Arg Asn Pro Asp Gln Leu

245 250 255

Glu Leu Leu Arg Ala Lys Pro Glu Leu Val Pro Asn Ala Val Glu Glu

260 265 270

Leu Leu Arg Phe Val Pro Leu Leu Ala Val Asp Gln Pro Ser Val Ala

275 280 285

Arg Glu Asp Val Thr Leu Gly Gly Val Leu Ile Arg Ala Gly Glu Thr

290 295 300

Val Val Pro Ser Met Asn Ser Ala Asn Arg Asp Ala Asp Val Phe Glu

305 310 315 320

Asn Pro Gln Arg Ile Asp Val Thr Arg Glu Asn Asn Pro His Leu Ala

325 330 335

Phe Ser His Gly Ala His His Cys Val Gly Ala Gln Leu Ala Arg Met

340 345 350

Glu Met Val Thr Ala Leu Arg Ser Leu Leu Val Arg Phe Pro Gly Leu

355 360 365

His Gln Ala Val Pro Asp Glu Glu Leu Arg Trp Lys Ala Gly Val Val

370 375 380

Leu Arg Gly Val Glu Ala Leu Pro Val Ala Trp

385 390 395

<210>13

<211>61

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Ser Gly Leu Cys Met Ser Leu Ala Pro Asn Val Phe Gly Glu Gly

1 5 10 15

Asp Asp His Arg Val Val Ala Leu Thr Asp Arg Ser Ala Pro Asp Gly

20 25 30

Arg Leu Leu Glu Ala Ala Glu Phe Cys Pro Ala Glu Ala Ile Ala Ile

35 40 45

Ser Ser Leu Ala Thr Gly Glu Thr Val Glu Gly Thr Gly

50 55 60

<210>14

<211>504

<212> PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Lys Arg Arg Thr Phe Leu Gly Thr Ala Ala Ala Ser Ala Ala Gly

1 5 10 15

Gly Leu Leu Leu Pro Ala Ala Thr Ala Ser Ala Gly Gly Pro Pro Trp

20 25 30

Asp Ala Leu Gln Arg Ser Leu Asp Gly Thr Val Val Leu Pro Gly Asp

35 40 45

Pro Gln Tyr Asp Thr Val Arg Ser Leu Ala Leu Arg Gln Phe Asp Ser

50 55 60

Val Arg Pro Gln Ala Val Val Arg Cys Thr Thr Val Glu Asp Val Cys

65 70 75 80

Glu Ala Val Arg Phe Ala Ala Arg His Arg Val Pro Ala Val Ala Arg

85 90 95

Ser Gly Gly His Ser Phe Ala Gly Tyr Ser Thr Thr Thr Gly Met Val

100 105 110

Ile Asp Leu Ser Leu Met Asn Ala Val Arg Leu Ala Gly Ser Val Ala

115 120 125

Arg Ile Gln Pro Gly Cys Gln Leu Val Asp Leu Glu Glu Ala Leu Ala

130 135 140

Val His Gly Val Ala Val Pro Thr Gly Trp Cys Pro Thr Val Ala Ile

145 150 155 160

Gly Gly Leu Ala Leu Gly Gly Gly Leu Gly Phe Leu Thr Arg Met Tyr

165 170 175

Gly Val Ala Ser Asp Arg Met Arg Arg Ala Gln Val Val Leu Ala Asp

180 185 190

Gly Arg Val Val Glu Ser Ser Ala His Gln His Ala Asp Leu Phe Trp

195 200 205

Ala Leu Arg Gly Gly Gly Gly Gly Asn Phe Gly Ile Val Thr Glu Tyr

210 215 220

Asp Phe Glu Pro Val Pro Ala Pro Asp Met Thr Ser Phe Thr Leu Thr

225 230 235 240

Trp Thr Trp Ala Ser Val Arg Ala Val Leu Ser Ala Trp Gln Arg Trp

245 250 255

Thr Ala Glu Ala Pro Asp Pro Leu Thr Pro Leu Leu Asn Ile Ser Thr

260 265 270

Tyr Gly Ala Asp Ala Gly Val Glu Pro Gly Val Thr Val Ser Gly Val

275 280 285

Trp Leu Gly Ser Pro Asp Gly Leu Gly Pro Leu Leu Asp Arg Leu Thr

290 295 300

Ala Ala Val Gly Thr Ala Pro Ala Thr Ser Glu Arg Arg Thr Asp Ser

305 310 315 320

Tyr Arg Phe Gly Met Arg His Trp Phe Gly Cys Asp Thr Leu Glu Pro

325 330 335

Ala Ala Cys His Arg Val Gly His Asn Pro Gln Ala Gln Leu Ala Arg

340 345 350

Tyr Gly Phe Ala Leu Ala Arg Gly Asn Phe Phe Asp Arg Pro Leu Asp

355 360 365

Ser Ala Gly Ile Asp Ala Val Leu Lys Ala Phe Thr Ala Ala Arg Ser

370 375 380

Glu Gly Glu Ala Arg Ser Phe Asp Leu Gln Gly Leu Gly Gly Ala His

385 390 395 400

Asn Arg Val Pro Ala Thr Ala Thr Ala Tyr Val His Arg Asn Ala Leu

405 410 415

Phe Tyr Ala Gly Trp Ser Val Gly Ile Asp Val Pro Glu Gly Glu Val

420 425 430

Leu Ala Pro Asp Arg Arg Arg Ala Cys Gln Glu Trp Val Asp Arg Ala

435 440 445

Tyr Ala Arg Val His Pro Trp Ser Ser Gly Gln Ala Tyr Gln Asn Tyr

450 455 460

Ile Asp Pro Asp Leu Ala Asp Trp Arg Glu Ala Tyr Tyr Gly Val Asn

465 470 475 480

Tyr Glu Arg Leu Ser Ala Val Lys Arg Ala Tyr Asp Pro Lys Gly Phe

485 490 495

Phe Arg Phe Ala Gln Ser Ile Thr

500

<210>15

<211>475

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Pro Gly Thr Pro Val Val Gln Asn Pro Ala Asp Arg Leu Ala Gly

1 5 10 15

Pro Ala Asp Pro Leu Gly Leu Arg Arg Leu Ser Ala Gly Leu Asn Leu

20 25 30

Leu Arg Arg Pro Leu Pro Phe Leu Gln Glu Leu Ala Ser Gly Tyr Gly

35 40 45

Asp Val Cys Ala Met Pro Gly Arg Asn Pro Arg Thr Val Phe Ala Phe

50 55 60

Gly Pro Asp Gln Ala Arg Gln Val Leu Thr Gly Lys Ser Phe Val Ser

65 70 75 80

Asp Ser Phe Arg His Leu Arg Leu Pro Pro Gly Ser Pro Met Met Leu

85 90 95

Leu Thr Ser Gly Leu Leu Arg Leu Asn Gly Thr Ile His Arg Lys His

100 105 110

Arg Gln Ala Met Gln Gln Ala Phe Ser Pro Arg His Val Thr Val Tyr

115 120 125

Thr Asp Thr Ile Val Arg Phe Ala Asp Asp Met Leu Asp Gly Trp Thr

130 135 140

Pro Gly Arg Ser Val Asp Leu His Glu Glu Leu Ala Arg Leu Val Thr

145 150 155 160

Arg Ile Ser Leu Ala Thr Met Ala Gly Leu Glu Asp Glu Ala Ala Ala

165 170 175

Arg His Leu Asn Asp Lys Met Val Glu Leu Ala Ala Ala Ala Gly His

180 185 190

Pro Leu Thr Ala Val Ile Gln Ala Pro Leu Pro Gly Thr Pro Phe Arg

195 200 205

Arg Met Thr Arg Ile Ala Gly Glu Ile Glu Asp Ile Leu Arg Ala Leu

210 215 220

Ile Ala Ala Arg Arg Ser Gly Ala Ala Ala Pro Asp Leu Leu Ser Lys

225 230 235 240

Leu Thr Val Pro Asp Ala Glu Ala Asp Ala Glu Glu Leu Ser Asp Glu

245 250 255

Glu Leu Leu Gly Glu Ala Tyr Thr Ala Leu Cys His Asp Ser Val Ala

260 265 270

Ser Ser Leu Phe Trp Thr Leu Phe Leu Leu Asp Gln His Arg Glu Trp

275 280 285

Trp Glu Lys Leu Arg Ala Glu Val Ala Glu Val Ala Gly Asp Ala Ala

290 295 300

Pro Gly Pro Asp Val Leu Val Arg Met Pro Val Leu Asp Arg Ile Val

305 310 315 320

Lys Glu Ser Val Arg Leu Met Pro Pro Ala Gly Phe Gly Val Arg Tyr

325 330 335

Ala Glu Glu Gly Ala Val Ile Asp Gly His Pro Ile Pro Lys Gly Ala

340 345 350

Met Thr Ile Phe Ser Ser Ser Phe Val Thr His Arg Asp Ala Gly Thr Phe

355 360 365

Pro Glu Pro Leu Arg Phe Arg Pro Glu Arg Trp Glu Thr Ala Gln Pro

370 375 380

Ser Gln His Glu Tyr Phe Pro Phe Gly Leu Gly Gln His Asn Cys Ile

385 390 395 400

Gly Arg Asn Leu Ala Leu Leu Glu Ser Lys Leu Val Leu Thr Arg Ile

405 410 415

Met Gln Arg Ala Pro Val Val Leu Pro Ala Gly Thr Val Val Asp Pro

420 425 430

Val Val Arg Ile Ser Thr Val Pro Asp Gly His Val Arg Ala Leu Val

435 440 445

Ala Gly Pro Asp Gly Pro Ala Pro Arg Ala Tyr Arg Val Thr Gly Ser

450 455 460

Val Asn Glu Ile Ile Glu Leu Pro Glu His Pro

465 470 475

<210>16

<211>1836

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Asp Glu Asp Ser Ser Leu Tyr Pro Thr Ala Ala Arg Pro Ser Arg

