US20020164742A1

US20020164742A1 - DNA encoding methymycin and pikromycin

Info

Publication number: US20020164742A1
Application number: US09/860,846
Authority: US
Inventors: David Sherman; Hung-Wen Liu; Yongquan Xue; Lishan Zhao
Original assignee: University of Minnesota
Current assignee: University of Minnesota
Priority date: 1998-06-26
Filing date: 2001-05-18
Publication date: 2002-11-07
Also published as: US20020110897A1; US6265202B1; WO2000000620A3; CA2332129A1; JP2002536959A; US20030087405A1; EP1090125A2; AU4719999A; WO2000000620A2

Abstract

A novel pathway for the synthesis of polyhydroxyalkanoates is provided. A method of synthesizing a recombinant polyhydroxyalkanoate monomer synthase is also provided. These recombinant polyhydroxyalkanoate synthases are derived from multifunctional fatty acid synthases or polyketide synthases and generate hydroxyacyl acids capable of polymerization by a polyhydroxyalkanoate synthase. Also provided is a biosynthetic gene cluster for methymycin and pikomycin as well as a biosynthetic gene cluster for desosamine.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. application Ser. No. 60/008,847, filed Dec. 19, 1995, the disclosure of which is incorporated by reference herein.[0001]

STATEMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with a grant from the Government of the United States of America (grants GM48562, GM35906 and GM54346 from the National Institutes of Health and a grant from the Office of Naval Research). The Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Polyhydroxyalkanoates (PHAs) are one class of biodegradable polymers. The first identified member of the PHAs thermoplastics was polyhydroxybutyrate (PHB), the polymeric ester of D(−)-3-hydroxybutyrate. The biosynthetic pathway of PHB in the gram negative bacterium Alcaligenes eutrophus is depicted in FIG. 1. PHAs related to PHB differ in the structure of the pendant arm, R (FIG. 2). For example, R=CH₃in PHB, while R=CH₂CH₃in polyhydroxyvalerate, and R=(CH₂)₄CH₃in polyhydroxyoctanoate.

The genes responsible for PHB synthesis in A. eutrophus have been cloned and sequenced. (Peoples et al., J. Biol. Chem., 264, 15293 (1989); Peoples et al., J. Biol. Chem., 264, 15298 (1989)). Three enzymes: β-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), and PHB synthase (phbC) are involved in the conversion of acetyl-CoA to PHB. The PHB synthase gene encodes a protein of M_r=63,900 which is active when introduced into E. coli (Peoples et al., J. Biol Chem., 264, 15298 (1989)).

Although PHB represents the archetypical form of a biodegradable thermoplastic, its physical properties preclude significant use of the homopolymer form. Pure PHB is highly crystalline and, thus, very brittle. However, unique physical properties resulting form the structural characteristics of the R groups in a PHA copolymer may result in a polymer with more desirable characteristics. These characteristics include altered crystallinity, UV weathering resistance, glass to rubber transition temperature (T _g), melting temperature of the crystalline phase, rigidity ard durability (Holmes et al., EPO 00052 459; Anderson et al., Microbiol. Rev., 54, 450 (1990)). Thus, these polyesters behave as thermoplastics, with melting temperatures of 50-180° C., which can be processed by conventional extension and molding equipment.

Traditional strategies for producing random PHA copolymers involve feeding short- and long-chain fatty acid monomers to bacterial cultures. However, this technology is limited by the monomer units which can be incorporated into a polymer by the endogenous PHA synthase and the expense of manufacturing PHAs by existing fermentation methods (Haywood et al., FEMS Microbiol, Lett, 51, 1 (1989); Poi et al., Int. J. Biol. Macromol., 12, 106 (1990); Steinbuchel et al., In: Novel Biomaterials from Biological Sources. D. Byron (ed.), MacMillan, NY (1991); Valentin et al., Appl. Microbiol. Biotechnical, 36, 507 (1992)).

The production of diverse hydroxyacylCoA monomers for homo- and co-polymeric PHAs also occurs in some bacteria through the reduction and condensation pathway of fatty acids. This pathway employs a fatty acid synthase (FAS) which condenses malonate and acetate. The resulting β-keto group undergoes three processing steps, β-keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit. However, this pathway provides only a limited array of PHA monomers which vary in alkyl chain length but not in the degree of alkyl group branching, saturation, or functionalization along the acyl chain.

The biosynthesis of polyketides, such as erythromycin, is mechanistically related to formation of long-chain fatty acids. However, polyketides, in contrast to FASs, retain ketone, hydroxyl, or olefinic finctions and contain methyl or ethyl side groups interspersed along an acyl chain comparable in length to that of common fatty acids. This asymmetry in structure implies that the polyketide synthase (PKS), the enzyme system responsible for formation of these molecules, although mechanistically related to a FAS, results in an end product that is structurally very different than that of a long-chain fatty acid.

Because PHAs are biodegradable polymers that have the versatility to replace petrochemical-based thermoplastics, it is desirable that new, more economical methods be provided for the production of defined PHAs. Thus, what is needed are methods to produce recombinant PHA monomer synthases for the generation of PHA polymers.

SUMMARY OF THE INVENTION

The present invention provides a method of preparing a polyhydroxyalkanoate synthase. The method comprises introducing an expression cassette into a non-plant eukaryotic cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate synthase, e.g., a polyhydroxybutyrate synthase, operably linked to a promoter functional in the non-plant eukaryotic cell. The DNA molecule may be obtained from a bacterium such as Alcaligenes eutrophus. The DNA molecule encoding the polyhydroxyalkanoate synthase is then expressed in the cell. Thus, another embodiment of the invention provides a purified recombinant polyhydroxybutyrate synthase isolated from a host cell which expresses the synthase.

Another embodiment of the invention is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first expression cassette and a second expression cassette into a eukaryotic cell. The first expression cassette comprises a DNA segment encoding a fatty acid synthase in which the dehydrase activity has been inactivated that is operably linked to a promoter functional in the eukaryotic cell, e.g., an insect cell. The inactivation preferably is via a mutation in the catalytic site of the dehydrase. The second expression cassette comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the eukaryotic cell. The expression cassettes may be on the same or separate molecules. The DNA segments in the expression cassettes are expressed in the cell so as to yield a polyhydroxyalkanoate polymer.

Another embodiment of the invention is a baculovirus expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in an insect cell. Preferably, the nucleic acid molecule is obtained from a bacterium, e.g., Alcaligenes eutrophus.

The present invention also provides an expression cassette comprising a nucleic acid molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell. The nucleic acid molecule comprises a plurality of DNA segments. Thus, the nucleic acid molecule comprises at least a first and a second DNA segment. No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. The first DNA segment encodes a first module and the second DNA segment encodes a second module, wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. The source of at least one DNA segment is preferably bacterial DNA. It is preferred that the first DNA segment encodes the first module form the vep gene cluster and the second DNA segment encodes module 7 from the tyl P gene cluster. The nucleic acid molecule may optionally further comprise a third DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second nucleic acid molecule encoding a polyhydroxyalkanoate synthase may be introduced into the host cell.

Also provided is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a first module and the second DNA segment encodes a second module. No more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. Also, it is preferred that no more than one module is derived from the gene cluster from Streptomyces hygroscopicus that encodes rapamycin or the gene cluster that encodes spiramycin. Together the DNA segments encode a recombinant polyhydroxyalkanoate monomer synthase. A preferred embodiment of the invention employs a first DNA segment derived from the vep gene cluster of Streptomyces. Another preferred embodiment of the invention employs a second DNA segment derived from the tyl gene cluster of Streptomyces. A further preferred embodiment of the isolated DNA molecule of the invention includes a DNA segment encoding a polyhydroxyalkanoate synthase.

Yet another preferred embodiment is an isolated DNA molecule of the invention wherein the second DNA segment comprises a DNA encoding a thioesterase which is located at the 3′ end of the second DNA segment. More preferably, the second DNA segment comprises a DNA encoding an acyl carrier protein which is located 5′ to the DNA encoding the thioesterase. Even more preferably, the second DNA segment comprises a DNA encoding a linker region, wherein the DNA encoding the linker region is located between the DNA encoding the acyl carrier protein and the DNA encoding the thioesterase.

Another embodiment of the isolated DNA molecule of the invention comprises a first DNA segment comprising DNA encoding two acyl transferases, wherein the DNA encoding the first acyl transferase is 5′ to the DNA encoding the second acyl transferase. Preferably, the second acyl transferase adds acyl groups to malonylCoA.

Other embodiments of the isolated DNA molecule include a first DNA segment comprising a DNA encoding a dehydrase, a first DNA segment comprising a DNA encoding a dehydrase and an enoyl reductase, a second DNA segment comprising a DNA encoding an inactive dehydrase, or a first DNA segment comprising a DNA encoding an acyl transferase. A preferred acyl transferase binds an acyl CoA substrate.

A further embodiment of the isolated DNA molecule includes a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. Also preferably, at least one DNA segment is derived from the vep gene cluster or the tyl gene cluster. In one preferred embodiment, the first DNA segment encodes the first module from the vep gene cluster and the second DNA segment encodes module 7 from the tyl gene cluster.

Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing a DNA molecule into a host cell. The DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase, which synthase comprises at least a first module and a second module, is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Preferably, the first DNA segment encodes the first module from the vep gene cluster and the second DNA segment encodes module 7 from the tyl P gene cluster. Also preferably, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase.

Also provided is a method of preparing a polyhydroxyalkanoate polymer. The method comprises introducing a first DNA molecule and a second DNA molecule into a host cell. The first DNA molecule comprises a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase. The recombinant polyhydroxyalkanoate monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first module and a second module. The first DNA molecule is operably linked to a promoter functional in a host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The DNAs encoding the recombinant polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate synthase are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.

Yet another embodiment of the invention is an isolated and purified DNA molecule. The DNA molecule comprises a plurality of DNA segments. That is, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase and the second DNA segment encodes a module of a polyketide synthase. A preferred embodiment of the invention employs a second DNA segment encoding a module which comprises a β-ketoacyl synthase amino-terminal to an acyltransferase which is amino-terminal to a ketoreductase which is amino-terminal to an acyl carrier protein which is amino-terminal to a thioesterase. Other preferred embodiments of the invention include a second DNA segment that is 3′ to the DNA encoding the fatty acid synthase, a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module of a polyketide synthase, or a second DNA segment that is separated from the first DNA segment by a DNA encoding a linker region. Preferred linker regions include the linker region from tyl ORF1 ACP ₁-KS₂, tyl ORF1 ACP₂-KS₃, tyl ORF3 ACP₅-KS₆, eryA ORF1 ACP₁-KS₁, eryA ORF1 ACP₂-KS₂, eryA ORF2 ACP₃-KS₄, and eryA ORF2 ACP₅-KS₆.

The invention also provides a method of preparing a polyhydroxyalkanoate monomer. The method comprises introducing a DNA molecule comprising a plurality of DNA segments into a host cell, e.g., an insect cell, a Streptomyces cell or a Pseudomonas cell. Thus, the DNA molecule comprises at least a first and a second DNA segment. The first DNA segment encodes a fatty acid synthase operably linked to a promoter functional in the host cell. Preferably, the fatty acid synthase is eukaryotic in origin. Alternatively, the fatty acid synthase is prokaryotic in origin. The second DNA segment encodes a polyketide synthase. Preferably, the second DNA segment encodes the tyl module F. The second DNA segment is located 3′ to the first DNA segment. The first DNA segment is linked to the second DNA segment so that the encoded protein is expressed as a fusion protein. The DNA molecule is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.

Another embodiment of the invention is an expression cassette comprising a DNA molecule comprising a DNA segment encoding a fatty acid synthase and a polyhydroxyalkanoate synthase.

Also provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette into a host cell. The expression cassette comprises a DNA molecule encoding a polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The monomer synthase comprises a plurality of modules. Thus, the monomer synthase comprises at least a first and second module which together encode the monomer synthase. Optionally, the expression cassette further comprises a second DNA molecule encoding a polyhydroxyalkanoate synthase.

A further embodiment of the invention is an isolated and purified DNA molecule comprising a DNA segment which encodes a Streptomyces venezuelae polyketide synthase, e.g., a polyhydroxyalkanoate monomer synthase, a biologically active variant or subunit (fragment) thereof. Preferably, the DNA segment encodes polypeptide having an amino acid sequence comprising SEQ ID NO:2. Preferably, the DNA segment comprises SEQ ID NO:1. The DNA molecules of the invention are double stranded or single stranded. A preferred embodiment of the invention is a DNA molecule that has at least about 70%, more preferably at least about 80%, and even more preferably at least about 90%, but less than 100%, contiguous sequence identity to the DNA segment comprising SEQ ID NO:1, e.g., a “variant” DNA molecule. A variant DNA molecule of the invention can be prepared by methods well known to the art, including oligonucleotide-mediated mutagenesis. See Adelman et al., DNA, 2, 183 (1983) and Sambrook et al., Molecular Cloning: A Laboratory Manual (1989).

The invention also provides an isolated, purified polyhydroxyalkanoate monomer synthase, e.g., a polypeptide having an amino acid sequence comprising SEQ ID NO:2, a biologically active subunit, or a biologically active variant thereof. Thus, the invention provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:2. A preferred variant polypeptide, or a subunit of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 10%, more preferably at least about 50%, and even more preferably at least about 90%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:2. Preferably, a variant polypeptide of the invention has one or more conservative amino acid substitutions relative to the polypeptide having the amino acid sequence comprising SEQ ID NO:2. For example, conservative substitutions include aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as basic amino acids; leucine/isoleucine, methionine/valine, alanine/valine as hydrophobic amino acids; serine/glycine/alanine/threonine as hydrophilic amino acids. The biological activity of a polypeptide of the invention can be measured by methods well known to the art, including but not limited to, methods described hereinbelow.

The invention also provides an isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a sugar (desosamine) biosynthetic gene cluster, a biologically active variant or fragment thereof, wherein the nucleic acid sequence is not derived from the eryC gene cluster of Saccharopolyspora erythraea. As described hereinbelow, the desosamine biosynthetic gene cluster from Streptomycyes venezuelae was isolated, cloned and sequenced. The isolated nucleic acid segment comprising the gene cluster preferably includes a nucleic acid sequence comprising SEQ ID NO:3, or a fragment or variant thereof. The cluster was found to encode nine polypeptides including DesI (e.g., SEQ ID NO:8 encoded by SEQ ID NO:7), DesII (e.g., SEQ ID NO:10 encoded by SEQ ID NO:9), DesII (e.g., SEQ ID NO:12 encoded by SEQ ID NO:11), DesIV (e.g., SEQ ID NO:14 encoded by SEQ ID NO:13), DesV (e.g., SEQ ID NO:16 encoded by SEQ ID NO:15), DesVI (e.g., SEQ ID NO:18 encoded by SEQ ID NO:17), DesVII (e.g., SEQ ID NO:20 encoded by SEQ ID NO: 19), DesVII (e.g., SEQ ID NO:22 encoded by SEQ ID NO:21), and DesR (e.g., SEQ ID NO:24 encoded by SEQ ID NO:23) (see FIG. 24). It is also preferred that the nucleic acid segment of the invention encoding DesR is not derived from the eryB gene cluster of Saccharopolyspora erythraea or the oleD gene from Streptomyces antibioticus.

The invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or a fragment thereof. A preferred variant polypeptide, or a subunit or fragment of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ iD NO:20, SEQ ID NO:22, or SEQ ID NO:24. Thus, for example, the glycosyltransferase activity of a polypeptide of SEQ ID NO:20 can be compared to a variant of SEQ ID NO:20 having at least one amino acid substitution, insertion, or deletion relative to SEQ ID NO:20.

A variant nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1 1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or a fragment thereof.

Also provided is an expression cassette comprising a nucleic acid sequence comprising a desosamine biosynthetic gene cluster, a biologically active variant or fragment thereof operably linked to a promoter functional in a host cell, as well as host cells comprising an expression cassette of the invention. Thus, the expression cassettes of the invention are useful to express individual genes within the cluster, e.g., the desR gene which encodes a glycosidase or the des VII gene which encodes a glycosyltransferase having relaxed substrate specificity for polyketides and deoxysugars, i.e., the glycosyltransferase processes sugar substrates other than TDP-desosamine. Thus, the des VII gene can be employed in combinatorial biology approaches to synthesize a library of macrolide compounds having various polyketide and deoxysugar structures. Moreover, the expression of a glycosylase in a host cell which synthesizes a macrolide antibiotic may be useful in a method to reduce toxicity of, e.g., inactivate, the antibiotic. For example, a host cell which produces the antibiotic is transformed with an expression cassette encoding the glycosyltransferase. The recombinant glycosyltransferase is expressed in an amount that reversibly inactivates the antibiotic. To activate the antibiotic, the antibiotic, preferably the isolated antibiotic which is recovered from the host cell, is contacted with an appropriate native or recombinant glycosidase.

Preferably, the nucleic acid segment encoding desosamine in the expression cassette of the invention is not derived form the eryC gene cluster of Saccharopolyspora erythraea. Preferred host cells are prokaryotic cells, although eukaryotic host cells are also envisioned. These host cells are useful to express desosamine, analogs or derivatives thereof. Also provided is an expression cassette or host cell comprising antisense sequences from at least a portion of the desosamine biosynthetic gene cluster.

Another embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of a nucleic acid sequence encoding desosamine in the host chromosome is disrupted, e.g., deleted or interrupted (e.g., by an insertion) with heterologous sequences, or substituted with a variant nucleic acid sequence of the invention, preferably so as to result in a decrease or lack of desosamine synthesis, and/or so as to result in the synthesis of an analog or derivative of desosamine. Preferably, the nucleic acid sequence which is disrupted is not derived from the eryC gene cluster of Saccharopolyspora erythraea. Thus, the recombinant host cell of the invention has at least one gene, i.e., desI, desII, desIII, desIV, desV, desVI, des VII, desVIII or desR, which is disrupted. One embodiment of the invention includes a recombinant host cell in which the desVI gene, which encodes an N-methyltransferase, is disrupted, for example, by replacement with an antibiotic resistance gene. Preferably, such a host cell produces an aglycone having an N-acetylated aminodeoxy sugar, 10-deoxy-methylonide, a compound of formula (7), a compound of formula (8), or a combination thereof. Thus, the deletion or disruption of the desVI gene may be useful in a method for preparing novel sugars.

Another preferred embodiment of the invention is a recombinant bacterial host cell in which the desR gene, which encodes a glycosidase such as β-glucosidase, is disrupted. Preferably, the host cell synthesizes C-2′ β-glucosylated macrolide antibiotics, for example, a compound of formula (13), a compound of formula (14), or a combination thereof. Therefore, the invention further provides a compound of formula (8), (9), (13) or (14). It will be appreciated by those skilled in the art that each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.

Further provided is an isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a macrolide biosynthetic gene cluster (the “met/pik” or “pik” gene cluster) encoding methymycin, pikomycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof. It is preferred that the nucleic acid segment comprises SEQ ID NO:5, or a fragment or variant thereof. It is also preferred that the isolated and purified nucleic acid segment is from Streptomyces sp., such as Streptomyces venezuelae (e.g., ATCC 15439, MCRL 0306, SC 2366 or 3629), Streptomyces narbonensis, Streptomyces eurocidicus, Streptomyces zaomyceticus (MCRL 0405), Streptomyces flavochromogens, Streptomyces sp. AM400, and Streptomyces felleus, although isolated and purified nucleic acid from other organisms which produce methymycin, narbomycin, neomethymycin and/or pikomycin are also within the scope of the invention. The cloned genes can be introduced into an expression system and genetically manipulated so as to yield novel macrolide antibiotics, e.g., ketolides, as well as monomers for polyhydroxyalkanoate (PHA) biopolymers. Preferably, the nucleic acid sequence encodes PikR1 (e.g., SEQ ID NO:27 encoded by SEQ ID NO:26), PikR2 (e.g., SEQ ID NO:29 encoded by SEQ ID NO:28), PikAl (e.g., SEQ ID NO:31 encoded by SEQ ID NO:30), PikAII (e.g., SEQ ID NO:33 encoded by SEQ ID NO:32), PikAIII (e.g., SEQ ID NO:35 encoded by SEQ ID NO:34), PikAIV (e.g., SEQ ID NO:37 encoded by SEQ ID NO:36), PikB (which is the desosamine gene cluster described above), PikC (e.g., SEQ ID NO:39 encoded by SEQ ID NO:38), and PikD (e.g., SEQ ID NO:41 encoded by SEQ ID NO:40), a variant or a fragment thereof.

The invention also provides a variant polypeptide having at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to the polypeptide having an amino acid sequence comprising SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, or a fragment thereof. A preferred variant polypeptide, or a subunit of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, or SEQ ID NO:41. The activities of polypeptides of the macrolide biosynthetic pathway of the invention are described below.

A variant nucleic acid sequence of the pik biosynthetic gene cluster of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:5, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, or a fragment thereof.

The pikA gene encodes a polyketide synthase which synthesizes macrolactone 10-deoxymethonolide and narbolide, pikB encodes desosamine synthases which catalyze the formation and transfer of a deoxysugar moiety onto aglycones, the pikC gene encodes a P450 hydoxylase which catalyzes the conversion of YC-17 and narbomycin into methymycin, neomethymycin, and pikromycin, and the pikR1, pikR2 (possibly one for a 12-membered ring and the other for a 14-membered ring) and desR genes which encode enzymes associated with bacterial self-protection. Thus, the isolated nucleic acid molecule of the invention encodes four active macrolide antibiotics two of which have a 12-membered ring while the other two have a 14-membered ring. The regulation of the synthesis of 12- or 14-membered rings may be the result of the sequences in the spacer region between

modules

5 and 6, as discussed below. Thus, the genetic mechanism underlying the alternative termination of polyketide synthesis may be useful to prepare novel antibiotics and PHA monomers.

The invention further provides isolated and purified nucleic acid segments, e.g., in the form of an expression cassette, for each of the individual genes in the macrolide biosynthetic gene cluster. For example, the invention provides an isolated and purified pikAV gene that encodes a thioesterase II. In particular, the thioesterase is useful to enhance the structural diversity of antibiotics and in PHA production, as the thioesterase modulates chain release and cyclization. For example, a thioesterase II gene having acyl-ACP coenzyme A transferase activity (e.g., a mutant pik TEII, bacterial, fungal or plant medium-chain-length thioesterase, an animal fatty acid thioesterase or a thioesterase from a polyketide synthase) is introduced at the end of a recombinant monomer synthase (see FIG. 36), which, in the presence of a PHA synthase, e.g., phaC1, produces a novel polyhydroxyalkanoate polymer. Alternatively, in the absence of a TEII domain, a fusion of a portion of PKS gene cluster with a PHA synthase may result in the transfer of an acyl chain from the PHA to the polymerase.

Also provided is a pikC gene that encodes a hydroxylase which is active at two positions on a 12-membered ring or at one position on a 14-membered ring. Such a gene may be particularly useful to prepare novel compounds through bioconversion or biotransformation.

The invention also provides an expression cassette comprising a nucleic acid segment comprising a macrolide biosynthetic gene cluster encoding methymycin, pikomycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof, operably linked to a promoter functional in a host cell. Further provided is a host cell comprising the nucleic acid segment encoding methymycin, pikomycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof. Moreover, the invention provides isolated and purified polypeptides of the invention, preferably obtained from host cells having the nucleic acid molecules of the invention. In addition, expression cassettes and host cells comprising antisense sequences of at least a portion of the macrolide biosynthetic gene cluster of the invention are envisioned.

Yet another embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which a portion of the macrolide biosynthetic gene cluster of the invention is disrupted or replaced with a heterologous sequence or a variant nucleic acid segment of the invention, preferably so as to result in a decrease or lack of methymycin, pikomycin, neomethymycin, narbomycin, or a combination thereof, and/or so as to result in the synthesis of novel macrolides. Therefore, the invention provides a recombinant host cell in which a pikAI gene, a pikAII gene, a pikAIII gene (12-membered rings), a pikIV gene (14-membered rings), a pikB gene cluster, a pikAV gene, a pikC gene, a pikD gene, a pikR1 gene, a pikR2 gene, or a combination thereof, is disrupted or replaced. A preferred embodiment of the invention is a host cell wherein the pikB (e.g., the desVI and desV genes), pikA1, pikAV or pikC gene, is disrupted.

Moreover, as the nucleic acid segment comprising the macrolide biosynthetic gene cluster of the invention encodes a polyketide synthase, modules of that synthase are useful in methods to prepare recombinant polyhydroxyalkanoate monomer synthases and polymers in addition to macrolide antibiotics and derivatives thereof.

Thus, the invention provides an isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae. Preferably, no more than one DNA segment is derived from the eryA gene cluster of Saccharopolyspora erythraea. In one embodiment of the invention, the 3′ most DNA segment of the isolated DNA molecule of the invention encodes a thioesterase II. Also provided is an expression cassette comprising a nucleic acid molecule encoding the polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in a host cell.

Yet another embodiment of the invention is a method of providing a polyhydroxyalkanoate monomer. The method comprises introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell. The recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae. The DNA encoding the recombinant polyhydroxyalkanoate monomer synthase is then expressed in the host cell so as to generate a polyhydroxyalkanoate monomer. Optionally, a a second DNA molecule may be introduced into the host cell. The second DNA molecule comprises a DNA segment encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in the host cell. The two DNA molecules are expressed in the host cell so as to generate a polyhydroxyalkanoate polymer.

Another embodiment of the invention is an isolated and purified DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a module from the pikA gene cluster of Streptomyces venezuelae. Such a DNA molecule can be employed in a method of providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule comprising a first DNA segment encoding a fatty acid synthase and a second DNA segment encoding a polyketide synthase is introduced into a host cell. The first DNA segment is 5′ to the second DNA segment and the first DNA segment is operably linked to a promoter functional in the host cell. The first DNA segment is linked to the second DNA segment so that the linked DNA segments express a fusion protein. The DNA molecule is expressed in the host cell so as to generate a polyhydroxyalkanoate monomer.

Further provided is a method of providing a polyhydroxyalkanoate monomer synthase. The method comprises introducing an expression cassette comprising a DNA molecule encoding a polyhydroxyalkanoate synthase operably linked to a promoter functional in a host cell. The DNA molecule comprises a first DNA segment encoding a first module and a second DNA segment encoding a second module wherein the DNA segments together encode a polyhydroxyalkanoate monomer synthase. At least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae. The DNA molecule is expressed in the host cell. Optionally, the DNA molecule further comprises a DNA segment encoding a polyhydroxyalkanoate synthase. Alternatively, a second, separate DNA molecule encoding a polyhydroxyalkanoate synthase is introduced into the host cell.

Also provided is a method for directing the biosynthesis of specific glycosylation-modified polyketides by genetic manipulation of a polyketide-producing microorganism. The method comprises introducing into a polyketide-producing microorganism a DNA sequence encoding enzymes in desosamine biosynthesis, e.g., a DNA sequence comprising SEQ ID NO:3, a variant or fragment thereof, so as to yield a microorganism that produces specific glycosylation-modified polyketides. Alternatively, an anti-sense DNA sequence of the invention may be employed. Then the glycosylation-modified polyketides are isolated from the microorganism. It is preferred that the DNA sequence is modified so as to result in the inactivation of at least one enzymatic activity in sugar biosynthesis or in the attachment of the sugar to a polyketide.

Thus, the modules encoded by the nucleic acid segments of the invention may be employed in the methods described hereinabove to prepare polyhydroxyalkanoates of varied chain length or having various side chain substitutions and/or to prepare glycosylated biopolymers. Therefore, the compounds produced by the recombinant host cells of the invention are useful as biopolymers, e.g., in packaging or biomedical applications, or to engineer PHA monomer synthases; pharmaceuticals such as chemotherapeutic agents, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, cholesterol-lowering agents, or macrolide-based antibiotics which are active against a variety of organisms, e.g., bacteria, including multi-drug-resistant pneumococci and other respiratory pathogens, as well as viral and parasitic pathogens; or as crop protection agents (e.g., fingicides or insecticides) via expression of polyketides in plants. Methods employing these compounds, e.g., to treat a mammal, bird or fish in need of such therapy, such as a patient having a bacterial infection, are also envisioned.

As used herein, a “linker region” is an amino acid sequence present in a multifunctional protein which is less well conserved in an amino acid sequence than an amino acid sequence with catalytic activity.

As used herein, an “extender unit” catalytic or enzymatic domain is an acyl transferase in a module that catalyzes chain elongation by adding 2-4 carbon units to an acyl chain and is located carboxy-terminal to another acyl transferase. For example, an extender unit with methylmalonylCoA specificity adds acyl groups to a methylmalonylCoA molecule.

As used herein, a “polyhydroxyalkanoate” or “PHA” polymer includes, but is not limited to, linked units of related, preferably heterologous, hydroxyalkanoates such as 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate, 3-hydroxyheptanoate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy and 5-hydroxy counterparts.

As used herein, a “Type I polyketide synthase” is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides.

As used herein, a “recombinant” nucleic acid or protein molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.

A “recombinant” host cell of the invention has a genome that has been manipulated in vitro so as to alter, e.g., decrease or disrupt, or, alternatively, increase, the function or activity of at least one gene in the macrolide or desosamine biosynthetic gene cluster of the invention.

As used herein, a “multifunctional protein” is one where two or more enzymatic activities are present on a single polypeptide.

As used herein, a “module” is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase.

As used herein, a “premature termination product” is a product which is produced by a recombinant multiflnctional protein which is different than the product produced by the non-recombinant multifunctional protein. In general, the product produced by the recombinant multifunctional protein has fewer acyl groups.

As used herein, a DNA that is “derived from” a gene cluster is a DNA that has been isolated and purified in vitro from genomic DNA, or synthetically prepared on the basis of the sequence of genomic DNA.

As used herein, the pik gene cluster includes sequences encoding a polyketide synthase (pikA), desosamine biosynthetic enzymes (pikB, also referred to as des), a cytochrome P450 (pikC), regulatory factors (pikD) and enzymes for cellular self-resistance (pikR).

As used herein, the terms “isolated and/or purified” refer to in vitro isolation of a DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that is can be sequenced, replicated and/or expressed. Moreover, the DNA may encode more than one recombinant Type I polyketide synthase and/or fatty acid synthase. For example, “an isolated DNA molecule encoding a polyhydroxyalkanoate monomer synthase” is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that encode a biologically active polypeptide, fragment, or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of a polyhydroxyalkanoate monomer synthase RNA, or hybridizes to the RNA or DNA encoding the polyhydroxyalkanoate monomer synthase and remains stably bound under stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., supra.

An “antibiotic” as used herein is a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell.

An “antibiotic biosynthetic gene” is a nucleic acid, e.g., DNA, segment or sequence that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics.

An “antibiotic biosynthetic pathway” includes the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. These genes can be isolated by methods well known to the art, e.g., see U.S. Pat. No. 4,935,340.

Antibiotic-producing organisms include any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic.

An antibiotic resistance-conferring gene is a DNA segment that encodes an enzymatic or other activity which confers resistance to an antibiotic.

The term “polyketide” as used herein refers to a large and diverse class of natural products, including but not limited to antibiotic, antifungal, anticancer, and anti-helminthic compounds. Antibiotics include, but are not limited to anthracyclines and macrolides of different types (polyenes and avermectins as well as classical macrolides such as erythromycins). Macrolides are produced by, for example, S. erytheus, S. antibioticus, S. venezuelae, S. fradiae and S. narbonensis.

The term “glycosylated polyketide” refers to any polyketide that contains one or more sugar residues.

The term “glycosylation-modified polyketide” refers to a polyketide having a changed glycosylation pattern or configuration relative to that particular polyketide's unmodified or native state.

The term “polyketide-producing microorganism” as used herein includes any microorganism that can produce a polyketide naturally or after being suitably engineered (i.e., genetically). Examples of actinomycetes that naturally produce polyketides include but are not limited to Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora erythraea, Streptomyces antibioticus,, Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avernitilis, Streptomycesfradiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rimosus, Streptomyces peucetius, Streptomyces coelicolor, Streptomyces glaucescens, Streptomyces roseofulvus, Streptomyces cinnamonensis, Streptomyces curacoi, and Amycolatopsis mediterranei (see Hopwood, D. A. and Sherman, D. H., Annu. Rev. Genet., 24:37-66 (1990), incorporated herein by reference). Other examples of polyketide-producing microorganisms that produce polyketides naturally include various Actinomadura, Dactylosporangium and Nocardia strains.

The term “sugar biosynthesis genes” as used herein refers to nucleic acid sequences from organisms such as Streptomyces venezuelae that encode sugar biosynthesis enzymes and is intended to include sequences of DNA from other polyketide-producing microorganisms which are identical or analogous to those obtained from Streptomyces venezuelae.

The term “sugar biosynthesis enzymes” as used herein refers to polypeptides which are involved in the biosynthesis and/or attachment of polyketide-associated sugars and their derivatives and intermediates.

The term “polyketide-associated sugar” refers to a sugar that is known to attach to polyketides or that can be attached to polyketides by the processes described herein.

The term “sugar derivative” refers to a sugar which is naturally associated with a polyketide but which is altered relative to the unmodified or native state, including but not limited to, N-3-α-desdimethyl D-desosamine.

The term “sugar intermediate” refers to an intermediate compound produced in a sugar biosynthesis pathway.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The PHB biosynthetic pathway in [0075] A. eutrophus.
FIG. 2. Molecular structure of common bacterial PHAs. Most of the known PHAs are polymers of 3-hydroxy acids possessing the general formula shown. For example, R=CH[0076] ₃in PHB, T=CH₂CH₃in polyhydroxyvalerate (PHV), and R=(CH₂)₄CH₃in polyhydroxyoctanoate (PHO).
FIG. 3. Comparison of the natural and recombinant pathways for PHB synthesis. The three enzymatic steps of PHB synthesis in bacteria involving 3-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase are shown on the left. The two enzymatic steps involved in PHB synthesis in the pathway in Sf21 cells containing a rat fatty acid synthase with an inactivated dehydrase domain (ratFAS206) are shown on the right. [0077]
FIG. 4. Schematic diagram of the molecular organization of the tyl polyketide synthase (PKS) gene cluster. Open arrows correspond to individual open reading frames (ORFs) and numbers above an ORF denote a multifinctional module or synthase unit (SU). AT=acyltransferase; ACP-acyl carrier protein; KS=β-ketoacyl synthase; KR=ketoreductase; DH=dehydrase; ER=enoyl reductase; TE=thioesterase; MM=methylmalonylCoA; M=malonyl CoA; EM=ethylmalonyl CoA. [0078] Module 7 in tyl is also kncwn as Module F.
FIG. 5. Schematic diagram of the molecular organization of the met PKS gene cluster. [0079]
FIG. 6. Strategy for producing a recombinant PHA monomer synthase by domain replacement. [0080]
FIG. 7. (A) 10% SDS-PAGE gel showing samples from various stages of the purification of PHA synthase; [0081] lane 1, molecular weight markers; lane 2, total protein of uninfected insect cells; lane 3, total protein or insect cells expressing a rat FAS (200 kDa; Joshi et al., Biochem. J., 296, 143 (1993)); lane 4, total protein of insect cells expressing PHA synthase; lane 5, soluble protein from sample in lane 4; lane 6, pooled hydroxylapatite (HA) fractions containing PHA synthase. (B) Western analysis of an identical gel using rabbit-α-PHA synthase antibody as probe. Bands designated with arrows are: a, intact PHB synthase with N-terminal alanine at residue 7 and serine at residue 10 (A7/S10); b, 44 kDa fragment of PHB synthase with N-terminal alanine at residue 181 and asparagine at residue 185 (A181/N185); c, PHB synthase fragment of approximately 30 kDa apparently blocked based on resistance to Edman degradation; d, 22 kDa fragment with N-terminal glycine at residue 187 (G187). Band d apparently does not react with rabbit-α-PHB synthase antibody (B, lane 6). The band of similar size in B, lane 4 was not further identified.
FIG. 8. N-terminal analysis of PHA synthase purified from insect cells. (a) The expected N-[0082] terminal 25 amino acid sequence of A. eutrophus PHA synthase. (b&c) The two N-terminal sequences determined for the A. eutrophus PHA synthase produced in insect cells. The bolded sequences are the actual N-terrnini determined.
FIG. 9. Spectrophotometric scans of substrate, 3-hydroxybutyrate CoA (HBCOA) and product, CoA. The wavelength at which the direct spectrophotometric assays were carried out (232 nm) is denoted by the arrow; substrate, HBCoA () and product, CoA (∘). [0083]
FIG. 10. Velocity of the hydrolysis of HBCoA as a finction of substrate concentration. Assays were carried out in 40 or 200 μl assay volumes with enzyme concentration remaining constant at 0.95 mg/ml (3.8 μg/40 μl assay). Velocities were calculated from the linear portions of the assay curves subsequent to the characteristic lag period. The substrate concentration at half-optimal velocity, the apparent K[0084] _mvalue, was estimated to be 2.5 mM from this data.
FIG. 11. Double reciprocal plot of velocity versus substrate concentration. The concave upward shape of this plot is similar to results obtained by Fukui et al. ([0085] Arch. Microbiol, 110N, 149 (1976)) with granular PHA synthase from Z. ramigera.
FIG. 12. Velocity of the hydrolysis of HBCoA as a function of enzyme concentration. Assays were carried out in 40 μl assay volumes with the concentration HBCoA remaining constant at 8 μM. [0086]
FIG. 13. Specific activity of PHA synthase as a function of enzyme concentration. [0087]
FIG. 14. pH activity curve for soluble PHA synthase produced using the baculovirus system. Reactions were carried out in the presence of 200 mM P[0088] ₁. Buffers of pH<10 were prepared with potassium phosphate, while buffers of pH>10 were prepared with the appropriate proportion of Na₃PO₄.
FIG. 15. Assays of the hydrolysis of HBCoA with varying amounts of PHA synthase. Assays were carried out in 40 μl assay volumes with the concentration of HBCoA remaining constant at 8 μM. Initial A[0089] ₂₃₂values, originally between 0.62 and 0.77, were normalized to 0.70. Enzyme amounts used in these assays were, from the uppermost curve, 0.38, 0.76, 1.14, 1.52, 1.90, 2.28, 2.66, 3.02, 3.42, 7.6, and 15.2 μg, respectively.
FIG. 16. SDS/PAGE analysis of proteins synthesized at various time points during infection of Sf21 cells. Approximately 0.5 mg of total cellular protein from various samples was fractionated on a 10% polyacrylamide gel. Samples include: uninfected cells, lanes 1-4, [0090] days 0, 1, 2, 3, respectively; infection with BacPAK6::phbC alone, lanes 5-8, days, 0, 1, 2, 3, respectively, infection with baculoviral clone containing ratFAS206 alone, lanes 9-12, days 0, 1, 2, 3, respectively; and ratFAS206 and BacPAK6 infected cells, lanes 13-16, days 0, 1, 2, 3, respectively. A=mobility of FAS, B=mobility of PHA synthase. Molecular weight standard lanes are marked M.
FIG. 17. Gas chromatographic evidence for PHB accumulation in Sf21 cells. Gas chromatograms from various samples are superimposed. PHB standard (Sigma) is [0091] chromatogram #7 showing a propylhydroxybutyrate elution time of 10.043 minutes (s, arrow). The gas chromatograms of extracts of the uninfected (#1); singly infected with ratFAS206 (#2, day 3); and singly infected with PHA synthase (#3, day 3) are shown at the bottom of the figure. Gas chromatograms of extracts of dual-infected cells at day 1 (#4), 2 (#5), and 3 (#6) are also shown exhibiting a peak eluting at 10.096 minutes (x, arrow). The peak of dual-infected, day 3 extract (#6) was used for mass spectrometry (MS) analysis.
FIG. 18. Gas chromatography-mass spectrometry analysis of PHB. The characteristic fragmentation of propylhydroxybutyrate at m/z of 43, 60, 87, and 131 is shown. A) standard PHB from bacteria (Sigma), and B) peak X from ratFAS206 and BacPAK6: phbC baculovirus infected, day 3 (#6, FIG. 17) Sf21 cells expressing rat FAS dehydrase inactivated protein and PHA synthase. [0092]
FIG. 19. Map of the vep ([0093] Streptomyces venezuelae polyene encoding) gene cluster.
FIG. 20. Plasmid map of pDHS502. [0094]
FIG. 21. Plasmid map of pDHS505. [0095]
FIG. 22. Cloning protocol for pDHS505. [0096]
FIG. 23. Nucleotide sequence (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO:22) of vep ORFI. [0097]
FIG. 24. Schematic diagram of the desosamine biosynthetic pathway and the enzymatic activity associated with each of the desosamine biosynthetic polypeptides. [0098]
FIG. 25. Schematic of the conversion of the inactive (diglycosylated) form of methymycin and pikromycin to the active form of methymycin and pikromycin. [0099]
FIG. 26. Schematic diagram of the desosamine biosynthetic pathway. [0100]
FIG. 27. Pathway for the synthesis of a compound of [0101] formula 7 and 8 in desVI⁻ mutants of Streptomyces.
FIG. 28. The methymycin/pikromycin biosynthetic gene cluster and the structure and biosynthesis of methymycin, neomethymycin, narbomycin, and pikromycin in [0102] S. venezuelae. Methymycin: R₁=OH, R₂=H, neomethymycin: R₁=H, R₂=OH; narbomycin R₃=H, pikromycin R₃=OH. Each circle represents an enzymatic domain in PKS protein. ACP, acyl carrier protein; KS, β-ketoacyl-ACP synthase; KS^Q, a KS-like domain; AT, acyltransferase; KR, β-ketoacyl ACP reductase; DH, β-hydroxyl-thioester dehydratase; ER, enoyl reductase; TEI, thioesterase domain; TEII, type II thioesterase. Des represents all eight enzymes in desosamine synthesis and transfer which include DesI, DesII, DesIlI, DesIV, DesV, DesVI, DesVIII, and Des VII.
FIG. 29. Organization of the pik cluster in [0103] S. venezuelae. Each arrow represents an open reading frame (ORF). The direction of transcription and relative sizes of the ORFs deduced from nucleotide sequence are indicated. The cluster is composed of four genetic loci: piA, pikB (des), pikC, and pikR. Cosmid clones are denoted as overlapping lines.
FIG. 30. Conversion ofYC-17 and narbomycin by PikC P450 hydroxylase. [0104]
FIG. 31. Nucleotide sequence (SEQ ID NO:5) and inferred amino acid sequence (SEQ ID NO:6) of the pik gene cluster. [0105]
FIG. 32. Nucleotide sequence (SEQ ID NO:3) and inferred amino acid sequence (SEQ ID NO:4) of the desosamine gene cluster. [0106]
FIG. 33. [0107] S. venezuelae AX916 construct useful to prepare a polyketide having a shorter chain length compared to wild-type pikA. pik module 2 is fused to pik module 5, and module 3 and 4 are deleted, so as to encode a three module PKS which produces two macrolides, a triketide and a tetraketide.
FIG. 34. Recombinant PKS having a wild-type thioesterase II. [0108]
FIG. 35. pAX703 construct, an expression and complementation vector. The PikTEII gene can be replaced with an EcoRI-NsiI fragment. The phaC1 gene can be replaced with a PacI-Dral fragment. [0109]
FIG. 36. Strategy for C7 polymer production. mTEII is a mutant pikTEII, an acyl-ACP CoA transferase; phaCI is a [0110] PHA polymerase 1 from P. olivarus which may have racemase activity. In a strain having these constructs, AX916, a PHA polymer is produced.
FIG. 37. Strategy for C5 polymer production. A PHA polymerase gene phaC1 is directly fused to pik [0111] module 2, so as to result in a fusion that transfers an acyl chain from the PKS protein directly to the polymerase by the prosthetic group on the ACP domain of the PKS.
FIG. 38. Codons for specified amino acids. [0112]
FIG. 39. Exemplary and preferred amino acid substitutions.[0113]

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein can be used for the production of a diverse range of biodegradable PHA polymers through genetic redesign of DNA encoding a FAS or a PKS such as that found in Streptomyces spp. Type I PKS polypeptide to provide a recombinant PHA monomer synthase. Different PHA synthases can then be tested for their ability to polymerize the monomers produced by the recombinant PHA synthase into a biodegradable polymer. The invention also provides a method by which various PHA synthases can be tested for their specificity with respect to different monomer substrates. [0114]
The potential uses and applications of PHAs produced by PHA monomer synthases and PHA synthases include both medical and industrial applications. Medical applications of PHAs include surgical pins, sutures, staples, swabs, wound dressings, blood vessel replacements, bone replacements and plates, stimulation of bone growth by piezoelectric properties, and biodegradable carrier for long-term dosage of pharmaceuticals. Industrial applications of PHAs include disposable items such as baby diapers, packaging containers, bottles, wrappings, bags, and films, and biodegradable carriers for long-term dosage of herbicides, fungicides, insecticides, or fertilizers. [0115]
In animals, the biosynthesis of fatty acids de novo from malonyl-CoA is catalyzed by FAS. For example, the rat FAS is a homodimer with a subunit structure consisting of 2505 amino acid residues having a molecular weight of 272,340 Da. Each subunit consists of seven catalytic activities in separate physical domains (Amy et al., [0116] Proc. Natl. Acad. Sci. US A, 86, 3114 (1989)). The physical location of six of the catalytic activities, ketoacyl synthase (KS), malonyl/acetyltransferase (M/AT), enoyl reductase (ER), ketoreductase (KR), acyl carrier protein (ACP), and thioesterase (TE), has been established by (1) the identification of the various active site residues within the overall amino acid sequence by isolation of catalytically active fragments from limited proteolytic digests of the whole FAS, (2) the identification of regions within the FAS that exhibit sequence similarity with various monofunctional proteins, (3) expression of DNA encoding an amino acid sequence with catalytic activity to produce recombinant proteins, and (4) the identification of DNA that does not encode catalytic activity, i.e., DNA encoding a linker region. (Smith et al., Proc. Natl. Acad. Sci. USA, 73, 1184 (1976); Tsukamoto et al., J. Biol. Chem., 263, 16225 (1988); Rangan et al., J. Biol. Chem., 266, 19180 (1991)).
The seventh catalytic activity, dehydrase (DH), was identified as physically residing between AT and ER by an amino acid comparison of FAS with the amino acid sequences encoded by the three open reading frames of the eryA polyketide synthase (PKS) gene cluster of [0117] Saccharopolyspora erythraea. The three polypeptides that comprise this PKS are constructed from “modules” which resemble animal FAS, both in terms of their amino acid sequence and in the ordering of the constituent domains (Donadio et al., Gene, 111, 51 (1992); Benh et al., Eur. J. Biochem., 204, 39 (1992)).
One embodiment of the invention employs a FAS in which the DH is inactivated (FAS DH−). The FAS DH− employed in this embodiment of the invention is preferably a eukaryotic FAS DH− and, more preferably, a mammalian FAS DH−. The most preferred embodiment of the invention is a FAS where the active site in the DH has been inactivated by mutation. For example, Joshi et al. ([0118] J. Biol. Chem, 268, 22508 (1993)) changed the His⁸⁷⁸residue in the rat FAS to an alanine residue by site-directed mutagenesis. In vitro studies showed that a FAS with this change (ratFAS206) produced 3-hydroxybutyrylCoA as a premature termination product from acetyl-CoA, malonyl-CoA and NADPH.
As shown below, a FAS DH− effectively replaces the β-ketothiolase and acetoacetyl-CoA reductase activities of the natural pathway by producing D(−)-3-hydroxybutyrate as a premature termination product, rather than the usual 16-carbon product, palmitic acid. This premature termination product can then be incorporated into PHB by a PHB synthase (See Example 2). [0119]
Another embodiment of the invention employs a recombinant Streptomyces spp. PKS to produce a variety of β-hydroxyCoA esters that can serve as monomers for a PHA synthase. One example of a DNA encoding a Type I PKS is the eryA gene cluster, which governs the synthesis of erythromycin aglycone deoxyerythronolide B (DEB). The gene cluster encodes six repeated units, termed modules or synthase units (SUs). Each module or SU, which comprises a series of putative FAS-like activities, is responsible for one of the six elongation cycles required for DEB formation. Thus, the processive synthesis of asymmetric acyl chains found in complex polyketides is accomplished through the use of a programmed protein template, where the nature of the chemical reactions occurring at each point is determined by the specificities in each SU. [0120]
Two other Type I PKS are encoded by the tyl (tylosin) (FIG. 4) and met (methymycin) (FIG. 5) gene clusters. The macrolide multifunctional synthases encoded by tyl and met provide a greater degree of metabolic diversity than that found in the eryA gene cluster. The PKSs encoded by the eryA gene cluster only catalyze chain elongation with methylmalonylCoA, as opposed to tyl and met PKSs, which catalyze chain elongation with malonylCoA, methylmalonylCoA and ethylmalonylCoA. Specifically, the tyl PKS includes two malonylCoA extender units and one ethylmalonylCoA extender unit, and the met PKS includes one malonylCoA extender unit. Thus, a preferred embodiment of the invention includes, but is not limited to, replacing catalytic activities encoded in met PKS open reading frame 1 (ORF 1) to provide a DNA encoding a protein that possesses the required keto group processing capacity and short-chain acylCoA ester starter and extender unit specificity necessary to provide a saturated β-hydroxyhexanoylCoA or unsaturated β-hydroxyhexenoylCoA monomer. [0121]
In order to manipulate the catalytic specificities within each module, DNA encoding a catalytic activity must remain undisturbed. To identify the amino acid sequences between the amino acid sequences with catalytic activity, the “linker regions,” amino acid sequences of related modules, preferably those encoded by more than one gene cluster, are compared. Linker regions are amino acid sequences which are less well conserved than amino acid sequences with catalytic activity. Witkowski et al., [0122] Eur. J. Biochem., 198, 571 (1991).
In an alternative embodiment of the invention, to provide a DNA encoding a Type I PKS module with a TE and lacking a functional DH, a DNA encoding a module F, containing KS, MT, KR, ACP, and TE catalytic activities, is introduced at the 3′ end of a DNA encoding a first module (FIG. 6). Module F introduces the final (R)-3-hydroxyl acyl group at the final step of PHA monomer synthesis, as a result of the presence of a TE domain. DNA encoding a module F is not present in the eryA PKS gene cluster (Donadio et al., supra, 1991). [0123]
A DNA encoding a recombinant monomer synthase is inserted into an expression vector. The expression vector employed varies depending on the host cell to be transformed with the expression vector. That is, vectors are employed with transcription, translation and/or post-translational signals, such as targeting signals, necessary for efficient expression of the genes in various host cells into which the vectors are introduced. Such vectors are constructed and transformed into host cells by methods well known in the art. See Sambrook et al., [0124] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor (1989). Preferred host cells for the vectors of the invention include insect, bacterial, and plant cells. Preferred insect cells include Spodoptera frugiperda cells such as Sf21, and Trichoplusia ni cells. Preferred bacterial cells include Escherichia coli, Streptomyces and Pseudomonas. Preferred plant cells include monocot and dicot cells, such as maize, rice, wheat, tobacco, legumes, carrot, squash, canola, soybean, potato, and the like.
Moreover, the appropriate subcellular compartment in which to locate the enzyme in eukaryotic cells must be considered when constructing eukaryotic expression vectors. Two factors are important: the site of production of the acetyl-CoA substrate, and the available space for storage of the PHA polymer. To direct the enzyme to a particular subcellular location, targeting sequences may be added to the sequences encoding the recombinant molecules. [0125]
The baculovirus system is particularly amenable to the introduction of DNA encoding a recombinant FAS or a PKS monomer synthase because an increasing variety of transfer plasmids are becoming available which can accommodate a large insert, and the virus can be propagated to high titers. Moreover, insect cells are adapted readily to suspension culture, facilitating relatively large-scale recombinant protein production. Further, recombinant proteins tend to be produced exclusively as soluble proteins in insect cells, thus, obviating the need for refolding, a task that might be particularly daunting in the case of a large multifunctional protein. The Sf21/baculovirus system has routinely expressed milligram quantities of catalytically active recombinant fatty acid synthase. Finally, the baculovirus/insect cell system provides the ability to construct and analyze different synthase proteins for the ability to polymerize monomers into unique biodegradable polymers. [0126]
A further embodiment of the invention is the introduction of at least one DNA encoding a PHA synthase and a DNA encoding a PHA monomer synthase into a host cell. Such synthases include, but are not limited to, [0127] A. eutrophus 3-hydroxy, 4-hydroxy, and 5-hydroxy alkanoate synthases, Rhodococcus ruber C₃-C₅hydroxyalkanoate synthases, Pseudomonas oleororans C₆-C,₄hydroxyalkanoate synthases, P. putida C₆-C₁₄hydroxyalkanoate synthases, P. aeruginosa C₅-C₁₀hydroxyalkanoate synthases, P. resinovorans C₄-C₁₀hydroxyalkanoate synthases, Rhodospirillum rubrum C₄-C₇hydroxyalkanoate syntheses, R. gelatinorus C₄-C₇ , Thiocapsa pfennigii C₄-C₈hydroxyalkanoate synthases, and Bacillus megaterium C₄-C₅hydroxyalkanoate synthases.
The introduction of DNA(s) encoding more than one PHA synthase may be necessary to produce a particular PHA polymer due to the specificities exhibited by different PHA synthases. As multifunctional proteins are altered to produce unusual monomeric structures, synthase specificity may be problematic for particular substrates. Although the [0128] A. eutrophus PHB synthase utilizes only C4 and C5 compounds as substrates, it appears to be a good prototype synthase for initial studies since it is known to be capable of producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate (Kunioka et al., Macromolecules, 22, 694 (1989)) as well as copolymers of 3-hydroxyvalerate, 3-hydroxybutyrate, and 5-hydroxyvalerate (Doi et al., Macromolecules, 19, 2860 (1986)). Other synthases, especially those of Pseudomonas aeruginosa (Timm et al., Eur. J. Biochem., 209, 15 (1992)) and Rhodococcus ruber (Pieper et al., FEMS Microbiol. Lett., 96, 73 (1992)), can also be employed in the practice of the invention. Synthase specificity may be alterable through molecular biological methods.
In yet another embodiment of the invention, a DNA encoding a FAS and a PHA synthase can be introduced into a single expression vector, obviating the need to introduce the genes into a host cell individually. [0129]
A further embodiment of the invention is the generation of a DNA encoding a recombinant multifunctional protein, which comprises a FAS, of either eukaryotic or prokaryotic origin, and a PKS module F. Module F will carry out the final chain extension to include two additional carbons and the reduction of the P-keto group, which results in a (R)-3-hydroxy acyl CoA moiety. [0130]
To produce this recombinant protein, DNA encoding the FAS TE is replaced with a DNA encoding a linker region which is normally found in the ACP-KS interdomain region of bimodular ORFs. DNA encoding a module F is then inserted 3′ to the DNA encoding the linker region. Different linker regions, such as those described below which vary in length and amino acid composition, can be tested to determine which linker most efficiently mediates or allows the required transfer of the nascent saturated fatty acid intermediate to module F for the final chain elongation and keto reduction steps. The resulting DNA encoding the protein can then be tested for expression of long-chain P-hydroxy fatty acids in insect cells, such as Sf21 cells, or Streptomyces, or Pseudomonas. The expected 3-hydroxy C-18 fatty acid can serve as a potential substrate for PHA synthases which are able to accept long-chain alkyl groups. A preferred embodiment of the invention is a FAS that has a chain length specificity between 4-22 carbons. [0131]
Examples of linker regions that can be employed in this embodiment of the invention include, but are not limited to, the ACP-KS linker regions encoded by the tyl ORFI (ACP[0132] ₁-KS2; ACP₂-KS₃), and ORF3 (ACP₅-KS₆), and eryA ORFI (ACP₁-KS₁; ACP₂-KS₂), ORF2 (ACP₃-KS₄) and ORF3 (ACP₅-KS₆).
This approach can also be used to produce shorter chain fatty acid groups by limiting the ability of the FAS unit to generate long-chain fatty acids. Mutagenesis of DNA encoding various FAS catalytic activities, starting with the KS, may result in the synthesis of short-chain (R)-3-hydroxy fatty acids. [0133]
The PHA polymers are then recovered from the biomass. Large-scale solvent extraction can be used, but is expensive. An alternative method involving heat shock with subsequent enzymatic and detergent digestive processes is also available (Byron, [0134] Trends Biotechnical, 5, 246 (1987); Holmes, In: Developments in Crystalline Polymers, D. C. Bassett (ed.), pp. 1-65 (1988)). PHB and other PHAs are readily extracted from microorganisms by chlorinated hydrocarbons. Refluxing with chloroform has been extensively used; the resulting solution is filtered to remove debris and concentrated, and the polymer is precipitated with methanol or ethanol, leaving low-molecular-weight lipids in solution. Longer side-chain PHAs show a less restricted solubility than PHB and are, for example, soluble in acetone. Other strategies adopted include the use of ethylene carbonate and propylene carbonate as disclosed by Lafferty et al. (Chem. Rundscha, 30, 14 (1977)) to extract PHB from biomass. Scandola et al. (Int. J. Biol. Microbiol., 10, 373 (1988)) reported that 1 M HCl-chloroform extraction of Rhizobium meliloti yielded PHB of M_w=6×10⁴compared with 1.4×10⁶when acetone was used.
Methods are well known in the art for the determination of the PHB or PHA content of microorganisms, the composition of PHAs, and the distribution of the monomer units in the polymer. Gas chromatography and high-pressure liquid chromatography are widely used for quantitative PHB analysis. See Anderson et al., [0135] Microbiol. Rev., 54, 450 (1990) for a review of such methods. NMR techniques can also be used to determine polymer composition, and the distribution of monomer units.
Preparation of Variant Nucleic Acid Molecules and Variant Polypeptides of the Invention [0136]
The present invention also contemplates nucleic acid sequences which hybridize under stringent hybridization conditions to the nucleic acid sequences set forth herein. Stringent hybridization conditions are well known in the art and define a degree of sequence identity greater than about 80 to about 90%. Thus, nucleic acid sequences encoding variant polypeptides (FIG. 38), or nucleic acid sequences having conservative (silent) nucleotide substitutions (FIG. 37), are within the scope of the invention. Preferably, variant polypeptides encoded by the nucleic acid sequences of the invention are biologically active. The present invention also contemplates naturally occurring allelic variations and mutations of the nucleic acid sequences described herein. [0137]
As is well known in the art, because of the degeneracy of the genetic code, there are numerous other DNA and RNA molecules that can code for the same polypeptides as those encoded by the exemplified biosynthetic genes and fragments thereof. The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode the polypeptides of, for example, portions of SEQ ID NO:4 or SEQ ID NO:6. Having identified the amino acid residue sequence encoded by a sugar biosynthetic or macrolide biosynthetic gene, and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid, are within the scope of this invention. [0138]
The 20 common amino acids and their representative abbreviations, symbols and codons are well known in the art (see, for example, [0139] Molecular Biology of the Cell, Second Edition, B. Alberts et al., Garland Publishing Inc., New York and London, 1989). As is also well known in the art, codons constitute triplet sequences of nucleotides in mRNA molecules and as such, are characterized by the base uracil (U) in place of base thymidine (T) which is present in DNA molecules. A simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure of the encoded polypeptide. By way of example, it can be seen from SEQ ID NO:6 that a TCT codon for serine exists at nucleotide positions 1735-1737. However, it can also be seen from that same sequence that serine can be encoded by a TCA codon (see, e.g., nucleotide positions 1738-1740) and a TCC codon (see, e.g., nucleotide positions 1874-1876). Substitution of the latter codons for serine with the TCT codon for serine or vice versa, does not substantially alter the DNA sequence of SEQ ID NO:6 and results in production of the same polypeptide. In a similar manner, substitutions of the recited codons with other equivalent codons can be made in a like manner without departing from the scope of the present invention.
A nucleic acid molecule, segment or sequence of the present invention can also be an RNA molecule, segment or sequence. An RNA molecule contemplated by the present invention corresponds to, is complementary to or hybridizes under stringent conditions to any of the DNA sequences set forth herein. Exemplary and preferred RNA molecules are MRNA molecules that encode sugar biosynthetic or macrolide biosynthetic enzymes of this invention. [0140]
Mutations can be made to the native nucleic acid sequences of the invention and such mutants used in place of the native sequence, so long as the mutants are able to function with other sequences to collectively catalyze the synthesis of an identifiable polyketide or macrolides. Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene using restriction endonuclease digestion. (See, e.g., Kunkel, T. A. [0141] Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.) Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide sequence (generally cDNA corresponding to the RNA sequence), at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. Zoller and Smith, Methods Enzymol., (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al., Proc. Natl Acad. Sci. USA (1982) 79:6409. PCR mutagenesis will also find use for effecting the desired mutations.
Random mutagenesis of the nucleotide sequence can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into [0142] E. coli and propagated as a pool or library of mutant plasmids.
Large populations of random enzyme variants can be constructed in vivo using “recombination-enhanced mutagenesis.” This method employs two or more pools of, for example, 10[0143] ⁶mutants each of the wild-type encoding nucleotide sequence that are generated using any convenient mutagenesis technique and then inserted into cloning vectors.
The gene sequences can be inserted into one or more expression vectors, using methods known to those of skill in the art. Expression vectors may include control sequences operably linked to the desired genes. Suitable expression systems for use with the present invention include systems which function in eukaryotic and prokaryotic host cells. Prokaryotic systems are preferred, and in particular, systems compatible with Streptomyces spp. are of particular interest. Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites. Particularly useful promoters include control sequences derived from the gene clusters of the invention. However, other bacterial promoters, such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the expression cassettes encoding desosamine. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the P-lactamase (bla) promoter system, bacteriophage lambda PL, and T5. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433), which do not occur in nature, also function in bacterial host cells. [0144]
Other regulatory sequences may also be desirable which allow for regulation of expression of the genes relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. [0145]
Selectable markers can also be included in the recombinant expression vectors. A variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. Such markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid. Alternatively, several polyketides are naturally colored and this characteristic provides a built-in marker for selecting cells successfully transformed by the present constructs. [0146]
The various subunits of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter. The subunits can include flanking restriction sites to allow for the easy deletion and insertion of other subunits so that hybrid PKSs can be generated. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR. [0147]
For sequences generated by random mutagenesis, the choice of vector depends on the pool of mutant sequences, i.e., donor or recipient, with which they are to be employed. Furthermore, the choice of vector determines the host cell to be employed in subsequent steps of the claimed method. Any transducible cloning vector can be used as a cloning vector for the donor pool of mutants. It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell. Phagemids and cosmids, for example, are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in M13 phage and λ phage, respectively. Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles. Cosmids which will find use in this method generally include λ phage-based vectors into which cos sites have been inserted. Recipient pool cloning vectors can be any suitable plasmid. The cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction. [0148]
Thus, for example, the cloning vector employed may be a phagemid and the host cell may be [0149] E. coli. Upon infection of the host cell which contains a phagemid, single-stranded phagemid DNA is produced, packaged and extruded from the cell in the form of a transducing phage in a manner similar to other phage vectors. Thus, clonal amplification of mutant encoding nucleotide sequences carried by phagemids is accomplished by propagating the phagemids in a suitable host cell.
Following clonal amplification, the cloned donor pool of mutants is infected with a helper phage to obtain a mixture of phage particles containing either the helper phage genome or phagemids mutant alleles of the wild-type encoding nucleotide sequence. [0150]
Infection, or transfection, of host cells with helper phage is generally accomplished by methods well known in the art (see., e.g., Sambrook et al., supra; and Russell et al. (1986) [0151] Gene 45:333-338).
The helper phage may be any phage which can be used in combination with the cloning phage to produce an infective transducing phage. For example, if the cloning vector is a cosmid, the helper phage will necessarily be a λ phage. Preferably, the cloning vector is a phagemid and the helper phage is a filamentous phage, and preferably phage M13. [0152]
If desired after infecting the phagemid with helper phage and obtaining a mixture of phage particles, the transducing phage can be separated from helper phage based on size difference (Barnes et al. (1983) [0153] Methods Enzymol. 101:98-122), or other similarly effective technique.
The entire spectrum of cloned donor mutations can now be transduced into clonally amplified recipient cells into which has been transduced or transformed a pool of mutant encoding nucleotide sequences. Recipient cells which may be employed in the method disclosed and claimed herein may be, for example, [0154] E. coli, or other bacterial expression systems which are not recombination deficient. A recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as rec⁻ mutants of E. coli (see, Clark et al. (1965) Proc. Natl. Acad. Sci. USA 53:451-459).
These transductants can now be selected for the desired expressed protein property or characteristic and, if necessary or desirable, amplified. Optionally, if the phagemids into which each pool of mutants is cloned are constructed to express different genetic markers, as described above, transductants may be selected by way of their expression of both donor and recipient plasmid markers. [0155]
The recombinants generated by the above-described methods can then be subjected to selection or screening by any appropriate method, for example, enzymatic or other biological activity. [0156]
The above cycle of amplification, infection, transduction, and recombination may be repeated any number of times using additional donor pools cloned on phagemids. As above, the phagemids into which each pool of mutants is cloned may be constructed to express a different marker gene. Each cycle could increase the number of distinct mutants by up to a factor of 10[0157] ⁶. Thus, if the probability of occurrence of an inter-allelic recombination event in any individual cell is f (a parameter that is actually a function of the distance between the recombining mutations), the transduced culture from two pools of 10⁶allelic mutants will express up to 10¹²distinct mutants in a population of 10¹²/f cells.

I. Experimental Procedures

Materials and Methods [0158]
Materials. [0159]
Sodium R-(−)-3-hydroxybutyrate, coenzyme-A, ethylchloroformate, pyridine and diethyl ether were purchased from Sigma Chemical Co. Amberlite IR-120 was purchased from Mallinckrodt Inc. 6-O-(N-Heptylcarbamoyl)methyl α-D-glycopyranoside (Hecameg) was obtained from Vegatec (Villeejuif, France). Two-piece spectrophotometer cells with pathlengths of 0.1 (#20/0-Q-1) and 0.01 cm (#20/0-Q-0.1) were obtained from Stama Cells Inc. (Atascadero, Calif.). Rabbit anti-A. eutrophus PHA synthase antibody was a gracious gift from Dr. F. Srienc and S. Stoup (Biological Process Technology Institute, University of Minnesota). Sf21 cells and [0160] T. ni cells were kindly provided by Greg Franzen (R&D Systems, Minneapolis, Minn.) and Stephen Harsch (Department of Veterinary Pathobiology, University of Minnesota), respectively.
Plasmid pFAS206 and a recombinant baculoviral clone encoding FAS206 (Joshi et al., [0161] J. Biol. Chem., 268, 22508 (1993)) were generous gifts of A. Joshi and S. Smith. Plasmid pAet4l (Peoples et al., J. Biol. Chem., 264, 15298 (1989)), the source of the A. eutrophus PHB synthase, was obtained from A. Sinskey. Baculovirus transfer vector, pBacPAK9, and linearized baculoviral DNA, were obtained from Clontech Inc. (Palo Alto, CA). Restriction enzymes, T4 DNA ligase, E. coli DH5a competent cells, molecular weight standards, lipofectin reagent, Grace's insect cell medium, fetal bovine serum (FBS), and antibiotic/antimycotic reagent were obtained from GIBCO-BRL (Grand Island, N.Y.). Tissue culture dishes were obtained from Corning Inc. Spinner flasks were obtained from Bellco Glass Inc. Seaplaque agarose GTG was obtained from FMC Bioproducts Inc.
Methods [0162]
Preparation of -3HBCoA. [0163]
R-(−)-3 HBCoA was prepared by the mixed anhydride method described by Haywood et al., [0164] FEMS Microbiol. Lett., , 1 (1989). 60 mg (0.58 nmol) of R-(−)-3 hydroxybutyric acid was freeze dried and added to a solution of 72 mg of pyridine in 10 ml diethyl ether at 0° C. Ethylchloroformate (100 mg) was added, and the mixture was allowed to stand at 4° C. for 60 minutes. Insoluble pyridine hydrochloride was removed by centrifugation. The resulting anhydride was added, dropwise with mixing, to a solution of 100 mg coenzyme-A (0.13 mmol) in 4 ml 0.2 M potassium bicarbonate, pH 8.0 at 0° C. The reaction was monitored by the nitroprusside test of Stadtman, Meth. Enzymol., 3, 931 (1957), to ensure sufficient anhydride was added to esterify all the coenzyme-A. The concentration of R-3-HBCoA was determined by measuring the absorbance at 260 nm (e=16.8 nM⁻¹cm⁻¹; 18).
Construction of PBP-phbC. [0165]
The phbC gene (approximately 1.8 kb) was excised from pAet41 (Peoples et al., [0166] J. Biol. Chem., 264, 15293 (1989)) by digestion with BstBI and Stul, purified as described by Williams et al. (Gene, 109, 445 (1991)), and ligated to pBacPAK9 digested with BsiBI and StuI. This resulted in pBP-phbC, the baculovirus transfer vector used in formation of recombinant baculovirus particles carrying phbC.
Large-scale Expression of PHA Synthase. [0167]
A 1 L culture of [0168] T. ni cells (1.2×10⁶cells/ml) in logarithmic growth was infected by the addition of 50 ml recombinant viral stock solution (2.5×10⁸pfu/ml) resulting in a multiplicity of infection (MOI) of 10. This infected culture was split between two Bellco spinners (350 ml/500 ml spinner, 700 m/1 L spinner) to facilitate oxygenation of the culture. These cultures were incubated at 28° C. and stirred at 60 rpm for 60 hours. Infected cells were harvested by centrifugation at 1000× g for 10 minutes at 4° C. Cells were flash frozen in liquid N₂and stored in 4 equal aliquots, at −80° C. until purification.
Insect Cell Maintenance and Recombinant Baculovirus Formation. [0169]
Sf21 cells were maintained at 26-28° C. in Grace's insect cell medium supplemented with 10% FBS, 1.0% pluronic F68, and 1.0% antibiotic/antimycotic (GIBCO-BRL). Cells were typically maintained in suspension at 0.2-2.0×10[0170] ⁶/ml in 60 ml total culture volume in 100 ml spinner flasks at 55-65 rpm. Cell viability during the culture period was typically 95-100%. The procedures for use of the transfer vector and baculovirus were essentially those described by the manufacturer (Clontech, Inc.). Purified pBP-phbC and linearized baculovirus DNA were used for cotransfection of Sf21 cells using the liposome-mediated method (Felgner et al., Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)) utilizing Lipofectin (GIBCO-BRL). Four days later cotransfection supernatants were utilized for plaque purification. Recombinant viral clones were purified from plaque assay plates containing 1.5% Seaplaque GTG after 5-7 days at 28° C. Recombinant viral clone stocks were then amplified in T25-flask cultures (4 ml, 3×10⁶/ml on day 0) for 4 days; infected cells were determined by their morphology and size and then screened by SDS/PAGE using 10% polyacrylamide gels (Laemmli, Nature, 227, 680 (1970)) for production of PHA synthase.
Purification of PHA Synthase from BTI-TN-5RT-4 [0171] T. ni Cells.
Purification of PHA synthase was performed according to the method of Gerngross et al., [0172] Biochemistry, 33, 9311 (1994) with the following alterations. One aliquot (110 mg protein) of frozen cells was thawed on ice and resuspended in 10 mM KPi (pH 7.2), 5% glycerol, and 0.05% Hecameg (Buffer A) containing the following protease inhibitors at the indicated final concentrations: benzamidine (2 mM), phenylmethylsulfonyl fluoride (PMSF, 0.4 mM), pepstatin (2 mg/ml), leupeptin (2.5 mg/ml), and Na-p-tosyl-l-lysine chloromethyl ketone (TLCK, 2 MM). EDTA was omitted at this stage due to its incompatibility with hydroxylapatite (HA). This mixture was homogenized with three series of 10 strokes each in two Thomas homogenizers while partially submerged in an ice bath and then sonicated for 2 minutes in a Branson Sonifier 250 at 30% cycle, 30% power while on ice. All subsequent procedures were carried out at 4° C.
The lysate was immediately centrifuged at 100000× g in a Beckman 50.2Ti rotor for 80 minutes, and the resulting supernatant (10.5 ml, 47 mg) was immediately filtered through a 0.45 mm Uniflow filter (Schleicher and Schuell Inc., Keene, N.H.) to remove any remaining insoluble matter. Aliquots of the soluble fraction (1.5 ml, 7 mg) were loaded onto a 5 ml BioRad Econo-Pac HTP column that had been equilibrated with Buffer A (+protease inhibitor mix) attached to a BioRad Econo-system, and the column was washed with 30 ml Buffer A. All chromatographic steps were carried out at a flow rate of 0.8 ml/minute. PHA synthase was eluted form the HA column with a 32×32 ml linear gradient from 10 to 300 mM KPi. [0173]
Fraction collection tubes were prepared by addition of 30 ml of 100 mM EDTA to provide a metalloprotease inhibitor at 1 mM immediately after HA chromatography. PHA synthase was eluted in a broad peak between 110-180 mM KPi. Fractions (3 ml) containing significant PHA synthase activity were pooled and stored at 0° C. until the entire soluble fraction had been run through the chromatographic process. Pooled fractions then were concentrated at 4° C. by use of a Centriprep-30 concentrator (Amicon) to 3.8 mg/ml. Aliquots (0.5 ml) were either flash frozen and stored in liquid N[0174] ₂or glycerol was added to a final concentration of 50% and samples (1.9 mg/ml) were stored at −20° C.
Western Analysis. [0175]
Samples of [0176] T. ni cells were fractionated by SDS-PAGE on 10% polyacrylamide gels, and the proteins then were transferred to 0.2 mm nitrocellulose membranes using a BioRad Transblot SD Semi-Dry electrophoretic transfer cell according to the manufacturer. Proteins were transferred for 1 hour at 15 V. The membrane was rinsed with doubly distilled H₂O, dried, and treated with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tween) and 3% nonfat dry milk to block non-specific binding sites. Primary antibody (rabbit anti-PHA synthase) was applied in fresh blocking solution and incubated at 25° C. for 2 hours. Membranes were then washed four times for 10 minutes with PBS-Tween followed by the addition of horseradish peroxidase-conjugated goat-anti-rabbit antibody (Boehringer-Mannheim) diluted 10,000× in fresh blocking solution and incubated at 25° C. for 1 hour. Membranes were washed finally in three changes (10 minutes) of PBS, and the immobilized peroxidase label was detected using the chemiluminescent LumiGLO substrate kit (Kirkegaard and Perry, Gaithersburg, Md.) and X-ray film.
N-terminal Analysis. [0177]
Approximately 10 mg of purified PHA synthase was run on a 10% SDS-polyacrylamide gel, transferred to PVDF (Immobilon-PSQ, Millipore Corporation, Bedford, Mass.), stained with Amido Black, and sequenced on a 494 Procise Protein Sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.). [0178]
Double-infection Protocol. [0179]
Four 100 ml spinner flasks were each inoculated with 8×10[0180] ⁷cells in 50 ml of fresh insect medium. To flask 1, an additional 20 ml of fresh insect medium was added (uninfected control); to flask 2, 10 ml BacPAK6::phbC viral stock (1×10⁸pfu/ml) and 10 ml fresh insect medium were added; to flask 3, 10 ml BacPAK6::FAS206 viral stock (1×10⁸pfu/ml) and 10 ml fresh insect medium were added; and to flask 4, 10 ml BacPAK6::phbC viral stock (1×10⁸pfu/ml) and 10 ml BacPAK6::FAS206 viral stock (1×10⁸pfu/ml) were added. These viral infections were carried out at a multiplicity of infection of approximately 10. Cultures were maintained under normal growth conditions and 15 ml samples were removed at 24, 48, and 72 hour time points. Cells were collected by gentle centrifugation at 1000× g for 5 minutes, the medium was discarded, and the cells were immediately stored at −70° C.
PHA Synthase Assay. [0181]
Coenzyme A released by PHA synthase in the process of polymerization was monitored precisely as described by Gerngross et al. (supra) using 5,5′-dithiobis (2-nitrobenzoic acid, DTNB) (Ellman, [0182] Arch. Biochem. Biophy., 82, 70 (1959)).
The presence of HBCoA was monitored spectrophotometrically. Assays were performed at 25° C. in a Hewlett Packard 8452A diode array spectrophotometer equipped with a waterjacketed cell holder. Two-piece Stama Spectrosil spectrophotometer cells with pathlengths of 0.1 and 0.01 cm were employed to avoid errors arising from the compression of the absorbance scale at higher values. Absorbance was monitored at 232 nm, and E[0183] ₂₃₂nm of 4.5×10³M⁻¹cm⁻¹was used in calculations. One unit (U) of enzyme is the amount required to hydrolyze 1 mmol of substrate minute⁻¹. Buffer (0.15 M KPi, pH 7.2) and substrate were equilibrated to 25° C. and then combined in an Eppendorf tube also at 25° C. Enzyme was added and mixed once in the pipet tip used to transfer the entire mixture to the spectrophotometer cell. The two-piece cell was immediately assembled, placed in the spectrophotometer with the cell holder (type CH) adapted for the standard 10 mm pathlength cell holder of the spectrophotometer. Manipulations of sample, from mixing to initiation of monitoring, took only 10-15 seconds. Absorbance was continually monitored for up to 10 minutes. Calibration of reactions was against a solution of buffer and enzyme (no substrate) which led to absorbance values that represented substrate only.
PHB Assay. [0184]
PHB was assayed from Sf21 cell samples according to the propanolysis method of Riis et al., [0185] J. Chromo., 445, 285 (1988). Cell pellets were thawed on ice, resuspended in 1 ml cold ddH₂O and transferred to 5 ml screwtop test tubes with teflon seals. Two ml of ddH₂O were added, the cells were washed and centrifuged and then 3 ml of acetone were added and the cells washed and centrifuged. The samples were then desiccated by placing them in a 94° C. oven for 12 hours. The following day 0.5 ml of 1,2-dichloroethane, 0.5 ml acidified propanol (20 ml HCl, 80 ml 1-propanol) and 50 ml benzoic acid standard were added and the sealed tubes were heated to 100° C. in a boiling water bath for 2 hours with periodic vortexing. The tubes were cooled to room temperature and the organic phase was used for gas-chromatographic (GC) analysis using a Hewlett Packard 5890A gas chromatograph equipped with a Hewlett Packard 7673A automatic injector and a fused silica capillary column, DB-WAX 30W of 30 meter length. Positive samples were further subjected to GC-mass spectrometric (MS) analysis for the presence of propylhydroxybutyrate using a Kratos MS25 GC/MS. The following parameters were used: source temperature, 210° C.; voltage, 70 eV; and accelerating voltage, 4KeV.
Catalytic Activities [0186]
Ketoacyl synthase (KS) activity was assessed radiochemically by the condensation-[0187] ¹⁴CO₂exchange reaction (Smith et al., PNAS USA 73, 1184 (1976)).
Transferase (AT) activity was assayed, using malonyl-CoA as donor and pantetheine as acceptor, by determining spectrophotometrically the free CoA released in a coupled ATP citrate-lyase-malate dehydrogenase reaction (see, Rangen et al., [0188] J. Biol. Chem., 266, 19180 (1991).
Ketoreductase (KR) was assayed spectrophotometrically at 340 nm: assay systems contained 0.1 M potassium phosphate buffer (pH 7), 0.15 mM NADPH, enzyme and either 10 mM trans-1-decalone or 0.1 mM acetoacetyl-CoA substrate. [0189]
Dehydrase (DH) activity was assayed spectrophotometrically at 270 nm using S-DL-β-hydyroxybutyryl N-acetylcysteamine as substrate (Kumar et al., [0190] J. Biol. Chem., 245, 4732 (1970)).
Enoyl reductase (ER) activity was assayed spectrophotometrically at 340 nm essentially as described by Strom et al. ([0191] J. Biol. Chem., 254, 8159 (1979)); the assay system contained 0.1 M potassium phosphate buffer (pH 7), 0.15 mM NADPH, 0.375 nM crotonoyl-CoA, 20 μM CoA and enzyme.
Thioesterase (TE) activity was assessed radiochemically by extracting and assaying the [[0192] ¹⁴C]palmitic acid formed from [1-¹⁴C]palmitoyl-CoA during a 3 minute incubation Smith, Meth. Enzymol., 71C, 181(1981); the assay was in a final volume of 0.1 ml, 25 mM potassium phosphate buffer (pH 8), 20 μM [1-¹⁴C]palmitoyl-CoA (20 nCi) and enzyme.
Assay of overall fatty acid synthase activity was performed spectrophotometrically as described previously by Smith et al. ([0193] Meth. Enzymol., 35, 65 (1975)). All enzyme activities were assayed at 37° C. except the transferase, which was assayed at 20° C. Activity units indicate nmol of substrate consumed/minute. All assays were conducted, at a minimum, at two different protein concentrations with the appropriate enzyme and substrate blanks included.