1 5 10 15

Arg Pro Lys Glu Pro Arg Lys Met Val Leu Gly Asp Phe Phe Arg Glu

20 25 30

Ser Leu Met Val Leu Lys Glu Ile Leu Arg Arg Val Arg Ala Pro Phe

35 40 45

Ser Phe Gly Lys Ala Gly Phe Gly Ser Gly Leu Ser Pro His Pro Ala

50 55 60

Glu Arg Gly Val Cys His Arg Gln Thr Gly Gly Asp Thr Met Gln Ala

65 70 75 80

Ser Thr Gly Arg Glu Thr Val Leu Gly Ala Ile Arg Ala Val Cys Glu

85 90 95

Thr Asp Pro Gly Ala Thr Ala Ala Val Phe Pro Ala Gln Pro Ala Ile

100 105 110

Gly Leu Thr Glu Ala Val Thr Phe Ser Arg Glu Gly Leu Asp Arg Glu

115 120 125

Ala Arg Ala Leu Ala Val Lys Leu Arg Glu Val Ala Glu Gly Pro Val

130 135 140

Leu Leu Leu Met Gln Pro Gly Pro Asp Tyr Leu Lys Gly Phe Leu Ala

145 150 155 160

Thr Val Tyr Ala Gly Leu Pro Ala Ala Pro Val Tyr Pro Pro Asn Pro

165 170 175

Ala Asp Val Arg Arg Asp Phe Ile Arg Leu Gly Ala Ile Leu Ala Lys

180 185 190

Leu Pro Asp Ala Thr Leu Leu Thr Glu Pro Gly Leu Leu Glu Pro Leu

195 200 205

Arg Glu Leu Phe Ala Glu Arg Leu Pro Asp Val Arg Pro Glu Arg Leu

210 215 220

Val Asp Thr Ser Val Pro Ala Gly Ala Glu Asp Asp Trp Arg Ala Pro

225 230 235 240

Ala Val Arg Pro Glu Glu Pro Leu Phe Ile Gln Phe Thr Ser Gly Ser

245 250 255

Thr Gly Thr Pro Arg Gly Val Leu Val Ser His Arg Asn Leu Leu Ala

260 265 270

Asn Val Arg Ala Ile Thr Asp Arg Phe Gly Leu Asp Thr Ser Ser Ser Thr

275 280 285

Gly Ala Leu Trp Leu Pro Pro Tyr His Asp Met Gly Leu Val Gly Gly

290 295 300

Val Leu Thr Pro Leu Val Ser Gly Phe Pro Ile His Leu Leu Ser Pro

305 310 315 320

Leu Ser Phe Val Ser Asp Pro Met Gly Trp Leu Arg Leu Val Ser Glu

325 330 335

Thr Gly Ala Thr His Thr Gly Ala Pro Asn Phe Gly Tyr Ala Leu Ala

340 345 350

Thr Arg Arg Ala Arg Asp Glu Asp Val Ala Ala Leu Asp Leu Ser Arg

355 360 365

Leu Gln Val Ala Phe Ser Gly Ala Glu Pro Val Asp Ala Ser Thr Leu

370 375 380

Arg Ala Phe Ala Glu Arg Phe Ala Pro Ala Gly Leu Ser Pro Asp Val

385 390 395 400

Phe Leu Pro Cys Tyr Gly Leu Ala Glu Ser Thr Leu Ile Val Ser Gly

405 410 415

Gly Arg Pro Gly Ala Gly Leu Arg Thr His Arg Leu Asp Pro Glu Ala

420 425 430

Leu Ala Leu Gly Arg Ala Glu Pro Ala Arg Pro Gly Ala Pro Ala Thr

435 440 445

Glu Leu Val Ser Ser Gly Pro Val Val Ala Gly Thr Glu Val Arg Ile

450 455 460

Ala Asp Pro Val Thr Gly Ala Ala Ala Gly Pro Gly Glu Ile Gly Glu

465 470 475 480

Val Phe Val Ala Ser Asp Ser Val Ala Glu Gly Tyr Phe Glu Asp Pro

485 490 495

Glu Glu Thr Ala Arg Thr Phe Gly Ala Thr Leu Ala Gly Ser Ser Arg

500 505 510

Ser Trp Met Arg Thr Gly Asp Leu Gly Phe Leu Gly Ala Asp Gly Asp

515 520 525

Leu Val Pro Val Ala Arg Ile Lys Asp Val Ile Val Val Arg Gly Arg

530 535 540

Asn Leu His Pro Gln Asp Ile Glu Arg Thr Val Gln Asp Thr Asp Pro

545 550 555 560

Gly Ile Arg Lys Gly Cys Val Ala Ala Ala Gly Val Pro Gly Pro Asp

565 570 575

Gly Gly Glu Glu Ile Leu Val Val Ala Glu Leu Arg Pro Glu Ala Ala

580 585 590

Asp Asp Glu Ala Gln Ala Arg Arg Ile Ala Arg Glu Val Arg Ala Ala

595 600 605

Val Ala Arg Glu His Ser Ala Pro Val Arg Gly Ile His Leu Ile Arg

610 615 620

Pro Ser Thr Leu Pro Lys Thr Ser Ser Gly Lys Val Gln Arg Ser Ala

625 630 635 640

Ala Arg Ala Ala His Leu Asp Gly Ser Leu Ala Thr Val Leu Cys Asp

645 650 655

Thr Ala Asp Thr Ala Ala Pro Thr Gly Ala Pro Leu Pro Ala Gly Pro

660 665 670

Ala Ala Gly Gly Ser Asp Asp Leu Val Val Asn Leu Ala Ala Thr Val

675 680 685

Ile Arg His Phe Val Gly His Gly Gly Thr Thr Gly Ala Leu Asp Ser

690 695 700

Leu Gly Ala Ala Arg Leu Ala Gly Glu Ala Ser Gln Ile Phe Lys Val

705 710 715 720

Ala Leu Pro Val Thr Asp Ile Leu Ala Gly Val Asp Ala Glu Ala Leu

725 730 735

Ala Ala Leu Ile Arg Thr Ala Ala Pro Leu Gly Gly Pro Ala Ala Ala

740 745 750

Thr Gly Ser Ala Gly Leu Ala Glu Leu Pro Ala Thr Ala Arg Gln Glu

755 760 765

Thr Leu Cys Leu Leu Gln Glu Leu Asp Pro Ala Ala Pro Gly Leu Ala

770 775 780

Leu Gly Ile Ala Phe Ala Leu Pro Ala Thr Ala Asp Pro Gln Arg Val

785 790 795 800

Ala Leu Ala Leu Thr Ala Leu Val Ala Arg His Pro Ala Leu Arg Thr

805 810 815

Arg Leu Val Arg Arg Gly Glu His Trp Ser Arg Leu Val Asp Pro Ala

820 825 830

Asp Thr Ala Ala Gly Ala Pro Trp Gly Arg Tyr Cys Thr Leu Thr Arg

835 840 845

Leu Gly Gly Ile Gly Glu Arg Asp Leu Glu Glu Ala Leu Ala Glu Arg

850 855 860

Cys Arg Arg Val Pro Asp Leu Ala Glu Gly Pro Leu Phe Gln Ala Glu

865 870 875 880

Val Val Leu Ala Asp Gly His Gly Ala His Leu Val Ile Thr Val His

885 890 895

His Ala Val Ala Asp Leu Trp Ser Ile Gly Val Leu Ala Ala Glu Leu

900 905 910

Ala His Leu Leu Ala Ala Gly Asp Pro Glu Ala Gly Ala Leu Thr Leu

915 920 925

Pro Pro Ala Pro Glu Val Ser Cys Asp Val Pro Asp Asp Arg Arg Leu

930 935 940

Glu Arg Ala Trp Asn Phe Trp Gln Gly Leu Leu Thr Asp Gly Cys Gly

945 950 955 960

Pro Leu Gln Leu Pro Ser Ala Asp Gly Pro Ala Gln Asn Val Pro Ala

965 970 975

Gly Arg Ala Arg Gln Ala Ser His Ala Pro Leu Thr Leu Asp Ala Arg

980 985 990

Arg Thr Ala Ala Leu Thr Ala Leu Ala Lys Glu Cys Gly Val Thr Leu

995 1000 1005

Tyr Ala Val Leu Leu Ala Val Gln Ser Leu Thr Leu Ser Arg Leu

1010 1015 1020

Thr Gly Thr Asp Arg Val Pro Val Ala Val Pro Leu His Gly Arg

1025 1030 1035

Gly Ser Ala Thr His Arg Ala Val Asp Tyr Leu Val Ser Thr Val

1040 1045 1050

Thr Leu Pro Met Glu Thr Ala Thr Gly Thr Val Arg Asp Leu Val

1055 1060 1065

Gly Arg Ala Ala Asp Ala Leu Arg Gly Ala Leu Ala His Arg Thr

1070 1075 1080

Val Gly Tyr Pro Glu Leu Val Ala Leu Ser Ala Ala Arg Gly Gly

1085 1090 1095

Pro Asp Val Pro Ala Pro Asp Ala Ala Leu Leu Leu Gln Gln Asp

1100 1105 1110

Thr Pro Gly Ala Pro Arg Gly Val Gly Ala Gly Leu Leu Gly Gly

1115 1120 1125

Gly Val Arg Leu Gly Asp Val Glu Leu Gly Val Ala Val Val Pro

1130 1135 1140

Pro Ser Ile Gly Pro Phe Gly Leu Thr Thr Leu Leu Thr Glu Thr

1145 1150 1155

Asp Gly Thr Leu Thr Gly Arg Val Glu Val Asp Pro Ser Arg His

1160 1165 1170

Ala Asp Trp Leu Ala Gly Arg Phe Ala Asp Ala Phe Leu Ala Ile

1175 1180 1185

Ala Asp Ser Val Ala Ala Gly Ala Gly Leu Pro Leu Ala Glu Ala

1190 1195 1200

Ser Ala Val Gly Ala Glu Gln Ala Glu Arg Leu Arg Gly Trp Ser

1205 1210 1215

Arg Ser Pro Val Pro Glu Asp Gly Glu Gly Thr Leu His Asp Leu

1220 1225 1230

Val Leu Ser Ala Ala Ala Arg His Pro Arg Arg Thr Ala Val Val

1235 1240 1245

Ala Gly Asp Gly Ser Leu Ser Tyr Gly Glu Leu Ala His Arg Ser

1250 1255 1260

Ala Val Val Ala Ala Ala Leu Ala Ala Glu Gly Ala Gly Pro Gly

1265 1270 1275

Cys Thr Val Gly Val Leu Met Gln Arg Gly Arg Asp Leu Pro Ala

1280 1285 1290

Val Leu Leu Gly Val Leu Arg Ser Gly Ala Ala Tyr Leu Pro Leu

1295 1300 1305

Asp Ala Ala Thr Pro Pro Ala Arg Leu Ala Ala Val Val Glu Asp

1310 1315 1320

Ala Gly Cys Arg His Val Leu Val Gly Asp Val Pro Val Glu Arg

1325 1330 1335

Gly Gln Phe Phe Pro Val Arg Thr Leu Asp Val Asp Ala Val Leu

1340 1345 1350

Ala Ala Gly Pro Ala Glu Pro Val Pro Pro Arg Pro Leu Thr Thr

1355 1360 1365

Pro Asp Asp Pro Ala Tyr Leu Leu Phe Thr Ser Gly Ser Thr Gly

1370 1375 1380

Arg Pro Lys Gly Val Val Ile Pro His Arg Gly Pro Val Asn Leu

1385 1390 1395

Ile Arg Trp Ala Gly Arg Glu Phe Gly Thr Asp Ala Leu Ala Arg

1400 1405 1410

Thr Leu Ala Val Thr Pro Thr Thr Phe Asp Leu Ser Val Phe Glu

1415 1420 1425

Leu Phe Thr Pro Leu Ala His Gly Cys Glu Val Arg Leu Leu Asp

1430 1435 1440

Gly Val Leu Asp Leu Val Asp Ser Pro Ala His Ala Ala Asp Ala

1445 1450 1455

Thr Leu Leu Asn Thr Val Pro Ser Ala Val Ala Ser Leu Leu Glu

1460 1465 1470

Gln Asp Ala Leu Pro Ala Gly Leu Ser Val Val Asn Val Ala Gly

1475 1480 1485

Glu Pro Leu Thr Ala Glu Leu Val His Ser Val His Arg Arg Leu

1490 1495 1500

Pro Gly Val Arg Met Val Asn Leu Tyr Gly Pro Ser Glu Thr Thr

1505 1510 1515

Thr Tyr Ser Thr Tyr Ala Glu Leu Gly Pro Asp Thr Ser Gly Ala

1520 1525 1530

Val Pro Ile Gly Arg Pro Val Gly Gly Thr Thr Leu Ser Val Val

1535 1540 1545

Asp Ala Ser Leu Arg Pro Leu Pro Gln Gly Ala Thr Gly Glu Leu

1550 1555 1560

Leu Ile Gly Gly Ala Gly Val Ala Val Gly Tyr Ala Gly Arg Pro

1565 1570 1575

Gly Met Thr Ala Ala Arg Phe Leu Pro Asp Pro Glu His Pro Gly

1580 1585 1590

Arg Arg Leu Tyr Arg Thr Gly Asp Leu Val Arg Trp Arg Ala Asp

1595 1600 1605

Gly Leu Leu Glu Phe Leu Gly Arg Ser Asp His Gln Val Lys Val

1610 1615 1620

Arg Gly Phe Arg Ile Glu Leu Gly Asp Val Glu Arg Ala Leu Thr

1625 1630 1635

Gly Leu Asp Ala Val Arg Glu Ala Val Val Val Ala Leu Gly Gln

1640 1645 1650

Gly Thr Asp Arg Arg Leu Ala Ala Tyr Leu Val Pro Glu Arg Pro

1655 1660 1665

Leu Glu Gly Asp Pro Gly Ser Trp Leu Arg Gly Val Arg Arg Arg

1670 1675 1680

Leu Gly His Glu Leu Pro Gly Tyr Met Val Pro Gly Glu Phe Ala

1685 1690 1695

Val Leu Asp Glu Leu Pro Arg Asn Arg His Gly Lys Leu Asp Arg

1700 1705 1710

Gly Arg Leu Ala Ser Thr Ala Thr Val Pro Leu Val Thr Gly Ser

1715 1720 1725

Arg Ile Ala Pro Arg Asn Asp Ser Glu Arg Arg Val Ala Ala Cys

1730 1735 1740

Trp Ala Gln Ala Leu Pro Val Pro Glu Ile Gly Val Thr Asp Glu

1745 1750 1755

Phe Leu Asp Val Gly Gly His Ser Leu Met Leu Thr Thr Ile Ala

1760 1765 1770

His Leu Ile Ala Arg Glu Phe Ser Val Gln Val Pro Leu Thr Ala

1775 1780 1785

Leu Arg Thr His Thr Thr Val Ala Glu Gln Ala Arg Tyr Leu Asp

1790 1795 1800

Glu Leu Thr Ser Ala Ala Pro Pro His Ala Ala Gly Pro Arg Ser

1805 1810 1815

Val Arg Arg Leu Asp Arg Ser Arg Tyr Thr Ala Ala Gly Asn Gly

1820 1825 1830

Arg Thr Thr

1835

<210>17

<211>1082

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Asn Glu Met Ala Gly Lys Ala Tyr Gly Leu Ala Val Arg Leu Asp