II. EXAMPLES

Example 1

Expression of A. Eutrophus PHA Synthase Using a Baculovirus System

Recent work has shown that PHA synthase from [0194] A. eutrophus can be overexpressed in E. coli, in the absence of 3-ketothiolase and acetoacetyl-CoA reductase (Gerngross et al., supra) and can be expressed in plants (See Poirier et al., Biotech, 13, 142 (1995) for a review). Isolation of the soluble form of PHA synthase provides opportunities to examine the mechanistic details of the priming and initiation reactions. Because the baculovirus system has been successful for the expression of a number of prokaryotic genes as soluble proteins, and insect cells, unlike bacterial expression systems, carry out a wide array of post-translational modifications, the baculovirus expression system appeared ideal for the expression of large quantities of soluble PHA synthase, a protein that must be modified by phosphopantetheine in order to be catalytically active (Gerngross et al., supra).
Purification of PHA Synthase. [0195]
The purification procedure employed for PHA synthase is a modification of Gerngross et al. (supra) involving the elimination of the second liquid chromatographic step and inclusion of a protease-inhibitor cocktail in all buffers. All steps were carried out on ice or at 4° C. except where noted. Frozen cells were thawed on ice in 10 ml of Buffer A (10 mM KPi, pH 7.2, 05% glycerol, and 0.05% Hecameg) and then immediately homogenized prior to centrifugation and HA chromatography. [0196]
The results of these efforts are summarized in Table 1 and FIG. 7. A prominent band at 64 kDa is visible in total, soluble, and HA eluate protein samples fractionated by SDS/PAGE ([0197] lanes 4, 5, and 6 of FIG. 7, respectively). The initial specific activity of the isolated PHA synthase was 20-fold higher than previous attempts at expression and purification of this polypeptide. Approximately 1000 units of PHB synthase have been purified, based on calculations from the direct spectrophotometric assay detailed below, with an overall recovery of activity of 70%. The large proportion of synthase present in the membrane fraction, and the fact that over 90% of the initial activity was found in the soluble fraction, suggest either that the synthase in the membrane fraction is in an inactive form or that the direct assay is not applicable to the initial, 12 U/mg, crude extract.

TABLE 1

Purification of PHA Synthase

protein specific

sample total units vol (mL) (mg) (mg/ml) activity recovery

total 1430 11.5 113 9.8 12.7 100

protein

soluble 1340 10.5 47 4.5 28.6 93

protein

pooled 1020 7.9 30 3.8 34.2 71

HA

fractions
N-terminal sequencing of the 64 kDa protein confirmed its identity as PHA synthase (FIG. 8). Two prominent N-termini, at amino acid residue 7 (alanine) and residue 10 (serine) were obtained in a 3:2 ratio. This heterogeneous N-terminus presumably is the result of aminopeptidase activity. Western analysis using a rabbit-anti-PHA synthase antibody corroborated the results of the sequencing and indicated the presence of at least three bands that resulted from proteolysis of PHA synthase (FIG. 7B, lanes 4-6). The antibody was specific for PHA synthase since neither [0198] T. ni nor baculoviral proteins showed reactivity (FIG. 7B, lanes 2 and 3). N-terminal protein sequencing (FIG. 8) showed directly that the 44 kDa (band b) and 32 kDa (band d) proteins were derived from PHA synthase (fragments beginning at A181/N185 and at G387, respectively). The 35-40 kDa (band c) protein gave low sequencing yields and may contain a blocked N-terminus. Inspection of FIG. 7B suggests that most degradation occurs following cell disruption since the total protein sample of this gel (lane 4) was prepared by boiling intact cells directly in SDS sample buffer while the HA sample (lane 6) went through the purification procedure described above.
Assay of Synthase Activity. [0199]
Due to the significant level of expression obtained using the baculovirus system, the synthase activity could be assayed spectrophotometrically by monitoring hydrolysis of the thioester bond at 232 nm, the wavelength at which there is a maximum decrease in absorbance upon hydrolysis. The difference between substrate (HBCOA) and product (CoA) at this wavelength is shown in FIG. 9. Absorbance of HBCoA and CoA at 232 nm occurs at a trough between two well-separated peaks. Assays were carried out at pH 7.2 for comparative analysis with previous studies (Gerngross et al., supra). Substrate (R-(−)3-HBCoA) substrate for these studies was prepared using the mixed anhydride method (Haywood et al., supra), and its concentration was determined by measuring A[0200] ₂₆₀. The short pathlength cells (0.1 cm and 0.01 cm) allowed use of relatively high reaction concentrations while conserving substrate and enzyme. Assay results showed an initial lag period of 60 seconds prior to the linear decrease in A₂₃₂, and velocities were determined from the slope of these linear regions of the assay curves. The length of the lag period was variable and was inversely related to enzyme concentration. These data are consistent with those using PHA synthase purified from E. coli (Gerngross et al., supra).
FIGS. 10 and 11 show the V versus S and 1/V versus 1/S plots, respectively. The double reciprocal plot was concave upward which is similar to results obtained from studies of the granular PHA synthase from [0201] Zooglea ramigera (Fukui et al., Arch. Microbiol, 110, 149 (1976)) and suggests a complex reaction mechanism. Examinations of velocity and specific activity as a function of enzyme concentration are shown in FIGS. 12 and 13. These results confirm that specific activity of the synthase depends upon enzyme concentration. The pH activity curve for A. eutrophus PHA synthase purified from T. ni cells is shown in FIG. 14. The curve shows a broad activity maximum centered around pH 8.5. This result agrees well with prior work on the A. eutrophus PHB synthase although it is significantly different than results obtained for the PHB synthase from Z. ramigera for which the optimum was determined to be pH 7.0.
The effect of varying enzyme concentration in the presence of a fixed amount of substrate revealed an intriguing trend (FIG. 15). From these data it appears that the extent of polymerization is dependent on the amount of enzyme included in the reaction mixture. This could be explained if there is a “terminal length” limitation of the polymer, which, once reached, cannot be extended any further. If this is the case, it would also suggest that termination of the polymerization reaction, the release of the synthase from the polymer, and/or reinitiation of polymerization by the newly released synthase are relatively slow events since no evidence of these reactions are seen within the time course of these studies. The phenomenon observed in FIG. 15 is not the result of decay of the enzyme over the course of the assay since virtually identical results are obtained following a 10 minute preincubation of the synthase at 25° C. [0202]
It must also be noted that comparisons of the direct spectrophotometric assays used here and the more common assay involving the use of Ellman's reagent, DTNB, (Ellman, supra) in the formation of thiolate of coenzyme-A showed that the values determined by the direct method were approximately 70% of the values determined using Ellman's reagent. This may be due to phase separation occurring in the cuvettes as the relatively insoluble polymer is formed. In support of this notion, a faint haze or opalescence in the cuvette developed during the course of the reaction, particularly at higher substrate concentrations. [0203]
PHA synthase purified from insect cells appears to be relatively stable. Examination of activity following storage, in liquid N[0204] ₂and at −20° C. in the presence of 50% glycerol showed that approximately 50% of synthase activity remained after 7 weeks when stored in liquid N₂and approximately 75% of synthase activity remained after 7 weeks when stored at −20° C. in the presence of 50% glycerol.
The expression of PHA synthase from [0205] A. eutrophus in a baculovirus expression system results in the synthase constituting approximately 50% of total protein 60 hours post-infection; however, approximately 50-75% of the synthase is observed in the membrane-associated fraction. This elevated level of expression allowed purification of the soluble PHA synthase using a single chromatographic step on HA. The purity of this preparation is estimated to be approximately 90% (intact PHA synthase and 3 proteolysis products).
The initial specific activity of 12 U/mg was approximately 20-fold higher than the most successful previous efforts at overexpression of [0206] A. eutrophus PHA synthase. The synthase reported here was isolated from a 250 ml culture with 70% recovery which represents an improvement of 500-fold (1000 U/64 U×8 L/0.25 L) when compared to an 8 L E. coli culture with 40% recovery. This high expression level should provide sufficient PHA synthase for extensive structural, functional, and mechanistic studies. Furthermore, it is clear that the baculovirus expression system is an attractive option for isolation of other PHA synthases from various sources.
PHA synthase produced in the baculovirus system was of sufficient potency to allow direct spectrophotometric analysis of the hydrolysis of the thioester bond of HBCoA at 232 nm. These assays revealed a lag period of approximately 60 seconds, the length of which was variable and inversely related to enzyme concentration. Such a lag period presumably reflects a slow step in the reaction, perhaps correlating to dimerization of the enzyme, the priming, and/or initiation steps in formation of PHB. Size exclusion chromatographic examination of the PHB synthase native MW indicated two forms of the synthase. One form showed a MW of approximately 100-160 kDa and the other showed a MW of approximately 50-80 kDA; these two forms likely represent the dimer and monomer of PHA synthase, respectively. Similar results have been reported previously in which two forms of approximately 60 and 130 kDa were observed. Comparisons of the direct assay reported here and the indirect assay using DTNB revealed that the former resulted in values that were 70% of the values determined by the DTNB indirect assay. Although the reason for this difference has not been examined in detail, it is probable that the apparent phase separation that occurred upon PHB formation in the short pathlength cuvettes used, particularly with high [HBCoA], results in this discrepancy. [0207]
Enzymatic analyses of the PHA synthase have found that the enzyme has a broad pH optimum centered at pH 8.5; however, the studies described herein have been performed at pH 7.2 to provide comparative values with the results of others. Moreover, the specific activity of this enzyme is dependent upon enzyme concentration which confirms and extends earlier results (Gerngross et al., supra). [0208]
In studies intended to examine the dependence of activity upon enzyme concentration, it became apparent that the extent of the polymerization reaction is dependent on the amount of enzyme included in the reaction mixture. Specifically, decreasing the amount of enzyme leads not only to decreased velocity of reaction but also to a decreased extent of condensation (FIG. 15). One possible explanation is that the enzyme is thermally labile; however, identical assays in which the enzyme is preincubated at 25° C. for 10 minutes prior to initiation of the reaction had similar results. Another possibility is that a terminal-length of the polymer is reached precluding further condensations until the particular synthase molecule is released from the terminal-length polymer. [0209]
This work clearly demonstrates the value of the baculovirus expression system for the production of [0210] A. eutrophus PHA synthase and for the potential application to studies of other PHA synthases. Furthermore, the high level of expression obtained using the baculoviral system should allow convenient analysis for substrate-specificity and structure-function studies of PHA synthases from relatively crude insect cell extracts.

Example 2

Co-expression of Rat FAS Dehydrase Mutant cDNA and PHB Synthase Gene in Insect Cells

Expression of a rat FAS DH− cDNA in Sf9 cells has been reported previously (Rangan et al., [0211] J. Biol. Chem., 266, 19180 (1991); Joshi et al., Biochem. J., 296, 143 (1993)). Once activity of thephbC gene product had been established in insect cells (see Example 1), baculovirus clones containing the rat FAS DH− cDNA and BacPAK6::phbC were employed in a double-infection strategy to determine if PHB would be produced in insect cells. It was not known if an intracellular pool of R(−)-3-hydroxybutyrate would be stable or available as a substrate for the PHB synthase. In order for the R-(−)-3-hydroxybutyrylCoA to be available as a substrate, the R-(−)-3-hydroxybutyrylCoA released from rat FAS DH− protein must be trapped by the PHB synthase and incorporated into a polymer at a rate faster than β-oxidation, which would regenerate acetylCoA. It was also not known if the stereochemical configuration of the 3-hydroxyl group, which must be in the R form, would be recognized as a substrate by PHB synthase. Fortunately, previous biochemical studies on eukaryotic FASs indicated that the R form of 3-hydroxybutyrylCoA would be generated (Wakil et al., J. Biol. Chem., 237, 687 (1962)).
SDS-PAGE of protein samples from a time course of uninfected, single-infected, and dual-infected Sf21 cells was performed (FIG. 16). From these data, it is clear that the rat FAS DH mutant and PHB synthase polypeptides are efficiently co-expressed in Sf21 cells. However, co-expression results in ˜50% reduced levels of both polypeptides compared to Sf21 cells that are producing the individual proteins. Western analysis using anti-rat FAS (Rangan et al., supra) and anti-PHA synthase antibodies confirmed simultaneous production of the corresponding proteins. [0212]
To provide further evidence that PHB was being synthesized in insect cells, [0213] T. ni cells which had been infected with a baculovirus vector encoding rat FAS DH⁰and/or a baculovirus vector encoding PHA synthase were analyzed for the presence of granules. Infected cells were fixed in paraformaldehyde and incubated with anti-PHA synthase antibodies (Williams et al., Protein Exp. Purif., 7, 203 (1996)). Granules were observed only in doubly infected cells (Williams et al., App. Environ. Micro., 62, 2540 (1996)).
Characterization of PHB Production in Insect Cells. [0214]
In order to determine if de novo synthesis of PHB was occurring in Sf21 cells that co-express the rat FAS DH mutant and PHB synthase, fractions of these samples were extracted, the extract subjected to propanolysis, and analyzed for the presence of propylhydroxybutyrate by gas chromatography (FIG. 17). A unique peak with a retention time that coincided with a propylhydroxybutyrate standard was detected only in the double infection samples at 48 and 72 hours, in contrast to the individually expressed gene products and uninfected controls, which were negative. These samples were analyzed further by GC/MS to confirm the identity of the product. FIG. 18 shows mass spectroscopy data corresponding to the material obtained from peak 10.1 in the gas chromatograph compared to a propylhydroxybutyrate standard. The results show that PHB synthesis is occurring only in [0215] Sf2 1 cells co-expressing the rat FAS DH mutant cDNA and the phbC gene from A. eutrophus. Integration of the peak in the gas chromatograph corresponding to propylhydroxybutyrate revealed that approximately 1 mg of PHB was isolated from 1 liter culture of Sf21 cells (approximately 600 mg dry cell weight of Sf21 cells). Thus, the ratFAS206 protein effectively replaces the β-ketothiolase and acetoacetyl-CoA reductase functions, resulting in the production of PHB by a novel pathway.
The approach described here provides a new strategy to combine metabolic pathways that are normally engaged in primary anabolic functions for production of polyesters. The premature termination of the normal fatty acid biosynthetic pathway to provide suitably modified acylCoA monomers for use in PHA synthesis can be applied to both prokaryotic and eukaryotic expression since the formation of polymer will not be dependent on specialized feedstocks. Thus, once a recombinant PHA monomer synthase is introduced into a prokaryotic or eukaryotic system, and co-expressed with the appropriate PHA synthase, novel bipolymer formation can occur. [0216]

Example 3

Cloning and Sequencing of the Vep ORFI PKS Gene Cluster

The entire PKS cluster form [0217] Streptomyces venezuelae was cloned using a heterologous hybridization strategy. A 1.2 kb DNA fragment that hybridized strongly to a DNA encoding an eryA PKS β-ketoacyl synthase domain was cloned and used to generate a plasmid for gene disruption. This method generated a mutant strain blocked in the synthesis of the antibiotic. A S. venezuelae genomic DNA library was generated and used to clone a cosmid containing the complete methymycin aglycone PKS DNA. Fine-mapping analysis was performed to identify the order and sequence of catalytic domains along the multifunctional PKS (FIG. 19). DNA sequence analysis of the vep ORFI showed that the order of catalytic domains is KS^Q/AT/ACP/KS/AT/KR/ACP/KS/AT/DH/KR/ACP. The complete DNA sequence, and corresponding amino acid sequence, of the vep ORFI is shown in FIG. 23 (SEQ ID NO:1 and SEQ ID NO:2, respectively).
The sequence data indicated that the PKS gene cluster encodes a polyene of twelve carbons. The vep gene cluster contains 5 polyketide synthase modules, with a loading module at its 5′ end and an ending domain at its 3′ end. Each of the sequenced modules includes a keto-ACP (KS), an acyltransferase (AT), a dehvdratase (DH), a keto-reductase (KR), and an acyl carrier protein domain. The six acyltransferase domains in the cluster are responsible for the incorporation of six acetyl-CoA moieties into the product. The loading module contains a KS[0218] ^Q, an AT and an ACP domain. KS^Qrefers to a domain that is homologous to a KS domain except that the active site cysteine (C) is replaced by glutamine (Q). There is no counterpart to the KS^Qdomain in the PKS clusters which have been previously characterized.
The ending domain (ED) is an enzyme which is responsible for the attachment of the nascent polyketide chain onto another molecule. The amino acid sequence of ED resembles an enzyme, HetM, which is involved in Anabaena heterocyst formation. The homology between vep and HetM suggests that the polypeptide encoded by the vep gene cluster may synthesize a polyene-containing composition which is present in the spore coat or cell wall of its natural host, [0219] S. venezuelae.

Example 4

Preparation of a Vector Encoding a Saturated β-hydroxyhexanoyl CoA Monomer or an Unsaturated β-hydroxyhexanoyl CoA Monomer

To provide a recombinant monomer synthase that generates a saturated β-hydroxyhexanoylCoA or unsaturated β-hydroxyhexanoylCoA monomer, the linear correspondence between the genetic organization of the Type I macrolide PKS and the catalytic domain organization in the multifunctional proteins is assessed (Donadio et al., supra, 1991; Katz et al., [0220] Ann. Rev. Microbiol., 47, 875 (1993)). First, a DNA encoding a TE is added to the 3′ end of an ORFI of a Type I PKS, preferably the met ORF I (FIG. 6) as recently described by Cortes et al. (Science, 268, 1487 (1995)) in the erythromycin system. To ensure that the DNA encoding the TE is completely active, DNA encoding a linker region separating a normal ACP-TE region in a PKS, for example, the one found in met PKS ORF5 (FIG. 5), will be incorporated into the DNA. The resulting vector can be introduced into a host cell and the TE activity, rate of release of the CoA product, and identity of the fatty acid chain determined.
The acyl chain that is most likely to be released is the CoA ester, specifically the 3-hydroxy-4-methyl heptenoylCoA ester, since the fully elongated chain is presumably released in this form prior to macrolide cyclization. If the CoA form of the acyl chain is not observed, then a gene encoding a CoA ligase will be cloned and co-expressed in the host cell to catalyze formation of the desired intermediate. [0221]
There is clear precedent for release of the predicted premature termination products from mutant strains of macrolide-producing Streptomyces that produce intermediates in macrolide synthesis (Huber et al., [0222] Antimicrob. Agents Chemother., 34, 1535 (1990); Kinoshita et al., J. Chem. Soc., Chem. Comm., 14, 943 (1988)). The structure of these intermediates is consistent with the linear organization of functional domains in macrolide PKSs, particularly those related to eryA, tyl, and met. Other known PKS gene clusters include, but are not limited to, the gene cluster encoding 6-methylsalicylic acid synthase (Beck et al., Eur. J. Biochem., 192, 487 (1990)), soraphen A (Schupp et al., J. Bacteriol, 117, 3673 (1995)), and sterigmatocystin (Yu et al., J. Bacteriol., 177, 4792 (1995)).
Once the release of the 3-hydroxy-4-methyl heptenoylCoA ester is established, DNA encoding the extender unit AT in [0223] met module 1 is replaced to change the specificity from methylmalonylCoA to malonylCoA (FIGS. 4-6). This change eliminates methyl group branching in the β-hydroxy acyl chain. While comparison of known AT amino acid sequences shows high overall amino acid sequence conservation, distinct regions are readily apparent where significant deletions or insertions have occurred. For example, comparison of malonyl and methylmalonyl amino acid sequences reveals a 37 amino acid deletion in the central region of the malonyltransferase. Thus, to change the specificity of the methylmalonyl transferase to malonyl transferase, the met ORFI DNA encoding the 37 amino acid sequence of MMT will be deleted, and the resulting gene will be tested in a host cell for production of the desmethyl species, 3-hydroxyheptenoylCoA. Alternatively, the DNA encoding the entire MMT can be replaced with a DNA encoding an intact MT to affect the desired chain construction.
After replacing MMT with MT, DNA encoding DHIER will be introduced into DNA encoding met [0224] ORFI module 1. This modification results in a multifunctional protein that generates a methylene group at C-3 of the acyl chain (FIG. 6). The DNA encoding DH/ER will be PCR amplified from the available eryA or tyl PKS sequences, including the DNA encoding the required linker regions, employing a primer pair to conserved sequences 5′ and 3′ of the DNA encoding DH/ER. The PCR fragment will then be cloned into the met ORFI. The result is a DNA encoding a multifunctional protein (MT* DH/ER*TE*). This protein possesses the full complement of keto group processing steps and results in the production of heptenoylCoA.
The DNA encoding dehydrase in met [0225] module 2 is then inactivated, using site-directed mutagenesis in a scheme similar to that used to generate the rat FAS DH− described above (Joshi et al., J. Biol. Chem., 268, 22508 (1993)). This preserves the required (R)-3-hydroxy group which serves as the substrate for PHA synthases and results in (R)-3-hydroxyheptanoylCoA species.
The final domain replacement will involve the DNA encoding the starter unit acyltransferase in met module 1 (FIG. 5), to change the specificity from propionyl CoA to acetyl CoA. This shortens the (R)-3-hydroxy acyl chain from heptanoyl to hexanoyl. The DNA encoding the catalytic domain will need to be generated based on a FAS or 6-methylsalicylic acid synthase model (Beck et al., [0226] Eur. J. Biochem., 192, 487 (1990)) or by using site-directed mutagenesis to alter the specificity of the resident met PKS propionyltransferase sequence. Limiting the initiator species to acetylCoA can result in the use of this starter unit by the monomer synthase. Previous work with macrolide synthases have shown that some are able to accept a wide range of starter unit carboxylic acids. This is particularly well documented for avermectin synthase, where over 60 new compounds have been produced by altering the starter unit substrate in precursor feeding studies (Dutton et al., J. Antibiotics, 44, 357 (1991)).

Example 5

Preparation of a Vector Encoding a Recombinant Monomer Synthase that Synthesizes 3-hydroxyl-4-hexenoic Acid

To provide a recombinant monomer synthase that synthesizes 3-hydroxyl-4-hexenoic acid, a precursor for polyhydroxyhexenoate, the DNA segment encoding the loading and the first module of the vep gene cluster was linked to the DNA [0227] segment encoding module 7 of the tyl gene cluster so as to yield a recombinant DNA molecule encoding a fusion polypeptide which has no amino acid differences relative to the corresponding amino acid sequence of the parent modules. The fusion polypeptide catalyzes the synthesis of 3-hydroxyl-4-hexenoic acid. The recombinant DNA molecule was introduced into SCP2, a Streptomyces vector, under the control of the act promoter (pDHS502, FIG. 20). A polyhydroxyalkanoate polymerase gene, phaC1 from Pseudomonas oleavorans, was then introduced downstream of the recombinant PKS cluster (pDHS505; FIGS. 22 and 23). The DNA segment encoding the polyhydroxyalkanoate polymerase is linked to the DNA segment encoding the recombinant PKS synthase so as to yield a fusion polypeptide which synthesizes polyhydroxyhexenoate in Streptomyces. Polyhydroxyhexenoate, a biodegradable thermoplastic, is not naturally synthesized in Streptomyces, or as a major product in any other organism. Moreover, the unsaturated double bond in the side chain of polyhydroxyhexenoate may result in a polymer which has superior physical properties as a biodegradable thermoplastic over the known polyhydroxyalkanoates.

Example 6

Deletion of the desr Gene of the Desosamine Biosynthetic Gene Cluster

As some macrolides have more than one attached sugar moiety, the assignment of sugar biosynthetic genes to the appropriate sugar biosynthetic pathway can be quite difficult. Since methymycin (a compound of formula (1)) and neomethymycin (a compound of formula (2)) (FIG. 24) (Donin et al., 1953; Djerassi et al., 1956), two closely related macrolide antibiotics produced by [0228] Streptomyces venezuelae, contain desosamine as their sole sugar component, the organization of the sugar biosynthetic genes in the methymycin/neomethymycin gene cluster may be less complicated. Thus, this system was chosen for the study of the biosynthesis of desosamine, a N,N-dimethylamino-3,4,6-trideoxyhexose, which also exists in the erythromycin structure (Flinn et al., 1954).
To study the formation of this unusual sugar, a DNA library was constructed by partially digesting the genomic DNA of [0229] S. venezuelae (ATCC 15439) with Sau3A I into 35-40 kb fragments which were ligated into the cosmid vector pNJ1 (Tuan et al., 1990). The recombinant DNA was packaged into bacteriophage λ which was used to transfect E. coli DH5a. The resulting cosmid library was screened for desired clones using the tylA1 and tylA2 genes from the tylosin biosynthetic cluster as probes (Baltz et al., 1988; Merson-Davies et al., 1994). These two probes are specific for sugar biosynthetic genes whose products catalyze the first two steps universally followed by all unusual 6-deoxyhexoses studied thus far. The initial reaction involves conversion of glucose-1 -phosphate to TDP-D-glucose by α-D-glucose-1-phosphate thymidylyltransferase (TylA1) and subsequently, TDP-D-glucose is transformed to TDP-4-keto-6-deoxy-D-glucose by TDP-D-glucose 4,6-dehydratase (TylA2). Three cosmids were found to contain genes homologous to tylA1 and tylA2. Further analysis of these cosmids led to the identification of nine open reading frames (ORFs) downstream of the PKS genes (FIG. 24). Based on sequence similarities to other sugar biosynthetic genes, especially those derived form the erythromycin cluster (Gaisser et al., 1997; Summers et al., 1997), eight of these nine ORFs are believed to be involved in the biosynthesis of TDP-D-desosamine. Interestingly, the ery cluster lacks homologs of the tylA1 and tylA2 genes that are responsible for the first two steps in desosamine pathway. It is possible that the erythromycin biosynthetic machinery may rely on a general cellular pool of TDP-4-keto-6-deoxy-D-glucose for mycarose and desosamine formation. Depicted in FIG. 24 is a biosynthetic pathway for TDP-D-desosamine.
Although eight of the nine ORFs have been assigned to desosamine formation, the presence of desR, which shows strong sequence homology to β-glucosidases (as high as 39% identity and 46% similarity) (Castle et al., 1998), within the desosamine gene cluster is puzzling. To investigate the function of DesR relative to the biosynthesis of methymycin/neomethymycin, a disruption plasmid (pBL1005) derived from pKC1139 (containing an apramycin resistance marker) (Bierman et al., 1992) was constructed in which a 1.0 kb NcoI/XhoI fragment of the desR gene was deleted and replaced by the thiostrepton resistance (tsr) gene (1.1 kb) (Bibb et al., 1985) via blunt-end ligation. This plasmid was used to transform [0230] E. coli S17-1, which serves as the donor strain to introduce the pBL1005 construct through conjugal transfer into the wild-type S. venezuelae (Bierman et al., 1992). The double crossover mutants in which chromosomal desR had been replaced with the disrupted gene were selected according to their thiostrepton-resistant and apramycin-sensitive characteristics. Southern blot hybridization analysis was used to confirm the gene replacement.
The desired mutant was first grown at 29° C. in seed medium for 48 hours, and then inoculated and grown in vegetative medium for another 48 hours (Cane et al., 1993). After the fermentation broth was centrifuged at 10,000 g to remove cellular debris and mycelia, the supernatant was adjusted to pH 9.5 with concentrated KOH, and extracted with an equivolume of chloroform (four times). The organic layer was dried over sodium sulfate and evaporated to dryness. The amber oil-like crude products were first subjected to flash chromatography on silica gel using a gradient of 0-40% methanol in chloroform, followed by HPLC purification on a C[0231] ₁₈column eluted isocratically with 45% acetonitrile in 57 mM ammonium acetate (pH 6.7). In addition to methymycin (a compound of formula (1)) and neomethymycin (a compound of formula (2)), two new products were isolated. The yield of a compound of formula (13) and a compound of formula (14) was each in the range of 5-10 mg/L of fermentation broth. However, a compound of formula (1) and a compound of formula (2) remained to be the major products. High-resolution FAB-MS revealed that both compounds have identical molecular compositions that differ from methymycin/neomethymycin by an extra hexose. The chemical nature of these two new compounds were elucidated to be C-2′ β-glucosylated methymycin and neomethymycin (a compound of formula (13) and formula (14), respectively) by extensive spectral analysis.
The spectral data of (13): [0232] ¹H NMR (acetone-d₆) δ 6.56 (1H, d, J=16.0, 9-H), 6.46 (1H, d, J=16.0, 8-H), 4.67 (1H, dd, J=10.8, 2.0, 11-H), 4.39 (1H, d, J=7.5, 1′-H), 4.32 (1H, d, J=8.0, 1″-H), 3.99 (1H, dd, J=11.5, 2.5, 6″-H), 3.72 (1H, dd, J=11.5, 5.5, 6″-H), 3.56 (1H, m, 5′-H), 3.52 (1H, d, J=10.0, 3-H), 3.37 (1H, t, J=8.5, 3″-H), 3.33 (1H, m, 5″-H), 3.28 (1H, t, J=8.5, 4″-H), 3.23 (1H, dd, J=10.5, 7.5, 2′-H), 3.15 (1H, dd, J =8.5, 8.0, 2″-H), 3.10 (1H, m, 2-H), 2.75 (1H, 3′-H, buried under H₂O peak), 2.42 (1H, m, 6H), 2.28 (6H, s, NMe₂), 1.95 (1H, m, 12-H), 1.9 (1H, m, 5-H), 1.82 (1H, m, 4′-H), 1.50 (1H, m, 12-H), 1.44 (3H, d, J=7.0, 2-Me), 1.4 (1H, m, 5-H), 1.34 (3H, s, 10-Me), 1.3 (1H, m, 4-H), 1.25 (1H, m, 4′-H), 1.20 (3H, d, J=6.0, 5′-Me), 1.15 (3H, d, J=7.0, 6-Me), 0.95 (3H, d, J=6.0, 4-Me), 0.86 (3H, t, J=7.5, 12-Me). High-resolution FAB-MS: calc for C₃,H₅₄NO₁₂(M+H)⁺632.3646, found 632.3686.
Spectral data of (14): [0233] ¹H NMR (acetone-d₆) δ 6.69 (1H, dd, J=16.0, 5.5 Hz, 9-H), 6.55 (1H, dd, J=16.0, 1.3, 8-H), 4.71 (1H, dd, J=9.0, 2.0, 11-H), 4.37 (1H, d, J=7.0, 1 ′-H), 4.31 (1H, d, J=8.0, 1 ″-H), 3.97 (1H, dd, J=11.5, 2.5, 6″-H), 3.81 (1H, dq, J=9.0, 6.0, 12-H), 3.72 (1H, dd, J=11.5, 5.0, 6″-H), 3.56 (1H, m, 5′-H), 3.50 (1H, bd, J=10.0, 3-H), 3.36 (1H, t, J=8.5, 3″-H), 3.32 (1H, m, 5″-H), 3.30 (1H, t, J=8.5, 4″-H), 3.23 (1H, dd, J=10.2, 7.0,2′-H), 3.13, (1H, dd, J=8.5, 8.0, 2″-H), 3.09 (1H, m, 2-H), 3.08 (1H, m, 10-H), 2.77 (1H, ddd, J=12.5, 10.2, 4.5, 3′-H), 2.41 (1H, m, 6-H), 2.28 (6H, s, NMe₂), 1.89 (1H, t, J=13.0, 5-H), 1.83 (1H, ddd, J=12.5, 4.5, 1.5, 4′-H), 1.41 (3H, d, J=7.0, 2-Me), 1.3 (1H, m, 4-H), 1.25 (1H, m, 5-H), 1.2 (11H, m, 4′-H, 1.20 (3H, d, J=6.0, 5′-Me), 1.17 (6H, d, J=7.0, 6-Me, 10-Me), 1.12 (3H, d, J=6.0, 12-me), 0.96 (3H, d, J=6.0, 4-Me). ¹³C NMR (acetone-d₆) δ 204.1 (C-7), 175.8 (C-1), 148.2 (C-9), 126.7 (C-8), 108.3 (C-1″), 104.2 (C-1′), 85.1 (C-3), 83.0 (C-2′), 78.2 (C-3″), 78.1 (C-5″), 76.6 (C-2″), 76.4 (C-11), 71.8 (C-4″), 69.3 (C-5′), 66.1 (C-12), 66.0 (C-3′), 63.7 (C-6″), 46.2 (C-6), 44.4 (C-2), 40.8 (NMe₂), 36.4 (C-10), 34.7 (C-5), 34.0 (C-4), 29.5 (C-4′), 21.5 (5′-Me), 21.5 (12-Me), 17.9 (6-Me), 17.7 (4-Me), 17.2 (2-Me), 9.9 (10-Me). High-resolution FAB-MS: calc for C₃₁H₅₄NO₁₂(M+H)⁺632.3646, found 632.3648.
The coupling constant (d, J=8.0 Hz) of the anomeric hydrogen (1″-H) of the added glucose and the magnitude of the downfield shift (11.8 ppm) of C-2′ of desosamine are all consistent with the assigned C-2′ β-configuration (Seo et al., 1978). [0234]
The antibiotic activity of a compound of formula (13) and (14) against [0235] Streptococcus pyogenes was examined by separately applying 20 μL of each sample (1.6 mM in MeOH) to sterilized filter paper discs which were placed onto the surface of S. pyogenes grown on Mueller-Hinton agar plates (Mangahas, 1996). After being grown overnight at 37° C., the plates of the controls (a compound of formula (1) and (2)) showed clearly visible inhibition zones. In contrast, no such clearings were discernible around the discs of a compound of formula (13) and (14). Evidently, β-glucosylation at C-2′ of desosamine in methymycin/neomethymycin renders these antibiotics inactive.
It should be noted that similar phenomena involving inactivation of macrolide antibiotics by glycosylation are known (Celmer et al., 1985; Kuo et al., 1989; Sasaki et al., 1996). For example, it was found that when erythromycin was given to [0236] Streptomyces lividans, which contains a macrolide glycosyltransferase (MgtA), the bacterium was able to defend itself by glycosylating the drug (Cundliffe, 1992; Jenkins et al., 1991). Such a macrolide glycosyltransferase activity has been detected in 15 out of a total of 32 actinomycete strains producing various polyketide antibiotics (Sasaki et al., 1996). Interestingly, the co-existence of a macrolide glycosyltransferase (OleD) capable of deactivating oleandomycin by glucosylation (Hernandez et al., 1993), and an extracellular β-glucosidase capable of removing the added glucose from the deactivated oleandomycin in Streptomyces antibioticus (Vilches et al., 1992) has led to the speculation of glycosylation as a possible self-resistance mechanism in S. antibioticus. Although the genes of the aforementioned glycosyltransferases have been cloned in a few cases, such as mgtA of S. lividans and oleD of S. antibioticus, the whereabouts of macrolide β-glycosidase genes remain obscure. Interestingly, the recently released eryBI sequence, which is part of the erythromycin biosynthetic cluster, is highly homologous to desR (55% identity) (Gaisser et al., 1997).
The discovery of desR, a macrolide β-glucosidase gene, within the desosamine gene cluster is thus significant, and the accumulation of deactivated compounds of formula (13) and (14) after desR disruption provides direct molecular evidence indicating that a similar self-defense mechanism via glycosylation/deglycosylation may also be operative in [0237] S. venezuelae. However, because a significant amount of methymycin and neomethymycin also exist in the fermentation broth of the mutant strain, glucosylation of desosamine may not be the primary self-resistance mechanism in S. venezuelae. Indeed, an rRNA methyltransferase gene found upstream from the PKS genes in this cluster may confer the primary self-resistance protection. Thus, these results are consistent with the fact that antibiotic producing organisms generally have more than one defensive option (Cundliffe, 1989). In light of this observation, it is conceivable that methymycin/neomethymycin may be produced in part as the inert diglycosides (a compound of formula (13) or (14)), and the macrolide β-glucosidase encoded by desR is responsible for transforming methymycin/neomethymycin from their dormant state to their active form. Supporting this idea, the translated desR gene has a leader sequence characteristic of secretory proteins (von Heijne, 1986; von Heijne, 1989). Thus, DesR may be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them (FIG. 25).
Summary [0238]
Inspired by the complex assembly and the enzymology of aminodeoxy sugars that are frequently found as essential components of macrolide antibiotics, the entire desosamine biosynthetic gene cluster from the methymycin and neomethymycin producing strain [0239] Streptomyces venezuelae was cloned, sequenced, and mapped. Eight of the nine mapped genes were assigned to the biosynthesis of TDP-D-desosamine based on sequence similarities to those derived from the erythromycin cluster. The remaining gene, designated desR, showed strong sequence homology to β-glucosidases.
To investigate the function of the encoded protein (DesR), a disruption mutant was constructed in which a NcoI/XhoI fragment of the desR gene was deleted and replaced by the thiostrepton resistance (tsr) gene. In addition to methymycin and neomethymycin, two new products were isolated from the fermentation of the mutant strain. These two new compounds, which are biologically inactive, were found to be C-2′ β-glucosylated methymycin and neomethymycin. Since the translated desR gene has a leader sequence characteristic of secretory proteins, the DesR protein may be an extracellular β-glucosidase capable of removing the added glucose from the modified antibiotics to activate them. Thus, the occurrence of desR within the desosamine gene cluster and the accumulation of deactivated glucosylated methymycin/neomethymycin upon disruption of desR provide strong molecular evidence suggesting that a self-resistance mechanism via glucosylation may be operative in [0240] S. venezuelae.
Thus, the desR gene can be used as a probe to identify homologs in other antibiotic biosynthetic pathways. Deletion of the corresponding macrolide glycosidase gene in other antibiotic biosynthetic pathways may lead to the accumulation of the glycosylated products which may be used as prodrugs with reduced cytotoxicity. Glycosylation also holds promise as a tool to regulate and/or minimize the potential toxicity associated with new macrolide antibiotics produced by genetically engineered microorganisms. Moreover, the availability of macrolide glycosidases, which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be useful in the development of novel antibiotics using the combinatorial biosynthetic approach (Hopwood et al., 1990; Katz et al., 1993; Hutchinson et al., 1995; Carreras et al., 1997; Kramer et al., 1996; Khosla et al., 1996; Jacobsen et al., 1997; Marsden etal., 1998). [0241]

Example 7

Deletion of the desVI Gene of the Desosamine Biosynthetic Gene Cluster

The emergence of pathogenic bacteria resistant to many commonly used antibiotics poses a serious threat to human health and has been the impetus of the present resurgent search for new antimicrobial agents (Box et al., 1997; Davies, 1996; Service, 1995). Since the first report on using genetic engineering techniques to create “hybrid” polyketides (Hopwood et al., 1995), the potential of manipulating the genes governing the biosynthesis of secondary metabolites to create new bioactive compounds, especially macrolide antibiotics, has received much attention (Kramer et al., 1996; Khosla et al., 1996). This class of clinically important drugs consists of two essential structural components: a polyketide aglycone and the appended deoxy sugars (Omura, 1984). The aglycone is synthesized via sequential condensations of acyl thioesters catalyzed by a highly organized multi-enzyme complex, polyketide synthase (PKS) (Hopwood et al., 1990; Katz, 1993; Hutchinson et al., 1995; Carreras et al., 1997). Recent advances in the understanding of the polyketide biosynthesis have allowed recombination of the PKS genes to construct an impressive array of novel skeletons (Kramer et al., 1996; Khosla et al., 1996; Hopwood et al., 1990; Katz, 1993; Hutchinson et al., 1995; Carreras et al., 1997; Epp et al., 1989; Donadio et al., 1993; Arisawa et al., 1994; Jacobsen et al., 1997; Marsden et al., 1998). Without the sugar components, however, these new compounds are usually biologically impotent. Hence, if one plans to make new macrolide antibiotics by a combinatorial biosynthetic approach, two immediate challenges must be overcome: assembling a repertoire of novel sugar structures and then having the capacity to couple these sugars to the structurally diverse macrolide aglycones. [0242]
Unfortunately, knowledge of the formation of the unusual sugars in these antibiotics remains limited (Liu et al., 1994; Kirschning et al., 1997; Johnson et al., 1998). Part of the reason for this comes from the fact that the sugar genes are generally scattered at both ends of the PKS genes. Such an organization within the macrolide biosynthetic gene cluster makes it difficult to distinguish the sugar genes from those encoding regulatory proteins or aglycone modification enzymes that are also interspersed in the same regions. The task can be made even more formidable if the macrolides contain multiple sugar components. In view of the “scattered” nature of the sugar biosynthetic genes, the antibiotic methymycin (a compound of formula (1) in FIG. 24) and its co-metabolite, neomethymycin (a compound of formula (2) in FIG. 24)), of [0243] Streptomyces venezuelae present themselves as an attractive system to study the formation of deoxy sugars (Donin et al., 1953; Djerassi et al., 1956). First, they carry D-desosamine (a compound of formula (3)) a prototypical aminodeoxy sugar that also exists in erythromycin. Second, since desosamine is the only sugar attached to the macrolactone of formula (1) and (2), identification of the sugar biosynthetic genes within the methymycin/neomethymycin gene cluster should be possible with much more certainty.
A 10 kb stretch of DNA downstream from the methymycin/neomethymycin gene cluster, which is about 60 kb in length, was found to harbor the entire desosamine biosynthetic gene cluster (FIG. 26). Among the nine open reading frames (ORFs) mapped in this segment, eight are likely to be involved in desosamine formation, while the remaining one, desR, encodes a macrolide β-glycosidase that may be involved in a self-resistance mechanism. Their identities, shown in FIG. 26, are assigned based on sequence similarities to other sugar biosynthetic genes (Gaisser et al., 1997; Summers et al., 1997). The proposed pathway is well founded on literature precedent and mechanistic intuition for the construction of aminodeoxy sugars (Liu et al., 1994; Kirschning et al., 1997; Johnson et al., 1998). [0244]
To determine whether new methymycin/neomethymycin analogues carrying modified sugars could be generated by altering the desosamine biosynthetic genes, the desVI gene, which has been predicted to encode the N-methyltransferase, was chosen as a target (Gaisser et al., 1997; Summers et al., 1997). The deduced desVI product is most closely related to that of eryCVI from the erythromycin producing strain [0245] Saccharopolyspora erythraea (70% identity), and also strongly resembles the predicted products of rdmD from the rhodomycin cluster of Streptomyces purpurascens (Niemi et al., 1995), srmX from the spiromycin cluster of Streptomyces ambofaciens (Geistlich et al., 1992), and tylM1 from the tylosin cluster of Streptomyces fradiae (Gandecha et al., 1997). All of these enzymes contain the consensus sequence LLDV(I)ACGTG (SEQ ID NO:25) (Gaisser et al., 1997; Summers et al., 1997), near their N-terninus, which is part of the S-adenosylmethionine binding site (Ingrosso et al., 1989; Haydock et al., 1991).
The deletion of desVI should have little polar effect (Lin et al., 1984) on the expression of other desosamine biosynthetic genes because the ORF (desR) lying immediately downstream from desVI is not directly involved in desosamine formation, and those lying further downstream are transcribed in the opposite direction. Second, since N,N-dimethylation is almost certainly the last step in the desosamine biosynthetic pathway (Liu et al., 1994; Kirschning et al., 1997; Johnson et al., 1998; Gaisser et al., 1997; Summers et al., 1997), perturbing this step may lead to the accumulation of a compound of formula (4), which stands the best chance among all other intermediates of being recognized by the glycosyltransferase (DesVII) for successful linkage to the macrolactone of formula (6) (FIG. 25). Deletion and/or disruption of a single biosynthetic gene often affects the pathway at more than one specific step. In fact, disruption of eryCVI, the desVI equivalent in the erythromycin cluster, which has been predicted to encode a similar N-methylase to make desosamine in erythromycin (Gaisser et al., 1997; Summers et al., 1997), led to the accumulation of an intermediate devoid of the entire desosamine moiety (Summers et al., 1997). [0246]
A plasmid pBL3001, in which desVI was replaced by the thiostrepton gene (tsr) (Bibb et al., 1985), was constructed and introduced into wild type [0247] S. venezuelae by conjugal transfer using E. coli S17-1 (Bierman et al., 1992). Two identical double crossover mutants, KdesVI-21 and KdesVI-22 with phenotypes of thiostrepton resistance (Thio^R) and apamycin sensitivity (Apm^S) were obtained. Southern blot hybridization using tsr or a 1.1 kb HincII fragment from the desVII region further confirmed that the desVI gene was indeed replaced by tsr on the chromosome of these mutants. The KdesVI-21 mutant was first grown at 29° C. in seed medium (100 mL) for 48 hours, and then inoculated and grown in vegetative medium (3 L) for another 48 hours (Cane et al., 1993). The fermentation broth was centrifuged to remove the cellular debris and mycelia, and the supernatant was adjusted to pH 9.5 with concentrated KOH, followed by extraction with chloroform. No methymycin or neomethymycin was found; instead, the 1 0-deoxy-methynolide (6) (350 mg) (Lambalot et al., 1992) and two new macrolides containing an N-acetylated amino sugar, a compound of formula (7) (20 mg) and a compound of formula (8) (15 mg), were isolated. Their structures were determined by spectral analyses and high-resolution MS.
Spectral data of formula 7 are: [0248] ¹H NMR (CDCl₃) δ 6.62 (1H, d, J=16.0, H-9), 6.22 (1H, d, J=16.0, H-8), 5.75 (1H, d, J=7.5, N-H), 4.75 (1H, dd, J=10.8, 2.2, H-11), 4.28 (1H, d, J=7.5, H-1′), 3.95 (1H, m, H-3′), 3.64 (1H, d, J=10.5, H-3), 3.56 (1H, m, H-5′), 3.16 (1H, dd, J=10.0, 7.5, H-2′), 2.84 (1H, dq, J=10.5, 7.0, H-2), 2.55 (1H, m, H-6), 2.02 (3H, s, NAc), 1.95 (1H, m, H-12), 1.90 (11H, m, H-4′), 1.66 (1H, m, H-5), 1.50 (1H, m, H-12), 1.41 (3H, d, J=7.0, 2-Me), 1.40 (1H, m, H-5), 1.34 (3H, s, 10-Me), 1.25 (1H, m, H-4), 1.22 (1H, m, H-4′), 1.21 (3H, d, J=6.0, H-6′), 1.17 (3H, d, J=7.0, 6-Me), 1.01 (3H, d, J=6.5, 4-Me), 0.89 (3H, t, J=7.2, 12-Me); ¹³C NMR (CDCl₃) δ 204.3 (C-7), 175.1 (C-1), 171.8 (Me-C═O), 149.1 (C-9), 125.3 (C-8), 104.4 (C-1′), 85.4 (C-3), 76.3 (C-11), 75.4 (C-2′), 74.1 (C-10), 68.6 (C-5′), 51.9 (C-3′), 45.0 (C-6), 44.0 (C-2), 38.5 (C-4′), 33.8 (C-5), 33.3 (C-4), 23.1 (Me-C=O), 21.1 (C-1 2), 20.6 (C-6′), 19.2 (10-Me), 17.5 (6-Me), 17.2 (4-Me), 16.2 (2-Me), 10.6 (12-Me). High-resolution FABMS: calc for C₂₅H₄₃O₈N (M+H)⁺484.2910, found 484.2903.
Spectral data of formula 8 are: [0249] ¹H NMR (CDCl₃) δ 6.76 (1H, dd, J=16.0, 5.5, H-9), 6.44 (1H, dd, J=16.0, 1.5, H-8), 5.50 (1H, d, J=6.5, N—H), 4.80 (1H, dd, J=9.0,2.0, H-11), 4.28 (1H, d, J=7.5, H-1′), 3.95 (1H, m, H-3′), 3.88 (1H, m, H-12), 3.62 (1H, d, J=11.0, H-3), 3.57 (1H, m, H-5′), 3.18 (1H, dd, J=10.0, 7.5, H-2′), 3.06 (1H, m, H-10), 2.86 (1H, dq, J=11.0, 7.0, H-2),2.54 (1H, m, H-6),2.04 (3H, s, NAc), 1.98 (1H, m, H-4′), 1.67 (1H, m, H-5), 1.40 (1H, m, H-5), 1.39 (3H, d, J=7.0, 2-Me), 1.25 (1H, m, H-4), 1.22 (1H, m, H-4′), 1.22 (3H, d, J =6.0, H-6′), 1.21 (3H, d, J=6.0,6-Me), 1.19 (3H, d, J=7.0, 12-Me), 1.16 (3H, d, J=6.5, 10-Me), 1.01 (3H, d, J=6.5, 4-Me); ¹³C NMR (CDCl₃) δ 205.1 (C-7), 174.6 (C-1), 171.9 (Me-C═O), 147.2 (C-9), 126.2 (C-8), 104.4 (C-1′), 85.3 (C-3), 75.7 (C-11), 75.4 (C-2′), 68.7 (C-5′), 66.4 (C-12), 52.0 (C-3′), 45.1 (C-6), 43.8 (C-2), 38.6 (C-4′), 35.4 (C-10), 34.1 (C-5), 33.4 (C-4), 23.1 (Me-C═O), 21.0 (12-Me), 20.7 (C-6′), 17.7 (6-Me), 17.4 (4-Me), 16.1 (2-Me), 9.8 (10-Me). High-resolution FABMS: calc for C₂₅H₄₃O₈N (M+H)⁺484.2910, found 484.2892.
The fact that compounds of formula (7) and (8) bearing modified desosamine are produced by the desVI-deletion mutant is a thrilling discovery. However, this result is also somewhat surprising since the sugar component in the products is expected to be the aminodeoxy hexose (4). As illustrated in FIG. 27, it is possible that a compound of formula (7) and (8) are derived from the predicted compound of formula (9) and (10), respectively, by a post-synthetic nonspecific acetylation of the attached aminodeoxy sugar. It is also conceivable that N-acetylation of (4) occurs first, followed by coupling of the resulting sugar (11) to the 10-deoxymethynolide (6). Nevertheless, the lack of N-methylation of the sugar component in these new products provides convincing evidence sustaining the assignment of desVI as the N-methyltransferase gene. Most significantly, the production of a compound of formula (7) and (8) by the desVI-deletion mutant attests to the fact that the glycosyltransferase (DesVII) in methymycin/neomethymycin pathway is capable of recognizing and processing sugar substrates other than TDP-desosamine (5). [0250]
Since both compounds of formula (7) and (8) are new compounds synthesized in vivo by the [0251] S. venezuelae mutant strain, the observed N-acetylation might be a necessary step for self-protection (Cundliffe, 1989). In view of these results, the potential toxicity associated with new macrolide antibiotics produced by genetically engineered microorganisms can be minimized and newly formed antibiotics that have been deactivated (either deliberately or not) during production can be activated. Such an approach can be part of an overall strategy for the development of novel antibiotics using the combinatorial biosynthetic approach. Indeed, purified compounds of formula (7) and (8) are inactive against Streptococcus pyogenes grown on Mueller-Hinton agar plates (Mangahas, 1996), while the controls (a compound of formula (1) and (2)) show clearly visible inhibition zones.
It should be pointed out that a few glycosyltransferases involved in the biosynthesis of antibiotics have been shown to have relaxed specificity towards modified macrolactones (Jacobsen et al., 1997; Marsden et al., 1998; Weber et al., 1991). However, a similar relaxed specificity toward sugar substrates has only been reported for the daunorubicin glycosyltransferase, which is able to recognize a modified daunosamine and catalyze its coupling to the aglycone, β-rhodomycinone (Madduri et al., 1998). Thus, the fact that the methymycin/neomethymycin glycosyltransferase can also tolerate structural variants of its sugar substrate indicates that at least some glycosyltransferases in antibiotic biosynthetic pathways may be useful to create biologically active hybrid natural products via genetic engineering. [0252]
Summary [0253]
The appended sugars in macrolide antibiotics are indispensable to the biological activities of these clinically important drugs. Therefore, the development of new antibiotics via a biological combinatorial approach requires detailed knowledge of the biosynthesis of these unusual sugars, as well as the ability to manipulate the biosynthetic genes to create novel sugars that can be incorporated into the final macrolide structures. A targeted deletion of the desVI gene of [0254] Streptomyces venezuelae, which has been predicted to encode an N-methyltransferase based on sequence comparison, was prepared to determine whether new methymycin/neomethymycin analogues bearing modified sugars can be generated by altering the desosamine biosynthetic genes. Growth of the S. venezuelae deletion mutant strain resulted in the accumulation of a methymycin/neomethymycin analogue carrying an N-acetylated aminodeoxy sugar. Isolation and characterization of these derivatives not only provide the first direct evidence confirming the identity of desVI as the N-methyltransferase gene, but also demonstrate the feasibility of preparing novel sugars by the gene deletion approach. Most significantly, the results also revealed that the glycosyltransferase of methymycin/neomethymycin exhibits a relaxed specificity towards its sugar substrates.

Example 8

Cloning and Sequencing of the Met/Pik Biosynthetic Gene Cluster

Materials and Methods [0255]
Bacterial Strains and Media. [0256]
[0257] E. coli DH5α was used as a cloning host. E. coli LE392 was the host for a cosmid library derived from S. venezuelae genomic DNA. LB medium was used in E. coli propagation. Streptomyces venezuelae ATCC 15439 was obtained as a freeze-dried pellet from ATCC. Media for vegetative growth and antibiotic production were used as described (Lambalot et al., 1992). Briefly, SGGP liquid medium was for propagation of S. venezuelae mycelia. Sporulation agar (SPA) was used for production of S. venezuelae spores. Methymycin production was conducted in either SCM or vegetative medium and pikromycin production was performed in Suzuki glucose-peptone medium.
Vectors, DNA Manipulation and Cosmid Library Construction. [0258]
pUC119 was the routine cloning vector, and pNJ1 was the cosmid vector used for genomic DNA library construction. Plasmid vectors for gene disruption were either pGM160 (Muth et al., 1989) or pKC1 139 (Bierman et al., 1992). Plasmid, cosmid, and genomic DNA preparation, restriction digestion, fragment isolation, and cloning were performed using standard procedures (Sambrook et al., 1989; Hopwood et al., 1985). The cosmid library was made according to instructions from the Packagene λ-packaging system (Promega). [0259]
DNA Sequencing and Analysis. [0260]
An Exonuclease III (ExoIII) nested deletion series combined with PCR-based double stranded DNA sequencing was employed to sequence the pik cluster. The ExoIII procedure followed the Erase-a-Base protocol (Stratagene) and DNA sequencing reactions were performed using the Dye Primer Cycle Sequencing Ready Reaction Kit (Applied Biosystems). The nucleotide sequences were read from an ABI PRISM 377 sequencer on both DNA strands. DNA and deduced protein sequence analyses were performed using GeneWorks and GCG sequence analysis package. All analyses were performed using the specific program default parameters. [0261]
Gene Disruption. [0262]
A replicative plasmid-mediated homologous recombination approach was developed to conduct gene disruption in [0263] S. venezuelae. Plasmids for insertional inactivation were constructed by cloning a kanamycin resistance marker into target genes, and plasmid for gene deletion/replacement was constructed by replacing the target gene with a kanamycin or thiostrepton resistance gene in the plasmid. Disruption plasmids were introduced into S. venezuelae by either PEG-mediated protoplast transformation (Hopwood et al., 1985) or RK2-mediated conjugation (Bierman et al., 1992). Then, spores from individual transformants or transconjugants were cultured on non-selective plates to induce recombination. The cycle was repeated three times to enhance the opportunity for recombination. Double crossovers yielding targeted gene disruption mutants were selected and screened using the appropriate combination of antibiotics and finally confirmed by Southern hybridization.
Antibiotic Extraction and Analysis. [0264]
Methymycin, pikromycin, and related compounds were extracted following published procedures (Cane et al., 1993). Thin layer chromatography (TLC) was routinely used to detect methymycin, neomethymycin, narbomycin and pikromycin. Further purification was conducted using flash column chromatography and HPLC, and the purified compounds were analyzed by [0265] ¹H, ¹³C NMR spectroscopy and MS spectrometry.
Results [0266]
Cloning and Identification of the pik Cluster. [0267]
Heterologous hybridization was used to identify genes for methymycin, neomethymycin, narbomycin and pikromycin biosynthesis in [0268] S. venezuelae. Initial Southern blot hybridization analysis using a type I PKS DNA probe revealed two multifunctional PKS clusters of uncharacterized function in the genome. Since these four antibiotics are all comprised of an identical desosarnine residue, a tylAI α-D-glucose-1-phosphate thymidylyltransferase DNA probe (for mycaminose/mycorose/mycinose biosynthesis in the tylosin pathway) (Merson-Davies et al., 1994) was used to locate the corresponding biosynthetic gene cluster(s). This analysis established that only one of the PKS pathways contained a cluster of desosamine biosynthetic genes. Nine overlapping cosmid clones were isolated spanning over 80 kilobases (kb) on the bacterial chromosome that encompassed the entire gene cluster (pik) for methymycin, neomethymycin, narbomycin and pikromycin biosynthesis (FIG. 28). Through subsequent gene disruption, the other PKS cluster (vep, devoid of linked desosamine biosynthetic genes) was found to play no role in production of methymycin, neomethymycin, narbomycin or pikromycin.
Nucleotide Sequence of the pik Cluster. [0269]
The nucleotide sequence of the pik cluster was completely determined and shown to contain 18 open reading frames (ORFs) that span approximately 60 kb. Central to the cluster are four large ORFs, pikAI, pikAII, pikAIII, and pikAIV, encoding a multifunctional PKS (FIG. 28). Analysis of the six modules comprising the pik PKS indicated that it would specify production of narbonolide, the 14-membered ring aglycone precursor of narbomycin and pikromycin (FIG. 28). [0270]
Initial analysis unveiled two significant architectural differences in the pikA-encoded PKS. First, compared with eryA (Donadio et al., 1998) and oleA (Swan et al., 1994), two PKS clusters that produce 14-membered ring macrolides erythromycin and oleadomycin similar to pikromycin, the presence of separate ORFs, pikAIII and pikAIV, encoding [0271] Pik module 5 and Pik module 6 (as individual modules) as opposed to one bimodular protein as in eryAIII and oleAIII is striking. Secondly, the presence of a type II thioesterase immediately downstream of the type I PKS cluster is also unprecedented (FIG. 28). These two characteristics suggest that pikA may produce the 1 2-membered ring macrolactone 10-deoxymethynolide as well. Indeed, the domain organization of PikAI-AIII (module L-5) is consistent with the predicted biosynthesis of 10-deoxymethynolide except for the absence of a TE function at the C-terminus of Pik module 5 (PikAIII). The lack of a TE domain in PikAIII may be compensated by the type II TE (encoded by pikAV) immediately downstream of pikAIV. Consistent with the supposition that two distinct polyketide ring systems are assembled from the pik PKS, two macrolide-lincosamide-streptogramin B type resistant genes, pikR1 and pikR2, are found upstream of the pik PKS (FIG. 29), which presumably provide cellular self-protection for S. venezuelae.
The genetic locus for desosamine biosynthesis and glycosyl transfer are immediately downstream of pikA. Seven genes, desI, desII, desIII, desIV, desV, desVI, and desVIII, are responsible for the biosynthesis of the deoxysugar, and the eighth gene, desVII, encodes a glycosyltransferase that apparently catalyzes transfer of desosamine onto the alternate (12- and 14-membered ring) polyketide aglycones. The existence of only one set of desosamine genes indicates that DesVIII can accept both 10-deoxymethynolide and narbonolide as substrates (Jacobsen et al., 1997). The largest ORF in the des locus, desR, encodes a β-glycosidase that is involved in a drug inactivation-reactivation cycle for bacterial self-protection. [0272]
Just downstream of the des locus is a gene (pikC) encoding a cytochrome P450 hydroxylase similar to eryF (Andersen et al., 1992), and eryK (Stassi et al., 1993), PikC, and a gene (pikD) encoding a putative regulator protein, PikD (FIG. 28). Interestingly, PikC is the only P450 hydroxylase identified in the entirepik cluster, suggesting that the enzyme can accept both 12- and 14-membered ring macrolide substrates and, more remarkably, it is active on both C-10 and C-12 of the YC-17 (12-membered ring intermediate) to produce methymycin and neomethymycin (FIG. 30). PikD is a putative regulatory protein similar to ORFH in the rapamycin gene cluster (Schwecke et al., 1995). [0273]

The combined functionality coded by the eighteen genes in the pik cluster predicts biosynthesis of methymycin, neomethymycin, narbomycin and pikromycin (Table 2). Flanking the pik cluster locus are genes presumably involved in primary metabolism and genes that may be involved in both primary and secondary metabolism. An S-adenosyl-methionine synthase gene is located downstream of pikD that may help to provide the methyl group in desosamine synthesis. A threonine dehydratase gene was identified upstream of pikR1 that may provide precursors for polyketide biosynthesis. It is not apparent that any of these genes are dedicated to antibiotic biosynthesis and they are not directly linked to the pik cluster.

TABLE 2


Deduced function of ORFs in the pik cluster

Polypeptide	Amino	Proposed function or sequence
(ORF)	acids, no.	similarity detected

PikAI	4,613	PKS

Loading module		KS^QAT(P) ACP

Module
1		KS AT(P) KR ACP

Module
2		KS AT(A) DH KR ACP

PikAII	3,739	PKS

Module
3		KS AT(P) KR⁰ACP

Module 4		KS AT(P) DH ER KR ACP

PikAIII	1,562	PKS

Module
5		KS AT(P) KR ACP

PikAIV	1,346	PKS

Module
6		KS AT(P) ACP TE

PikAV	281	Thioesterase II (TEII)

DesI	415	4-Dehydrase

DesII	485	Reductase?

DesIII	292	α-D-Glucose-1-phosphate
		thymidylyltransferase

DesIV	337	TDP-glucose 4, 6-dehydratase

DesV
	379	Transaminase

DesVI	237	N,N-dimethyltransferase

DesVII	426	Glycosyl transferase

DesVIII	402	Tautomerase?

DesR	809	βGlucosidase (involved in
		resistance mechanism)

PikC	418	P450 hydroxylase

PikD	945?	Putative regulator

PikR1	336	rRNA methyltransferase
		(mls resistance)

PikR2	288?	rRNA methyltransferase
		(mls resistance)

TABLE 3


Summary of mutational analyses of the pik cluster

			Antibiotic production/
	Type of	Target	Intermediate accumulation

Mutant	mutation	gene	Met & neomethymycin	Pikromycin

AX903	Insertion	pikAI	No/No	No/No
LZ3001	Deletion/	desVI	No/10-deoxymethynolide	No/narbonolide
	replace-
	ment
LZ4001	Deletion/	desV	No/10-deoxymethynolide	No/narbonolide
	replace-
	ment
AX905	Deletion/	pikAV	<5%/No	<5%/No
	replace-
	ment
AX906	Insertion	pikC	No/YC-17	No/narbomycin

Mutational Analysis of the pik Cluster. [0276]
Extensive disruption of genes in the pik cluster were carried out to address the role of key enzymes in antibiotic production (Table 3). First, PikAI, the first putative enzyme involved in the biosynthesis of 10-deoxymethynolide and narbonolide was inactivated by insertional mutagenesis. The resulting mutant, AX903, produced neither methymycin or neomethymycin, nor narbomycin or pikromycin, indicating that pikA encodes a PKS required for both 12- and 14-membered ring macrolactone formation. [0277]
Second, deletion of both desVI and desV abolished methymycin, neomethymycin, narbomycin and pikromycin production, and the resulting mutants, LZ3001 and LZ4001, accumulate lO-deoxymethynolide and narbonolide in their culture broth, indicating that enzymes for desosamine synthesis and transfer are -also shared by the 12- and 14-membered ring macrolides. [0278]
In order to understand the mechanism of polyketide chain termination at PikAIII (PIKAII (module 5) is presumed to be the termination point in construction of 10-deoxymethynolide), the pik TEII gene, pikAV, was deleted. The deletion/replacement mutant, AX905, produces less than 5% of methymycin, neomethymycin, and less than 5% of pikromycin compared to wild type [0279] S. venezuelae. This abrogation in product formation occurs without significant accumulation of the expected aglycone intermediates, suggesting thatpik TEII is involved in the termination of 12- as well as 14-membered ring macrolides at PikAIII and PikAIV, respectively. Although the polar effects may influence the observed phenotype in AX905, this has been ruled out after the consideration of mutant LZ3001, in which mutation in an enzyme downstream of pikAV accumulated 10-deoxymethynolide and narbonolide. The fact that mutant AX905 failed to accumulate these intermediates suggested that the polyketide chains were not efficiently released from this PKS protein in the absence of Pik TEII. Therefore, Pik TEII plays a crucial role in polyketide chain release and cyclization, and it presumably provides the mechanism for alternative termination in pik polyketide biosynthesis.
Finally, disruption of pikC confirmed that PikC is the sole enzyme catalyzing hydroxylation of both YC-17 (at C-10 and C-12) and narbomycin (at C-12). The relaxed substrate specificity of PikC and its regional specificity at C-10 and C-12 provide another layer of metabolite diversity in the pik-encoded biosynthetic system. [0280]
Discussion [0281]
The work described herein has established that methymycin, neomethymycin, narbomycin and pikromycin biosynthesis is encoded by the pik cluster in [0282] S. venezuelae. Three key enzymes as well as the unique architecture of the cluster enable this relatively compact system to produce multiple macrolide antibiotics. Foremost, the presence of pik module 5 and 6 as separate proteins, PikAIII and PikAIV, and the activity of pik TEII enable the bacterium to terminate the polyketide chain at two different points of assembly, thereby producing two macrolactones of different ring size. Second, DesVII, the glycosyltransferase in the pik cluster, can accept both 12- and 14-membered ring macrolactones as substrates. Finally, PikC, the P450 hydroxylase, has a remarkable substrate and regiochemical specificity that introduces another layer of diversity into the system.
It is interesting to consider that pikA evolved in a line analogous to eryA and oleA since each of these PKSs specify the synthesis of 14-membered ring macrolactones. Therefore, pik may have acquired the capacity to generate methymycin when a mutation in the primordial pikAIII-pikAIV linker region caused splitting of [0283] Pik module 5 and 6 into two separate gene products. This notion is raised by two features of the nucleotide sequence. First, the intergenic region between pikAIII and pikAIV, which is 105 bp, may be the remanent of an intramodular linker peptide of 35 amino acids. Moreover, the potential for independently regulated expression of pikAIV is implied by the presence of a 100 nucleotide region at the 5′ end of the gene that is relatively AT-rich (62% as comparing 74% G+C content in coding region). Thus, as the mutation in an original ORF encoding the bimodular multifunctional protein (PikAIII-PikAIV) occurred, so too may have evolved a mechanism for regulated synthesis of the new gene product (PikAIV).
The role of Pik TEII in alternative termination of polyketide chain elongation intermediates provides a unique aspect of diversity generation in natural product biosynthesis. Engineered polyketides of different chain length are typically generated by moving the TE catalytic domain to alternate positions in a modular PKS (Cortes et al., 1995). Repositioning of the TE domain necessarily abolishes production of the original full-length polyketide so only one macrolide is produced each time. In contrast to the fixed-position TE domain, the independent Pik TEII polypeptide presumably has the flexibility to catalyze termination at different stages of polyketide assembly, therefore enabling the system to produce multiple products of variant chain length. Combinatorial biology technologies can now exploit this system for generating molecular diversity through construction of novel PKS systems with TElls for simultaneous production of several new molecules as opposed to the TE domains alone that limit catalysis to a single termination step. [0284]
It is noteworthy that sequences similar to Pik TEII are found in almost all known polyketide and non-ribosomal polypeptide biosynthetic systems (Marahiel et al., 1997). Currently, the pik TEII is the first to be characterized in a modular PKS. However, recent work on a TEII gene in the lipopeptide surfactin biosynthetic cluster (Schneider et al., 1998) demonstrated that srf-TEII plays an important role in polypeptide chain release, and may suggest that srf-TEII reacts at multiple stages in peptide assembly as well (Marahiel et al., 1997). [0285]
The enzymes involved in post-polyketide assembly of 10-deoxymethynolide and narbonolide are particularly intriguing, especially the glycosyltransferase, DesVII, and P450 hydroxylase, PikC. Both have the remarkable ability to accept substrates with significant structural variability. Moreover, disruption of desVI demonstrated that DesVII also tolerates variations in deoxysugar structure (Example 6). Likewise, PikC has recently been shown to convert YC-17 to methymycin/neomethymycin and narbomycin to pikromycin in vitro. [0286]
Targeted gene disruption of ORFI abolished both pikromycin and methymycin production, indicating that the single cluster is responsible for biosynthesis of both antibiotics. Deletion of the TE2 gene substantially reduced methymycin and pikromycin production, which demonstrates that TE2, in contrast to the position-fixed TE1 domain, has the capacity to release polyketide chain at different points during the assembly process, thereby producing polyketides of different chain length. [0287]
The results described above were unexpected in that it was surprising that one PKS cluster produces two macrolides which differ in the number of atoms in their ring structure, that [0288] module 5 and module 6 of the PKS are in ORFs that are separated by a spacer region, that PikAIII lacked TE, that there was a Type II thioesterase, that TEI domain was not separate, and that 2 resistance genes were identified which may be specific for either a 12- or 14-membered ring.
With eighteen genes spanning less than 60 kb of DNA capable of producing four active macrolide antibiotics, the pik cluster represents the least complex yet most versatile modular PKS system so far investigated. This simplicity provides the basis for a compelling expression system in which novel active ketoside products are engineered and produced with considerable facility for discovery of a diverse range of new biologically active compounds. [0289]
Summary [0290]
Complex polyketide synthesis follows a processive reaction mechanism, and each module within a PKS harbors a string of three to six enzymatic domains that catalyze reactions in nearly linear order as described in particular detail for the erythromycin-producing PKS (Katz, 1997; Khosla, 1997; Staunton et al. 1997). The combined set of PKS modules and catalytic domains along with genes that encode enzymes for post-polyketide tailoring (e.g., glycosyl transferases, hydroxylases) typically limits a biosynthetic system to the generation of a single polyketide product. [0291]
Combinatorial biology involves the genetic manipulation of multistep biosynthetic pathways to create molecular diversity in natural products for use in novel drug discovery. PKSs represent one of the most amenable systems for combinatorial technologies because of their inherent genetic organization and ability to produce polyketide metabolites, a large group of natural products generated by bacteria (primarily actinomycetes and myxobacteria) and fungi with diverse structures and biological activities. Complex polyketides are produced by multifunctional PKSs involving a mechanism similar to long-chain fatty acid synthesis in animals (Hopwood et al., 1990). Pioneering studies (Cortes et al., 1990; Donadio et al., 1991) on the erythromycin PKS in [0292] Saccharopolyspora erythraea revealed a modular organization. Characterization of this multidomain protein system, followed by molecular analysis of rapamycin (Aparicio et al., 1996), FK506 (Motamedi et al., 1997), soraphen A (Schupp et al., 1995), niddamycin (Kakavas et al., 1997), and rifamycin (August et al., 1998) PKSs, demonstrated a co-linear relationship between modular structure of a multifunctional bacterial PKS and the structure of its polyketide product.
In a survey of microbial systems capable of generating unusual metabolite structural variability, [0293] Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, while narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. The cloning and characterization of the biosynthetic gene cluster for these antibiotics reveals the key role of a type II thioesterase in forming a metabolic branch through which polyketides of different chain length are generated by the pikromycin multifunctional polyketide synthase (PKS). Immediately downstream of the PKS genes (pik4) are a set of genes for desosamine (des) biosynthesis and macrolide ring hydroxylation. The glycosyl transferase (encoded by desVIII) has the remarkable ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems.