1 5 10 15

Leu Arg Gly Ala Leu Glu Arg Thr Ala Leu Gln Ser Ala Leu Ser Ala

20 25 30

Val Val Glu Arg His Glu Ala Leu Arg Thr Gly Leu Arg Gln Ile Asp

35 40 45

Gly Thr Leu Thr Gln Val Val Val Pro Gly Val Thr Val Ser Leu Pro

50 55 60

Val Val Asp Leu Gly Gly Arg Gly Pro Asp Pro Ala Gln Leu Asp Arg

65 70 75 80

Glu Val Arg Arg Leu Ala Arg Gln Glu Ala Gln Arg Gly Trp Asn Leu

85 90 95

Ala Gln Pro Pro Leu Leu Arg Gly Leu Leu Ala Arg Leu Ala Asp Asp

100 105 110

His His Val Leu Leu Leu Cys Val His His Ala Val Cys Asp Gly Leu

115 120 125

Ser Leu Gln Ile Val Leu Arg Glu Leu Leu Glu Asn Tyr Thr Gly Gly

130 135 140

Gly Pro Ala Gly Ala Asp Glu Pro Leu Gln Phe Ala Asp Tyr Val Val

145 150 155 160

Trp Asn Asn Gly Gly Glu Glu Phe Pro Asp Pro Glu Trp Ser Ala Arg

165 170 175

Arg Gln Ala Ala Arg Glu His Trp Ser Ser Ser Thr Leu Ala Gly Ala Pro

180 185 190

Gln Val Leu Asp Leu Pro Thr Asp Arg Arg Arg Arg Pro Pro Leu Gln Ser

195 200 205

Tyr Ala Gly Ala Arg Val Pro Val Arg Leu Asp Thr Ala Phe Ala Asp

210 215 220

Arg Val Arg Asp Trp Ser Ala Gln Arg Gly Val Thr Pro Phe Thr Thr

225 230 235 240

Leu Leu Ala Ala Tyr Thr Val Val Leu Ala Arg Asn Gly Gly Ala Asp

245 250 255

Asp Leu Leu Val Gly Leu Pro Val Ala Asn Arg Ser His Ala Asp Leu

260 265 270

Ala Gly Thr Val Gly Tyr Leu Ala Asn Thr Cys Pro Leu Arg Ala Asp

275 280 285

Leu Arg Ala Asp Pro Thr Leu Gly Glu Leu Val Arg Asp Leu Tyr Asp

290 295 300

Arg Leu Thr Gly Val Leu Glu His Ala Asp Leu Pro Phe Gly Glu Leu

305 310 315 320

Val Glu Leu Leu Ala Pro Pro Arg Met Pro Glu Arg Asn Pro Val Phe

325 330 335

Gln Val Met Phe Gly Leu Gln Gln Asp Val Arg Arg Gly Trp Asp Leu

340 345 350

Pro Gly Leu Arg Val Asp Val Glu Asp Val Asp Cys Gly Asn Ala Arg

355 360 365

Val Asp Leu Ser Leu Phe Leu Phe Glu Glu Ala Asp Gly Ala Ile Asp

370 375 380

Gly Phe Leu Glu Tyr Ala Ser Ala Leu Phe Asp Arg Ala Thr Ala Glu

385 390 395 400

Arg Phe Ala Asp Gln Leu His Thr Val Leu Arg Gln Ile Leu Arg Asp

405 410 415

Ala Arg Ile Pro Val Ser Ala Val Asp Leu Val Gly Asn Gly Ser Ala

420 425 430

Thr Thr Ile Asp Ser Phe Leu Asp Gly Gly Pro Leu Glu Gln Pro Trp

435 440 445

Pro Leu Val Trp Pro Arg Ile Arg Glu Leu Ala Ala Arg Arg Pro Ser

450 455 460

Ala Glu Ala Val Arg Asp Asp Ala Glu Ala Leu Asp Tyr Ala Ser Leu

465 470 475 480

Val Asp Arg Val Asp Ala Ala Ala Ala Arg Leu Thr Ala Ala Gly Ala

485 490 495

Gly Pro Gly Asp Arg Val Ala Val Leu Ala Glu Arg Gly Val Arg Ala

500 505 510

Val Val Ala Met Leu Ala Cys Trp Arg Ala Gly Gly Val Tyr Val Pro

515 520 525

Val Asp Pro Ala Ala Pro Leu Pro Arg Arg Glu Leu Ile Leu Glu Gln

530 535 540

Ala Ala Pro Ala Val Leu Val Cys Glu Asp Pro Asp Glu Gln Pro Pro

545 550 555 560

His His Arg Ser Arg Ala Val Ala Ile Gly Asp Leu Thr Ala Glu Ala

565 570 575

Asp Ala Gly Ala Gly Thr Pro Ala Glu Pro Ala Pro Arg Pro His Asp

580 585 590

Pro Ala Tyr Leu Met Phe Thr Ser Gly Ser Thr Gly Arg Pro Lys Gly

595 600 605

Val Ala Val Ser His Ala Asn Leu Ser Ser Phe Leu His Ala Leu Thr

610 615 620

Gly Arg Leu Ala Leu Gly Pro Ala Asp Arg Leu Leu Ala Leu Thr Thr

625 630 635 640

Thr Ala Phe Asp Ile Ser Leu Leu Glu Leu Leu Gly Pro Leu Val Thr

645 650 655

Gly Gly Thr Val Val Val Ala Pro Ser Ser Ala Gln Arg Gly Ala Ala

660 665 670

Asp Leu Ala Ala Arg Leu Ser Ser Pro Gly Ile Thr Thr Ala Gln Ala

675 680 685

Thr Pro Ala Val Trp Arg Leu Ala Leu Ser Ala Gly Trp Arg Pro Arg

690 695 700

Glu Gly Phe Thr Leu Leu Cys Gly Gly Glu Ala Leu Pro Pro Asp Leu

705 710 715 720

Ala Asp Leu Leu Ala Ala Thr Pro Ala Glu Ala His Asn Leu Tyr Gly

725 730 735

Pro Thr Glu Thr Thr Ile Trp Ser Cys Ala Ala Arg Ile Arg Pro Gly

740 745 750

Glu Pro Val Thr Ile Gly Arg Pro Ile Pro Gly Thr Arg Val Leu Val

755 760 765

Ala Asp Ala Ala Leu Arg Pro Val Pro Pro Gly Val Cys Gly Glu Leu

770 775 780

Leu Val Gly Gly Pro Gly Val Ala Leu Gly Tyr Leu Asp Asp Pro Ala

785 790 795 800

Arg Thr Ala Ala Arg Phe Val Pro Asp Pro Tyr His Pro Gly Glu Arg

805 810 815

Leu Tyr Arg Thr Gly Asp Val Val Arg Leu Arg Ser Asp Gly Leu Ile

820 825 830

Glu Phe Val Gly Arg Val Asp Glu Gln Val Lys Val Arg Gly His Arg

835 840 845

Ile Glu Leu Gly Glu Ile Glu Ser Ala Leu Arg Ala Leu Pro Gly Val

850 855 860

Arg Asp Ala Ala Ala Thr Val Leu Asp Pro Arg Gly Asn Ala Arg Ile

865 870 875 880

Ala Gly Tyr Leu Val Ala Asp Asp Gly Ala Leu Asp Thr Ala Gly Arg

885 890 895

Ala Ala Arg Leu Arg Gln Asp Leu Ser Glu Ala Leu Pro Ala Ser Met

900 905 910

Val Pro Ser Glu Leu Tyr Ala Val Pro Ala Ile Pro Leu Asn Pro Asn

915 920 925

Gly Lys Val Asp Arg Arg Ala Leu Pro Gly Thr Gly Arg Arg Leu Glu

930 935 940

Gly Gly Ser Glu Arg Val Ala Pro Ser Thr Asp Ala Glu His Ala Val

945 950 955 960

Ala Ala Leu Trp Cys Glu Leu Leu Ser Leu Pro Glu Val Gly Val Arg

965 970 975

Glu Asp Phe Phe Gly Leu Gly Gly His Ser Leu Leu Ala Ala Asp Leu

980 985 990

Leu Gln Arg Leu Glu Arg Asp Leu Gly Ala Arg Val Pro Val Ala Glu

995 1000 1005

Phe Phe Met Glu Pro Thr Val Ala Arg Leu Ala Ala Thr Val Ser

1010 1015 1020

Gln Leu Ser Gly Ala Val His Gln Thr Pro Thr Gln Asp Pro Asp

1025 1030 1035

Gly Arg Ala Glu Gln Thr Ala Glu Pro Ser Pro Glu Asp Arg Gly

1040 1045 1050

Ala Asp Gly Asp Gly Trp Asp Phe Pro Thr Val Arg Arg Ser Val

1055 1060 1065

Thr Val Pro Gly Ser Asp Pro Val Pro Leu Glu Val Ser Pro

1070 1075 1080

<210>18

<211>1485

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Thr Arg His Glu Pro Thr Glu Thr Phe Thr Asp Thr Leu His Arg