Example 9

Strategies Employing Modular PKS as PHA Monomer Providers

One strategy to exploit modular PKSs, e.g., modules of pikA or a FAS, to provide PHA monomers is to harvest polyketide intermediates as CoA derivatives using a TEII which is converted to an acyl-CoA transferase (mTEII). PikTEII is a small enzyme (281 amino acids) encoded by pikAV in [0294] S. venezuelae. The primary function of the wild-type enzyme is to catalyze the release of a polyketide chain at the fifth module in the pikA pathway as 1 0-deoxymethonolide. The enzyme most likely binds to the fifth module (PikAIII) ACP (ACP5) and releases the acyl chain attached to it. This relationship, TEII and its cognate ACP5, can be exploited to produce a polyketide having different chain lengths by moving Pik ACP5 to a different position in the cluster. For example, by moving ACP5 into the second module in place of ACP2, a triketide instead of hexoketide may be produced by the cluster. Further, moving KR5 together with ACP5 into the second module, and replacing the DH, KR, and ACP domains, a 3-hydroxyl triketide is produced that is structurally suitable as PHA monomer. A mutant TEII (mTEII) catalyzes the release of the triketide as CoA form. The triketide-CoA, 3,5-dihydroxyl-4-methyl-heptonyl-CoA, is a substrate for PHA polymerase, e.g., PhaC1 from P. olivarus, which, in turn, can incorporate the monomer into a polymer.
A second strategy includes the harvesting of a polyketide intermediate as a CoA derivative using a TEI which has been converted to an acyl-CoA transferase (mTE). Thus, the second strategy for 3-hydroxyacyl-CoA monomer production is to exploit the TE domain (TEI) within the PKS module. It has been demonstrated that the TE domain can release polyketide intermediates attached to the ACP domain within the same module. Moving the TEI to a different position in a PKS cluster results in the production of a polyketide having a different chain length. Similarly, a mutant TEI (mTEI) (i.e., one which is an acyl-CoA transferase) releases the polyketide intermediate to acyl-CoA, which then is polymerized by PHA synthetase. Preferably, a mutant TE domain in the pika gene cluster is moved into [0295] pik module 1, fusing it immediately downstream of ACP 1. The recombinant enzyme produces 2-(S)-methyl-3(R)-hydroxylveleratyl-CoA, which is a suitable substrate for PHA polymerase PhaC1. Therefore, the coexpression of the polymerase with the recombinant PKS produces a polymer.
A third strategy is to directly collect polyketide intermediates as substrates for PHA synthesis by fusing a PHA polymerase with a polyketide synthase. The first two strategies produce 3-hydroxylacyl-CoA as a substrate for PHA synthesis by employing a mutant PKS enzyme (TEI or TEII). As PHA polymerase may be active on acyl-ACP itself if the acyl-ACP is properly oriented, the third strategy fuses a PHA polymerase downstream of an ACP in a PKS protein. The PHA synthetase then serves as a domain within the chimeric multifunctional enzyme in place of a TE domain. The PKS portion of the protein catalyzes the synthesis of a 3-hydroxylacyl-ACP intermediate and then the PHA synthetase domain accepts it as substrate and adds the 3-hydroxylacyl monomer to the growing polyhydroxyalkanoate chain. The process regenerates ACP function so that the reaction can go on repeatedly to synthesize a PHA of multiple units. For example, a phaC1 gene is fused directly downstream of pik ACP1 so as to produce a chimeric enzyme that catalyzes the synthesis of a polymer. [0296]
The strategies described above can produce PHAs of complex structure, and having superior properties. In addition, the structure can be easily fine-tuned by modifying the PKS gene, thus resulting in PHAs having desired properties or functions. [0297]

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The complete disclosure of all patents, patent documents and publications cited herein are incorporated herein by reference as if individually incorporated. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described for variations obvious to one skilled in the art will be included within the invention defined by the claims. [0373]
1 43 1 15872 DNA Streptomyces venezuelae 1 ttaattaagg aggaccatca tgaacgaggc catcgccgtc gtcggcatgt cctgccgcct 60 gccgaaggcc tcgaacccgg ccgccttctg ggagctgctg cggaacgggg agagcgccgt 120 caccgacgtg ccctccggcc ggtggacgtc ggtgctcggg ggagcggacg ccgaggagcc 180 ggcggagtcc ggtgtccgcc ggggcggctt cctcgactcc ctcgacctct tcgacgcggc 240 cttcttcgga atctcgcccc gtgaggccgc cgccatggac ccgcagcagc gactggtcct 300 cgaactcgcc tgggaggcgc tggaggacgc cggaatcgtc cccggcaccc tcgccggaag 360 ccgcaccgcc gtcttcgtcg gcaccctgcg ggacgactac acgagcctcc tctaccagca 420 cggcgagcag gccatcaccc agcacaccat ggcgggcgtg aaccggggcg tcatcgccaa 480 ccgcgtctcg taccacctcg gcctgcaggg cccgagcctc accgtcgacg ccgcgcagtc 540 gtcctcgctc gtcgccgtgc acctggcctg cgagtccctg cgcgccgggg agtccacgac 600 ggcgctcgtc gccggcgtga acctcaacat cctcgcggag agcgccgtga cggaggagcg 660 cttcggtgga ctctccccgg acggcaccgc ctacaccttc gacgcgcggg ccaacggatt 720 cgtccggggc gagggcggcg gagtcgtcgt actcaagccg ctctcccgcg ccctcgccga 780 cggcgaccgt gtccacggcg tcatccgcgc cagcgccgtc aacaacgacg gagccacccc 840 gggtctcacc gtgcccagca gggccgccca ggagaaggtg ctgcgcgagg cgtaccggaa 900 ggcggccctg gacccgtccg ccgtccagta cgtcgaactc cacggcaccg gaacccccgt 960 cggcgacccc atcgaggccg ccgcgctcgg cgccgtcctc ggctcggcgc gccccgcgga 1020 cgaacccctg ctcgtcggct cggccaagac gaacgtcggg cacctcgaag gcgccgccgg 1080 catcgtcggc ctcatcaaga cgctcctcgc gctcggccgg cgccggatcc cggcgagcct 1140 caacttccgt acgccccacc cggacatccc gctcgacacc ctcgggctcg acgtgcccga 1200 cggcctgcgg gagtggccgc acccggaccg cgaactcctc gccggcgtca gctcgttcgg 1260 catgggcggc accaacgccc acgtcgtcct cagcgaaggc cccgcccagg gcggcgagca 1320 gcccggcatc gatgaggaga cccccgtcga cagcggggcc gcactgccct tcgtcgtcac 1380 cggccgcggc ggcgaggccc tgcgcgccca ggcccggcgc ctgcacgagg ccgtcgaagc 1440 ggacccggag ctcgcgcccg ccgcactcgc ccggtcgctg gtcaccaccc gtacggtctt 1500 cacgcaccgg tcggtcgtcc tcgccccgga ccgcgcccgc ctcctcgacg gcctcggcgc 1560 cctcgccgcc gggacgcccg cgcccggcgt ggtcaccggc acccccgccc ccgggcgcct 1620 cgccgtcctg ttcagcggcc agggtgccca acgtacgggc atgggcatgg agttgtacgc 1680 cgcccacccc gccttcgcga cggccttcga cgccgtcgcc gccgaactgg accccctcct 1740 cgaccggccc ctcgccgaac tcgtcgcggc gggcgacacc ctcgaccgca ccgtccacac 1800 acagcccgcg ctcttcgccg tggaggtcgc cctccaccgc ctcgtcgagt cctggggcgt 1860 cacgcccgac ctgctcgccg gccactccgt cggcgagatc agcgccgccc acgtcgccgg 1920 ggtcctgtcg ctgcgcgacg ccgcccgcct cgtcgcggcg cgcggccgcc tcatgcaggc 1980 gctccccgag ggcggcgcga tggtcgcggt cgaggcgagc gaggaggaag tgcttccgca 2040 cctcgcggga cgcgagcggg agctctccct cgcggccgtg aacggccccc gcgcggtcgt 2100 cctcgcgggc gccgagcgcg ccgtcctcga cgtcgccgag ctgctgcgcg aacagggccg 2160 ccggacgaag cggctcagcg tctcgcacgc cttccactcg ccgctcatgg agccgatgct 2220 cgacgacttc cgccgggtcg tcgaagagct ggacttccag gagccccgcg tcgacgtcgt 2280 gtccacggtg acgggcctgc ctgtcacagc gggccaatgg accgatcccg agtactgggt 2340 ggaccaggtc cgcaggcccg tacgcttcct cgacgccgta cgcaccctgg aggaatcggg 2400 cgccgacacc ttcctggagc tcggtcccga cggggtctgc tccgcgatgg cggcggactc 2460 cgtacgcgac caggaggccg ccacggcggt ctccgccctg cgcaagggcc gcccggagcc 2520 ccagtcgctg ctcgccgcac tcaccaccgt cttcgtccgg ggccacgacg tcgactggac 2580 cgccgcgcac gggagcaccg gcacggtcag ggtgcccctg ccgacctacg ccttccagcg 2640 cgaacgccac tggttcgacg gcgccgcgcg aacggcggcg ccgctcacgg cgggccgatc 2700 gggcaccggt gcgggcaccg gcccggccgc gggtgtgacg tcgggcgagg gcgagggcga 2760 gggcgagggc gcgggtgcgg gtggcggtga tcggccggct cgccacgaga cgaccgagcg 2820 cgtgcgcgca cacgtcgccg ccgtcctcga gtacgacgac ccgacccgcg tcgaactcgg 2880 cctcaccttc aaggagctgg gcttcgactc cctcatgtcc gtcgagctgc ggaacgcgct 2940 cgtcgacgac acgggactgc gcctgcccag cggactgctc ttcgaccacc cgacgccgcg 3000 cgccctcgcc gcccacctgg gcgacctgct caccggcggc agcggcgaga ccggatcggc 3060 cgacgggata ccgcccgcga ccccggcgga caccaccgcc gagcccatcg cgatcatcgg 3120 catggcctgc cgctaccccg gcggcgtcac ctcccccgag gacctgtggc ggctcgtcgc 3180 cgaggggcgc gacgccgtct cggggctgcc caccgaccgc ggctgggacg aggacctctt 3240 cgacgccgac cccgaccgca gcggcaagag ctcggtccgc gagggcggat tcctgcacga 3300 cgccgccctg ttcgacgccg gcttcttcgg gatatcgccc cgcgaggccc tcggcatgga 3360 cccgcagcag cggctgctcc tggagacggc atgggaggcc gtggagcgcg cagggctcga 3420 ccccgaaggc ctcaagggca gccggacggc cgtcttcgtc ggcgccaccg ccctggacta 3480 cggcccgcgc atgcacgacg gcgccgaggg cgtcgagggc cacctcctga ccgggaccac 3540 gcccagcgtg atgtcgggcc gcatcgccta ccagctcggc ctcaccggtc ctgcggtcac 3600 cgtcgacacg gcctgctcgt cctcgctcgt cgcgctgcac ctggccgtcc gttcgctgcg 3660 gcagggcgag tcgagcctcg cgctcgccgg cggagcgacc gtcatgtcga caccgggcat 3720 gttcgtcgag ttctcgcggc agcgcggcct cgccgccgac ggccgctcca aggccttctc 3780 cgactccgcc gacggcacct cctgggccga gggcgtcggc ctcctcgtcg tcgagcggct 3840 ctcggacgcc gagcgcaacg gccaccccgt gctcgccgtg atccggggca gcgcggtcaa 3900 ccaggacggc gcctccaacg ggctcaccgc ccccaacggc ccgtcccagc agcgcgtcat 3960 ccgacaggcc ctggccgacg ccgggctcac cccggccgac gtcgacgccg tcgaggcgca 4020 cggtacgggt acccggctcg gcgaccccat cgaggccgag gcgatcctcg gcacctacgg 4080 ccgggaccgg ggcgagggcg ctccgctcca gctcggctcg ctgaagtcga acatcggcca 4140 cgcgcaggcc gccgcgggcg tgggcgggct catcaagatg gtcctcgcga tgcgccacgg 4200 cgtcctgccc aggacgctcc acgtggaccg gcccaccacc cgcgtcgact gggaggccgg 4260 cggcgtcgag ctcctcaccg aggagcggga gtggccggag acgggccgcc cgcgccgcgc 4320 ggcgatctcc tccttcggca tcagcggcac caacgcccac atcgtggtcg aacaggcccc 4380 ggaagccggg gaggcggcgg tcaccaccac cgccccggaa gcaggggaag ccggggaagc 4440 ggcggacacc accgccacca cgacgccggc cgcggtcggc gtccccgaac ccgtacgcgc 4500 ccccgtcgtg gtctccgcgc gggacgccgc cgccctgcgc gcccaggccg ttcggctgcg 4560 gaccttcctc gacggccgac cggacgtcac cgtcgccgac ctcggacgct cgctggccgc 4620 ccgtaccgcc ttcgagcaca aggccgccct caccaccgcc accagggacg agctgctcgc 4680 cgggctcgac gccctcggcc gcggggagca agccacgggc ctggtcaccg gcgaaccggc 4740 cagggccgga cgcacggcct tcctgttcac cggccaggga gcgcagcgcg tcgccatggg 4800 cgaggaactg cgcgccgcgc accccgtgtt cgccgccgcc ctcgacaccg tgtacgcggc 4860 cctcgaccgt cacctcgacc ggccgctgcg ggagatcgtc gccgccgggg aggagctgga 4920 cctcaccgcg tacacccagc ccgccctctt cgccttcgag gtggcgctgt tccgcctcct 4980 cgaacaccac ggcctcgtcc ccgacctgct caccggccac tccgtcggcg agatcgccgc 5040 cgcgcacgtc gccggtgtcc tctccctcga cgacgccgca cgtctcgtca ccgcccgcgg 5100 ccggctcatg cagtcggccc gcgagggcgg cgcgatgatc gccgtgcagg cgggcgaggc 5160 cgaggtcgtc gagtccctga agggctacga gggcagggtc gccgtcgccg ccgtcaacgg 5220 acccaccgcc gtggtcgtct ccggcgacgc ggacgccgcc gaggagatcc gcgccgtatg 5280 ggcgggacgc ggccggcgca cccgcaggct gcgcgtcagc cacgccttcc actccccgca 5340 catggacgac gtcctcgacg agttcctccg ggtcgccgag ggcctgacct tcgaggagcc 5400 gcggatcccc gtcgtctcca cggtcaccgg cgcgctcgtc acgtccggcg agctcacctc 5460 gcccgcgtac tgggtcgacc agatccggcg gcccgtgcgc ttcctggacg ccgtccgcac 5520 cctggccgcc caggacgcga ccgtcctcgt cgagatcggc cccgacgccg tcctcacggc 5580 actcgccgag gaggctctcg cgcccggcac ggacgccccg gacgcccggg acgtcacggt 5640 cgtcccgctg ctgcgcgcgg ggcgccccga gcccgagacc ctcgccgccg gtctcgcgac 5700 cgcccatgtc cacggcgcac ccttggaccg ggcgtcgttc ttcccggacg ggcgccgcac 5760 ggacctgccc acgtacgcct tccggcgcga gcactactgg ctgacgcccg aggcccgtac 5820 ggacgcccgc gcactcggct tcgacccggc gcggcacccg ctgctgacga ccacggtcga 5880 ggtcgccggc ggcgacggcg tcctgctgac cggccgtctc tccctgaccg accagccctg 5940 gctggccgac cacatggtca acggcgccgt cctgttgccg gccaccgcct tcctggagct 6000 cgccctcgcg gcgggcgacc acgtcggggc ggtccgggtg gaggaactca ccctcgaagc 6060 gccgctcgtc ctgcccgagc ggggcgccgt ccgcatccag gtcggcgtga gcggcgacgg 6120 cgagtcgccg gccgggcgca ccttcggtgt gtacagcacc cccgactccg gcgacaccgg 6180 tgacgacgcg ccccgggagt ggacccgcca tgtctccggc gtactcggcg aaggggaccc 6240 ggccacggag tcggaccacc ccggcaccga cggggacggt tcagcggcct ggccgcctgc 6300 ggcggcgacc gccacacccc tcgacggcgt ctacgaccgg ctcgcggagc tcggctacgg 6360 atacggtccg gccttccagg gcctgacggg gctgtggcgc gacggcgccg acacgctcgc 6420 cgagatccgg ctgcccgcgg cgcagcacga gagcgcgggg ctcttcggcg tacacccggc 6480 gctgctcgac gcggcgctcc acccgatcgt cctggagggc aactcagctg ccggtgcctg 6540 tgacgccgat accgacgcga ccgaccggat ccggctgccg ttcgcgtggg cgggggtgac 6600 cctccacgcc gaaggggcca ccgcgctccg cgtacggatc acacccaccg gcccggacac 6660 ggtcacgctc cgcctcaccg acaccaccgg tgcgcccgtg gccaccgtgg agtccctgac 6720 cctgcgcgcg gtggcgaagg accggctggg caccaccgcc gggcgcgtcg acgacgccct 6780 gttcacggtc gtgtggacgg agaccggcac accggaaccc gcagggcgcg gagccgtgga 6840 ggtcgaggaa ctcgtcgacc tcgccggcct cggcgacctc gtggagctcg gcgccgcgga 6900 cgtcgtcctc cgggccgacc gctggacgct cgacggggac ccgtccgccg ccgcgcgcac 6960 agccgtccgg cgcaccctcg ccatcgtcca ggagttcctg tccgagccgc gcttcgacgg 7020 ctcgcgactg gtgtgcgtca ccaggggcgc ggtcgccgca ctccccggcg aggacgtcac 7080 ctccctcgcc accggccccc tctggggcct cgtccgctcc gcccagtccg agaacccggg 7140 acgcctgttc ctcctggacc tgggtgaagg cgaaggcgag cgcgacggag ccgaggagct 7200 gatccgcgcg gccacggccg gggacgagcc gcagctcgcg gcacgggacg gccgactgct 7260 cgcgccgagg ctggcccgta ccgccgccct ttcgagtgag gacaccgccg gcggcgccga 7320 ccgtttcggc cccgacggca ccgtcctcgt caccgggggc accggaggcc tcggagcgct 7380 cctcgcccgc cacctcgtgg agcgtcacgg ggtgcgccgg ctgctgctgg tgagccgccg 7440 cggggccgac gcccccggcg cggccgacct gggcgaggac ctcgcgggcc tcggcgcgga 7500 ggtggcgttc gccgccgccg acgccgccga ccgcgagagc ctggcgcggg cgatcgccac 7560 cgtgcccgcc gagcatccgc tgacggccgt cgtgcacacg gcgggagtcg tcgacgacgc 7620 gacggtggag gcgctcacac cggaacggct ggacgcggta ctgcgcccga aggtcgacgc 7680 cgcgtggaac ctgcacgagc tcaccaagga cctgcggctc gacgccttcg tcctcttctc 7740 ctccgtctcc ggcatcgtcg gcaccgccgg ccaggccaac tacgcggcgg ccaacacggg 7800 cctcgacgcc ctcgccgccc accgcgccgc cacgggcctg gccgccacgt cgctggcctg 7860 gggcctctgg gacggcacgc acggcatggg cggcacgctc ggcgccgccg acctcgcccg 7920 ctggagccgg gccggaatca ccccgctcac cccgctgcag ggcctcgcgc tcttcgacgc 7980 cgcggtcgcc agggacgacg ccctcctcgt acccgccggg ctccgtccca ccgcccaccg 8040 gggcacggac ggacagcctc ctgcgctgtg gcgcggcctc gtccgggcgc gcccgcgccg 8100 tgccgcgcgg acggccgccg aggcggcgga cacgaccggc ggctggctga gcgggctcgc 8160 cgcacagtcc cccgaggagc ggcgcagcac agccgtcacg ctcgtgacgg gtgtcgtcgc 8220 ggacgtcctc gggcacgccg actccgccgc ggtcggggcg gagcggtcct tcaaggacct 8280 cggcttcgac tccctggccg gggtggagct ccgcaaccgg ctgaacgccg ccaccggcct 8340 gcggctcccc gcgaccacgg tcttcgacca tccctcgccg gccgcgctcg cgtcccatct 8400 cctcgcccag gtgcccgggt tgaaggaggg gacggcggcg accgcgaccg tcgtggccga 8460 gcggggcgct tccttcggtg accgtgcgac cgacgacgat ccgatcgcga tcgtgggcat 8520 ggcatgccgc tatccgggtg gtgtgtcgtc gccggaggac ctgtggcggc tggtggccga 8580 ggggacggac gcgatcagcg agttccccgt caaccgcggc tgggacctgg agagcctcta 8640 cgacccggat cccgagtcga agggcaccac gtactgccgg gagggcgggt tcctggaagg 8700 cgccggtgac ttcgacgccg ccttcttcgg catctcgccg cgcgaggccc tggtgatgga 8760 cccgcagcag cggctgctgc tggaggtgtc ctgggaggcg ctggaacgcg cgggcatcga 8820 cccgtcctcg ctgcgcggca gccgcggtgg tgtctacgtg ggcgccgcgc acggctcgta 8880 cgcctccgat ccccggctgg tgcccgaggg ctcggagggc tatctgctga ccggcagcgc 8940 cgacgcggtg atgtccggcc gcatctccta cgcgctcggt ctcgaaggac cgtccatgac 9000 ggtggagacg gcctgctcct cctcgctggt ggcgctgcat ctggcggtac gggcgctgcg 9060 gcacggcgag tgcgggctcg cgctggcggg cggggtggcg gtgatggccg atccggcggc 9120 gttcgtggag ttctcccggc agaaggggct ggccgccgac ggccgctgca aggcgttctc 9180 ggccgccgcc gacggcaccg gctgggccga gggcgtcggc gtgctcgtcc tggagcggct 9240 gtcggacgcg cgccgcgcgg ggcacacggt cctcggcctg gtcaccggca ccgcggtcaa 9300 ccaggacggt gcctccaacg ggctgaccgc gcccaacggc ccagcccagc aacgcgtcat 9360 cgccgaggcg ctcgccgacg ccgggctgtc cccggaggac gtggacgcgg tcgaggcgca 9420 cggcaccggc acccggctcg gcgaccccat cgaggccggg gcgctgctcg ccgcctccgg 9480 acggaaccgt tccggcgacc acccgctgtg gctcggctcg ctgaagtcca acatcgggca 9540 tgcccaggcc gccgccggtg tcggcggcgt catcaagatg ctccaggcgc tgcggcacgg 9600 cttgctgccc cgcaccctcc acgccgacga gccgaccccg catgccgact ggagctccgg 9660 ccgggtacgg ctgctcacct ccgaggtgcc gtggcagcgg accggccggc cccggcggac 9720 cggggtgtcc gccttcggcg tcggcggcac caatgcccat gtcgtcctcg aagaggcacc 9780 cgccccgccc gcgccggaac cggccgggga ggcccccggc ggctcccgcg ccgcagaagg 9840 ggcggaaggg cccctggcct gggtggtctc cggacgcgac gagccggccc tgcggtccca 9900 ggcccggcgg ctccgcgacc acctctcccg cacccccggg gcccgcccgc gtgacatcgc 9960 cttctccctc gccgccacgc gcgcagcctt tgaccaccgc gccgtgctga tcggctcgga 10020 cggggccgaa ctcgccgccg ccctggacgc gttggccgaa ggacgcgacg gtccggcggt 10080 ggtgcgcgga gtccgcgacc gggacggcag gatggccttc ctcttcaccg ggcagggcag 10140 ccagcgcgcc gggatggccc acgacctgca tgccgcccat accttcttcg cgtccgccct 10200 cgacgaggtg acggaccgtc tcgacccgct gctcggccgg ccgctcggcg cgctgctgga 10260 cgcccgaccc ggctcgcccg aagcggcact cctggaccgg accgagtaca cccagccggc 10320 gctcttcgcc gtcgaggtgg cgctccaccg gctgctggag cactggggga tgcgccccga 10380 cctgctgctg gggcactcgg tgggcgaact ggcggccgcc cacgtcgcgg gtgtgctcga 10440 tctcgacgac gcctgcgcgc tggtggccgc ccgcggcagg ctgatgcagc gcctgccgcc 10500 cggcggcgcg atggtctccg tgcgggccgg cgaggacgag gtccgcgcac tgctggccgg 10560 ccgcgaggac gccgtctgcg tcgccgcggt gaacggcccc cggtcggtgg tgatctccgg 10620 cgcggaggaa gcggtggccg aggcggcggc gcagctcgcc ggacgaggcc gccgcaccag 10680 gcggctccgc gtcgcgcacg ccttccactc acccctgatg gacggcatgc tcgccggatt 10740 ccgggaggtc gccgccggcc tgcgctaccg ggaaccggag ctgacggtcg tctccacggt 10800 cacggggcgg cccgcccgcc ccggtgaact caccggcccc gactactggg tggcccaggt 10860 ccgtgagccc gtgcgcttcg cggacgcggt ccgcacggca caccgcctcg gagcccgcac 10920 cttcctggag accggcccgg acggcgtgct gtgcggcatg gcagaggagt gcctggagga 10980 cgacaccgtg gccctgctgc cggcgatcca caagcccggc accgcgccgc acggtccggc 11040 ggctcccggc gcgctgcggg cggccgccgc cgcgtacggc cggggcgccc gggtggactg 11100 ggccgggatg cacgccgacg gccccgaggg gccggcccgc cgcgtcgaac tgcccgtcca 11160 cgccttccgg caccgccgct actggctcgc cccgggccgc gcggcggaca ccgacgactg 11220 gatgtaccgg atcggctggg accggctgcc ggctgtgacc ggcggggccc ggaccgccgg 11280 ccgctggctg gtgatccacc ccgacagccc gcgctgccgg gagctgtccg gccacgccga 11340 acgcgcgctg cgcgccgcgg gcgcgagccc cgtaccgctg cccgtggacg ctccggccgc 11400 cgaccgggcg tccttcgcgg cactgctgcg ctccgccacc ggacctgaca cacgaggtga 11460 cacagccgcg cccgtggccg gtgtgctgtc gctgctgtcc gaggaggatc ggccccatcg 11520 ccagcacgcc ccggtacccg ccggggtcct ggcgacgctg tccctgatgc aggctatgga 11580 ggaggaggcg gtggaggctc gcgtgtggtg cgtctcccgc gccgcggtcg ccgccgccga 11640 ccgggaacgg cccgtcggcg cgggcgccgc cctgtggggg ctggggcggg tggccgccct 11700 ggaacgcccc acccggtggg gcggtctcgt ggacctgccc gcctcgcccg gtgcggcgca 11760 ctgggcggcc gccgtggaac ggctcgccgg tcccgaggac cagatcgccg tgcgcgcgtc 11820 cggcagttgg ggccggcgcc tcaccaggct gccgcgcgac ggcggcggcc ggacggccgc 11880 acccgcgtac cggccgcgcg gcacggtgct cgtcaccggt ggcaccggcg cgctcggcgg 11940 gcatctcgcc cgctggctcg ccgcggcggg cgccgaacac ctggcgctca ccagccgccg 12000 gggcccggac gcgcccggcg ccgccggact cgaggccgaa ctcctcctcc tgggcgccaa 12060 ggtgacgttc gccgcctgcg acaccgccga ccgcgacggc ctcgcccggg tcctgcgggc 12120 gataccggag gacaccccgc tcaccgcggt gttccacgcc gcgggcgtac cgcaggtcac 12180 gccgctgtcc cgtacctcgc ccgagcactt cgccgacgtg tacgcgggca aggcggcggg 12240 cgccgcgcac ctggacgaac tgacccgcga actcggcgcc ggactcgacg cgttcgtcct 12300 ctactcctcc ggcgccggcg tctggggcag cgccggccag ggtgcctacg ccgccgccaa 12360 cgccgccctg gacgcgctcg cccggcgccg tgcggcggac ggactccccg ccacctccat 12420 cgcctggggc gtgtggggcg gcggcggtat gggggccgac gaggcgggcg cggagtatct 12480 gggccggcgc ggtatgcgcc ccatggcacc ggtctccgcg ctccgggcga tggccaccgc 12540 catcgcctcc ggggaaccct gccccaccgt cacccacacc gactgggagc gcttcggcga 12600 gggcttcacc gccttccggc ccagccctct gatcgcgggg ctcggcacgc cgggcggcgg 12660 ccgggcggcg gagacccccg aggaggggaa cgccaccgct gcggcggacc tcaccgccct 12720 gccgcccgcc gaactccgca ccgcgctgcg cgagctggtg cgagcccgga ccgccgcggc 12780 gctcggcctc gacgacccgg ccgaggtcgc cgagggcgaa cggttccccg ccatgggctt 12840 cgactccctg gccaccgtac ggctgcgccg cggactcgcc tcggccacgg gcctcgacct 12900 gccccccgat ctgctcttcg accgggacac cccggccgcg ctcgccgccc acctggccga 12960 actgctcgcc accgcacggg accacggacc cggcggcccc gggaccggtg ccgcgccggc 13020 cgatgccgga agcggcctgc cggccctcta ccgggaggcc gtccgcaccg gccgggccgc 13080 ggaaatggcc gaactgctcg ccgccgcttc ccggttccgc cccgccttcg ggacggcgga 13140 ccggcagccg gtggccctcg tgccgctggc cgacggcgcg gaggacaccg ggctcccgct 13200 gctcgtgggc tgcgccggga cggcggtggc ctccggcccg gtggagttca ccgccttcgc 13260 cggagcgctg gcggacctcc cggcggcggc cccgatggcc gcgctgccgc agcccggctt 13320 tctgccggga gaacgagtcc cggccacccc ggaggcattg ttcgaggccc aggcggaagc 13380 gctgctgcgc tacgcggccg gccggccctt cgtgctgctg gggcactccg ccggcgccaa 13440 catggcccac gccctgaccc gtcatctgga ggcgaacggt ggcggccccg cagggctggt 13500 gctcatggac atctacaccc ccgccgaccc cggcgcgatg ggcgtctggc ggaacgacat 13560 gttccagtgg gtctggcggc gctcggacat ccccccggac gaccaccgcc tcacggccat 13620 gggcgcctac caccggctgc ttctcgactg gtcgcccacc cccgtccgcg cccccgtact 13680 gcatctgcgc gccgcggaac ccatgggcga ctggccaccc ggggacaccg gctggcagtc 13740 ccactgggac ggcgcgcaca ccaccgccgg catccccgga aaccacttca cgatgatgac 13800 cgaacacgcc tccgccgccg cccggctcgt gcacggctgg ctcgcggaac ggaccccgtc 13860 cgggcagggc gggtcaccgt cccgcgcggc ggggagagag gagaggccgt gaacacggca 13920 gccggcccga ccggcaccgc cgccggcggc accaccgccc cggcggcggc acacgacctg 13980 tcccgcgccg gacgcaggct ccaactcacc cgggccgcac agtggttcgc cggcaaccag 14040 ggagacccct acgggatgat cctgcgcgcc ggcaccgccg acccggcacc gtacgaggaa 14100 gagatccccg ggtaccgagc tcgaattctt aattaaggag gtcgtagatg agtaacaaga 14160 acaacgatga gctgcagcgg caggcctcgg aaaacaccct ggggctgaac ccggtcatcg 14220 gtatccgccg caaagacctg ttgagctcgg cacgcaccgt gctgcgccag gccgtgcgcc 14280 aaccgctgca cagcgccaag catgtggccc actttggcct ggagctgaag aacgtgctgc 14340 tgggcaagtc cagccttgcc ccggaaagcg acgaccgtcg cttcaatgac ccggcatgga 14400 gcaacaaccc actttaccgc cgctacctgc aaacctatct ggcctggcgc aaggagctgc 14460 aggactggat cggcaacagc gacctgtcgc cccaggacat cagccgcggc cagttcgtca 14520 tcaacctgat gaccgaagcc atggctccga ccaacaccct gtccaacccg gcagcagtca 14580 aacgcttctt cgaaaccggc ggcaagagcc tgctcgatgg cctgtccaac ctggccaagg 14640 acctggtcaa caacggtggc atgcccagcc aggtgaacat ggacgccttc gaggtgggca 14700 agaacctggg caccagtgaa ggcgccgtgg tgtaccgcaa cgatgtgctg gagctgatcc 14760 agtacaagcc catcaccgag caggtgcatg cccgcccgct gctggtggtg ccgccgcaga 14820 tcaacaagtt ctacgtattc gacctgagcc cggaaaagag cctggcacgc tactgcctgc 14880 gctcgcagca gcagaccttc atcatcagct ggcgcaaccc gaccaaagcc cagcgcgaat 14940 ggggcctgtc cacctacatc gacgcgctca aggaggcggt cgacgcggtg ctggcgatta 15000 ccggcagcaa ggacctgaac atgctcggtg cctgctccgg cggcatcacc tgcacggcat 15060 tggtcggcca ctatgccgcc ctcggcgaaa acaaggtcaa tgccctgacc ctgctggtca 15120 gcgtgctgga caccaccatg gacaaccagg tcgccctgtt cgtcgacgag cagactttgg 15180 aggccgccaa gcgccactcc taccaggccg gtgtgctcga aggcagcgag atggccaagg 15240 tgttcgcctg gatgcgcccc aacgacctga tctggaacta ctgggtcaac aactacctgc 15300 tcggcaacga gccgccggtg ttcgacatcc tgttctggaa caacgacacc acgcgcctgc 15360 cggccgcctt ccacggcgac ctgatcgaaa tgttcaagag caacccgctg acccgcccgg 15420 acgccctgga ggtttgcggc actccgatcg acctgaaaca ggtcaaatgc gacatctaca 15480 gccttgccgg caccaacgac cacatcaccc cgtggcagtc atgctaccgc tcggcgcacc 15540 tgttcggcgg caagatcgag ttcgtgctgt ccaacagcgg ccacatccag agcatcctca 15600 acccgccagg caaccccaag gcgcgcttca tgaccggtgc cgatcgcccg ggtgacccgg 15660 tggcctggca ggaaaacgcc accaagcatg ccgactcctg gtggctgcac tggcaaagct 15720 ggctgggcga gcgtgccggc gagctggaaa aggcgccgac ccgcctgggc aaccgtgcct 15780 atgccgctgg cgaggcatcc ccgggcacct acgttcacga gcgttgagct gcagcgccgt 15840 ggccacctgc gggacgccac ggtgttgaat tc 15872 2 5215 PRT Streptomyces venezuelae 2 Met Asn Glu Ala Ile Ala Val Val Gly Met Ser Cys Arg Leu Pro Lys 1 5 10 15 Ala Ser Asn Pro Ala Ala Phe Trp Glu Leu Leu Arg Asn Gly Glu Ser 20 25 30 Ala Val Thr Asp Val Pro Ser Gly Arg Trp Thr Ser Val Leu Gly Gly 35 40 45 Ala Asp Ala Glu Glu Pro Ala Glu Ser Gly Val Arg Arg Gly Gly Phe 50 55 60 Leu Asp Ser Leu Asp Leu Phe Asp Ala Ala Phe Phe Gly Ile Ser Pro 65 70 75 80 Arg Glu Ala Ala Ala Met Asp Pro Gln Gln Arg Leu Val Leu Glu Leu 85 90 95 Ala Trp Glu Ala Leu Glu Asp Ala Gly Ile Val Pro Gly Thr Leu Ala 100 105 110 Gly Ser Arg Thr Ala Val Phe Val Gly Thr Leu Arg Asp Asp Tyr Thr 115 120 125 Ser Leu Leu Tyr Gln His Gly Glu Gln Ala Ile Thr Gln His Thr Met 130 135 140 Ala Gly Val Asn Arg Gly Val Ile Ala Asn Arg Val Ser Tyr His Leu 145 150 155 160 Gly Leu Gln Gly Pro Ser Leu Thr Val Asp Ala Ala Gln Ser Ser Ser 165 170 175 Leu Val Ala Val His Leu Ala Cys Glu Ser Leu Arg Ala Gly Glu Ser 180 185 190 Thr Thr Ala Leu Val Ala Gly Val Asn Leu Asn Ile Leu Ala Glu Ser 195 200 205 Ala Val Thr Glu Glu Arg Phe Gly Gly Leu Ser Pro Asp Gly Thr Ala 210 215 220 Tyr Thr Phe Asp Ala Arg Ala Asn Gly Phe Val Arg Gly Glu Gly Gly 225 230 235 240 Gly Val Val Val Leu Lys Pro Leu Ser Arg Ala Leu Ala Asp Gly Asp 245 250 255 Arg Val His Gly Val Ile Arg Ala Ser Ala Val Asn Asn Asp Gly Ala 260 265 270 Thr Pro Gly Leu Thr Val Pro Ser Arg Ala Ala Gln Glu Lys Val Leu 275 280 285 Arg Glu Ala Tyr Arg Lys Ala Ala Leu Asp Pro Ser Ala Val Gln Tyr 290 295 300 Val Glu Leu His Gly Thr Gly Thr Pro Val Gly Asp Pro Ile Glu Ala 305 310 315 320 Ala Ala Leu Gly Ala Val Leu Gly Ser Ala Arg Pro Ala Asp Glu Pro 325 330 335 Leu Leu Val Gly Ser Ala Lys Thr Asn Val Gly His Leu Glu Gly Ala 340 345 350 Ala Gly Ile Val Gly Leu Ile Lys Thr Leu Leu Ala Leu Gly Arg Arg 355 360 365 Arg Ile Pro Ala Ser Leu Asn Phe Arg Thr Pro His Pro Asp Ile Pro 370 375 380 Leu Asp Thr Leu Gly Leu Asp Val Pro Asp Gly Leu Arg Glu Trp Pro 385 390 395 400 His Pro Asp Arg Glu Leu Leu Ala Gly Val Ser Ser Phe Gly Met Gly 405 410 415 Gly Thr Asn Ala His Val Val Leu Ser Glu Gly Pro Ala Gln Gly Gly 420 425 430 Glu Gln Pro Gly Ile Asp Glu Glu Thr Pro Val Asp Ser Gly Ala Ala 435 440 445 Leu Pro Phe Val Val Thr Gly Arg Gly Gly Glu Ala Leu Arg Ala Gln 450 455 460 Ala Arg Arg Leu His Glu Ala Val Glu Ala Asp Pro Glu Leu Ala Pro 465 470 475 480 Ala Ala Leu Ala Arg Ser Leu Val Thr Thr Arg Thr Val Phe Thr His 485 490 495 Arg Ser Val Val Leu Ala Pro Asp Arg Ala Arg Leu Leu Asp Gly Leu 500 505 510 Gly Ala Leu Ala Ala Gly Thr Pro Ala Pro Gly Val Val Thr Gly Thr 515 520 525 Pro Ala Pro Gly Arg Leu Ala Val Leu Phe Ser Gly Gln Gly Ala Gln 530 535 540 Arg Thr Gly Met Gly Met Glu Leu Tyr Ala Ala His Pro Ala Phe Ala 545 550 555 560 Thr Ala Phe Asp Ala Val Ala Ala Glu Leu Asp Pro Leu Leu Asp Arg 565 570 575 Pro Leu Ala Glu Leu Val Ala Ala Gly Asp Thr Leu Asp Arg Thr Val 580 585 590 His Thr Gln Pro Ala Leu Phe Ala Val Glu Val Ala Leu His Arg Leu 595 600 605 Val Glu Ser Trp Gly Val Thr Pro Asp Leu Leu Ala Gly His Ser Val 610 615 620 Gly Glu Ile Ser Ala Ala His Val Ala Gly Val Leu Ser Leu Arg Asp 625 630 635 640 Ala Ala Arg Leu Val Ala Ala Arg Gly Arg Leu Met Gln Ala Leu Pro 645 650 655 Glu Gly Gly Ala Met Val Ala Val Glu Ala Ser Glu Glu Glu Val Leu 660 665 670 Pro His Leu Ala Gly Arg Glu Arg Glu Leu Ser Leu Ala Ala Val Asn 675 680 685 Gly Pro Arg Ala Val Val Leu Ala Gly Ala Glu Arg Ala Val Leu Asp 690 695 700 Val Ala Glu Leu Leu Arg Glu Gln Gly Arg Arg Thr Lys Arg Leu Ser 705 710 715 720 Val Ser His Ala Phe His Ser Pro Leu Met Glu Pro Met Leu Asp Asp 725 730 735 Phe Arg Arg Val Val Glu Glu Leu Asp Phe Gln Glu Pro Arg Val Asp 740 745 750 Val Val Ser Thr Val Thr Gly Leu Pro Val Thr Ala Gly Gln Trp Thr 755 760 765 Asp Pro Glu Tyr Trp Val Asp Gln Val Arg Arg Pro Val Arg Phe Leu 770 775 780 Asp Ala Val Arg Thr Leu Glu Glu Ser Gly Ala Asp Thr Phe Leu Glu 785 790 795 800 Leu Gly Pro Asp Gly Val Cys Ser Ala Met Ala Ala Asp Ser Val Arg 805 810 815 Asp Gln Glu Ala Ala Thr Ala Val Ser Ala Leu Arg Lys Gly Arg Pro 820 825 830 Glu Pro Gln Ser Leu Leu Ala Ala Leu Thr Thr Val Phe Val Arg Gly 835 840 845 His Asp Val Asp Trp Thr Ala Ala His Gly Ser Thr Gly Thr Val Arg 850 855 860 Val Pro Leu Pro Thr Tyr Ala Phe Gln Arg Glu Arg His Trp Phe Asp 865 870 875 880 Gly Ala Ala Arg Thr Ala Ala Pro Leu Thr Ala Gly Arg Ser Gly Thr 885 890 895 Gly Ala Gly Thr Gly Pro Ala Ala Gly Val Thr Ser Gly Glu Gly Glu 900 905 910 Gly Glu Gly Glu Gly Ala Gly Ala Gly Gly Gly Asp Arg Pro Ala Arg 915 920 925 His Glu Thr Thr Glu Arg Val Arg Ala His Val Ala Ala Val Leu Glu 930 935 940 Tyr Asp Asp Pro Thr Arg Val Glu Leu Gly Leu Thr Phe Lys Glu Leu 945 950 955 960 Gly Phe Asp Ser Leu Met Ser Val Glu Leu Arg Asn Ala Leu Val Asp 965 970 975 Asp Thr Gly Leu Arg Leu Pro Ser Gly Leu Leu Phe Asp His Pro Thr 980 985 990 Pro Arg Ala Leu Ala Ala His Leu Gly Asp Leu Leu Thr Gly Gly Ser 995 1000 1005 Gly Glu Thr Gly Ser Ala Asp Gly Ile Pro Pro Ala Thr Pro Ala Asp 1010 1015 1020 Thr Thr Ala Glu Pro Ile Ala Ile Ile Gly Met Ala Cys Arg Tyr Pro 1025 1030 1035 1040 Gly Gly Val Thr Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Glu Gly 1045 1050 1055 Arg Asp Ala Val Ser Gly Leu Pro Thr Asp Arg Gly Trp Asp Glu Asp 1060 1065 1070 Leu Phe Asp Ala Asp Pro Asp Arg Ser Gly Lys Ser Ser Val Arg Glu 1075 1080 1085 Gly Gly Phe Leu His Asp Ala Ala Leu Phe Asp Ala Gly Phe Phe Gly 1090 1095 1100 Ile Ser Pro Arg Glu Ala Leu Gly Met Asp Pro Gln Gln Arg Leu Leu 1105 1110 1115 1120 Leu Glu Thr Ala Trp Glu Ala Val Glu Arg Ala Gly Leu Asp Pro Glu 1125 1130 1135 Gly Leu Lys Gly Ser Arg Thr Ala Val Phe Val Gly Ala Thr Ala Leu 1140 1145 1150 Asp Tyr Gly Pro Arg Met His Asp Gly Ala Glu Gly Val Glu Gly His 1155 1160 1165 Leu Leu Thr Gly Thr Thr Pro Ser Val Met Ser Gly Arg Ile Ala Tyr 1170 1175 1180 Gln Leu Gly Leu Thr Gly Pro Ala Val Thr Val Asp Thr Ala Cys Ser 1185 1190 1195 1200 Ser Ser Leu Val Ala Leu His Leu Ala Val Arg Ser Leu Arg Gln Gly 1205 1210 1215 Glu Ser Ser Leu Ala Leu Ala Gly Gly Ala Thr Val Met Ser Thr Pro 1220 1225 1230 Gly Met Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Ala Asp Gly 1235 1240 1245 Arg Ser Lys Ala Phe Ser Asp Ser Ala Asp Gly Thr Ser Trp Ala Glu 1250 1255 1260 Gly Val Gly Leu Leu Val Val Glu Arg Leu Ser Asp Ala Glu Arg Asn 1265 1270 1275 1280 Gly His Pro Val Leu Ala Val Ile Arg Gly Ser Ala Val Asn Gln Asp 1285 1290 1295 Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln Arg 1300 1305 1310 Val Ile Arg Gln Ala Leu Ala Asp Ala Gly Leu Thr Pro Ala Asp Val 1315 1320 1325 Asp Ala Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro Ile 1330 1335 1340 Glu Ala Glu Ala Ile Leu Gly Thr Tyr Gly Arg Asp Arg Gly Glu Gly 1345 1350 1355 1360 Ala Pro Leu Gln Leu Gly Ser Leu Lys Ser Asn Ile Gly His Ala Gln 1365 1370 1375 Ala Ala Ala Gly Val Gly Gly Leu Ile Lys Met Val Leu Ala Met Arg 1380 1385 1390 His Gly Val Leu Pro Arg Thr Leu His Val Asp Arg Pro Thr Thr Arg 1395 1400 1405 Val Asp Trp Glu Ala Gly Gly Val Glu Leu Leu Thr Glu Glu Arg Glu 1410 1415 1420 Trp Pro Glu Thr Gly Arg Pro Arg Arg Ala Ala Ile Ser Ser Phe Gly 1425 1430 1435 1440 Ile Ser Gly Thr Asn Ala His Ile Val Val Glu Gln Ala Pro Glu Ala 1445 1450 1455 Gly Glu Ala Ala Val Thr Thr Thr Ala Pro Glu Ala Gly Glu Ala Gly 1460 1465 1470 Glu Ala Ala Asp Thr Thr Ala Thr Thr Thr Pro Ala Ala Val Gly Val 1475 1480 1485 Pro Glu Pro Val Arg Ala Pro Val Val Val Ser Ala Arg Asp Ala Ala 1490 1495 1500 Ala Leu Arg Ala Gln Ala Val Arg Leu Arg Thr Phe Leu Asp Gly Arg 1505 1510 1515 1520 Pro Asp Val Thr Val Ala Asp Leu Gly Arg Ser Leu Ala Ala Arg Thr 1525 1530 1535 Ala Phe Glu His Lys Ala Ala Leu Thr Thr Ala Thr Arg Asp Glu Leu 1540 1545 1550 Leu Ala Gly Leu Asp Ala Leu Gly Arg Gly Glu Gln Ala Thr Gly Leu 1555 1560 1565 Val Thr Gly Glu Pro Ala Arg Ala Gly Arg Thr Ala Phe Leu Phe Thr 1570 1575 1580 Gly Gln Gly Ala Gln Arg Val Ala Met Gly Glu Glu Leu Arg Ala Ala 1585 1590 1595 1600 His Pro Val Phe Ala Ala Ala Leu Asp Thr Val Tyr Ala Ala Leu Asp 1605 1610 1615 Arg His Leu Asp Arg Pro Leu Arg Glu Ile Val Ala Ala Gly Glu Glu 1620 1625 1630 Leu Asp Leu Thr Ala Tyr Thr Gln Pro Ala Leu Phe Ala Phe Glu Val 1635 1640 1645 Ala Leu Phe Arg Leu Leu Glu His His Gly Leu Val Pro Asp Leu Leu 1650 1655 1660 Thr Gly His Ser Val Gly Glu Ile Ala Ala Ala His Val Ala Gly Val 1665 1670 1675 1680 Leu Ser Leu Asp Asp Ala Ala Arg Leu Val Thr Ala Arg Gly Arg Leu 1685 1690 1695 Met Gln Ser Ala Arg Glu Gly Gly Ala Met Ile Ala Val Gln Ala Gly 1700 1705 1710 Glu Ala Glu Val Val Glu Ser Leu Lys Gly Tyr Glu Gly Arg Val Ala 1715 1720 1725 Val Ala Ala Val Asn Gly Pro Thr Ala Val Val Val Ser Gly Asp Ala 1730 1735 1740 Asp Ala Ala Glu Glu Ile Arg Ala Val Trp Ala Gly Arg Gly Arg Arg 1745 1750 1755 1760 Thr Arg Arg Leu Arg Val Ser His Ala Phe His Ser Pro His Met Asp 1765 1770 1775 Asp Val Leu Asp Glu Phe Leu Arg Val Ala Glu Gly Leu Thr Phe Glu 1780 1785 1790 Glu Pro Arg Ile Pro Val Val Ser Thr Val Thr Gly Ala Leu Val Thr 1795 1800 1805 Ser Gly Glu Leu Thr Ser Pro Ala Tyr Trp Val Asp Gln Ile Arg Arg 1810 1815 1820 Pro Val Arg Phe Leu Asp Ala Val Arg Thr Leu Ala Ala Gln Asp Ala 1825 1830 1835 1840 Thr Val Leu Val Glu Ile Gly Pro Asp Ala Val Leu Thr Ala Leu Ala 1845 1850 1855 Glu Glu Ala Leu Ala Pro Gly Thr Asp Ala Pro Asp Ala Arg Asp Val 1860 1865 1870 Thr Val Val Pro Leu Leu Arg Ala Gly Arg Pro Glu Pro Glu Thr Leu 1875 1880 1885 Ala Ala Gly Leu Ala Thr Ala His Val His Gly Ala Pro Leu Asp Arg 1890 1895 1900 Ala Ser Phe Phe Pro Asp Gly Arg Arg Thr Asp Leu Pro Thr Tyr Ala 1905 1910 1915 1920 Phe Arg Arg Glu His Tyr Trp Leu Thr Pro Glu Ala Arg Thr Asp Ala 1925 1930 1935 Arg Ala Leu Gly Phe Asp Pro Ala Arg His Pro Leu Leu Thr Thr Thr 1940 1945 1950 Val Glu Val Ala Gly Gly Asp Gly Val Leu Leu Thr Gly Arg Leu Ser 1955 1960 1965 Leu Thr Asp Gln Pro Trp Leu Ala Asp His Met Val Asn Gly Ala Val 1970 1975 1980 Leu Leu Pro Ala Thr Ala Phe Leu Glu Leu Ala Leu Ala Ala Gly Asp 1985 1990 1995 2000 His Val Gly Ala Val Arg Val Glu Glu Leu Thr Leu Glu Ala Pro Leu 2005 2010 2015 Val Leu Pro Glu Arg Gly Ala Val Arg Ile Gln Val Gly Val Ser Gly 2020 2025 2030 Asp Gly Glu Ser Pro Ala Gly Arg Thr Phe Gly Val Tyr Ser Thr Pro 2035 2040 2045 Asp Ser Gly Asp Thr Gly Asp Asp Ala Pro Arg Glu Trp Thr Arg His 2050 2055 2060 Val Ser Gly Val Leu Gly Glu Gly Asp Pro Ala Thr Glu Ser Asp His 2065 2070 