1 5 10 15

Val Leu Glu Glu Arg Gly Ala Glu Ser Ala Thr Pro Leu Ser Val Glu

20 25 30

Gln Arg Arg Leu Trp Leu Leu Gly Gly Ile Ala Asp Gly Ser Trp Ala

35 40 45

Val Val Thr Gly Arg Tyr Arg Leu Gln Gly Val Pro Asp Ala Ala Arg

50 55 60

Leu Gln Leu Arg Leu Ala Ser Leu Val Ser Arg His Glu Ala Leu Arg

65 70 75 80

Ser Val Phe Val Gln Val Ala Glu Arg Pro Val Arg Leu Val Leu Pro

85 90 95

Phe Ala Glu Val Ala Leu Arg Thr Val Asn Ala Pro Asp Ser Thr Asp

100 105 110

Pro Ala Arg Ala Asp Glu His Val Arg His Leu Val Glu Glu Glu Phe

115 120 125

Ser Leu Gly His Gly Pro Leu Leu Arg Ala Leu Leu Leu Leu Arg Ser Ala

130 135 140

Asp Gly Gly Ala Ala Glu Leu Val Leu Val Gly His Arg Leu Val Leu

145 150 155 160

Asp Ala Thr Ser Leu Asp Leu Leu Val Ala Glu Leu Leu Gly Glu Asp

165 170 175

Ala Pro His Gly Arg Glu Gly Ala Ala Asp Thr Ala Gly Ala Leu Glu

180 185 190

Thr Thr Leu Ala Ala Glu Arg Glu Arg Leu Ala Asp Pro Ala Leu Thr

195 200 205

Gln Ala Val Thr Gly Arg Ala Ala Glu Leu Ala Leu Pro Ala Ala Thr

210 215 220

Glu Ile Pro Gly Tyr Gly Arg Arg Pro Glu Ile Lys Gly Thr Ser Tyr

225 230 235 240

Ala Ser Val Ala Leu Pro Leu Pro Leu Ala Leu Ala Pro Arg Ala Ala

245 250 255

Glu Ala Ala Asp Trp Glu Ala Ala Val Ala Ala Ala Trp Leu Val Val

260 265 270

Leu Met Arg Ser Gln Ala Thr Gly Ser Ala Val Cys Gly Val Arg Thr

275 280 285

Ala Arg Gly Ala Glu Gln Ala Gly Ile Val Gly Pro Leu Asp Gly Leu

290 295 300

Arg Leu Val Arg Val Asp Asp Ala Asp Asp Ala Pro Leu Ser Asp Leu

305 310 315 320

Leu Ala Ala Val Gly Arg Gln Leu Arg Ala Pro Ala Val Asp Val Pro

325 330 335

Leu Ala His Leu Leu Glu Val Ala Pro Pro Arg Arg Asp Ile Ser Arg

340 345 350

Thr Pro Tyr Ala Gln Thr Val Val Arg Ala Val Asp Ala Arg Glu Pro

355 360 365

Leu Gly Gly Ala Ser Gly Thr Gly Arg Gln Ile Gly Gly Ala Ala Ser

370 375 380

Gly Thr Glu Tyr Asp Ile Glu Val Thr Val Arg Leu Gln Pro Gly Leu

385 390 395 400

Ala Val Ala Gln Ile Asp Tyr Asp Val Gln Leu Tyr Ser Glu Glu Arg

405 410 415

Val Arg Ala Leu Gly Asn Gln Leu Ala Ala Val Leu Asp Ala Ile Leu

420 425 430

Pro Gly Gly Thr Pro Glu Val Thr Ala Thr Val Val Pro Leu Leu Asp

435 440 445

Ala Glu Gly Ala Arg Leu Ala Leu Glu Ala Gly Arg Gly Glu Arg Thr

450 455 460

Ala Pro Asp Thr Arg Ser Leu Val Asp Leu Val Glu Ala Gln Val Ala

465 470 475 480

Ala Ala Pro Asp Ala Val Ala Leu Trp Gln Gly Asp Thr Arg Val Thr

485 490 495

Tyr Ala Gln Leu Trp Ala Asp Ala Thr Arg Leu Ala Asp Glu Leu Ala

500 505 510

Ala Arg Gly Val Arg Pro Gly Asp Arg Val Ala Val Trp Leu Arg Arg

515 520 525

Gly Pro Ser Thr Val Thr Ala Leu Leu Ala Val Leu Ala Ala Gly Ala

530 535 540

Ala Phe Val Pro Val Asp Ala Ala Tyr Pro Glu Glu Arg Val Arg Tyr

545 550 555 560

Leu Leu Ser Asp Ser Arg Pro Ser Leu Val Val Thr Glu Ser Ser Val

565 570 575

His Leu Leu Gly Glu Leu Gly Leu Pro Thr Leu Leu Leu Asp Glu Leu

580 585 590

Ser Gly Ala Pro Ala Ala Val Asp Gly Ala Arg Arg Pro Asp Arg Val

595 600 605

Ala Ala Asp Thr Pro Ala Tyr Leu Ile Tyr Thr Ser Gly Thr Thr Gly

610 615 620

Arg Pro Lys Gly Val Val Val Arg His Ser Ser Val Val Asn Asn Ile

625 630 635 640

Ala Trp Arg Gln Ala Asn Trp Gln Leu Thr Glu Asp Asp Arg Val Leu

645 650 655

His Asn His Ser Phe Cys Phe Asp Pro Ser Val Trp Ala Ala Phe Trp

660 665 670

Pro Leu Ala Thr Gly Ala Ala Ile Val Leu Ala Thr Glu Glu Gln Met

675 680 685

Lys Asp Pro Gly Glu Met Ile Thr Thr Leu Arg Asp His Gln Val Thr

690 695 700

Val Leu Gly Gly Val Pro Ser Leu Leu Ser Leu Leu Leu Asp His Arg

705 710 715 720

Asp Ala Gly Thr Cys Thr Arg Val Arg Leu Val Leu Ser Gly Gly Glu

725 730 735

Pro Leu Thr Asp Thr Leu Leu Glu Ser Val Glu Ser Thr Trp Ser Ala

740 745 750

Glu Val Ala Asn Leu Tyr Gly Pro Thr Glu Ala Thr Ile Asp Ala Thr

755 760 765

Gly His Arg Val Pro Arg Gly Asp Arg Thr Val Pro Val Pro Ile Gly

770 775 780

Arg Ala Val Ser Asn Thr Ala Val His Val Val Asp Ala Glu Leu Arg

785 790 795 800

Pro Val Pro Glu Gly Val Pro Gly Glu Ile Val Val Thr Gly Ala Gly

805 810 815

Val Ala Val Gly Tyr His Asp Arg Pro Ala Leu Thr Ala Ala Arg Phe

820 825 830

Leu Pro Ala Pro Phe Ala Asp Ala Ser Asp Asp Pro Gly Ala Thr Leu

835 840 845

Tyr Arg Thr Gly Asp Leu Gly Arg Arg Leu Pro Asp Gly Ser Val Gln

850 855 860

Phe Phe Gly Arg Val Asp Asp Gln Val Lys Ile Arg Gly His Arg Val

865 870 875 880

Glu Val Ser Glu Val Glu Ser Val Leu Lys Ala Leu Ala Gly Val Gln

885 890 895

Asp Ala Ala Val Val Ala Leu Asp Ala Gly Thr Glu Asn Ala Arg Leu

900 905 910

Ala Ala Ala Leu Val Leu Pro Ala Gly Ser Asp Ala Pro Ser Leu Glu

915 920 925

Asp Val Arg Ser Ala Leu Ala Gly Glu Leu Pro Asp Tyr Leu Val Pro

930 935 940

Asp Arg Phe Ala Val Val Asp Glu Leu Pro Leu Thr Ala Asn Gly Lys

945 950 955 960

Thr Asp Arg Arg Gly Val Ala Glu Leu Leu Ser Arg Gln Ala Ala Ala

965 970 975

Pro Val Ala Gly Gln Asp Gly Pro Thr Glu Pro Arg Asn Ala Leu Glu

980 985 990

Arg Ser Val Ala Glu Ala Phe Ala Ser Val Leu Arg Leu Pro Ala Ile

995 1000 1005

Asp Ile His Ala Asp Phe Phe Asp Val Gly Gly Thr Ser Leu Ile

1010 1015 1020

Leu Ala Lys Leu Ala Ser Leu Leu Gly Arg Lys His Asp Val Glu

1025 1030 1035

Ile Pro Leu His Glu Phe Phe Arg Thr Pro Thr Val Ala Gly Val

1040 1045 1050

Ser Glu Thr Ile Glu Val Tyr Arg Arg Glu Gly Leu Ala Gly Val

1055 1060 1065

Leu Gly Arg Lys His Ala Ala Thr Leu Glu Gly Asp Gly Thr Leu

1070 1075 1080

Asp Pro Ser Ile Ser Pro Glu Gly Leu Pro Glu Ala Glu Trp Glu

1085 1090 1095

Asn Pro Arg Arg Val Phe Leu Thr Gly Ala Thr Gly Tyr Leu Gly

1100 1105 1110

Leu His Leu Val Glu Gln Leu Leu Arg Arg Thr Asp Ala Glu Val

1115 1120 1125

Val Thr Leu Cys Arg Ala Arg Asp Glu Gln His Ala Leu Glu Arg

1130 1135 1140

Leu Lys Glu Gly Phe Ala Leu Tyr Glu Ile Asp Val Glu Asp Gln

1145 1150 1155

Leu His Arg Ile Ser Ala Val Ile Gly Asp Leu Ala Glu Pro Arg

1160 1165 1170

Leu Gly Leu Thr Gln Glu Gln Trp Asp Asp Leu Ala Ala Thr Val

1175 1180 1185

Asp Val Ile Tyr His Asn Gly Ala Leu Val Asn Phe Val Tyr Pro

1190 1195 1200

Tyr Ser Ala Leu Lys Ala Ala Asn Val Gly Gly Thr Gln Arg Val

1205 1210 1215

Leu Glu Leu Ala Cys Thr Thr Arg Leu Lys Ala Val His His Val

1220 1225 1230

Ser Thr Ile Asp Thr Leu Leu Ala Thr His Met Pro Arg Pro Phe

1235 1240 1245

Leu Glu Asn Asp Ala Pro Leu His Ser Ala Val Gly Val Pro Ala

1250 1255 1260

Gly Tyr Thr Gly Ser Lys Trp Val Ala Glu Lys Val Val Asp Glu

1265 1270 1275

Ala Arg Arg Arg Gly Ile Pro Val Thr Val Phe Arg Pro Gly Leu

1280 1285 1290

Ile Leu Gly His Thr Lys Asn Gly Ala Thr Gln Thr Ile Asp Tyr

1295 1300 1305

Leu Leu Val Ala Leu Arg Gly Phe Leu Pro Met Arg Ile Leu Pro

1310 1315 1320

Asp Tyr Pro Arg Ile Phe Asp Val Ile Pro Val Asp Tyr Val Ala

1325 1330 1335

Ser Ala Ile Val His Ile Ser Arg Lys Arg Glu Ala Ile Asp Gly

1340 1345 1350

Phe Tyr His Leu Phe Asn Pro Ala Pro Val Pro Leu Leu Thr Phe

1355 1360 1365

Cys Asp Trp Ile Lys Ser Tyr Gly Tyr Glu Phe Asp Ile Val Pro

1370 1375 1380

Phe Glu Glu Gly Arg Arg Arg Ala Leu Gly Val Gly Pro Ser His

1385 1390 1395

Leu Leu Tyr Pro Leu Val Pro Leu Ile Lys Asp Ala Glu Ala Glu

1400 1405 1410

Pro His Arg Ala Leu Asp Pro Lys Tyr Met Asp Glu Val Gln Pro

1415 1420 1425

Ala Leu Glu Cys Ala Glu Thr Leu Arg Met Leu Ala Gly Ser Asp

1430 1435 1440

Ile Ala Cys Pro Pro Thr Thr Glu Ala Asp Ala His Ala Val Met

1445 1450 1455

Asp Tyr Leu Val Arg Thr Gly Phe Met Pro Ala Pro Ala Asp Val

1460 1465 1470

Val His Asp Glu Pro Ser Ser Thr Leu Glu Glu Arg

1475 1480 1485

<210>19

<211>365

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Thr Ala Pro Ala Asp Thr Val His Pro Ala Gly Gln Pro Asp Tyr

1 5 10 15

Val Ala Gln Val Ala Thr Val Pro Phe Arg Leu Gly Arg Pro Glu Glu

20 25 30

Leu Pro Gly Thr Leu Asp Glu Leu Arg Ala Ala Val Ser Ala Arg Ala

35 40 45

Gly Glu Ala Val Arg Gly Leu Asn Arg Pro Gly Ala Arg Thr Asp Leu

50 55 60

Ala Ala Leu Leu Ala Ala Thr Glu Arg Thr Arg Ala Ala Leu Ala Pro

65 70 75 80

Val Gly Ala Gly Pro Val Gly Asp Asp Pro Ser Glu Ser Glu Ala Asn

85 90 95

Arg Asp Asn Asp Leu Ala Phe Gly Ile Val Arg Thr Arg Gly Pro Val

100 105 110

Ala Glu Leu Leu Val Asp Ala Ala Leu Ala Ala Leu Ala Gly Ile Leu

115 120 125

Glu Val Ala Val Asp Arg Gly Ser Asp Leu Glu Asp Ala Ala Trp Gln

130 135 140

Arg Phe Ile Gly Gly Phe Asp Ala Leu Leu Gly Trp Leu Ala Asp Pro

145 150 155 160

His Ser Ala Pro Arg Pro Ala Thr Val Pro Gly Ala Gly Pro Ala Gly

165 170 175

Pro Pro Val His Gln Asp Ala Leu Arg Arg Trp Val Arg Gly His His

180 185 190

Val Phe Met Val Leu Ala Gln Gly Cys Ala Leu Ala Thr Ala Cys Leu

195 200 205

Arg Asp Ser Ala Ala Arg Gly Asp Leu Pro Gly Ala Glu Ala Ser Ala

210 215 220

Ala Ala Ala Glu Ala Leu Met Arg Gly Cys Gln Gly Ala Leu Leu Tyr

225 230 235 240

Ala Gly Asp Ala Asn Arg Glu Gln Tyr Asn Glu Gln Ile Arg Pro Thr

245 250 255

Leu Met Pro Pro Val Ala Pro Pro Lys Met Ser Gly Leu His Trp Arg

260 265 270

Asp His Glu Val Leu Ile Lys Glu Leu Ala Gly Ser Arg Asp Ala Trp

275 280 285

Glu Trp Leu Ser Ala Gln Gly Ser Glu Arg Pro Ala Thr Phe Arg Ala

290 295 300

Ala Leu Ala Glu Thr Tyr Asp Ser His Ile Gly Val Cys Gly His Phe

305 310 315 320

Val Gly Asp Gln Ser Pro Ser Leu Leu Ala Ala Gln Gly Ser Thr Arg

325 330 335

Ser Ala Val Gly Val Ile Gly Gln Phe Arg Lys Ile Arg Leu Ser Ala

340 345 350

Leu Pro Glu Gln Pro Ala Thr Gln Gln Gly Glu Pro Ser

355 360 365

<210>20

<211>789

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Asn Arg Gly Ser Arg Ser Leu Arg Glu Ser Leu Ala Arg Tyr Gly

1 5 10 15

Pro Ala Ala Ala Ala Cys Ala Ala Ala Ala Tyr Ala Gly Thr Ser Leu

20 25 30

Thr Ser Pro Arg Pro Arg Pro Ala Ser Ala Pro Gln Glu Gln Phe Ser

35 40 45

Ala Glu Arg Ala Met Arg His Val Arg Ala Val Ala Ala Glu Pro His

50 55 60

Pro Val Gly Ser Arg Ala Ala Ala Arg Val Arg Asp Tyr Leu Leu Ala

65 70 75 80

Glu Leu Lys Asp Leu Gly Phe Glu Thr Glu Val Gln Glu Ala Val Ala

85 90 95

Ser His Asp Leu Gly Pro Thr Pro Tyr Gly Pro Arg Tyr Leu Thr Gly

100 105 110

Gly Val Val Arg Ash Val Ile Gly Arg Leu Pro Gly Ser Ile Pro Gly

115 120 125

His Ala Val Ala Leu Met Thr His Tyr Asp Ser Val Ser Gln Gly Pro

130 135 140

Gly Ala Ser Asp Ala Gly Val Pro Val Ala Ala Leu Leu Glu Ala Ala

145 150 155 160

Arg Ala Leu Arg Thr Asp Gly Val Gln Pro Val Asn Asp Leu Leu Val

165 170 175

Val Phe Thr Asp Gly Glu Glu Ala Gly Leu Leu Gly Ala Arg Ala Phe

180 185 190

Phe Asp Arg His Pro Leu Ala Lys Thr Val Gly Ala Ala Phe Asn Phe

195 200 205

Glu Ala Arg Gly Thr Glu Gly Pro Val Leu Met Phe Glu Ala Gly Pro

210 215 220

Gly Asn Gly Pro Met Leu Glu Glu Leu Ala Arg Thr Gly Val Pro Val

225 230 235 240

Phe Ala Ser Ser Leu Phe Asp Ala Ile Tyr Arg Arg Met Pro Asn Ala

245 250 255

Thr Asp Phe Ala Leu Val Lys Glu Arg Gly Ile Pro Gly Leu Asn Phe

260 265 270

Ala His Ile Gly Gly Phe Ala Ala Tyr His Gly Pro Leu Asp Asp Ile

275 280 285

Asp His Val Glu Pro Ser Ala Leu Gln His Gln Gly Glu Leu Ala Leu

290 295 300

Ala Leu Ala Arg Arg Leu Gly Ser Ala Asp Leu Ala Glu Leu Thr Gly

305 310 315 320

Glu Asp Ala Val Phe Phe Pro Ala Gly Arg Gly Lys Leu Ile Arg Val

325 330 335

Pro Gly Arg Ala Val Val Pro Leu Ala Val Ala Ala Gly Leu Leu Gly

340 345 350

Ala Gly Gly Leu Gly His Ala Leu Arg Lys Gly Gly Leu Thr Gly Arg

355 360 365

Arg Leu Ala Gly Thr Ser Ala Leu Leu Leu Gly Arg Ile Ala Ala Gly

370 375 380

Ala Ala Ala Gly Thr Ala Phe Thr Ala Ala Met Gly Lys Leu Ala Pro

385 390 395 400

Glu Phe Arg Arg His Gly Asp Phe His Gly Ser Gly Asp Ile Tyr Ala

405 410 415

Ala Val Ala Gly Leu Ala Cys Ala Ala Val Leu Ala Gly Pro Gly Asp

420 425 430

Ala Pro Ala Ala Ala Ala Arg Thr Ala Ala Ala Thr Leu Pro Leu Ala

435 440 445

Ala Ala Ser Ala Ala Leu Ala Ser Gly Leu Pro Gly Gly Ser Tyr Leu

450 455 460

Thr Thr Trp Pro Leu Ala Gly Ala Gly Leu Gly Leu Arg Leu Leu Asp

465 470 475 480

Ser Ala Gln Pro Thr Gly Thr Gly Arg Val Pro Ser Val Arg His Ala

485 490 495

Val Gly Ala Ala Ala Ala Ala Ala Pro Ala Ala Ala Leu Leu Val Pro

500 505 510

Leu Ser Arg Leu Leu Phe Lys Gly Leu Thr Pro Arg Leu Ala Gly Thr

515 520 525

Ala Val Val Ala Leu Gln Leu Cys Gly Glu Leu Ala Ala Pro Ala Leu

530 535 540

Ala Gly Leu Pro Arg Arg Thr Arg Arg Ala Ala Ala Ala Gly Gly Leu

545 550 555 560

Leu Leu Ala Gly Gly Val Ala Ala Arg Arg Phe Leu Arg Arg Gly Lys

565 570 575

Gly Ala Pro Pro Pro Glu Thr Ile Ser Tyr Leu Leu Asp Pro Val His

580 585 590

Ser Thr Ala Leu Trp Ile Ser Ser Asp Glu Ser Ala Ser Glu Arg Thr

595 600 605

Arg Ala Phe Leu Gly Glu Gln Pro Glu Lys Gly Arg Leu Pro Glu His

610 615 620

Phe Pro Gly Trp Gln Arg Asp Phe Leu His Ala Pro Ala Pro Arg Leu

625 630 635 640

Asp Leu Pro Ala Pro Glu Val Thr Val Leu Glu Asp Gly Ala Gly Glu

645 650 655

Ala Gly Arg Arg Arg Leu Leu Leu His Leu Arg Ser Pro Arg Gly Ala

660 665 670

Arg Gln Leu Ser Leu Ala Val Val Gly Ala Gly Val Arg Arg Trp Arg

675 680 685

Val Glu Asp Gly Pro Trp Ser Glu Pro Ser Pro Ala Glu Asp Asp Ser

690 695 700

Trp Glu Leu Trp Leu His Ala Val Pro Asp Pro Gly Ile Arg Val Glu

705 710 715 720

Leu Glu Leu Pro Ser Ser Gly Thr Arg Leu Arg Val Leu Asp Arg Ile

725 730 735

Asp Gly Leu Pro Gln Glu Leu Ala Ala Ala Pro Gly Thr Asp Pro Ala

740 745 750

Thr Gly Ile Pro Glu His Ile Ser Pro Ala Ala Ala Leu Asp Val Asp

755 760 765

Thr Trp Gly Asn Ala Ser Leu Val Val Arg Thr Val His Leu Pro Ala

770 775 780

Gln Glu Gly Pro Ala

785

<210>21

<211>73

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Thr Thr Lys Gly Val Thr Gly Leu Ala Thr Phe Leu Gly Asp Asp

1 5 10 15

Asp Leu Val Pro Cys Ala Val Val Val Asn Asp Ala Ala Gln Tyr Ser

20 25 30

Ile Trp Pro Gln Asp Arg Pro Val Pro Ala Gly Trp Arg Pro Ala Gly

35 40 45

Phe Glu Gly Pro Arg Ser Ala Cys Leu Glu His Ile Glu Thr Val Trp

50 55 60

Ala Gly Pro Thr Pro Ala Pro Ala Ala

65 70

<210>22

<211>366

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Thr Ile Ser Leu Glu Asn Thr Thr Val Gly Gln Asn Pro Ala Gly

1 5 10 15

Gly Pro Pro Thr Gly Lys Ala Pro Leu Asp Met Glu Gly Leu Ala Trp

20 25 30

Ile Leu Phe Gly Ala Ser Ala Phe Gln Tyr Leu Asn Ala Ala Cys Glu

35 40 45

Leu Asn Leu Phe Glu Leu Leu Glu Asn Lys Pro Gly Leu Thr Lys Pro

50 55 60

Gln Ile Gly Ala Glu Leu Gly Leu Ala Asp Arg Ala Asn Asp Ile Leu

65 70 75 80

Leu Leu Gly Ala Thr Ala Thr Gly Met Leu Thr Val Glu Asp Gly Arg

85 90 95

Tyr Gln Leu Ala Thr Val Leu Ala Glu Leu Leu Lys Thr Asp Asp Trp

100 105 110

Gln Arg Phe Lys Asp Thr Val Gly Phe Glu Gln Tyr Val Cys Tyr Glu

115 120 125

Gly Gln Ile Asp Phe Thr Glu Ser Leu Arg Ser Asn Ser Asn Val Gly

130 135 140

Leu Arg Arg Val Arg Gly Ser Gly Arg Asp Leu Tyr His Arg Leu His

145 150 155 160

Glu Asn Pro Gln Met Glu Gln Ala Phe Tyr Lys Tyr Met Arg Ser Trp

165 170 175

Ser Glu Leu Ala Asn Gln His Leu Val Glu Val Leu Asp Leu Ser Gly

180 185 190

Thr Ser Lys Leu Leu Asp Cys Gly Gly Gly Asp Ala Val Asn Ser Ile

195 200 205

Ala Leu Ala Gln Ala Asn Pro His Ile Glu Ala Gly Ile Leu Glu Ile

210 215 220

Pro Pro Thr Ala Pro Leu Thr Glu Lys Lys Ile Ala Glu Ala Gly Leu

225 230 235 240

Ser Asp Arg Ile Thr Val Lys Pro Gly Asp Met His Thr Asp Glu Phe

245 250 255

Pro Thr Gly Tyr Asp Thr Val Met Phe Ala His Gln Leu Val Ile Trp

260 265 270

Thr Pro Glu Glu Asn Thr Ala Leu Leu Arg Lys Ala Tyr Asn Ala Leu

275 280 285

Pro Glu Gly Gly Arg Val Ile Ile Phe Asn Ser Met Ser Asn Asp Glu

290 295 300

Gly Asp Gly Pro Val Val Ala Ala Leu Asp Ser Val Tyr Phe Ala Ala

305 310 315 320

Leu Pro Ala Glu Gly Gly Met Ile Tyr Ser Trp Ala Thr Tyr Glu Glu

325 330 335

Ser Leu Thr Lys Ala Gly Phe Asn Pro Glu Thr Phe Gln Arg Ile Asp

340 345 350

Phe Pro Gly Trp Thr Pro His Gly Val Ile Ile Ala Thr Lys

355 360 365

<210>23

<211>178

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Leu Thr Thr Pro Asp Glu Asp Lys Val Arg Ile Val Arg Trp Pro

1 5 10 15

Leu Glu Thr Ala Lys Arg Glu Arg Cys Arg Glu Ala Gly Ala Leu Arg

20 25 30

Ile Leu Leu Val Glu Asn Gly Ala Lys Pro Pro Leu Thr Ala Asp Leu

35 40 45

Arg Glu Asp Trp Val Arg Ala Pro Val Ser Arg Glu Asp Leu Ala Ala

50 55 60

Arg Val Thr Ala Leu Arg Ala Lys Ala Gly Ala Tyr Arg Val Pro Thr

65 70 75 80

Leu Asp Val Asp Gly Glu Leu Arg Phe Asp Gly Arg Ser Val Val Leu

85 90 95

Ser Arg Ala Glu Val Ala Ile Val Ser Val Leu Val Arg Asn Tyr Gly

100 105 110

Ser Leu Val Ala Arg Glu Ser Leu Ala His Leu Pro Asp Ala Pro Arg

115 120 125

Ala Gly Ser Arg Asn Ala Leu Asp Leu His Val Met Arg Ile Arg Arg

130 135 140

Arg Ile Ala Pro Leu Gly Leu Gly Ile Ser Thr Ala Trp Gly Arg Gly

145 150 155 160

Tyr Leu Leu Gln Glu Leu Thr Gln Glu Ala Gln Gly Gln Asp Ala Gly

165 170 175

Gly Thr

<210>24

<211>389

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Thr Ser Ile Gly Asn Thr Glu Asp Ala Gly Ser Leu Ile Ser Val

1 5 10 15

Thr Val Ile Gly Thr Gly Leu Ile Gly Thr Ser Ile Gly Leu Ala Leu

20 25 30

Thr Glu Tyr Gly Val Arg Val His Leu Gln Asp Val His Ala Glu Ser

35 40 45

Val Arg Ile Ala Glu Ala Arg Gly Ala Gly Thr Ala Leu Ala Pro Glu

50 55 60

Arg Gln Thr Asp Leu Ala Val Ile Ala Val Pro Pro Gln Ser Val Arg

65 70 75 80

Thr Ala Leu Ala Asp Ala Gln Arg Arg Arg Ile Ala Arg His Tyr Thr

85 90 95

Asp Val Ala Ser Val Lys Ala Ala Leu Pro Ala Ala Gly Ala Asp Leu

100 105 110

Gly Trp Asp Ala Ser Arg Phe Ile Gly Gly His Pro Ile Ala Gly Arg

115 120 125

Glu His Pro Gly Pro Gly Ala Ala Arg Ser Asp Met Phe Arg Ala Lys

130 135 140

Pro Trp Ile Leu Thr Pro Ser Ala Asp Thr Ser Gln Glu Thr Ile Ala

145 150 155 160

Thr Ala Arg Arg Leu Ile Gly Leu Cys Gly Ala Asp Val Val Thr Met

165 170 175

Gln Pro Glu Ala His Asp Arg Ala Val Ala Met Thr Ser His Leu Pro

180 185 190

His Leu Val Ser Ser Leu Met Ala Arg Leu Leu Arg Ser Tyr Asp His

195 200 205

Arg Ala Leu Glu Leu Ser Gly Ser Gly Leu Arg Asp Phe Thr Arg Ile

210 215 220

Ala Ala Gly Asp Pro Asp Leu Trp Thr Asp Ile Leu Gly Ser Asn Ala

225 230 235 240

Thr Ala Val Leu Gly Ala Leu Glu Asn Leu Gln His Asp Leu Glu Met

245 250 255

Thr Arg Arg Ile Leu His Ala Leu Ala Ala Ser Pro Gly Ala Ala Glu

260 265 270

Ala Ala Glu Ala Pro Ala Tyr Arg Glu Leu Leu Arg Arg Leu Leu Glu

275 280 285

Glu Gly Arg Ala Gly Gln Leu Leu Ile Pro Gln Lys Tyr Gly Ala Ala

290 295 300

Gly Arg Ala Tyr Asp Lys Leu Gln Val Leu Leu Thr Asp Arg Glu Gly

305 310 315 320

Glu Leu Ala Arg Leu Leu Ala Asp Val Ser Glu Leu Asn Val Asn Val

325 330 335

Glu Asp Leu Trp Ile Glu Asp Ser Ala Asp Ser Pro Val Gly Leu Ala

340 345 350

Val Leu Ser Val Asp Ala Gly Met Ala Ser Met Leu Ser Asp Trp Leu

355 360 365

Ala Glu Arg Gly Trp Pro Val Leu Gly Glu Ser Met Ser Ala Asp Pro

370 375 380

Val Ala Gly Arg Val

385

<210>25

<211>484

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Thr Asn Leu Ser Pro Ala Asp Phe Lys Val Ala Asp Leu Ser Leu