2075 2080 Pro Gly Thr Asp Gly Asp Gly Ser Ala Ala Trp Pro Pro Ala Ala Ala 2085 2090 2095 Thr Ala Thr Pro Leu Asp Gly Val Tyr Asp Arg Leu Ala Glu Leu Gly 2100 2105 2110 Tyr Gly Tyr Gly Pro Ala Phe Gln Gly Leu Thr Gly Leu Trp Arg Asp 2115 2120 2125 Gly Ala Asp Thr Leu Ala Glu Ile Arg Leu Pro Ala Ala Gln His Glu 2130 2135 2140 Ser Ala Gly Leu Phe Gly Val His Pro Ala Leu Leu Asp Ala Ala Leu 2145 2150 2155 2160 His Pro Ile Val Leu Glu Gly Asn Ser Ala Ala Gly Ala Cys Asp Ala 2165 2170 2175 Asp Thr Asp Ala Thr Asp Arg Ile Arg Leu Pro Phe Ala Trp Ala Gly 2180 2185 2190 Val Thr Leu His Ala Glu Gly Ala Thr Ala Leu Arg Val Arg Ile Thr 2195 2200 2205 Pro Thr Gly Pro Asp Thr Val Thr Leu Arg Leu Thr Asp Thr Thr Gly 2210 2215 2220 Ala Pro Val Ala Thr Val Glu Ser Leu Thr Leu Arg Ala Val Ala Lys 2225 2230 2235 2240 Asp Arg Leu Gly Thr Thr Ala Gly Arg Val Asp Asp Ala Leu Phe Thr 2245 2250 2255 Val Val Trp Thr Glu Thr Gly Thr Pro Glu Pro Ala Gly Arg Gly Ala 2260 2265 2270 Val Glu Val Glu Glu Leu Val Asp Leu Ala Gly Leu Gly Asp Leu Val 2275 2280 2285 Glu Leu Gly Ala Ala Asp Val Val Leu Arg Ala Asp Arg Trp Thr Leu 2290 2295 2300 Asp Gly Asp Pro Ser Ala Ala Ala Arg Thr Ala Val Arg Arg Thr Leu 2305 2310 2315 2320 Ala Ile Val Gln Glu Phe Leu Ser Glu Pro Arg Phe Asp Gly Ser Arg 2325 2330 2335 Leu Val Cys Val Thr Arg Gly Ala Val Ala Ala Leu Pro Gly Glu Asp 2340 2345 2350 Val Thr Ser Leu Ala Thr Gly Pro Leu Trp Gly Leu Val Arg Ser Ala 2355 2360 2365 Gln Ser Glu Asn Pro Gly Arg Leu Phe Leu Leu Asp Leu Gly Glu Gly 2370 2375 2380 Glu Gly Glu Arg Asp Gly Ala Glu Glu Leu Ile Arg Ala Ala Thr Ala 2385 2390 2395 2400 Gly Asp Glu Pro Gln Leu Ala Ala Arg Asp Gly Arg Leu Leu Ala Pro 2405 2410 2415 Arg Leu Ala Arg Thr Ala Ala Leu Ser Ser Glu Asp Thr Ala Gly Gly 2420 2425 2430 Ala Asp Arg Phe Gly Pro Asp Gly Thr Val Leu Val Thr Gly Gly Thr 2435 2440 2445 Gly Gly Leu Gly Ala Leu Leu Ala Arg His Leu Val Glu Arg His Gly 2450 2455 2460 Val Arg Arg Leu Leu Leu Val Ser Arg Arg Gly Ala Asp Ala Pro Gly 2465 2470 2475 2480 Ala Ala Asp Leu Gly Glu Asp Leu Ala Gly Leu Gly Ala Glu Val Ala 2485 2490 2495 Phe Ala Ala Ala Asp Ala Ala Asp Arg Glu Ser Leu Ala Arg Ala Ile 2500 2505 2510 Ala Thr Val Pro Ala Glu His Pro Leu Thr Ala Val Val His Thr Ala 2515 2520 2525 Gly Val Val Asp Asp Ala Thr Val Glu Ala Leu Thr Pro Glu Arg Leu 2530 2535 2540 Asp Ala Val Leu Arg Pro Lys Val Asp Ala Ala Trp Asn Leu His Glu 2545 2550 2555 2560 Leu Thr Lys Asp Leu Arg Leu Asp Ala Phe Val Leu Phe Ser Ser Val 2565 2570 2575 Ser Gly Ile Val Gly Thr Ala Gly Gln Ala Asn Tyr Ala Ala Ala Asn 2580 2585 2590 Thr Gly Leu Asp Ala Leu Ala Ala His Arg Ala Ala Thr Gly Leu Ala 2595 2600 2605 Ala Thr Ser Leu Ala Trp Gly Leu Trp Asp Gly Thr His Gly Met Gly 2610 2615 2620 Gly Thr Leu Gly Ala Ala Asp Leu Ala Arg Trp Ser Arg Ala Gly Ile 2625 2630 2635 2640 Thr Pro Leu Thr Pro Leu Gln Gly Leu Ala Leu Phe Asp Ala Ala Val 2645 2650 2655 Ala Arg Asp Asp Ala Leu Leu Val Pro Ala Gly Leu Arg Pro Thr Ala 2660 2665 2670 His Arg Gly Thr Asp Gly Gln Pro Pro Ala Leu Trp Arg Gly Leu Val 2675 2680 2685 Arg Ala Arg Pro Arg Arg Ala Ala Arg Thr Ala Ala Glu Ala Ala Asp 2690 2695 2700 Thr Thr Gly Gly Trp Leu Ser Gly Leu Ala Ala Gln Ser Pro Glu Glu 2705 2710 2715 2720 Arg Arg Ser Thr Ala Val Thr Leu Val Thr Gly Val Val Ala Asp Val 2725 2730 2735 Leu Gly His Ala Asp Ser Ala Ala Val Gly Ala Glu Arg Ser Phe Lys 2740 2745 2750 Asp Leu Gly Phe Asp Ser Leu Ala Gly Val Glu Leu Arg Asn Arg Leu 2755 2760 2765 Asn Ala Ala Thr Gly Leu Arg Leu Pro Ala Thr Thr Val Phe Asp His 2770 2775 2780 Pro Ser Pro Ala Ala Leu Ala Ser His Leu Leu Ala Gln Val Pro Gly 2785 2790 2795 2800 Leu Lys Glu Gly Thr Ala Ala Thr Ala Thr Val Val Ala Glu Arg Gly 2805 2810 2815 Ala Ser Phe Gly Asp Arg Ala Thr Asp Asp Asp Pro Ile Ala Ile Val 2820 2825 2830 Gly Met Ala Cys Arg Tyr Pro Gly Gly Val Ser Ser Pro Glu Asp Leu 2835 2840 2845 Trp Arg Leu Val Ala Glu Gly Thr Asp Ala Ile Ser Glu Phe Pro Val 2850 2855 2860 Asn Arg Gly Trp Asp Leu Glu Ser Leu Tyr Asp Pro Asp Pro Glu Ser 2865 2870 2875 2880 Lys Gly Thr Thr Tyr Cys Arg Glu Gly Gly Phe Leu Glu Gly Ala Gly 2885 2890 2895 Asp Phe Asp Ala Ala Phe Phe Gly Ile Ser Pro Arg Glu Ala Leu Val 2900 2905 2910 Met Asp Pro Gln Gln Arg Leu Leu Leu Glu Val Ser Trp Glu Ala Leu 2915 2920 2925 Glu Arg Ala Gly Ile Asp Pro Ser Ser Leu Arg Gly Ser Arg Gly Gly 2930 2935 2940 Val Tyr Val Gly Ala Ala His Gly Ser Tyr Ala Ser Asp Pro Arg Leu 2945 2950 2955 2960 Val Pro Glu Gly Ser Glu Gly Tyr Leu Leu Thr Gly Ser Ala Asp Ala 2965 2970 2975 Val Met Ser Gly Arg Ile Ser Tyr Ala Leu Gly Leu Glu Gly Pro Ser 2980 2985 2990 Met Thr Val Glu Thr Ala Cys Ser Ser Ser Leu Val Ala Leu His Leu 2995 3000 3005 Ala Val Arg Ala Leu Arg His Gly Glu Cys Gly Leu Ala Leu Ala Gly 3010 3015 3020 Gly Val Ala Val Met Ala Asp Pro Ala Ala Phe Val Glu Phe Ser Arg 3025 3030 3035 3040 Gln Lys Gly Leu Ala Ala Asp Gly Arg Cys Lys Ala Phe Ser Ala Ala 3045 3050 3055 Ala Asp Gly Thr Gly Trp Ala Glu Gly Val Gly Val Leu Val Leu Glu 3060 3065 3070 Arg Leu Ser Asp Ala Arg Arg Ala Gly His Thr Val Leu Gly Leu Val 3075 3080 3085 Thr Gly Thr Ala Val Asn Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala 3090 3095 3100 Pro Asn Gly Pro Ala Gln Gln Arg Val Ile Ala Glu Ala Leu Ala Asp 3105 3110 3115 3120 Ala Gly Leu Ser Pro Glu Asp Val Asp Ala Val Glu Ala His Gly Thr 3125 3130 3135 Gly Thr Arg Leu Gly Asp Pro Ile Glu Ala Gly Ala Leu Leu Ala Ala 3140 3145 3150 Ser Gly Arg Asn Arg Ser Gly Asp His Pro Leu Trp Leu Gly Ser Leu 3155 3160 3165 Lys Ser Asn Ile Gly His Ala Gln Ala Ala Ala Gly Val Gly Gly Val 3170 3175 3180 Ile Lys Met Leu Gln Ala Leu Arg His Gly Leu Leu Pro Arg Thr Leu 3185 3190 3195 3200 His Ala Asp Glu Pro Thr Pro His Ala Asp Trp Ser Ser Gly Arg Val 3205 3210 3215 Arg Leu Leu Thr Ser Glu Val Pro Trp Gln Arg Thr Gly Arg Pro Arg 3220 3225 3230 Arg Thr Gly Val Ser Ala Phe Gly Val Gly Gly Thr Asn Ala His Val 3235 3240 3245 Val Leu Glu Glu Ala Pro Ala Pro Pro Ala Pro Glu Pro Ala Gly Glu 3250 3255 3260 Ala Pro Gly Gly Ser Arg Ala Ala Glu Gly Ala Glu Gly Pro Leu Ala 3265 3270 3275 3280 Trp Val Val Ser Gly Arg Asp Glu Pro Ala Leu Arg Ser Gln Ala Arg 3285 3290 3295 Arg Leu Arg Asp His Leu Ser Arg Thr Pro Gly Ala Arg Pro Arg Asp 3300 3305 3310 Ile Ala Phe Ser Leu Ala Ala Thr Arg Ala Ala Phe Asp His Arg Ala 3315 3320 3325 Val Leu Ile Gly Ser Asp Gly Ala Glu Leu Ala Ala Ala Leu Asp Ala 3330 3335 3340 Leu Ala Glu Gly Arg Asp Gly Pro Ala Val Val Arg Gly Val Arg Asp 3345 3350 3355 3360 Arg Asp Gly Arg Met Ala Phe Leu Phe Thr Gly Gln Gly Ser Gln Arg 3365 3370 3375 Ala Gly Met Ala His Asp Leu His Ala Ala His Thr Phe Phe Ala Ser 3380 3385 3390 Ala Leu Asp Glu Val Thr Asp Arg Leu Asp Pro Leu Leu Gly Arg Pro 3395 3400 3405 Leu Gly Ala Leu Leu Asp Ala Arg Pro Gly Ser Pro Glu Ala Ala Leu 3410 3415 3420 Leu Asp Arg Thr Glu Tyr Thr Gln Pro Ala Leu Phe Ala Val Glu Val 3425 3430 3435 3440 Ala Leu His Arg Leu Leu Glu His Trp Gly Met Arg Pro Asp Leu Leu 3445 3450 3455 Leu Gly His Ser Val Gly Glu Leu Ala Ala Ala His Val Ala Gly Val 3460 3465 3470 Leu Asp Leu Asp Asp Ala Cys Ala Leu Val Ala Ala Arg Gly Arg Leu 3475 3480 3485 Met Gln Arg Leu Pro Pro Gly Gly Ala Met Val Ser Val Arg Ala Gly 3490 3495 3500 Glu Asp Glu Val Arg Ala Leu Leu Ala Gly Arg Glu Asp Ala Val Cys 3505 3510 3515 3520 Val Ala Ala Val Asn Gly Pro Arg Ser Val Val Ile Ser Gly Ala Glu 3525 3530 3535 Glu Ala Val Ala Glu Ala Ala Ala Gln Leu Ala Gly Arg Gly Arg Arg 3540 3545 3550 Thr Arg Arg Leu Arg Val Ala His Ala Phe His Ser Pro Leu Met Asp 3555 3560 3565 Gly Met Leu Ala Gly Phe Arg Glu Val Ala Ala Gly Leu Arg Tyr Arg 3570 3575 3580 Glu Pro Glu Leu Thr Val Val Ser Thr Val Thr Gly Arg Pro Ala Arg 3585 3590 3595 3600 Pro Gly Glu Leu Thr Gly Pro Asp Tyr Trp Val Ala Gln Val Arg Glu 3605 3610 3615 Pro Val Arg Phe Ala Asp Ala Val Arg Thr Ala His Arg Leu Gly Ala 3620 3625 3630 Arg Thr Phe Leu Glu Thr Gly Pro Asp Gly Val Leu Cys Gly Met Ala 3635 3640 3645 Glu Glu Cys Leu Glu Asp Asp Thr Val Ala Leu Leu Pro Ala Ile His 3650 3655 3660 Lys Pro Gly Thr Ala Pro His Gly Pro Ala Ala Pro Gly Ala Leu Arg 3665 3670 3675 3680 Ala Ala Ala Ala Ala Tyr Gly Arg Gly Ala Arg Val Asp Trp Ala Gly 3685 3690 3695 Met His Ala Asp Gly Pro Glu Gly Pro Ala Arg Arg Val Glu Leu Pro 3700 3705 3710 Val His Ala Phe Arg His Arg Arg Tyr Trp Leu Ala Pro Gly Arg Ala 3715 3720 3725 Ala Asp Thr Asp Asp Trp Met Tyr Arg Ile Gly Trp Asp Arg Leu Pro 3730 3735 3740 Ala Val Thr Gly Gly Ala Arg Thr Ala Gly Arg Trp Leu Val Ile His 3745 3750 3755 3760 Pro Asp Ser Pro Arg Cys Arg Glu Leu Ser Gly His Ala Glu Arg Ala 3765 3770 3775 Leu Arg Ala Ala Gly Ala Ser Pro Val Pro Leu Pro Val Asp Ala Pro 3780 3785 3790 Ala Ala Asp Arg Ala Ser Phe Ala Ala Leu Leu Arg Ser Ala Thr Gly 3795 3800 3805 Pro Asp Thr Arg Gly Asp Thr Ala Ala Pro Val Ala Gly Val Leu Ser 3810 3815 3820 Leu Leu Ser Glu Glu Asp Arg Pro His Arg Gln His Ala Pro Val Pro 3825 3830 3835 3840 Ala Gly Val Leu Ala Thr Leu Ser Leu Met Gln Ala Met Glu Glu Glu 3845 3850 3855 Ala Val Glu Ala Arg Val Trp Cys Val Ser Arg Ala Ala Val Ala Ala 3860 3865 3870 Ala Asp Arg Glu Arg Pro Val Gly Ala Gly Ala Ala Leu Trp Gly Leu 3875 3880 3885 Gly Arg Val Ala Ala Leu Glu Arg Pro Thr Arg Trp Gly Gly Leu Val 3890 3895 3900 Asp Leu Pro Ala Ser Pro Gly Ala Ala His Trp Ala Ala Ala Val Glu 3905 3910 3915 3920 Arg Leu Ala Gly Pro Glu Asp Gln Ile Ala Val Arg Ala Ser Gly Ser 3925 3930 3935 Trp Gly Arg Arg Leu Thr Arg Leu Pro Arg Asp Gly Gly Gly Arg Thr 3940 3945 3950 Ala Ala Pro Ala Tyr Arg Pro Arg Gly Thr Val Leu Val Thr Gly Gly 3955 3960 3965 Thr Gly Ala Leu Gly Gly His Leu Ala Arg Trp Leu Ala Ala Ala Gly 3970 3975 3980 Ala Glu His Leu Ala Leu Thr Ser Arg Arg Gly Pro Asp Ala Pro Gly 3985 3990 3995 4000 Ala Ala Gly Leu Glu Ala Glu Leu Leu Leu Leu Gly Ala Lys Val Thr 4005 4010 4015 Phe Ala Ala Cys Asp Thr Ala Asp Arg Asp Gly Leu Ala Arg Val Leu 4020 4025 4030 Arg Ala Ile Pro Glu Asp Thr Pro Leu Thr Ala Val Phe His Ala Ala 4035 4040 4045 Gly Val Pro Gln Val Thr Pro Leu Ser Arg Thr Ser Pro Glu His Phe 4050 4055 4060 Ala Asp Val Tyr Ala Gly Lys Ala Ala Gly Ala Ala His Leu Asp Glu 4065 4070 4075 4080 Leu Thr Arg Glu Leu Gly Ala Gly Leu Asp Ala Phe Val Leu Tyr Ser 4085 4090 4095 Ser Gly Ala Gly Val Trp Gly Ser Ala Gly Gln Gly Ala Tyr Ala Ala 4100 4105 4110 Ala Asn Ala Ala Leu Asp Ala Leu Ala Arg Arg Arg Ala Ala Asp Gly 4115 4120 4125 Leu Pro Ala Thr Ser Ile Ala Trp Gly Val Trp Gly Gly Gly Gly Met 4130 4135 4140 Gly Ala Asp Glu Ala Gly Ala Glu Tyr Leu Gly Arg Arg Gly Met Arg 4145 4150 4155 4160 Pro Met Ala Pro Val Ser Ala Leu Arg Ala Met Ala Thr Ala Ile Ala 4165 4170 4175 Ser Gly Glu Pro Cys Pro Thr Val Thr His Thr Asp Trp Glu Arg Phe 4180 4185 4190 Gly Glu Gly Phe Thr Ala Phe Arg Pro Ser Pro Leu Ile Ala Gly Leu 4195 4200 4205 Gly Thr Pro Gly Gly Gly Arg Ala Ala Glu Thr Pro Glu Glu Gly Asn 4210 4215 4220 Ala Thr Ala Ala Ala Asp Leu Thr Ala Leu Pro Pro Ala Glu Leu Arg 4225 4230 4235 4240 Thr Ala Leu Arg Glu Leu Val Arg Ala Arg Thr Ala Ala Ala Leu Gly 4245 4250 4255 Leu Asp Asp Pro Ala Glu Val Ala Glu Gly Glu Arg Phe Pro Ala Met 4260 4265 4270 Gly Phe Asp Ser Leu Ala Thr Val Arg Leu Arg Arg Gly Leu Ala Ser 4275 4280 4285 Ala Thr Gly Leu Asp Leu Pro Pro Asp Leu Leu Phe Asp Arg Asp Thr 4290 4295 4300 Pro Ala Ala Leu Ala Ala His Leu Ala Glu Leu Leu Ala Thr Ala Arg 4305 4310 4315 4320 Asp His Gly Pro Gly Gly Pro Gly Thr Gly Ala Ala Pro Ala Asp Ala 4325 4330 4335 Gly Ser Gly Leu Pro Ala Leu Tyr Arg Glu Ala Val Arg Thr Gly Arg 4340 4345 4350 Ala Ala Glu Met Ala Glu Leu Leu Ala Ala Ala Ser Arg Phe Arg Pro 4355 4360 4365 Ala Phe Gly Thr Ala Asp Arg Gln Pro Val Ala Leu Val Pro Leu Ala 4370 4375 4380 Asp Gly Ala Glu Asp Thr Gly Leu Pro Leu Leu Val Gly Cys Ala Gly 4385 4390 4395 4400 Thr Ala Val Ala Ser Gly Pro Val Glu Phe Thr Ala Phe Ala Gly Ala 4405 4410 4415 Leu Ala Asp Leu Pro Ala Ala Ala Pro Met Ala Ala Leu Pro Gln Pro 4420 4425 4430 Gly Phe Leu Pro Gly Glu Arg Val Pro Ala Thr Pro Glu Ala Leu Phe 4435 4440 4445 Glu Ala Gln Ala Glu Ala Leu Leu Arg Tyr Ala Ala Gly Arg Pro Phe 4450 4455 4460 Val Leu Leu Gly His Ser Ala Gly Ala Asn Met Ala His Ala Leu Thr 4465 4470 4475 4480 Arg His Leu Glu Ala Asn Gly Gly Gly Pro Ala Gly Leu Val Leu Met 4485 4490 4495 Asp Ile Tyr Thr Pro Ala Asp Pro Gly Ala Met Gly Val Trp Arg Asn 4500 4505 4510 Asp Met Phe Gln Trp Val Trp Arg Arg Ser Asp Ile Pro Pro Asp Asp 4515 4520 4525 His Arg Leu Thr Ala Met Gly Ala Tyr His Arg Leu Leu Leu Asp Trp 4530 4535 4540 Ser Pro Thr Pro Val Arg Ala Pro Val Leu His Leu Arg Ala Ala Glu 4545 4550 4555 4560 Pro Met Gly Asp Trp Pro Pro Gly Asp Thr Gly Trp Gln Ser His Trp 4565 4570 4575 Asp Gly Ala His Thr Thr Ala Gly Ile Pro Gly Asn His Phe Thr Met 4580 4585 4590 Met Thr Glu His Ala Ser Ala Ala Ala Arg Leu Val His Gly Trp Leu 4595 4600 4605 Ala Glu Arg Thr Pro Ser Gly Gln Gly Gly Ser Pro Ser Arg Ala Ala 4610 4615 4620 Gly Arg Glu Glu Arg Pro Met Ile Leu Arg Ala Gly Thr Ala Asp Pro 4625 4630 4635 4640 Ala Pro Tyr Glu Glu Glu Ile Pro Gly Tyr Arg Ala Arg Ile Leu Asn 4645 4650 4655 Met Ser Asn Lys Asn Asn Asp Glu Leu Gln Arg Gln Ala Ser Glu Asn 4660 4665 4670 Thr Leu Gly Leu Asn Pro Val Ile Gly Ile Arg Arg Lys Asp Leu Leu 4675 4680 4685 Ser Ser Ala Arg Thr Val Leu Arg Gln Ala Val Arg Gln Pro Leu His 4690 4695 4700 Ser Ala Lys His Val Ala His Phe Gly Leu Glu Leu Lys Asn Val Leu 4705 4710 4715 4720 Leu Gly Lys Ser Ser Leu Ala Pro Glu Ser Asp Asp Arg Arg Phe Asn 4725 4730 4735 Asp Pro Ala Trp Ser Asn Asn Pro Leu Tyr Arg Arg Tyr Leu Gln Thr 4740 4745 4750 Tyr Leu Ala Trp Arg Lys Glu Leu Gln Asp Trp Ile Gly Asn Ser Asp 4755 4760 4765 Leu Ser Pro Gln Asp Ile Ser Arg Gly Gln Phe Val Ile Asn Leu Met 4770 4775 4780 Thr Glu Ala Met Ala Pro Thr Asn Thr Leu Ser Asn Pro Ala Ala Val 4785 4790 4795 4800 Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser 4805 4810 4815 Asn Leu Ala Lys Asp Leu Val Asn Asn Gly Gly Met Pro Ser Gln Val 4820 4825 4830 Asn Met Asp Ala Phe Glu Val Gly Lys Asn Leu Gly Thr Ser Glu Gly 4835 4840 4845 Ala Val Val Tyr Arg Asn Asp Val Leu Glu Leu Ile Gln Tyr Lys Pro 4850 4855 4860 Ile Thr Glu Gln Val His Ala Arg Pro Leu Leu Val Val Pro Pro Gln 4865 4870 4875 4880 Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Glu Lys Ser Leu Ala 4885 4890 4895 Arg Tyr Cys Leu Arg Ser Gln Gln Gln Thr Phe Ile Ile Ser Trp Arg 4900 4905 4910 Asn Pro Thr Lys Ala Gln Arg Glu Trp Gly Leu Ser Thr Tyr Ile Asp 4915 4920 4925 Ala Leu Lys Glu Ala Val Asp Ala Val Leu Ala Ile Thr Gly Ser Lys 4930 4935 4940 Asp Leu Asn Met Leu Gly Ala Cys Ser Gly Gly Ile Thr Cys Thr Ala 4945 4950 4955 4960 Leu Val Gly His Tyr Ala Ala Leu Gly Glu Asn Lys Val Asn Ala Leu 4965 4970 4975 Thr Leu Leu Val Ser Val Leu Asp Thr Thr Met Asp Asn Gln Val Ala 4980 4985 4990 Leu Phe Val Asp Glu Gln Thr Leu Glu Ala Ala Lys Arg His Ser Tyr 4995 5000 5005 Gln Ala Gly Val Leu Glu Gly Ser Glu Met Ala Lys Val Phe Ala Trp 5010 5015 5020 Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Trp Val Asn Asn Tyr Leu 5025 5030 5035 5040 Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Leu Phe Trp Asn Asn Asp 5045 5050 5055 Thr Thr Arg Leu Pro Ala Ala Phe His Gly Asp Leu Ile Glu Met Phe 5060 5065 5070 Lys Ser Asn Pro Leu Thr Arg Pro Asp Ala Leu Glu Val Cys Gly Thr 5075 5080 5085 Pro Ile Asp Leu Lys Gln Val Lys Cys Asp Ile Tyr Ser Leu Ala Gly 5090 5095 5100 Thr Asn Asp His Ile Thr Pro Trp Gln Ser Cys Tyr Arg Ser Ala His 5105 5110 5115 5120 Leu Phe Gly Gly Lys Ile Glu Phe Val Leu Ser Asn Ser Gly His Ile 5125 5130 5135 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Lys Ala Arg Phe Met Thr 5140 5145 5150 Gly Ala Asp Arg Pro Gly Asp Pro Val Ala Trp Gln Glu Asn Ala Thr 5155 5160 5165 Lys His Ala Asp Ser Trp Trp Leu His Trp Gln Ser Trp Leu Gly Glu 5170 5175 5180 Arg Ala Gly Glu Leu Glu Lys Ala Pro Thr Arg Leu Gly Asn Arg Ala 5185 5190 5195 5200 Tyr Ala Ala Gly Glu Ala Ser Pro Gly Thr Tyr Val His Glu Arg 5205 5210 5215 3 13613 DNA Streptomyces venezuelae 3 ggatccggcg cttccacccc gcgccgaaca gcgcggtgcg gctggtctgc ctgccgcacg 60 ccggcggctc cgccagctac ttcttccgct tctcggagga gctgcacccc tccgtcgagg 120 ccctgtcggt gcagtatccg ggccgccagg accggcgtgc cgagccgtgt ctggagagcg 180 tcgaggagct cgccgagcat gtggtcgcgg ccaccgaacc ctggtggcag gagggccggc 240 tggccttctt cgggcacagc ctcggcgcct ccgtcgcctt cgagacggcc cgcatcctgg 300 aacagcggca cggggtacgg cccgagggcc tgtacgtctc cggtcggcgc gccccgtcgc 360 tggcgccgga ccggctcgtc caccagctgg acgaccgggc gttcctggcc gagatccggc 420 ggctcagcgg caccgacgag cggttcctcc aggacgacga gctgctgcgg ctggtgctgc 480 ccgcgctgcg cagcgactac aaggcggcgg agacgtacct gcaccggccg tccgccaagc 540 tcacctgccc ggtgatggcc ctggccggcg accgtgaccc gaaggcgccg ctgaacgagg 600 tggccgagtg gcgtcggcac accagcgggc cgttctgcct ccgggcgtac tccggcggcc 660 acttctacct caacgaccag tggcacgaga tctgcaacga catctccgac cacctgctcg 720 tcacccgcgg cgcgcccgat gcccgcgtcg tgcagccccc gaccagcctt atcgaaggag 780 cggcgaagag atggcagaac ccacggtgac cgacgacctg acgggggccc tcacgcagcc 840 cccgctgggc cgcaccgtcc gcgcggtggc cgaccgtgaa ctcggcaccc acctcctgga 900 gacccgcggc atccactgga tccacgccgc gaacggcgac ccgtacgcca ccgtgctgcg 960 cggccaggcg gacgacccgt atcccgcgta cgagcgggtg cgtgcccgcg gcgcgctctc 1020 cttcagcccg acgggcagct gggtcaccgc cgatcacgcc ctggcggcga gcatcctctg 1080 ctcgacggac ttcggggtct ccggcgccga cggcgtcccg gtgccgcagc aggtcctctc 1140 gtacggggag ggctgtccgc tggagcgcga gcaggtgctg ccggcggccg gtgacgtgcc 1200 ggagggcggg cagcgtgccg tggtcgaggg gatccaccgg gagacgctgg agggtctcgc 1260 gccggacccg tcggcgtcgt acgccttcga gctgctgggc ggtttcgtcc gcccggcggt 1320 gacggccgct gccgccgccg tgctgggtgt tcccgcggac cggcgcgcgg acttcgcgga 1380 tctgctggag cggctccggc cgctgtccga cagcctgctg gccccgcagt ccctgcggac 1440 ggtacgggcg gcggacggcg cgctggccga gctcacggcg ctgctcgccg attcggacga 1500 ctcccccggg gccctgctgt cggcgctcgg ggtcaccgca gccgtccagc tcaccgggaa 1560 cgcggtgctc gcgctcctcg cgcatcccga gcagtggcgg gagctgtgcg accggcccgg 1620 gctcgcggcg gccgcggtgg aggagaccct ccgctacgac ccgccggtgc agctcgacgc 1680 ccgggtggtc cgcggggaga cggagctggc gggccggcgg ctgccggccg gggcgcatgt 1740 cgtcgtcctg accgccgcga ccggccggga cccggaggtc ttcacggacc cggagcgctt 1800 cgacctcgcg cgccccgacg ccgccgcgca cctcgcgctg caccccgccg gtccgtacgg 1860 cccggtggcg tccctggtcc ggcttcaggc ggaggtcgcg ctgcggaccc tggccgggcg 1920 tttccccggg ctgcggcagg cgggggacgt gctccgcccc cgccgcgcgc ctgtcggccg 1980 cgggccgctg agcgtcccgg tcagcagctc ctgagacacc ggggccccgg tccgcccggc 2040 cccccttcgg acggaccgga cggctcggac cacggggacg gctcagaccg tcccgtgtgt 2100 ccccgtccgg ctcccgtccg ccccatcccg cccctccacc ggcaaggaag gacacgacgc 2160 catgcgcgtc ctgctgacct cgttcgcaca tcacacgcac tactacggcc tggtgcccct 2220 ggcctgggcg ctgctcgccg ccgggcacga ggtgcgggtc gccagccagc ccgcgctcac 2280 ggacaccatc accgggtccg ggctcgccgc ggtgccggtc ggcaccgacc acctcatcca 2340 cgagtaccgg gtgcggatgg cgggcgagcc gcgcccgaac catccggcga tcgccttcga 2400 cgaggcccgt cccgagccgc tggactggga ccacgccctc ggcatcgagg cgatcctcgc 2460 cccgtacttc catctgctcg ccaacaacga ctcgatggtc gacgacctcg tcgacttcgc 2520 ccggtcctgg cagccggacc tggtgctgtg ggagccgacg acctacgcgg gcgccgtcgc 2580 cgcccaggtc accggtgccg cgcacgcccg ggtcctgtgg gggcccgacg tgatgggcag 2640 cgcccgccgc aagttcgtcg cgctgcggga ccggcagccg cccgagcacc gcgaggaccc 2700 caccgcggag tggctgacgt ggacgctcga ccggtacggc gcctccttcg aagaggagct 2760 gctcaccggc cagttcacga tcgacccgac cccgccgagc ctgcgcctcg acacgggcct 2820 gccgaccgtc gggatgcgtt atgttccgta caacggcacg tcggtcgtgc cggactggct 2880 gagtgagccg cccgcgcggc cccgggtctg cctgaccctc ggcgtctccg cgcgtgaggt 2940 cctcggcggc gacggcgtct cgcagggcga catcctggag gcgctcgccg acctcgacat 3000 cgagctcgtc gccacgctcg acgcgagtca gcgcgccgag atccgcaact acccgaagca 3060 cacccggttc acggacttcg tgccgatgca cgcgctcctg ccgagctgct cggcgatcat 3120 ccaccacggc ggggcgggca cctacgcgac cgccgtgatc aacgcggtgc cgcaggtcat 3180 gctcgccgag ctgtgggacg cgccggtcaa ggcgcgggcc gtcgccgagc agggggcggg 3240 gttcttcctg ccgccggccg agctcacgcc gcaggccgtg cgggacgccg tcgtccgcat 3300 cctcgacgac ccctcggtcg ccaccgccgc gcaccggctg cgcgaggaga ccttcggcga 3360 ccccaccccg gccgggatcg tccccgagct ggagcggctc gccgcgcagc accgccgccc 3420 gccggccgac gcccggcact gagccgcacc cctcgcccca ggcctcaccc ctgtatctgc 3480 gccgggggac gcccccggcc caccctccga aagaccgaaa gcaggagcac cgtgtacgaa 3540 gtcgaccacg ccgacgtcta cgacctcttc tacctgggtc gcggcaagga ctacgccgcc 3600 gaggcctccg acatcgccga cctggtgcgc tcccgtaccc ccgaggcctc ctcgctcctg 3660 gacgtggcct gcggtacggg cacgcatctg gagcacttca ccaaggagtt cggcgacacc 3720 gccggcctgg agctgtccga ggacatgctc acccacgccc gcaagcggct gcccgacgcc 3780 acgctccacc agggcgacat gcgggacttc cggctcggcc ggaagttctc cgccgtggtc 3840 agcatgttca gctccgtcgg ctacctgaag acgaccgagg aactcggcgc ggccgtcgcc 3900 tcgttcgcgg agcacctgga gcccggtggc gtcgtcgtcg tcgagccgtg gtggttcccg 3960 gagaccttcg ccgacggctg ggtcagcgcc gacgtcgtcc gccgtgacgg gcgcaccgtg 4020 gcccgtgtct cgcactcggt gcgggagggg aacgcgacgc gcatggaggt ccacttcacc 4080 gtggccgacc cgggcaaggg cgtgcggcac ttctccgacg tccatctcat caccctgttc 4140 caccaggccg agtacgaggc cgcgttcacg gccgccgggc tgcgcgtcga gtacctggag 4200 ggcggcccgt cgggccgtgg cctcttcgtc ggcgtccccg cctgagcacc gcccaagacc 4260 ccccggggcg ggacgtcccg ggtgcaccaa gcaaagagag agaaacgaac cgtgacaggt 4320 aagacccgaa taccgcgtgt ccgccgcggc cgcaccacgc ccagggcctt caccctggcc 4380 gtcgtcggca ccctgctggc gggcaccacc gtggcggccg ccgctcccgg cgccgccgac 4440 acggccaatg ttcagtacac gagccgggcg gcggagctcg tcgcccagat gacgctcgac 4500 gagaagatca gcttcgtcca ctgggcgctg gaccccgacc ggcagaacgt cggctacctt 4560 cccggcgtgc cgcgtctggg catcccggag ctgcgtgccg ccgacggccc gaacggcatc 4620 cgcctggtgg ggcagaccgc caccgcgctg cccgcgccgg tcgccctggc cagcaccttc 4680 gacgacacca tggccgacag ctacggcaag gtcatgggcc gcgacggtcg cgcgctcaac 4740 caggacatgg tcctgggccc gatgatgaac aacatccggg tgccgcacgg cggccggaac 4800 tacgagacct tcagcgagga ccccctggtc tcctcgcgca ccgcggtcgc ccagatcaag 4860 ggcatccagg gtgcgggtct gatgaccacg gccaagcact tcgcggccaa caaccaggag 4920 aacaaccgct tctccgtgaa cgccaatgtc gacgagcaga cgctccgcga gatcgagttc 4980 ccggcgttcg aggcgtcctc caaggccggc gcggcctcct tcatgtgtgc ctacaacggc 5040 ctcaacggga agccgtcctg cggcaacgac gagctcctca acaacgtgct gcgcacgcag 5100 tggggcttcc agggctgggt gatgtccgac tggctcgcca ccccgggcac cgacgccatc 5160 accaagggcc tcgaccagga gatgggcgtc gagctccccg gcgacgtccc gaagggcgag 5220 ccctcgccgc cggccaagtt cttcggcgag gcgctgaaga cggccgtcct gaacggcacg 5280 gtccccgagg cggccgtgac gcggtcggcg gagcggatcg tcggccagat ggagaagttc 5340 ggtctgctcc tcgccactcc ggcgccgcgg cccgagcgcg acaaggcggg tgcccaggcg 5400 gtgtcccgca aggtcgccga gaacggcgcg gtgctcctgc gcaacgaggg ccaggccctg 5460 ccgctcgccg gtgacgccgg caagagcatc gcggtcatcg gcccgacggc cgtcgacccc 5520 aaggtcaccg gcctgggcag cgcccacgtc gtcccggact cggcggcggc gccactcgac 5580 accatcaagg cccgcgcggg tgcgggtgcg acggtgacgt acgagacggg tgaggagacc 5640 ttcgggacgc agatcccggc ggggaacctc agcccggcgt tcaaccaggg ccaccagctc 5700 gagccgggca aggcgggggc gctgtacgac ggcacgctga ccgtgcccgc cgacggcgag 5760 taccgcatcg cggtccgtgc caccggtggt tacgccacgg tgcagctcgg cagccacacc 5820 atcgaggccg gtcaggtcta cggcaaggtg agcagcccgc tcctcaagct gaccaagggc 5880 acgcacaagc tcacgatctc gggcttcgcg atgagtgcca ccccgctctc cctggagctg 5940 ggctgggtga cgccggcggc ggccgacgcg acgatcgcga aggccgtgga gtcggcgcgg 6000 aaggcccgta cggcggtcgt cttcgcctac gacgacggca ccgagggcgt cgaccgtccg 6060 aacctgtcgc tgccgggtac gcaggacaag ctgatctcgg ctgtcgcgga cgccaacccg 6120 aacacgatcg tggtcctcaa caccggttcg tcggtgctga tgccgtggct gtccaagacc 6180 cgcgcggtcc tggacatgtg gtacccgggc caggcgggcg ccgaggccac cgccgcgctg 6240 ctctacggtg acgtcaaccc gagcggcaag ctcacgcaga gcttcccggc cgccgagaac 6300 cagcacgcgg tcgccggcga cccgacaagc tacccgggcg tcgacaacca gcagacgtac 6360 cgcgagggca tccacgtcgg gtaccgctgg ttcgacaagg agaacgtcaa gccgctgttc 6420 ccgttcgggc acggcctgtc gtacacctcg ttcacgcaga gcgccccgac cgtcgtgcgt 6480 acgtccacgg gtggtctgaa ggtcacggtc acggtccgca acagcgggaa gcgcgccggc 6540 caggaggtcg tccaggcgta cctcggtgcc agcccgaacg tgacggctcc gcaggcgaag 6600 aagaagctcg tgggctacac gaaggtctcg ctcgccgcgg gcgaggcgaa gacggtgacg 6660 gtgaacgtcg accgccgtca gctgcagacc ggttcgtcct ccgccgacct gcggggcagc 6720 gccacggtca acgtctggtg acgtgacgcc gtgaaagcgg cggtgcccgc cacccgggag 6780 ggtggcgggc accgcttttt cggcctgctg ggtctaccgg accacctgac taggcctggt 6840 cgacccgctc ggcccattcg cgcacggcgt cgatcacccg cagcgcctgc gggcgctcca 6900 ggtgcgggcc gatcggcagg ctgaggacct gccgcgcgaa gctctcggcc cgcgggagcg 6960 agccttccgg cggtgcctcg cccgcgtagg cgggcgagag gtgcacgggt accgggtagt 7020 gcgtgagggt gtcgatgccg cgggcgtcga ggtggctgcg cagctcgtcg cggcgctcgg 7080 tgcgcacggt gaagaggtgc cagaccgggt cggtgtcggg cgcggtcacc ggcaggccga 7140 tgccgggcag tccggcgagc ccggagaggt actccgcggc cagcgccgac ctgcggccgt 7200 tccagctgtc caggtgggcg agccggatcc gcagcacggc ggcctgcatc tcgtccaggc 7260 gggagttggt gcccttcgtc tcgtggctgt acttctgccg cgagccgtag ttgcggagca 7320 tccggagccg ttcggcgagc tcggggtcgc cggtgacgac ggcgccgccg tcgccgaagc 7380 agccgaggtt cttgcccggg tagaagctga acgcggccac cgacgacccg gcgccgatcc 7440 gccggccccg gtagcgggcg ccgtgggcct gcgcggcgtc ctcgacgatg tgcaggccgt 7500 gccggtccgc gagctcgcgg agggcgtcca tgtcggcggg gtgcccgtag aggtggacgg 7560 ggaggagcgc ccgggtgcgg ggggtgatcg ccttctcgac gagcagcggg tccagggtgg 7620 ggtggtcctc gtgcggctcg acgggcacgg gggtcgcgcc ggtggcggac accgcgagcc 7680 agctggcgat gtacgtgtgc gaggggacga tcacctcgtc cccgggtccg atgccgaggc 7740 cgcggagggc gagctggagg gcgtccatcc cgctgttcac gccgacggcg tggtccgtct 7800 cgcagtacgc ggcgaactcc gcctcgaatc cttcgagttc gggtccgagg aggtagcgcc 7860 ccgagtcgag gacgcgggcg atcgcggcgt cggtctccgc gcggagctcc tcgtaggcgg 7920 ccttgaggtc gaggaagggg acgcgggggg tctcggcgcg gctgctcacg cggacacctc 7980 cacggcggtg gcgggcagct gcggggcggt cgccttgagc ggctcccacc agccgcggtt 8040 ctcccggtac cagcggacgg tccgcgcgag gccgtccgcg aaggagacct gcgggcggta 8100 gccgagctcg cgctcgatct cgccgccgtc gagggagtag cgcaggtcgt ggcccttgcg 8160 gtcggcgacc ttccggaccg aggaccagtc ggcgccgagc gagtccagga ggatgccggt 8220 gagttcgcgg ttggtcagct ccaggccgcc gccgatgtgg tagatctcgc cggcccggcc 8280 gcccgcgagg acgagcgcga tgccccggca gtggtcgtcg gtgtgcaccc actcgcggac 8340 gttcgcgccg tcgccgtaca gcgggagcgt cccgccgtcg aggaggttcg tcacgaagag 8400 ggggatgagc ttctcggggt gctggtacgg cccgtagttg ttgcagcagc gggtgatccg 8460 tacgtcgagg ccgtacgtcc ggtggtaggc gcgggcaacg aggtcggagc cggccttgga 8520 cgccgcgtag ggcgagttgg gctccagcgg gctgctctcg gtccaggagc cggagtcgat 8580 cgacccgtac acctcgtcgg tggagacgtg cacgacccgg ccgacgccgg cgtcgacggc 8640 gcactggagc agcgtctgcg tgccctgcac gttggtctcg gtgaacacgg acgcgcccgc 8700 gatggagcgg tccacgtggc tctcggccgc gaagtggacg atggcgtcca cgccgcgcag 8760 ttcccgggcg aggaggccgg cgtcgcggat gtcgccgtgg acgaagcgca gtcgcgggtc 8820 cgcgtccacc ggggcgaggt tggcgcggtt gcccgcgtag gtgaggctgt ccaggacgat 8880 cacctcatcg gcgggcacgt cggggtacgc cccggcgagg agctgccgca cgaagtgcga 8940 gccgatgaag cccgcacctc cggtcaccag aagccgcact gccgtcttcc tttcggtcgc 9000 gctgtaggtc gcggtgtggg tcgcactgtc ggtggcggtg cgggtcgcgg tgtgggtcgc 9060 actgtcggtg gcgctgtcgg tcgtgggaac gcgtcggccg cgaggtgccc tcacggggct 9120 ccctcgcggc cggcgatctc catcagatag ctgccgtact cggtgcggga gaggccttct 9180 cccaggccgt gacaggcctc ggcgtcgatg aagcccatgc ggaaggcgat ctcctcaagg 9240 cccgcgatcc agacgccctg ccgctcctcc aggacctgga cgtactgggc ggcccgcagg 9300 agcgagtcgt gggtgccggt gtccagccag gcgaagccgc ggcccaggtt gacgagttcg 9360 gcccggcccc gctccaggta gacgcggttg acgtcggtga tctccagctc gccgcgcggc 9420 gagggccgga tgttcttggc gatgtcgacg acgtcgttgt cgtagaggta gaggccggtg 9480 acggcgaggt tggagcgcgg cttgacgggc ttctcgacga ggtcggtcag ccggcccgtc 9540 gcgtccacct cggcgacgcc gtaccgctcg gggtccttga ccgggtagcc gaagagcacg 9600 cagccgtcga ggcgcgcgat gctgtcccgc aggagcgtgt agaggccggg cccgtggaag 9660 atgttgtcgc ccaggatcag ggcgcaggtg tcgtcgccga tgtgctcggc tccgacgaga 9720 agtgcgtccg cgattcctgc gggctctttc tggaccgcat agtcgagttc tattcccagg 9780 tgcctgccgt ttccgagaag cgactggaag agttcgatgt gctggggggt cgagatgatt 9840 tgaatctcgc gaataccgcc gagcatgaga accgacagcg gatagtagat catcggtttg 9900 ttgtagaccg gaagaatctg cttcgaaatg accgaggtcg ccggatgcag ccgagttccg 9960 ctcccgccgg ccaggactat tcccttcatt ctcggaaact agcagcaggg cgccggtgat 10020 aacggtcggc gtggcgagtt aggggggcgc taggggctgc gcagggggag tgtcaccacc 10080 cctttggggg gtgggaaaac accgagggcc cggccggacg gccgggccct caggtggggg 10140 gatcgtgggg gggggatcgg ggggatcggg gcgggtgcgg gtcagcgcag gaagccgcgg 10200 gcctcctccc agccgtccgc ggcgtcgcgc tccagctggt tcaggcgggc ggtgacgacc 10260 tgatcgaagc cgtccatgaa gtactcgtcg ccgtcgacgg ccgccacctc gccgccgcgc 10320 tcgacgaagt ccctgacgac ctcggtgagg gaggtgtcgg gggtcacgcg gcccgcgatg 10380 tagcgggtcg cgccgtccag gtcggggaag ccggcctcgc ggtacaggta cacgtcgccg 10440 aggagatcga cctgcaccgc gacctgcggg tgcgcggtgg gccgcatggt ggcgggcttg 10500 atccgcagca gttcggcgtc ggccccggtg cgcaggctgt tcagggcgta gccgtagtcg 10560 atgtggagtc cgggggtgcg ctcgcggacc cgctcctcga aggcgttgag ggcctcctgg 10620 agctcggccc gctcctcctg cggcagcttg ccgtcgtcac ggccgctgta gtcctcgcga 10680 atgttgacga agtcgatcgt cctgccctgc ccggcgtcgt tgaggtcggc gatgaagtcg 10740 accaggtcga gcaggcggga ggcacggccc gggagcacga tgtaggcgaa gccgaggttg 10800 atcggcgact cgcgctcggc gcgcagctgc tggaagcggc gcaggttctc gcggacgcgg 10860 cggaaggcgg ccttcttgcc ggtggtctgc tcgtactcct cgtcgttgag gccgtagagc 10920 gaggtgcgga tggcgtgcag gccccagagg ccgggctggc gctccagggt gcgctcggtg 10980 agcgcgaagg agttcgtgta gacggtgggc cgcaggccgt ggtcggtggc gtgcgcggcc 11040 aggctcccga ggccggggtt ggtgagcggc tccaggccgc cggagaagta catcgccgag 11100 gggttgcccg cgggtatctc gtcgatgacc gaccggaaca tggcgttgcc ggcgtcgagg 11160 gcggacgggt cgtagcgggc gccggtcaca cggacgcaga agtggcagcg gaacatgcag 11220 gtcgggccgg ggtagaggcc gacgctgtac gggaagacgg gcttcctggc gagcgccgcg 11280 tcgaagacgc cgcgctgttc gagcgggagc agggtgttct tccagtacgc cccggcgggg 11340 ccggtctcga ccgcggtgcg gagctccggg acctgcccga acagggcgag gaggcgccgg 11400 aaggcgtccc ggtcgacgcc caggtcgtgg cgggcctcct ccagcggggt gaaggggctg 11460 ttgccgtagc gcacggcgag ccggacgagg tggcgggcgg tcgttccggc ctcgtcgggc 11520 ggcacgaggc cgccggcggc gagggtctgg ccgacggcgt ggaccgccgc ccccagatcg 11580 gctccggggt gcgcgcagcg ttcggccggg