1 5 10 15

Ala Ala Phe Gly Arg Lys Glu Ile Thr Leu Ala Glu His Glu Met Pro

20 25 30

Gly Leu Met Ser Ile Arg Lys Glu Tyr Ala Ala Ala Gln Pro Leu Ala

35 40 45

Gly Ala Arg Ile Thr Gly Ser Leu His Met Thr Val Gln Thr Ala Val

50 55 60

Leu Ile Glu Thr Leu Val Ala Leu Gly Ala Asp Val Arg Trp Ala Ser

65 70 75 80

Cys Asn Ile Phe Ser Thr Gln Asp His Ala Ala Ala Ala Ile Ala Val

85 90 95

Gly Pro Asp Gly Thr Pro Glu Asn Pro Gln Gly Val Pro Val Phe Ala

100 105 110

Trp Lys Gly Glu Thr Leu Glu Glu Tyr Trp Trp Cys Thr Glu Gln Ala

115 120 125

Leu Thr Trp Pro Asn Thr Pro Thr Gly Gly Pro Asn Met Ile Leu Asp

130 135 140

Asp Gly Gly Asp Ala Thr Leu Leu Val His Lys Gly Val Glu Phe Glu

145 150 155 160

Lys Ala Gly Ala Ala Pro Asp Pro Ser Thr Ala Asp Ser Glu Glu Tyr

165 170 175

Gly His Ile Leu Thr Leu Leu Asn Arg Thr Leu Gly Glu Thr Pro Gln

180 185 190

Lys Trp Thr Gln Leu Ala Ser Glu Ile Arg Gly Val Thr Glu Glu Thr

195 200 205

Thr Thr Gly Val His Arg Leu Tyr Glu Met Met Ala Glu Gly Thr Leu

210 215 220

Leu Phe Pro Ala Ile Asn Val Asn Asp Ala Val Thr Lys Ser Lys Phe

225 230 235 240

Asp Asn Lys Tyr Gly Cys Arg His Ser Leu Ile Asp Gly Ile Asn Arg

245 250 255

Ala Thr Asp Val Leu Ile Gly Gly Lys Val Ala Val Val Cys Gly Tyr

260 265 270

Gly Asp Val Gly Lys Gly Cys Ala Glu Ser Leu Arg Gly Gln Gly Ala

275 280 285

Arg Val Ile Val Thr Glu Ile Asp Pro Ile Cys Ala Leu Gln Ala Ala

290 295 300

Met Asp Gly Tyr Gln Val Ala Thr Leu Asp Asp Val Val Glu Ile Ala

305 310 315 320

Asp Ile Phe Ile Thr Thr Thr Gly Asn Lys Asp Ile Ile Met Ala Ser

325 330 335

Asp Met Ala Lys Met Lys His Gln Ala Ile Val Gly Asn Ile Gly His

340 345 350

Phe Asp Asn Glu Ile Asp Met Ala Gly Leu Ala Lys Ile Glu Gly Ile

355 360 365

Val Lys Asp Glu Val Lys Pro Gln Val His Thr Trp Lys Phe Pro Asp

370 375 380

Gly Lys Val Leu Ile Val Leu Ser Glu Gly Arg Leu Leu Asn Leu Gly

385 390 395 400

Asn Ala Thr Gly His Pro Ser Phe Val Met Ser Asn Ser Phe Ala Asp

405 410 415

Gln Thr Leu Ala Gln Ile Glu Leu Phe Thr Lys Pro Ser Glu Tyr Pro

420 425 430

Thr Asp Val Tyr Val Leu Pro Lys His Leu Asp Glu Lys Val Ala Arg

435 440 445

Leu His Leu Asp Ala Leu Gly Val Lys Leu Thr Thr Leu Arg Pro Glu

450 455 460

Gln Ala Ala Tyr Ile Gly Val Gln Val Glu Gly Pro Tyr Lys Pro Asp

465 470 475 480

His Tyr Arg Tyr

<210>26

<211>313

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Ala Ala Glu Thr Ala Glu Phe Pro Tyr Arg Ala Arg Pro Gly Arg

1 5 10 15

Leu Gly Asp Leu Leu Arg Thr Gly Gln Lys Ser Tyr Ser Phe Glu Phe

20 25 30

Phe Pro Pro Lys Thr Ala Ala Gly Glu Arg Thr Leu Trp Arg Ala Ile

35 40 45

Arg Arg Ile Glu Ala Phe Ser Pro Ser Phe Val Ser Val Thr Tyr Gly

50 55 60

Ala Gly Gly Ser Ser Arg Asp Arg Thr Val Glu Val Thr Lys Arg Ile

65 70 75 80

Ala Ala Asp Thr Thr Leu Arg Pro Val Ala His Leu Thr Ala Val Gly

85 90 95

His Ser Ile Ala Gln Leu Arg His Ile Ile Gly Gln Tyr Ala Asp Ala

100 105 110

Gly Ile Arg Asp Val Leu Ala Leu Arg Gly Asp Pro Pro Gly Asp Pro

115 120 125

Lys Gly Glu Trp Val Pro His Pro Asp Gly Leu Arg His Ala Ala Glu

130 135 140

Leu Val Ser Leu Thr Arg Glu Leu Gly Asp Phe Thr Val Gly Val Ala

145 150 155 160

Ala Phe Pro Glu Arg His Pro Arg Ser Pro Asp Trp Asp Ser Asp Ile

165 170 175

Arg His Phe Val Ala Lys Cys Arg Ala Gly Ala Asp Tyr Ala Ile Thr

180 185 190

Gln Met Phe Phe Arg Val Asp Asp Tyr Leu Arg Leu Arg Asp Arg Val

195 200 205

Ala Val Val Gly Cys Asn Thr Pro Ile Ile Pro Ala Ile Met Pro Ala

210 215 220

Thr Asp Val Arg Gln Ile Arg Arg Phe Ala Glu Leu Ser Asn Ala Ser

225 230 235 240

Phe Pro Asp Glu Leu Ala Arg Arg Leu Asp Ala Ala Arg Asp Asp Pro

245 250 255

Ser Glu Gly His Arg Ile Gly Val Asp His Ala Thr Ala Met Ala Asp

260 265 270

Arg Leu Leu Ala Glu Gly Ala Pro Gly Leu His Tyr Ile Thr Leu Asn

275 280 285

Arg Ser Thr Ala Thr Val Glu Ile His Arg Asn Leu Gly Leu Ser Pro

290 295 300

Ala Arg Gln Pro Ala Ala Ala Arg His

305 310

<210>27

<211>1127

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Glu Asp Phe Gln Gln Leu Glu Gly Cys Asn Glu Val Leu Asn Val

1 5 10 15

Thr Arg Pro Asp Ile Val Ala Asn Val His Arg Glu Tyr Phe Ala Val

20 25 30

Gly Val Asp Cys Val Glu Thr Asn Thr Phe Gly Ala Asn Phe Ala Ala

35 40 45

Leu Ala Glu Tyr Asp Ile Pro Glu Arg Asn Phe Glu Leu Ser Glu Ala

50 55 60

Gly Ala Arg Ile Ala Arg Gln Val Ala Asp Glu Phe Thr Ala Ser Thr

65 70 75 80

Gly Arg Gln Arg Trp Val Leu Gly Ser Met Gly Pro Gly Thr Lys Leu

85 90 95

Pro Thr Leu Gly His Ile Asp Tyr Ala Gln Ile Arg Asp Ala Tyr Gln

100 105 110

Val Asn Ala Glu Gly Leu Ile Ser Gly Gly Ala Asp Ala Leu Leu Val

115 120 125

Glu Thr Thr Gln Asp Leu Leu Gln Thr Lys Ala Ala Ile Ile Gly Ala

130 135 140

Arg Arg Ala Leu Ser Asn Cys Gly Glu Asp Met Pro Val Ile Cys Ser

145 150 155 160

Val Thr Val Glu Ala Thr Gly Thr Met Leu Leu Gly Ser Glu Ile Gly

165 170 175

Ala Ala Leu Thr Ala Leu Glu Pro Leu Gly Ile Asp Met Ile Gly Leu

180 185 190

Asn Cys Ala Thr Gly Pro Ala Glu Met Ser Glu His Leu Arg Tyr Leu

195 200 205

Ala Arg Asn Ala Arg Ile Pro Leu Ser Cys Met Pro Asn Ala Gly Leu

210 215 220

Pro Val Leu Thr Lys Gln Gly Ala His Tyr Pro Leu Ser Ala Ala Glu

225 230 235 240

Leu Ala Asp Ala Gln Glu Thr Phe Val Arg Glu Tyr Gly Leu Ser Leu

245 250 255

Val Gly Gly Cys Cys Gly Thr Thr Pro Glu His Leu Arg Gln Val Val

260 265 270

Glu Arg Val Arg Gly Thr Ala Val Thr Glu Arg Ser Pro Gln Pro Glu

275 280 285

Pro Gly Ala Ala Ser Leu Tyr Gln Thr Val Pro Phe Arg Gln Asp Thr

290 295 300

Ser Tyr Met Ala Ile Gly Glu Arg Thr Asn Ala Asn Gly Ser Lys Lys

305 310 315 320

Phe Arg Glu Ala Met Leu Glu Ala Arg Trp Asp Asp Cys Val Glu Met

325 330 335

Ala Arg Asp Gln Ile Arg Glu Gly Ala His Met Leu Asp Leu Cys Val

340 345 350

Asp Tyr Val Gly Arg Asp Gly Val Ala Asp Met Gln Glu Leu Ala Gly

355 360 365

Arg Phe Ala Thr Ala Ser Thr Leu Pro Ile Val Leu Asp Ser Thr Glu

370 375 380

Val Pro Val Ile Gln Ala Gly Leu Glu Lys Leu Gly Gly Arg Ala Val

385 390 395 400

Ile Asn Ser Val Asn Tyr Glu Asp Gly Asp Gly Pro Glu Ser Arg Phe

405 410 415

Ala Lys Val Thr Arg Leu Ala Gln Glu His Gly Ala Ala Leu Ile Ala

420 425 430

Leu Thr Ile Asp Glu Glu Gly Gln Ala Arg Ser Val Glu His Lys Val

435 440 445

Ala Ile Ala Glu Arg Leu Ile Glu Asp Leu Thr Ser Asn Trp Gly Ile

450 455 460

Arg Glu Ser Asp Ile Leu Ile Asp Thr Leu Thr Phe Thr Ile Cys Thr

465 470 475 480

Gly Gln Glu Glu Ser Arg Lys Asp Gly Ile Ala Thr Ile Glu Ser Ile

485 490 495

Arg Glu Leu Lys Arg Arg His Pro Glu Val Gln Thr Thr Leu Gly Leu

500 505 510

Ser Asn Ile Ser Phe Gly Leu Asn Pro Ala Ala Arg Val Leu Leu Asn

515 520 525

Ser Val Phe Leu Asp Glu Cys Val Lys Ala Gly Leu Asp Ser Ala Ile

530 535 540

Val His Ala Ser Lys Ile Leu Pro Ile Ala Arg Phe Asp Glu Glu Gln

545 550 555 560

Val Ser Thr Ala Leu Asp Leu Ile Tyr Asp Arg Arg Glu Gly Asp Tyr

565 570 575

Asp Pro Leu Gln Lys Leu Met Ala Leu Phe Glu Gly Val Asn Thr Lys

580 585 590

Ser Leu Lys Ala Gly Arg Ala Glu Glu Leu Leu Ala Leu Pro Leu Asp

595 600 605

Glu Arg Leu Gln Arg Arg Ile Ile Asp Gly Glu Lys Asn Gly Leu Glu

610 615 620

Ala Asp Leu Asp Glu Ala Leu Ala Ser Arg Pro Ala Leu Asp Ile Val

625 630 635 640

Asn Asp Thr Leu Leu Glu Gly Met Lys Val Val Gly Glu Leu Phe Gly

645 650 655

Ser Gly Gln Met Gln Leu Pro Phe Val Leu Gln Ser Ala Glu Val Met

660 665 670

Lys Thr Ala Val Ala Tyr Leu Glu Pro His Met Glu Lys Thr Asp Ala

675 680 685

Asp Gly Lys Gly Thr Ile Val Leu Ala Thr Val Arg Gly Asp Val His

690 695 700

Asp Ile Gly Lys Asn Leu Val Asp Ile Ile Leu Thr Asn Asn Gly Tyr

705 710 715 720

Asn Val Val Asn Ile Gly Ile Lys Gln Pro Val Ser Ala Ile Leu Glu

725 730 735

Ala Ala Gln Glu His Lys Ala Asp Val Ile Gly Met Ser Gly Leu Leu

740 745 750

Val Lys Ser Thr Val Ile Met Lys Glu Asn Leu Glu Glu Leu Asn Gln

755 760 765

Arg Lys Leu Ala Ala Asp Tyr Pro Val Ile Leu Gly Gly Ala Ala Leu

770 775 780

Thr Arg Ala Tyr Val Glu Gln Asp Leu His Glu Ile Tyr Glu Gly Glu

785 790 795 800

Val Arg Tyr Ala Arg Asp Ala Phe Glu Gly Leu Arg Leu Met Asp Ala

805 810 815

Leu Ile Ala Val Lys Arg Gly Ile Pro Gly Ala Glu Leu Pro Pro Leu

820 825 830

Lys Gln Arg Arg Val Ala Lys Arg Asp Thr Pro Ala Leu Gln Val Glu

835 840 845

Glu Pro Glu Glu Thr Gly Gly Arg Ser Asp Val Ala Val Asp Asn Pro

850 855 860

Val Pro Thr Pro Pro Phe Trp Gly Thr Arg Val Ile Lys Gly Ile Pro

865 870 875 880

Leu Lys Asp Tyr Ala Ser Trp Leu Asp Glu Gly Ala Leu Phe Lys Gly

885 890 895

Gln Trp Gly Leu Lys Gln Ala Arg Ala Gly Gly Ala Thr Tyr Glu Glu

900 905 910

Leu Val Glu Ser Glu Gly Arg Pro Arg Leu Arg Gly Leu Leu Glu Lys

915 920 925

Leu His Thr Glu Asn Leu Leu Glu Ala Ala Val Val Tyr Gly Tyr Phe

930 935 940

Pro Cys Val Ser Lys Gly Glu Asp Leu Ile Ile Leu Asp Glu Gln Gly

945 950 955 960

Asn Glu Arg Thr Arg Phe Thr Phe Pro Arg Gln Arg Arg Gly Arg Arg

965 970 975

Leu Cys Leu Ala Asp Phe Phe Arg Pro Glu Glu Ser Gly Glu Thr Asp

980 985 990

Val Ile Gly Leu Gln Val Val Thr Val Gly Ser Arg Ile Gly Glu Ala

995 1000 1005

Thr Ala Lys Leu Phe Glu Ala Asp Ser Tyr Arg Glu Tyr Leu Glu

1010 1015 1020

Leu His Gly Leu Ser Val Gln Leu Ala Glu Ala Leu Ala Glu Tyr

1025 1030 1035

Trp His Ala Arg Val Arg Ala Glu Leu Gly Phe Gly Gly Glu Asp

1040 1045 1050

Pro Glu Lys Val Glu Asp Met Phe Asp Leu Lys Tyr Arg Gly Ala

1055 1060 1065

Arg Phe Ser Leu Gly Tyr Gly Ala Cys Pro Asp Leu Glu Asp Arg

1070 1075 1080

Ala Lys Ile Ala Asp Leu Leu Gln Pro Glu Arg Ile Gly Val His

1085 1090 1095

Leu Ser Glu Glu Phe Gln Leu His Pro Glu Gln Ser Thr Asp Ala

1100 1105 1110

Ile Val Ile His His Pro Asp Ala Thr Tyr Phe Asn Ala Arg

1115 1120 1125

<210>28

<211>325

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Arg Ile Ala Ile Thr Gly Ser Ile Ala Thr Asp His Leu Thr Thr

1 5 10 15

Phe Pro Gly Arg Phe Ala Glu Gln Leu Ile Pro Asp Arg Leu Asp Thr

20 25 30

Ile Ser Leu Ser Phe Leu Val Asp Asp Leu Glu Val Arg Arg Gly Gly

35 40 45

Val Ala Ala Asn Ile Ala Phe Gly Leu Gly Arg Leu Gly Phe Ser Pro

50 55 60

Leu Leu Val Gly Ala Ala Gly Thr Asp Phe Pro Glu Tyr Asp Ser Trp

65 70 75 80

Leu Arg Glu His Asp Thr Gly Ser Val Arg Val Ser Ala Thr Arg His

85 90 95

Thr Ala Arg Phe Met Cys Thr Thr Asp Ala Asp Gln Ash Gln Ile Ala

100 105 110

Ser Phe Tyr Thr Gly Ala Met Val Glu Ala Arg Glu Ile Asp Leu Ala

115 120 125

Asp Val Val Arg Arg Ser Gly Pro Gln Asp Arg Val Val Val Ala Pro

130 135 140

Asp Asp Pro Glu Ala Met Leu Arg His Ser Ala Lys Thr Arg Glu Leu

145 150 155 160

Gly Ile Pro Leu Ala Ala Asp Pro Ser Gln Gln Leu Ala Arg Leu Asn

165 170 175

Gly Asp Glu Thr Arg Arg Leu Val Asp Gly Ala Glu Leu Leu Phe Thr

180 185 190

Asn Glu Tyr Glu Ala Ala Leu Leu Gln Glu Cys Thr Gly Trp Thr Ala

195 200 205

His Gln Val Leu Gln Arg Val Gly Thr Trp Ile Ile Thr Cys Gly Gly

210 215 220

Asp Gly Val Thr Ile His Asn Thr Asp Thr Pro Ser Val Ser Val Pro

225 230 235 240

Ala Val Pro Ala Lys Asn Val Ala Asp Pro Thr Gly Val Gly Asp Gly

245 250 255

Phe Arg Ala Gly Tyr Leu Ala Gly Ile Gly Gln Gly Leu Ser Gln Glu

260 265 270

Arg Ala Ala Gln Leu Gly Cys Ala Leu Ala Thr Thr Val Leu Glu Ser

275 280 285

Val Gly Thr Gln Glu Tyr Arg Leu Asp Arg Ser Ser Leu Thr Ala Arg

290 295 300

Ile Ala Asp Ala Tyr Gly Asp Gly Pro Ala Ala Glu Ile Thr Pro His

305 310 315 320

Leu Gln Ala Lys Pro

325

<210>29

<211>401

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Gly Arg Leu Phe Thr Ser Glu Ser Val Thr Glu Gly His Pro Asp

1 5 10 15

Lys Ile Ala Asp Arg Ile Ser Asp Thr Ile Leu Asp Ala Leu Leu Arg

20 25 30

Glu Asp Pro Ser Ser Arg Val Ala Val Glu Thr Leu Ile Thr Thr Gly

35 40 45

Gln Val His Val Ala Gly Glu Val Thr Thr Lys Ala Tyr Ala Pro Ile

50 55 60

Ala Gln Leu Val Arg Asp Ala Ile Leu Asp Ile Gly Tyr Asp Ser Ser

65 70 75 80

Ala Lys Gly Phe Asp Gly Ala Ser Cys Gly Val Ser Ile Ser Ile Gly

85 90 95

Ala Gln Ser Pro Asp Ile Ala Gln Gly Val Asp Thr Ala Tyr Glu Gln

100 105 110

Arg Val Glu Gly Glu Gly Asp Glu Leu Asp Lys Gln Gly Ala Gly Asp

115 120 125

Gln Gly Leu Met Phe Gly Tyr Ala Cys Asp Glu Thr Pro Glu Leu Met

130 135 140

Pro Leu Pro Ile His Leu Ala His Arg Leu Ser Arg Arg Leu Ser Glu

145 150 155 160

Val Arg Lys Asp Gly Thr Ile Pro Tyr Leu Arg Pro Asp Gly Lys Thr

165 170 175

Gln Val Thr Ile Glu Tyr Asp Gly Asp Arg Ala Val Arg Leu Asp Thr

180 185 190

Val Val Val Ser Thr Gln His Ala Ala Asp Val Asp Leu Asp Ala Leu

195 200 205

Leu Thr Leu Asp Val Arg Glu His Val Val Glu His Val Leu Lys Gln

210 215 220

Leu Val Gln Glu Gly Ile Lys Leu Asp Thr Asp Gly Tyr Arg Leu Leu

225 230 235 240

Val Asn Pro Thr Gly Arg Phe Glu Ile Gly Gly Pro Met Gly Asp Ala

245 250 255

Gly Leu Thr Gly Arg Lys Ile Ile Ile Asp Thr Tyr Gly Gly Met Ala

260 265 270

Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp

275 280 285

Arg Ser Ala Ala Tyr Ala Met Arg Trp Val Ala Lys Asn Val Val Ala

290 295 300

Ala Gly Leu Ala Ser Arg Cys Glu Val Gln Val Ala Tyr Ala Ile Gly

305 310 315 320

Lys Ala Glu Pro Val Gly Leu Phe Val Glu Thr Phe Gly Thr Ala Lys

325 330 335

Val Asp Thr Ser Lys Ile Glu Glu Ala Ile Gly Gln Val Phe Asp Leu

340 345 350

Arg Pro Ala Ala Ile Ile Arg Asp Leu Asp Leu Leu Arg Pro Ile Tyr

355 360 365

Ala Gln Thr Ala Ala Tyr Gly His Phe Gly Arg Glu Leu Thr Asp Phe

370 375 380

Thr Trp Glu Arg Thr Asp Arg Ala Asp Ala Leu Arg Arg Val Ala Gly

385 390 395 400

Val

<210>30

<211>334

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Met Arg Arg Met Leu Tyr Ala Gln Leu Pro Ser Arg Ala Leu Val Val

1 5 10 15

Val Ala Gln Leu Gly Ile Ala Asp Ile Leu Ala Glu Gly Pro Ala Asp

20 25 30

Ile Ser Thr Leu Ala Glu Arg Thr Ser Thr Asp Ala Val Ala Leu Ala

35 40 45

Arg Leu Leu Arg Gly Leu Ala Val Phe Gly Val Phe Glu Glu Gly Ala

50 55 60

Glu Gln Val Tyr Ser Leu Thr Pro Leu Gly Glu Ala Leu Thr Ser Gly

65 70 75 80

His Pro Ala Ser Ala Leu Pro Ser Ala Thr Leu Val Ala Gly Gln Phe

85 90 95

Gly Ala Ala Trp Gly Asp Leu Leu Glu Thr Val Arg Thr Gly Gln Ser

100 105 110

Pro Phe Glu Arg Ser Arg Gly Val Ser Leu Phe Thr His Met Glu Gln

115 120 125

Asp Glu Glu Leu Arg Ala Val Phe Asp Asp Ser Gln Gly Arg Gly Leu

130 135 140

Ala Leu Glu Leu Asp Glu Ile Leu Arg Ala Ile Asp Phe Ser Ala Tyr

145 150 155 160

Pro Thr Val Val Asp Val Gly Gly Ser Asp Gly Thr Phe Leu Arg Arg

165 170 175

Ile Leu Ser Ala His Pro Asp Ile Ser Gly Ile Val Phe Asp Leu Pro

180 185 190

Gly Ser Thr Ser Leu Gln Ala Glu Arg Pro Thr Ala Asp Pro Leu Glu

195 200 205

Gly Arg Tyr Ser Val Ala Thr Gly Asp Phe Phe Asp Ser Leu Pro Glu

210 215 220

Gly Gly Asp Leu Tyr Leu Leu Ser His Ile Leu His Asp Trp Asp Asp

225 230 235 240

Asp Arg Ala Val Gln Ile Leu Arg Thr Cys Arg Ala Ala Met Ser Asp

245 250 255

Asp Ala Thr Leu Met Val Val Asp Leu Ile Ala Ala Asn Arg Gly Gln

260 265 270

Arg Asp Glu Arg Leu His Thr Ala Ala Leu Met Asp Leu Tyr Met Leu

275 280 285

Ser Leu Phe Gly Gly Asn Gly Gly Gln Glu Arg Thr Ala Ala Gln Val

290 295 300

Glu Val Leu Leu Ser Lys Ala Gly Phe Arg Ile Thr Arg Val Asp Ser

305 310 315 320

Leu Pro Ser Gly Met Asn Val Ile Arg Ala Val Arg Ala Ala

325 330

<210>31

<211>376

<212>PRT

<213>Streptomyces lavendulae 314(NRRL11002)

<400>1

Val Thr Glu Asn Pro Arg Arg Pro Pro Leu Val Val Gly Ala Ser Ala

1 5 10 15

Ala Gly Leu Thr Val Ala His Glu Leu Ala Arg Arg Gly Leu Pro Val

20 25 30

Arg Val Ile Glu Ala Ser Ala Gly Pro Ala Thr Thr Gly Pro Ala Val

35 40 45

Thr Leu Gln Pro Arg Thr Leu Glu Val Leu Asp Gln Met Ala Val Ile

50 55 60

Asp Gly Phe Leu Arg His Gly Arg Lys Gly Glu Thr Leu Ala Asp His

65 70 75 80

Thr Thr Leu Pro Thr Leu His Pro Tyr Thr Leu Val Ile Asp Arg Ala

85 90 95

Arg Ala Ala Ala Leu Leu Arg Glu Ala Val Val Asp Leu Gly Val Ser

100 105 110

Val Glu Gly Gly Ala Arg Asp Arg His Pro Ala Ala Pro Ala Cys Leu

115 120 125

Ile Thr Cys Asp Gly Phe Pro Gln Arg Ser Asp Pro Val Gly Arg Arg

130 135 140

Leu Thr Val Val Ala Asp His Asp Pro Asp Thr Gly Ile Arg Gln Ala

145 150 155 160

Tyr Asn Leu Ala Trp Lys Leu Ala Met Val Glu Gln Gly Leu Val Gly

165 170 175

Pro Arg Leu Leu Asp Thr Tyr Asp Glu Glu His Pro Ala Glu Arg Gly

180 185 190

His Arg Pro Gly Ile Arg Ala Leu Leu Asn Ala Val Pro Ala Leu Arg

195 200 205

Arg Thr His Pro Ser His Arg Ser Gly Leu Gly Leu Thr Tyr Arg Asn

210 215 220

Ser Ser Leu Thr Thr Arg Ash Thr Leu Asp Phe Leu Ser Gly Tyr His

225 230 235 240

Val Gly Ala Val Gly Phe Glu Pro Thr Pro Val Pro Ala Pro Gly Ser

245 250 255

Arg Val Thr Glu Ala Gly Pro Ala Cys Asn Leu Leu Arg Ser Glu Leu

260 265 270

Arg Asp Pro Arg Trp Ser Leu Leu Phe Ala Thr Asp Pro Ala Gly Thr

275 280 285

Gln Gly Ser Pro Asp Ser Val Ala Ala Thr Ala Ala Ala Gln Tyr Ser

290 295 300

Ala Tyr Leu Ser Val Arg Ile Leu Gly Ala Ala Ser Leu Pro Asp Pro

305 310 315 320

Leu Cys Arg Leu Arg Ala Ala Leu Gly Thr Pro Pro Gly Gly Trp Leu

325 330 335

Leu Ile Arg Pro Asp Gly Tyr Val Ala Ala Arg Gly Ser Leu Leu Thr

340 345 350

Ser Gln Ala Leu Asp Arg Ala Leu Ser Pro Leu Ser Val Ala Pro Leu

355 360 365

Arg Ile Gly Ala Asn Pro Cys Gly

370 375

Claims (10)

1. the biosynthetic gene cluster of the antibiotic-saframycin (Saframycin) of a kind of antitumor activity, it is characterized in that 30 genes involved in coding saframycin biosynthesis are specifically: 1) Non-ribosomal polypeptide synthase genes, namely sfmA, sfmB, and sfmC, a total of 3 genes: sfmA is located at bases 16971-22481 of the nucleotide sequence of the gene cluster, with a length of 5511 base pairs, encoding a non-ribosomal polypeptide synthetase, 1836 amino acids; sfmB is located at the 22618-25866th base of the gene cluster nucleotide sequence, with a length of 3249 base pairs, encoding a non-ribosomal polypeptide synthetase, 1082 amino acids; sfmC is located at the 25863-30320th base of the nucleotide sequence of the gene cluster, with a length of 4458 base pairs, encoding a non-ribosomal polypeptide synthetase, 1485 amino acids; 2) The biosynthetic genes of the precursor molecule 3-hydroxy-5-methyl-O-methyl-tyrosine, namely 4 genes sfmO5, sfmM2, sfmM3, and sfmD: sfmO5 is located at the 37069-35900th base of the nucleotide sequence of the gene cluster, with a length of 1170 base pairs, encoding prephenate dehydrogenase, and 389 amino acids; sfmM2 is located at the 34031-35131 base of the nucleotide sequence of the gene cluster, with a length of 1101 base pairs, encoding a methylase, and 366 amino acids; sfmM3 is located at bases 46245-45241 in the nucleotide sequence of the gene cluster, with a length of 1005 base pairs, encoding a methylase, and 334 amino acids; sfmD is located at the 30317th-31414th base of the gene cluster nucleotide sequence, with a length of 1098 base pairs, encoding 5-methyl-O-methyl-tyrosine-3-hydroxylase, 365 amino acids; 3) Peptide backbone cyclase genes, namely sfmcy1 and sfmCy2, a total of 2 genes: sfmCy1 is located at the 3166-4704th base of the gene cluster nucleotide sequence, with a length of 1539 base pairs, encoding redox cyclase, 512 amino acids; sfmCy2 is located at bases 15388-13874 of the nucleotide sequence of the gene cluster, with a length of 1515 base pairs, encoding redox cyclase, and 504 amino acids; 4) Modified genes of safromycin, namely sfmM1, sfmO1, sfmO2, sfmO3, sfmK, sfmO4, sfmO6, a total of 7 genes: sfmM1 is located at bases 11059-10460 in the nucleotide sequence of the gene cluster, with a length of 600 base pairs, encoding N-methylase, and 199 amino acids; sfmO1 is located at the 1370th-2284th base of the nucleotide sequence of the gene cluster, with a length of 915 base pairs, encoding an oxidoreductase, and 304 amino acids; sfmO2 is located at the 10384-8819th base of the nucleotide sequence of the gene cluster, with a length of 1566 base pairs, encoding a monooxygenase, and 521 amino acids; sfmO3 is located at bases 12372-13559 of the nucleotide sequence of the gene cluster, with a length of 1188 base pairs, encoding cytochrome P-450 hydroxylase, and 395 amino acids; sfmK is located at the 13613-13798th base of the gene cluster nucleotide sequence, with a length of 186 base pairs, encoding ferredoxin, and 61 amino acids; sfmO4 is located at bases 16947-15520 of the nucleotide sequence of the gene cluster, with a length of 1428 base pairs, encoding cytochrome P-450 hydroxylase, and 475 amino acids; sfmO6 is located at bases 46553-47683 of the nucleotide sequence of the gene cluster, with a length of 1131 base pairs, encoding a FAD-dependent oxidase, and 376 amino acids; 5) S-adenylated methionine synthesis-related enzyme genes, namely sfmS1, sfmS2, sfmS3, sfmS4, sfmS5, a total of 5 genes: sfmS1 is located at the 38550-37096th base of the gene cluster nucleotide sequence, with a length of 1455 base pairs, encoding S-adenosyl-L-homocysteine hydrolase, 484 amino acids; sfmS2 is located at the 39521-38580th base of the nucleotide sequence of the gene cluster, with a length of 942 base pairs, encoding methylenetetrahydrofolate reductase, and 313 amino acids; sfmS3 is located at the 42940-39557th base of the gene cluster nucleotide sequence, with a length of 3384 base pairs, encoding 5-methyltetrahydrofolate-homocysteine S-methyltransferase, 1127 amino acids; sfmS4 is located at the 44013-43036th base of the nucleotide sequence of the gene cluster, with a length of 978 base pairs, encoding a carbohydrate kinase, and 325 amino acids; sfmS5 is located at the 45220-44015th base of the nucleotide sequence of the gene cluster, with a length of 1206 base pairs, encoding S-adenosylmethionine synthetase, 401 amino acids; 6) Regulatory genes, namely sfmR1, sfmR2, and sfmR3, a total of 3 genes: sfmR1 is located at bases 786-1373 of the nucleotide sequence of the gene cluster, with a length of 588 base pairs, encoding TetR family transcription regulators, and 195 amino acids; sfmR2 is located at the 2487-2996th base of the nucleotide sequence of the gene cluster, with a length of 510 base pairs, encoding a transcriptional regulator, and 169 amino acids; sfmR3 is located at the 35223-35759th base of the nucleotide sequence of the gene cluster, with a length of 537 base pairs, encoding a transcriptional regulator, and 178 amino acids; 7) Resistance genes, namely sfmG and sfmH, a total of 2 genes: sfmG is located at the 4738-6177th base of the nucleotide sequence of the gene cluster, with a length of 1440 base pairs, encoding a translocase, and 479 amino acids; sfmH is located at the 8748-6289th base of the nucleotide sequence of the gene cluster, with a length of 2460 base pairs, encoding an ultraviolet repair protein, and 819 amino acids; 8) Also includes 4 genes with unclear functions, namely srmE, sfmF, sfmI, sfmJ: sfmE is located at the 31411-33780th base of the nucleotide sequence of the gene cluster, with a length of 2370 base pairs, encoding a peptidase, and 789 amino acids; sfmF is located at the 33777-33998th base of the nucleotide sequence of the gene cluster, with a length of 222 base pairs, encoding a MbtH-like protein, and 73 amino acids; sfmI is located at bases 11694-11212 in the nucleotide sequence of the gene cluster, with a length of 483 base pairs, encoding an unknown functional protein, and 160 amino acids; sfmJ is located at bases 12188-11691 of the nucleotide sequence of the gene cluster, with a length of 498 base pairs, encoding a pyridoxamine-5'-phosphate oxidase-related protein with 165 amino acids. 2. The biosynthetic gene cluster of safromycin according to claim 1, characterized in that the encoded non-ribosomal polypeptide synthetase comprises modules or domains: acyl-CoA ligase domain AL, peptidyl condensing enzyme Domain C, adenylase domain A, peptidyl carrier protein PCP, anoreductase domain RE. 3. A use of the biosynthetic gene cluster of safromycin according to claim 1, its encoded protein catalyzes the synthesis of antibiotic safromycin. 4. The purposes of the biosynthetic gene cluster of safromycin according to claim 3, its encoded protein catalyzes the synthesis of S-adenylated methionine. 5. The purposes of the biosynthetic gene cluster of safromycin according to claim 3, its encoded protein catalyzes the synthesis of double tetrahydroisoquinoline alkaloid core skeleton structure. 6. the purposes of the biosynthetic gene cluster of safromycin according to claim 3, its coding protein catalyzes the synthesis of 3-hydroxyl-5-methyl-O-methyltyrosine or 5-methyl-O- Methyltyrosine. 7. the purposes of the biosynthetic gene cluster of saffron according to claim 3, wherein gene carries out heterologous expression in Pseudomonas fluorescens A2-2 and can catalyze safracin B (safracin B ) biotransformation to obtain the oxidized product aminated safromycin S, which can be treated with potassium cyanide to obtain safromycin Y3. 8. The purposes of the biosynthetic gene cluster of safrothromycin according to claim 3, the mutants obtained by genetic modification of the genes therein make the output of safrothromycin change through biological fermentation. 9. The purposes of the biosynthetic gene cluster of safromycin according to claim 3, wherein the NRPS gene obtains heterologous expression in Escherichia coli, and the recombinant proteins SfmA, SfmB and SfmC obtained through separation and purification recognize and activate respectively Alanine, Glycine and 3-Hydroxy-5-methyl-O-methyltyrosine. 10. the purposes of the biosynthetic gene cluster of safromycin according to claim 3, wherein oxidoreductase, cyclosynthase gene sfmO1, sfmO2, sfmO3, sfmO4, sfmO6, sfmCy1, sfmCy2, methylase gene sfmM1, sfmM2, sfmM3 and 5-methyl-O-methyl-tyrosine-3-hydroxylase gene sfmD were all heterologously expressed in Escherichia coli, and the recombinant proteins SfmO1, SfmO3, SfmO4, SfmM2, SfmM3, and SfmD.

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