gcggtggcgg aaagggcggg ggcggtcatc 11640 gggagcgtcc aatcgtgggc gtggatgtct ggggggccgc gagcggggcg ggggccgtgt 11700 cgcggtggcg cgcggtcagt tcgcggccgc gggtcgcgca gagacgcagc aggtcggcga 11760 cccggcggat gtcgtcgtcg ccgatggcgg tgccggtcgg cagggacagc acgcgcgcgg 11820 cgaggcgttc ggtgtgcggc agcggggcgt gcggctgccc gcggtacggc tccagctcgt 11880 ggcagcccgg cgagaagtag gcgcgggtgt gcacgccttc ggccttcagg acctccatga 11940 cgaggtcgcg gtggatgccg gtggtggcct cgtcgatctc gacgatcacg tactggtggt 12000 tgttgaggcc gtggcggtcg tggtcggcga cgaggacgcc ggggaggtcc gcgaggtgct 12060 cgcggtaggc ggcgtggttg cgccggttcc ggtcgatgac ctcgggaaac gcgtcgaggg 12120 aggtgaggcc catggcggcg gcggcctcgc tcatcttggc gttggtcccg ccggcggggc 12180 tgccgccggg caggtcgaag ccgaagttgt ggagggcgcg gatccgggcg gcgaggtcgg 12240 cgtcgtcggt gacgacggcg ccgccctcga aggcgttgac ggccttggtg gcgtggaagc 12300 tgaagacctc ggcgtcgccg aggctgccgg cgggccggcc gtcgaccgcg cagccgaggg 12360 cgtgcgcggc gtcgaagtac agccgcaggc cgtgctcgtc ggcgaccttc cgcagctggt 12420 cggcggcgca ggggcggccc cagaggtgga cgccgacgac ggccgaggtg cggggtgtga 12480 ccgcggcggc cacctggtcc gggtcgaggt tgccggtgtc cgggtcgatg tcggcgaaga 12540 ccggggtgag gccgatccag cgcagtgcgt gcggggtggc ggcgaacgtc atcgacggca 12600 tgatcacttc gccggtgagg ccggcggcgt gcgcgaggag ctggagcccg gccgtggcgt 12660 tgcaggtggc cacggcatgc cggaccccgg cgagcccggc gacgcgctcc tcgaactcgc 12720 ggacgagcgg gccgccgttg gacagccact ggctgtcgag ggcccggtcg agccgctcgt 12780 acagcctggc gcggtcgatg cggttgggcc gccccacgag gagcggctgg tcgaaagcgg 12840 cggggccgcc gaagaatgcg aggtcggata aggcgctttt cacggatgtt ccctccgggc 12900 caccgtcacg aaatgattcg ccgatccggg aatcccgaac gaggtcgccg cgctccaccg 12960 tgacgtacga cgagatggtc gattgtggtg gtcgatttcg gggggactct aatccgcgcg 13020 gaacgggacc gacaagagca cgctatgcgc tctcgatgtg cttcggatca catccgcctc 13080 cggggtattc catcggcggc ccgaatgtga tgatccttga caggatccgg gaatcagccg 13140 agccgccggg agggccgggg cgcgctccgc ggaagagtac gtgtgagaag tcccgttcct 13200 cttcccgttt ccgttccgct tccggcccgg tctggagttc tccgtgcgcc gtacccagca 13260 gggaacgacc gcttctcccc cggtactcga cctcggggcc ctggggcagg atttcgcggc 13320 cgatccgtat ccgacgtacg cgagactgcg tgccgagggt ccggcccacc gggtgcgcac 13380 ccccgagggg gacgaggtgt ggctggtcgt cggctacgac cgggcgcggg cggtcctcgc 13440 cgatccccgg ttcagcaaga ctggcgcaac tccacgactc ccctgaccga agccgaagcc 13500 gcgctcaacc acaacatgct gagttccgaa cccgccgcgg cacacccggc tgcgccagct 13560 ggtggcccgt gagttcacca tgcgccggtg cgagttgctg ccgccccggg tcc 13613 4 3782 PRT Streptomyces venezuelae 4 Met Thr Asp Asp Leu Thr Gly Ala Leu Thr Gln Pro Pro Leu Gly Arg 1 5 10 15 Thr Val Arg Ala Val Ala Asp Arg Glu Leu Gly Thr His Leu Leu Glu 20 25 30 Thr Arg Gly Ile His Trp Ile His Ala Ala Asn Gly Asp Pro Tyr Ala 35 40 45 Thr Val Leu Arg Gly Gln Ala Asp Asp Pro Tyr Pro Ala Tyr Glu Arg 50 55 60 Val Arg Ala Arg Gly Ala Leu Ser Phe Ser Pro Thr Gly Ser Trp Val 65 70 75 80 Thr Ala Asp His Ala Leu Ala Ala Ser Ile Leu Cys Ser Thr Asp Phe 85 90 95 Gly Val Ser Gly Ala Asp Gly Val Pro Val Pro Gln Gln Val Leu Ser 100 105 110 Tyr Gly Glu Gly Cys Pro Leu Glu Arg Glu Gln Val Leu Pro Ala Ala 115 120 125 Gly Asp Val Pro Glu Gly Gly Gln Arg Ala Val Val Glu Gly Ile His 130 135 140 Arg Glu Thr Leu Glu Gly Leu Ala Pro Asp Pro Ser Ala Ser Tyr Ala 145 150 155 160 Phe Glu Leu Leu Gly Gly Phe Val Arg Pro Ala Val Thr Ala Ala Ala 165 170 175 Ala Ala Val Leu Gly Val Pro Ala Asp Arg Arg Ala Asp Phe Ala Asp 180 185 190 Leu Leu Glu Arg Leu Arg Pro Leu Ser Asp Ser Leu Leu Ala Pro Gln 195 200 205 Ser Leu Arg Thr Val Arg Ala Ala Asp Gly Ala Leu Ala Glu Leu Thr 210 215 220 Ala Leu Leu Ala Asp Ser Asp Asp Ser Pro Gly Ala Leu Leu Ser Ala 225 230 235 240 Leu Gly Val Thr Ala Ala Val Gln Leu Thr Gly Asn Ala Val Leu Ala 245 250 255 Leu Leu Ala His Pro Glu Gln Trp Arg Glu Leu Cys Asp Arg Pro Gly 260 265 270 Leu Ala Ala Ala Ala Val Glu Glu Thr Leu Arg Tyr Asp Pro Pro Val 275 280 285 Gln Leu Asp Ala Arg Val Val Arg Gly Glu Thr Glu Leu Ala Gly Arg 290 295 300 Arg Leu Pro Ala Gly Ala His Val Val Val Leu Thr Ala Ala Thr Gly 305 310 315 320 Arg Asp Pro Glu Val Phe Thr Asp Pro Glu Arg Phe Asp Leu Ala Arg 325 330 335 Pro Asp Ala Ala Ala His Leu Ala Leu His Pro Ala Gly Pro Tyr Gly 340 345 350 Pro Val Ala Ser Leu Val Arg Leu Gln Ala Glu Val Ala Leu Arg Thr 355 360 365 Leu Ala Gly Arg Phe Pro Gly Leu Arg Gln Ala Gly Asp Val Leu Arg 370 375 380 Pro Arg Arg Ala Pro Val Gly Arg Gly Pro Leu Ser Val Pro Val Ser 385 390 395 400 Ser Ser Met Arg Val Leu Leu Thr Ser Phe Ala His His Thr His Tyr 405 410 415 Tyr Gly Leu Val Pro Leu Ala Trp Ala Leu Leu Ala Ala Gly His Glu 420 425 430 Val Arg Val Ala Ser Gln Pro Ala Leu Thr Asp Thr Ile Thr Gly Ser 435 440 445 Gly Leu Ala Ala Val Pro Val Gly Thr Asp His Leu Ile His Glu Tyr 450 455 460 Arg Val Arg Met Ala Gly Glu Pro Arg Pro Asn His Pro Ala Ile Ala 465 470 475 480 Phe Asp Glu Ala Arg Pro Glu Pro Leu Asp Trp Asp His Ala Leu Gly 485 490 495 Ile Glu Ala Ile Leu Ala Pro Tyr Phe His Leu Leu Ala Asn Asn Asp 500 505 510 Ser Met Val Asp Asp Leu Val Asp Phe Ala Arg Ser Trp Gln Pro Asp 515 520 525 Leu Val Leu Trp Glu Pro Thr Thr Tyr Ala Gly Ala Val Ala Ala Gln 530 535 540 Val Thr Gly Ala Ala His Ala Arg Val Leu Trp Gly Pro Asp Val Met 545 550 555 560 Gly Ser Ala Arg Arg Lys Phe Val Ala Leu Arg Asp Arg Gln Pro Pro 565 570 575 Glu His Arg Glu Asp Pro Thr Ala Glu Trp Leu Thr Trp Thr Leu Asp 580 585 590 Arg Tyr Gly Ala Ser Phe Glu Glu Glu Leu Leu Thr Gly Gln Phe Thr 595 600 605 Ile Asp Pro Thr Pro Pro Ser Leu Arg Leu Asp Thr Gly Leu Pro Thr 610 615 620 Val Gly Met Arg Tyr Val Pro Tyr Asn Gly Thr Ser Val Val Pro Asp 625 630 635 640 Trp Leu Ser Glu Pro Pro Ala Arg Pro Arg Val Cys Leu Thr Leu Gly 645 650 655 Val Ser Ala Arg Glu Val Leu Gly Gly Asp Gly Val Ser Gln Gly Asp 660 665 670 Ile Leu Glu Ala Leu Ala Asp Leu Asp Ile Glu Leu Val Ala Thr Leu 675 680 685 Asp Ala Ser Gln Arg Ala Glu Ile Arg Asn Tyr Pro Lys His Thr Arg 690 695 700 Phe Thr Asp Phe Val Pro Met His Ala Leu Leu Pro Ser Cys Ser Ala 705 710 715 720 Ile Ile His His Gly Gly Ala Gly Thr Tyr Ala Thr Ala Val Ile Asn 725 730 735 Ala Val Pro Gln Val Met Leu Ala Glu Leu Trp Asp Ala Pro Val Lys 740 745 750 Ala Arg Ala Val Ala Glu Gln Gly Ala Gly Phe Phe Leu Pro Pro Ala 755 760 765 Glu Leu Thr Pro Gln Ala Val Arg Asp Ala Val Val Arg Ile Leu Asp 770 775 780 Asp Pro Ser Val Ala Thr Ala Ala His Arg Leu Arg Glu Glu Thr Phe 785 790 795 800 Gly Asp Pro Thr Pro Ala Gly Ile Val Pro Glu Leu Glu Arg Leu Ala 805 810 815 Ala Gln His Arg Arg Pro Pro Ala Asp Ala Arg His Met Tyr Glu Val 820 825 830 Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly Arg Gly Lys Asp 835 840 845 Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val Arg Ser Arg Thr 850 855 860 Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly Thr Gly Thr His 865 870 875 880 Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala Gly Leu Glu Leu 885 890 895 Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu Pro Asp Ala Thr 900 905 910 Leu His Gln Gly Asp Met Arg Asp Phe Arg Leu Gly Arg Lys Phe Ser 915 920 925 Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu Lys Thr Thr Glu 930 935 940 Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His Leu Glu Pro Gly 945 950 955 960 Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu Thr Phe Ala Asp 965 970 975 Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly Arg Thr Val Ala 980 985 990 Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr Arg Met Glu Val 995 1000 1005 His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg His Phe Ser Asp 1010 1015 1020 Val His Leu Ile Thr Leu Phe His Gln Ala Glu Tyr Glu Ala Ala Phe 1025 1030 1035 1040 Thr Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly Gly Pro Ser Gly 1045 1050 1055 Arg Gly Leu Phe Val Gly Val Pro Ala Met Thr Gly Lys Thr Arg Ile 1060 1065 1070 Pro Arg Val Arg Arg Gly Arg Thr Thr Pro Arg Ala Phe Thr Leu Ala 1075 1080 1085 Val Val Gly Thr Leu Leu Ala Gly Thr Thr Val Ala Ala Ala Ala Pro 1090 1095 1100 Gly Ala Ala Asp Thr Ala Asn Val Gln Tyr Thr Ser Arg Ala Ala Glu 1105 1110 1115 1120 Leu Val Ala Gln Met Thr Leu Asp Glu Lys Ile Ser Phe Val His Trp 1125 1130 1135 Ala Leu Asp Pro Asp Arg Gln Asn Val Gly Tyr Leu Pro Gly Val Pro 1140 1145 1150 Arg Leu Gly Ile Pro Glu Leu Arg Ala Ala Asp Gly Pro Asn Gly Ile 1155 1160 1165 Arg Leu Val Gly Gln Thr Ala Thr Ala Leu Pro Ala Pro Val Ala Leu 1170 1175 1180 Ala Ser Thr Phe Asp Asp Thr Met Ala Asp Ser Tyr Gly Lys Val Met 1185 1190 1195 1200 Gly Arg Asp Gly Arg Ala Leu Asn Gln Asp Met Val Leu Gly Pro Met 1205 1210 1215 Met Asn Asn Ile Arg Val Pro His Gly Gly Arg Asn Tyr Glu Thr Phe 1220 1225 1230 Ser Glu Asp Pro Leu Val Ser Ser Arg Thr Ala Val Ala Gln Ile Lys 1235 1240 1245 Gly Ile Gln Gly Ala Gly Leu Met Thr Thr Ala Lys His Phe Ala Ala 1250 1255 1260 Asn Asn Gln Glu Asn Asn Arg Phe Ser Val Asn Ala Asn Val Asp Glu 1265 1270 1275 1280 Gln Thr Leu Arg Glu Ile Glu Phe Pro Ala Phe Glu Ala Ser Ser Lys 1285 1290 1295 Ala Gly Ala Ala Ser Phe Met Cys Ala Tyr Asn Gly Leu Asn Gly Lys 1300 1305 1310 Pro Ser Cys Gly Asn Asp Glu Leu Leu Asn Asn Val Leu Arg Thr Gln 1315 1320 1325 Trp Gly Phe Gln Gly Trp Val Met Ser Asp Trp Leu Ala Thr Pro Gly 1330 1335 1340 Thr Asp Ala Ile Thr Lys Gly Leu Asp Gln Glu Met Gly Val Glu Leu 1345 1350 1355 1360 Pro Gly Asp Val Pro Lys Gly Glu Pro Ser Pro Pro Ala Lys Phe Phe 1365 1370 1375 Gly Glu Ala Leu Lys Thr Ala Val Leu Asn Gly Thr Val Pro Glu Ala 1380 1385 1390 Ala Val Thr Arg Ser Ala Glu Arg Ile Val Gly Gln Met Glu Lys Phe 1395 1400 1405 Gly Leu Leu Leu Ala Thr Pro Ala Pro Arg Pro Glu Arg Asp Lys Ala 1410 1415 1420 Gly Ala Gln Ala Val Ser Arg Lys Val Ala Glu Asn Gly Ala Val Leu 1425 1430 1435 1440 Leu Arg Asn Glu Gly Gln Ala Leu Pro Leu Ala Gly Asp Ala Gly Lys 1445 1450 1455 Ser Ile Ala Val Ile Gly Pro Thr Ala Val Asp Pro Lys Val Thr Gly 1460 1465 1470 Leu Gly Ser Ala His Val Val Pro Asp Ser Ala Ala Ala Pro Leu Asp 1475 1480 1485 Thr Ile Lys Ala Arg Ala Gly Ala Gly Ala Thr Val Thr Tyr Glu Thr 1490 1495 1500 Gly Glu Glu Thr Phe Gly Thr Gln Ile Pro Ala Gly Asn Leu Ser Pro 1505 1510 1515 1520 Ala Phe Asn Gln Gly His Gln Leu Glu Pro Gly Lys Ala Gly Ala Leu 1525 1530 1535 Tyr Asp Gly Thr Leu Thr Val Pro Ala Asp Gly Glu Tyr Arg Ile Ala 1540 1545 1550 Val Arg Ala Thr Gly Gly Tyr Ala Thr Val Gln Leu Gly Ser His Thr 1555 1560 1565 Ile Glu Ala Gly Gln Val Tyr Gly Lys Val Ser Ser Pro Leu Leu Lys 1570 1575 1580 Leu Thr Lys Gly Thr His Lys Leu Thr Ile Ser Gly Phe Ala Met Ser 1585 1590 1595 1600 Ala Thr Pro Leu Ser Leu Glu Leu Gly Trp Val Thr Pro Ala Ala Ala 1605 1610 1615 Asp Ala Thr Ile Ala Lys Ala Val Glu Ser Ala Arg Lys Ala Arg Thr 1620 1625 1630 Ala Val Val Phe Ala Tyr Asp Asp Gly Thr Glu Gly Val Asp Arg Pro 1635 1640 1645 Asn Leu Ser Leu Pro Gly Thr Gln Asp Lys Leu Ile Ser Ala Val Ala 1650 1655 1660 Asp Ala Asn Pro Asn Thr Ile Val Val Leu Asn Thr Gly Ser Ser Val 1665 1670 1675 1680 Leu Met Pro Trp Leu Ser Lys Thr Arg Ala Val Leu Asp Met Trp Tyr 1685 1690 1695 Pro Gly Gln Ala Gly Ala Glu Ala Thr Ala Ala Leu Leu Tyr Gly Asp 1700 1705 1710 Val Asn Pro Ser Gly Lys Leu Thr Gln Ser Phe Pro Ala Ala Glu Asn 1715 1720 1725 Gln His Ala Val Ala Gly Asp Pro Thr Ser Tyr Pro Gly Val Asp Asn 1730 1735 1740 Gln Gln Thr Tyr Arg Glu Gly Ile His Val Gly Tyr Arg Trp Phe Asp 1745 1750 1755 1760 Lys Glu Asn Val Lys Pro Leu Phe Pro Phe Gly His Gly Leu Ser Tyr 1765 1770 1775 Thr Ser Phe Thr Gln Ser Ala Pro Thr Val Val Arg Thr Ser Thr Gly 1780 1785 1790 Gly Leu Lys Val Thr Val Thr Val Arg Asn Ser Gly Lys Arg Ala Gly 1795 1800 1805 Gln Glu Val Val Gln Ala Tyr Leu Gly Ala Ser Pro Asn Val Thr Ala 1810 1815 1820 Pro Gln Ala Lys Lys Lys Leu Val Gly Tyr Thr Lys Val Ser Leu Ala 1825 1830 1835 1840 Ala Gly Glu Ala Lys Thr Val Thr Val Asn Val Asp Arg Arg Gln Leu 1845 1850 1855 Gln Thr Gly Ser Ser Ser Ala Asp Leu Arg Gly Ser Ala Thr Val Asn 1860 1865 1870 Val Trp Met Ser Ser Arg Ala Glu Thr Pro Arg Val Pro Phe Leu Asp 1875 1880 1885 Leu Lys Ala Ala Tyr Glu Glu Leu Arg Ala Glu Thr Asp Ala Ala Ile 1890 1895 1900 Ala Arg Val Leu Asp Ser Gly Arg Tyr Leu Leu Gly Pro Glu Leu Glu 1905 1910 1915 1920 Gly Phe Glu Ala Glu Phe Ala Ala Tyr Cys Glu Thr Asp His Ala Val 1925 1930 1935 Gly Val Asn Ser Gly Met Asp Ala Leu Gln Leu Ala Leu Arg Gly Leu 1940 1945 1950 Gly Ile Gly Pro Gly Asp Glu Val Ile Val Pro Ser His Thr Tyr Ile 1955 1960 1965 Ala Ser Trp Leu Ala Val Ser Ala Thr Gly Ala Thr Pro Val Pro Val 1970 1975 1980 Glu Pro His Glu Asp His Pro Thr Leu Asp Pro Leu Leu Val Glu Lys 1985 1990 1995 2000 Ala Ile Thr Pro Arg Thr Arg Ala Leu Leu Pro Val His Leu Tyr Gly 2005 2010 2015 His Pro Ala Asp Met Asp Ala Leu Arg Glu Leu Ala Asp Arg His Gly 2020 2025 2030 Leu His Ile Val Glu Asp Ala Ala Gln Ala His Gly Ala Arg Tyr Arg 2035 2040 2045 Gly Arg Arg Ile Gly Ala Gly Ser Ser Val Ala Ala Phe Ser Phe Tyr 2050 2055 2060 Pro Gly Lys Asn Leu Gly Cys Phe Gly Asp Gly Gly Ala Val Val Thr 2065 2070 2075 2080 Gly Asp Pro Glu Leu Ala Glu Arg Leu Arg Met Leu Arg Asn Tyr Gly 2085 2090 2095 Ser Arg Gln Lys Tyr Ser His Glu Thr Lys Gly Thr Asn Ser Arg Leu 2100 2105 2110 Asp Glu Met Gln Ala Ala Val Leu Arg Ile Arg Leu Ala His Leu Asp 2115 2120 2125 Ser Trp Asn Gly Arg Arg Ser Ala Leu Ala Ala Glu Tyr Leu Ser Gly 2130 2135 2140 Leu Ala Gly Leu Pro Gly Ile Gly Leu Pro Val Thr Ala Pro Asp Thr 2145 2150 2155 2160 Asp Pro Val Trp His Leu Phe Thr Val Arg Thr Glu Arg Arg Asp Glu 2165 2170 2175 Leu Arg Ser His Leu Asp Ala Arg Gly Ile Asp Thr Leu Thr His Tyr 2180 2185 2190 Pro Val Pro Val His Leu Ser Pro Ala Tyr Ala Gly Glu Ala Pro Pro 2195 2200 2205 Glu Gly Ser Leu Pro Arg Ala Glu Ser Phe Ala Arg Gln Val Leu Ser 2210 2215 2220 Leu Pro Ile Gly Pro His Leu Glu Arg Pro Gln Ala Leu Arg Val Ile 2225 2230 2235 2240 Asp Ala Val Arg Glu Trp Ala Glu Arg Val Asp Gln Ala Met Arg Leu 2245 2250 2255 Leu Val Thr Gly Gly Ala Gly Phe Ile Gly Ser His Phe Val Arg Gln 2260 2265 2270 Leu Leu Ala Gly Ala Tyr Pro Asp Val Pro Ala Asp Glu Val Ile Val 2275 2280 2285 Leu Asp Ser Leu Thr Tyr Ala Gly Asn Arg Ala Asn Leu Ala Pro Val 2290 2295 2300 Asp Ala Asp Pro Arg Leu Arg Phe Val His Gly Asp Ile Arg Asp Ala 2305 2310 2315 2320 Gly Leu Leu Ala Arg Glu Leu Arg Gly Val Asp Ala Ile Val His Phe 2325 2330 2335 Ala Ala Glu Ser His Val Asp Arg Ser Ile Ala Gly Ala Ser Val Phe 2340 2345 2350 Thr Glu Thr Asn Val Gln Gly Thr Gln Thr Leu Leu Gln Cys Ala Val 2355 2360 2365 Asp Ala Gly Val Gly Arg Val Val His Val Ser Thr Asp Glu Val Tyr 2370 2375 2380 Gly Ser Ile Asp Ser Gly Ser Trp Thr Glu Ser Ser Pro Leu Glu Pro 2385 2390 2395 2400 Asn Ser Pro Tyr Ala Ala Ser Lys Ala Gly Ser Asp Leu Val Ala Arg 2405 2410 2415 Ala Tyr His Arg Thr Tyr Gly Leu Asp Val Arg Ile Thr Arg Cys Cys 2420 2425 2430 Asn Asn Tyr Gly Pro Tyr Gln His Pro Glu Lys Leu Ile Pro Leu Phe 2435 2440 2445 Val Thr Asn Leu Leu Asp Gly Gly Thr Leu Pro Leu Tyr Gly Asp Gly 2450 2455 2460 Ala Asn Val Arg Glu Trp Val His Thr Asp Asp His Cys Arg Gly Ile 2465 2470 2475 2480 Ala Leu Val Leu Ala Gly Gly Arg Ala Gly Glu Ile Tyr His Ile Gly 2485 2490 2495 Gly Gly Leu Glu Leu Thr Asn Arg Glu Leu Thr Gly Ile Leu Leu Asp 2500 2505 2510 Ser Leu Gly Ala Asp Trp Ser Ser Val Arg Lys Val Ala Asp Arg Lys 2515 2520 2525 Gly His Asp Leu Arg Tyr Ser Leu Asp Gly Gly Glu Ile Glu Arg Glu 2530 2535 2540 Leu Gly Tyr Arg Pro Gln Val Ser Phe Ala Asp Gly Leu Ala Arg Thr 2545 2550 2555 2560 Val Arg Trp Tyr Arg Glu Asn Arg Gly Trp Trp Glu Pro Leu Lys Ala 2565 2570 2575 Thr Ala Pro Gln Leu Pro Ala Thr Ala Val Glu Val Ser Ala Met Lys 2580 2585 2590 Gly Ile Val Leu Ala Gly Gly Ser Gly Thr Arg Leu His Pro Ala Thr 2595 2600 2605 Ser Val Ile Ser Lys Gln Ile Leu Pro Val Tyr Asn Lys Pro Met Ile 2610 2615 2620 Tyr Tyr Pro Leu Ser Val Leu Met Leu Gly Gly Ile Arg Glu Ile Gln 2625 2630 2635 2640 Ile Ile Ser Thr Pro Gln His Ile Glu Leu Phe Gln Ser Leu Leu Gly 2645 2650 2655 Asn Gly Arg His Leu Gly Ile Glu Leu Asp Tyr Ala Val Gln Lys Glu 2660 2665 2670 Pro Ala Gly Ile Ala Asp Ala Leu Leu Val Gly Ala Glu His Ile Gly 2675 2680 2685 Asp Asp Thr Cys Ala Leu Ile Leu Gly Asp Asn Ile Phe His Gly Pro 2690 2695 2700 Gly Leu Tyr Thr Leu Leu Arg Asp Ser Ile Ala Arg Leu Asp Gly Cys 2705 2710 2715 2720 Val Leu Phe Gly Tyr Pro Val Lys Asp Pro Glu Arg Tyr Gly Val Ala 2725 2730 2735 Glu Val Asp Ala Thr Gly Arg Leu Thr Asp Leu Val Glu Lys Pro Val 2740 2745 2750 Lys Pro Arg Ser Asn Leu Ala Val Thr Gly Leu Tyr Leu Tyr Asp Asn 2755 2760 2765 Asp Val Val Asp Ile Ala Lys Asn Ile Arg Pro Ser Pro Arg Gly Glu 2770 2775 2780 Leu Glu Ile Thr Asp Val Asn Arg Val Tyr Leu Glu Arg Gly Arg Ala 2785 2790 2795 2800 Glu Leu Val Asn Leu Gly Arg Gly Phe Ala Trp Leu Asp Thr Gly Thr 2805 2810 2815 His Asp Ser Leu Leu Arg Ala Ala Gln Tyr Val Gln Val Leu Glu Glu 2820 2825 2830 Arg Gln Gly Val Trp Ile Ala Gly Leu Glu Glu Ile Ala Phe Arg Met 2835 2840 2845 Gly Phe Ile Asp Ala Glu Ala Cys His Gly Leu Gly Glu Gly Leu Ser 2850 2855 2860 Arg Thr Glu Tyr Gly Ser Tyr Leu Met Glu Ile Ala Gly Arg Glu Gly 2865 2870 2875 2880 Ala Pro Met Thr Ala Pro Ala Leu Ser Ala Thr Ala Pro Ala Glu Arg 2885 2890 2895 Cys Ala His Pro Gly Ala Asp Leu Gly Ala Ala Val His Ala Val Gly 2900 2905 2910 Gln Thr Leu Ala Ala Gly Gly Leu Val Pro Pro Asp Glu Ala Gly Thr 2915 2920 2925 Thr Ala Arg His Leu Val Arg Leu Ala Val Arg Tyr Gly Asn Ser Pro 2930 2935 2940 Phe Thr Pro Leu Glu Glu Ala Arg His Asp Leu Gly Val Asp Arg Asp 2945 2950 2955 2960 Ala Phe Arg Arg Leu Leu Ala Leu Phe Gly Gln Val Pro Glu Leu Arg 2965 2970 2975 Thr Ala Val Glu Thr Gly Pro Ala Gly Ala Tyr Trp Lys Asn Thr Leu 2980 2985 2990 Leu Pro Leu Glu Gln Arg Gly Val Phe Asp Ala Ala Leu Ala Arg Lys 2995 3000 3005 Pro Val Phe Pro Tyr Ser Val Gly Leu Tyr Pro Gly Pro Thr Cys Met 3010 3015 3020 Phe Arg Cys His Phe Cys Val Arg Val Thr Gly Ala Arg Tyr Asp Pro 3025 3030 3035 3040 Ser Ala Leu Asp Ala Gly Asn Ala Met Phe Arg Ser Val Ile Asp Glu 3045 3050 3055 Ile Pro Ala Gly Asn Pro Ser Ala Met Tyr Phe Ser Gly Gly Leu Glu 3060 3065 3070 Pro Leu Thr Asn Pro Gly Leu Gly Ser Leu Ala Ala His Ala Thr Asp 3075 3080 3085 His Gly Leu Arg Pro Thr Val Tyr Thr Asn Ser Phe Ala Leu Thr Glu 3090 3095 3100 Arg Thr Leu Glu Arg Gln Pro Gly Leu Trp Gly Leu His Ala Ile Arg 3105 3110 3115 3120 Thr Ser Leu Tyr Gly Leu Asn Asp Glu Glu Tyr Glu Gln Thr Thr Gly 3125 3130 3135 Lys Lys Ala Ala Phe Arg Arg Val Arg Glu Asn Leu Arg Arg Phe Gln 3140 3145 3150 Gln Leu Arg Ala Glu Arg Glu Ser Pro Ile Asn Leu Gly Phe Ala Tyr 3155 3160 3165 Ile Val Leu Pro Gly Arg Ala Ser Arg Leu Leu Asp Leu Val Asp Phe 3170 3175 3180 Ile Ala Asp Leu Asn Asp Ala Gly Gln Gly Arg Thr Ile Asp Phe Val 3185 3190 3195 3200 Asn Ile Arg Glu Asp Tyr Ser Gly Arg Asp Asp Gly Lys Leu Pro Gln 3205 3210 3215 Glu Glu Arg Ala Glu Leu Gln Glu Ala Leu Asn Ala Phe Glu Glu Arg 3220 3225 3230 Val Arg Glu Arg Thr Pro Gly Leu His Ile Asp Tyr Gly Tyr Ala Leu 3235 3240 3245 Asn Ser Leu Arg Thr Gly Ala Asp Ala Glu Leu Leu Arg Ile Lys Pro 3250 3255 3260 Ala Thr Met Arg Pro Thr Ala His Pro Gln Val Ala Val Gln Val Asp 3265 3270 3275 3280 Leu Leu Gly Asp Val Tyr Leu Tyr Arg Glu Ala Gly Phe Pro Asp Leu 3285 3290 3295 Asp Gly Ala Thr Arg Tyr Ile Ala Gly Arg Val Thr Pro Asp Thr Ser 3300 3305 3310 Leu Thr Glu Val Val Arg Asp Phe Val Glu Arg Gly Gly Glu Val Ala 3315 3320 3325 Ala Val Asp Gly Asp Glu Tyr Phe Met Asp Gly Phe Asp Gln Val Val 3330 3335 3340 Thr Ala Arg Leu Asn Gln Leu Glu Arg Asp Ala Ala Asp Gly Trp Glu 3345 3350 3355 3360 Glu Ala Arg Gly Phe Leu Arg Met Lys Ser Ala Leu Ser Asp Leu Ala 3365 3370 3375 Phe Phe Gly Gly Pro Ala Ala Phe Asp Gln Pro Leu Leu Val Gly Arg 3380 3385 3390 Pro Asn Arg Ile Asp Arg Ala Arg Leu Tyr Glu Arg Leu Asp Arg Ala 3395 3400 3405 Leu Asp Ser Gln Trp Leu Ser Asn Gly Gly Pro Leu Val Arg Glu Phe 3410 3415 3420 Glu Glu Arg Val Ala Gly Leu Ala Gly Val Arg His Ala Val Ala Thr 3425 3430 3435 3440 Cys Asn Ala Thr Ala Gly Leu Gln Leu Leu Ala His Ala Ala Gly Leu 3445 3450 3455 Thr Gly Glu Val Ile Met Pro Ser Met Thr Phe Ala Ala Thr Pro His 3460 3465 3470 Ala Leu Arg Trp Ile Gly Leu Thr Pro Val Phe Ala Asp Ile Asp Pro 3475 3480 3485 Asp Thr Gly Asn Leu Asp Pro Asp Gln Val Ala Ala Ala Val Thr Pro 3490 3495 3500 Arg Thr Ser Ala Val Val Gly Val His Leu Trp Gly Arg Pro Cys Ala 3505 3510 3515 3520 Ala Asp Gln Leu Arg Lys Val Ala Asp Glu His Gly Leu Arg Leu Tyr 3525 3530 3535 Phe Asp Ala Ala His Ala Leu Gly Cys Ala Val Asp Gly Arg Pro Ala 3540 3545 3550 Gly Ser Leu Gly Asp Ala Glu Val Phe Ser Phe His Ala Thr Lys Ala 3555 3560 3565 Val Asn Ala Phe Glu Gly Gly Ala Val Val Thr Asp Asp Ala Asp Leu 3570 3575 3580 Ala Ala Arg Ile Arg Ala Leu His Asn Phe Gly Phe Asp Leu Pro Gly 3585 3590 3595 3600 Gly Ser Pro Ala Gly Gly Thr Asn Ala Lys Met Ser Glu Ala Ala Ala 3605 3610 3615 Ala Met Gly Leu Thr Ser Leu Asp Ala Phe Pro Glu Val Ile Asp Arg 3620 3625 3630 Asn Arg Arg Asn His Ala Ala Tyr Arg Glu His Leu Ala Asp Leu Pro 3635 3640 3645 Gly Val Leu Val Ala Asp His Asp Arg His Gly Leu Asn Asn His Gln 3650 3655 3660 Tyr Val Ile Val Glu Ile Asp Glu Ala Thr Thr Gly Ile His Arg Asp 3665 3670 3675 3680 Leu Val Met Glu Val Leu Lys Ala Glu Gly Val His Thr Arg Ala Tyr 3685 3690 3695 Phe Ser Pro Gly Cys His Glu Leu Glu Pro Tyr Arg Gly Gln Pro His 3700 3705 3710 Ala Pro Leu Pro His Thr Glu Arg Leu Ala Ala Arg Val Leu Ser Leu 3715 3720 3725 Pro Thr Gly Thr Ala Ile Gly Asp Asp Asp Ile Arg Arg Val Ala Asp 3730 3735 3740 Leu Leu Arg Leu Cys Ala Thr Arg Gly Arg Glu Leu Thr Ala Arg His 3745 3750 3755 3760 Arg Asp Thr Ala Pro Ala Pro Leu Ala Ala Pro Gln Thr Ser Thr Pro 3765 3770 3775 Thr Ile Gly Arg Ser Arg 3780 5 36778 DNA Streptomyces venezuelae 5 ggatccgacc gtgggtgtga atctccgggt gctcgcctcg tcctgccccg ttacctgtcc 60 gcctcccgct ccagaccagc gggaggcgga caggggcatg cccgccgggc ggctaacggc 120 ccgtgcggcg tccgtacgac gagcctcgcg cgccctggcg gcccttggtc tgccggacct 180 gtgcgcgggg tgcgcagggt tcgccgccgc gcgtggggcc gtatctgcgg ctcccgggca 240 cggcggccct gctcgtctcc gagtcatagt ccctgccgcc ggcgccaccg ccctggcccg 300 gcatgcgcgt gccgggcgcc cccggcgcgt aactcggctg ggaggcctgg aaaagggcga 360 tccattgggt gagcgtgagg tccttcggca gtccgccgtc cggaattccg tggcggtcgg 420 cgagggaacg gtaggtccgc ttggggatgt ggcgccggag gatctccgcg aggccccgtc 480 cggggccggt gaagacggct tcggcgaagt tctggaaggc gcggctcgcg ctctcgggca 540 gcaggggctg ggggcgtcgc ctgatcgtca ggacgccgcc gtcgacgcgg ggcatcggac 600 ggaacgacga ggcgcggacg cggtcgtgga ccgcgaactc gtaccagggg gcccaggagg 660 tcgtgaggag cgatccgccg ctgcgaccgg cgcgtttgcg ggcgacctcc cactgcacta 720 tcagggccgc cgactgccag ttcgtcgatt ccaggagact ccggagaatc tgggtcgtga 780 tgccgaaggg aacgtttccg acgacggtgt cgatatcgcg cggaatgcgg aagtcgagga 840 aatcaccctg gaatacggtg accctctccc cttcgaattt ccgccgcaca tgcgcggccc 900 agtgcgggtc catctccacg accgtcacgg tgtcgaagga gcgcaccaac tcctcggtta 960 tcgcgccctt tccggggccg atttcgagaa cgttcctacc gtccccctcg acatgcgtga 1020 cgagattgcg cacggctctg tcgtcctgaa ggaagttctg gcctaattcg cggcgaaggg 1080 tgtcgcggtc cgctcgcctc ggtatggagt cgcgcattgc catgaacgat cccctccctg 1140 gatgccgtgg tcaatggact tggcacggac catacctcac ggtccgtcgg acgaccggag 1200 aagaagttca cgcacgggcg ttccggagta cgggagttgt gaacggccgc gacgaagtcg 1260 gtcgcggctc ggcgggcggt gacgagcgag gtccggagga acgcgacgaa gcagccgaac 1320 cccaagtgag gtgcgacgga gtgacattgg gggcatacgg agggttgtcg tacggagcgc 1380 actcaacgag gctccaggag ggaggggttg aacccgccgc cgactggcct tcgccgcccg 1440 cgcggccgga gtatgtcatg tcgggggtga aatcaagcca ttcccccggg atcggctgtt 1500 acccatccct ttacctggcg tggatttccc aacccttggt atagagcggg agacgacgcg 1560 acaccatgga gaccacgcac accacgagcg ccaccccccg gccatcccga caaggggggt 1620 ccggctcgcc tcccgacacc catggcctgg ggtacacgcc aggtataggg ggaacgtagg 1680 gggagcatag ggggggtgcc ctggggttgg gtgaaagcgc ggcttccgga gacggagccg 1740 gatgtcttca gccggaatta ccaggaccgg tgcgagaaca ccggtgacag ggcgtggggc 1800 ggcagcgtgg gacacggggg aagtgcgggt ccgacggggg ttgccccctg ccggccccga 1860 tcatgcggag cactccttct ctcgtgctcc taccggtgat gtgcgcgccg aattgattcg 1920 tggagagatg tcgacagtgt ccaagagtga gtccgaggaa ttcgtgtccg tgtcgaacga 1980 cgccggttcc gcgcacggca cagcggaacc cgtcgccgtc gtcggcatct cctgccgggt 2040 gcccggcgcc cgggacccga gagagttctg ggaactcctg gcggcaggcg gccaggccgt 2100 caccgacgtc cccgcggacc gctggaacgc cggcgacttc tacgacccgg accgctccgc 2160 ccccggccgc tcgaacagcc ggtggggcgg gttcatcgag gacgtcgacc ggttcgacgc 2220 cgccttcttc ggcatctcgc cccgcgaggc cgcggagatg gacccgcagc agcggctcgc 2280 cctggagctg ggctgggagg ccctggagcg cgccgggatc gacccgtcct cgctcaccgg 2340 cacccgcacc ggcgtcttcg ccggcgccat ctgggacgac tacgccaccc tgaagcaccg 2400 ccagggcggc gccgcgatca ccccgcacac cgtcaccggc ctccaccgcg gcatcatcgc 2460 gaaccgactc tcgtacacgc tcgggctccg cggccccagc atggtcgtcg actccggcca 2520 gtcctcgtcg ctcgtcgccg tccacctcgc gtgcgagagc ctgcggcgcg gcgagtccga 2580 gctcgccctc gccggcggcg tctcgctcaa cctggtgccg gacagcatca tcggggcgag 2640 caagttcggc ggcctctccc ccgacggccg cgcctacacc ttcgacgcgc gcgccaacgg 2700 ctacgtacgc ggcgagggcg gcggtttcgt cgtcctgaag cgcctctccc gggccgtcgc 2760 cgacggcgac ccggtgctcg ccgtgatccg gggcagcgcc gtcaacaacg gcggcgccgc 2820 ccagggcatg acgacccccg acgcgcaggc gcaggaggcc gtgctccgcg aggcccacga 2880 gcgggccggg accgcgccgg ccgacgtgcg gtacgtcgag ctgcacggca ccggcacccc 2940 cgtgggcgac ccgatcgagg ccgctgcgct cggcgccgcc ctcggcaccg gccgcccggc 3000 cggacagccg ctcctggtcg gctcggtcaa gacgaacatc ggccacctgg agggcgcggc 3060 cggcatcgcc ggcctcatca aggccgtcct ggcggtccgc ggtcgcgcgc tgcccgccag 3120 cctgaactac gagaccccga acccggcgat cccgttcgag gaactgaacc tccgggtgaa 3180 cacggagtac ctgccgtggg agccggagca cgacgggcag cggatggtcg tcggcgtgtc 3240 ctcgttcggc atgggcggca cgaacgcgca tgtcgtgctc gaagaggccc ccgggggttg 3300 tcgaggtgct tcggtcgtgg agtcgacggt cggcgggtcg gcggtcggcg gcggtgtggt 3360 gccgtgggtg gtgtcggcga agtccgctgc cgcgctggac gcgcagatcg agcggcttgc 3420 cgcgttcgcc tcgcgggatc gtacggatgg tgtcgacgcg ggcgctgtcg atgcgggtgc 3480 tgtcgatgcg ggtgctgtcg ctcgcgtact ggccggcggg cgtgctcagt tcgagcaccg 3540 ggccgtcgtc gtcggcagcg ggccggacga tctggcggca gcgctggccg cgcctgaggg 3600 tctggtccgg ggcgtggctt ccggtgtcgg gcgagtggcg ttcgtgttcc ccgggcaggg 3660 cacgcagtgg gccggcatgg gtgccgaact gctggactct tccgcggtgt tcgcggcggc 3720 catggccgaa tgcgaggccg cactctcccc gtacgtcgac tggtcgctgg aggccgtcgt 3780 acggcaggcc cccggtgcgc ccacgctgga gcgggtcgat gtcgtgcagc ctgtgacgtt 3840 cgccgtcatg gtctcgctgg ctcgcgtgtg gcagcaccac ggggtgacgc cccaggcggt 3900 cgtcggccac tcgcagggcg agatcgccgc cgcgtacgtc gccggtgccc tgagcctgga 3960 cgacgccgct cgtgtcgtga ccctgcgcag caagtccatc gccgcccacc tcgccggcaa 4020 gggcggcatg ctgtccctcg cgctgagcga ggacgccgtc ctggagcgac tggccgggtt 4080 cgacgggctg tccgtcgccg ctgtgaacgg gcccaccgcc accgtggtct ccggtgaccc 4140 cgtacagatc gaagagcttg ctcgggcgtg tgaggccgat ggggtccgtg cgcgggtcat 4200 tcccgtcgac tacgcgtccc acagccggca ggtcgagatc atcgagagcg agctcgccga 4260 ggtcctcgcc gggctcagcc cgcaggctcc gcgcgtgccg ttcttctcga cactcgaagg 4320 cgcctggatc accgagcccg tgctcgacgg cggctactgg taccgcaacc tgcgccatcg 4380 tgtgggcttc gccccggccg tcgagaccct ggccaccgac gagggcttca cccacttcgt 4440 cgaggtcagc gcccaccccg tcctcaccat ggccctcccc gggaccgtca ccggtctggc 4500 gaccctgcgt cgcgacaacg gcggtcagga ccgcctagtc gcctccctcg ccgaagcatg 4560 ggccaacgga ctcgcggtcg actggagccc gctcctcccc tccgcgaccg gccaccactc 4620 cgacctcccc acctacgcgt tccagaccga gcgccactgg ctgggcgaga tcgaggcgct 4680 cgccccggcg ggcgagccgg cggtgcagcc cgccgtcctc cgcacggagg cggccgagcc 4740 ggcggagctc gaccgggacg agcagctgcg cgtgatcctg gacaaggtcc gggcgcagac 4800 ggcccaggtg ctggggtacg cgacaggcgg gcagatcgag gtcgaccgga ccttccgtga 4860 ggccggttgc acctccctga ccggcgtgga cctgcgcaac cggatcaacg ccgccttcgg 4920 cgtacggatg gcgccgtcca tgatcttcga cttccccacc cccgaggctc tcgcggagca 4980 gctgctcctc gtcgtgcacg gggaggcggc ggcgaacccg gccggtgcgg agccggctcc 5040 ggtggcggcg gccggtgccg tcgacgagcc ggtggcgatc gtcggcatgg cctgccgcct 5100 gcccggtggg gtcgcctcgc cggaggacct gtggcggctg gtggccggcg gcggggacgc 5160 gatctcggag ttcccgcagg accgcggctg ggacgtggag gggctgtacc acccggatcc 5220 ggagcacccc ggcacgtcgt acgtccgcca gggcggtttc atcgagaacg tcgccggctt 5280 cgacgcggcc ttcttcggga tctcgccgcg cgaggccctc gccatggacc cgcagcagcg 5340 gctcctcctc gaaacctcct gggaggccgt cgaggacgcc gggatcgacc cgacctccct 5400 gcggggacgg caggtcggcg tcttcactgg ggcgatgacc cacgagtacg ggccgagcct 5460 gcgggacggc ggggaaggcc tcgacggcta cctgctgacc ggcaacacgg ccagcgtgat 5520 gtcgggccgc gtctcgtaca cactcggcct tgagggcccc gccctgacgg tggacacggc 5580 ctgctcgtcg tcgctggtcg ccctgcacct cgccgtgcag gccctgcgca agggcgaggt 5640 cgacatggcg ctcgccggcg gcgtggccgt gatgcccacg cccgggatgt tcgtcgagtt 5700 cagccggcag cgcgggctgg ccggggacgg ccggtcgaag gcgttcgccg cgtcggcgga 5760 cggcaccagc tggtccgagg gcgtcggcgt cctcctcgtc gagcgcctgt cggacgcccg 5820 ccgcaacgga caccaggtcc tcgcggtcgt ccgcggcagc gccttgaacc aggacggcgc 5880 gagcaacggc ctcacggctc cgaacgggcc ctcgcagcag cgcgtcatcc ggcgcgcgct 5940 ggcggacgcc cggctgacga cctccgacgt ggacgtcgtc gaggcacacg gcacgggcac 6000 gcgactcggc gacccgatcg aggcgcaggc cctgatcgcc acctacggcc agggccgtga 6060 cgacgaacag ccgctgcgcc tcgggtcgtt gaagtccaac atcgggcaca cccaggccgc 6120 ggccggcgtc tccggtgtca tcaagatggt ccaggcgatg cgccacggac tgctgccgaa 6180 gacgctgcac gtcgacgagc cctcggacca gatcgactgg tcggctggcg ccgtggaact 6240 cctcaccgag gccgtcgact ggccggagaa gcaggacggc gggctgcgcc gggccgccgt 6300 ctcctccttc gggatcagcg gcaccaatgc gcatgtggtg ctcgaagagg ccccggtggt 6360 tgtcgagggt gcttcggtcg tcgagccgtc ggttggcggg tcggcggtcg gcggcggtgt 6420 gacgccttgg gtggtgtcgg cgaagtccgc tgccgcgctc gacgcgcaga tcgagcggct 6480 tgccgcattc gcctcgcggg atcgtacgga tgacgccgac gccggtgctg tcgacgcggg 6540 cgctgtcgct cacgtactgg ctgacgggcg tgctcagttc gagcaccggg ccgtcgcgct 6600 cggcgccggg gcggacgacc tcgtacaggc gctggccgat ccggacgggc tgatacgcgg 6660 aacggcttcc ggtgtcgggc gagtggcgtt cgtgttcccc ggtcagggca cgcagtgggc 6720 tggcatgggt gccgaactgc tggactcttc cgcggtgttc gcggcggcca tggccgagtg 6780 tgaggccgcg ctgtccccgt acgtcgactg gtcgctggag gccgtcgtac ggcaggcccc 6840 cggtgcgccc acgctggagc gggtcgatgt cgtgcagcct gtgacgttcg ccgtcatggt 6900 ctcgctggct cgcgtgtggc agcaccacgg tgtgacgccc caggcggtcg tcggccactc 6960 gcagggcgag atcgccgccg cgtacgtcgc cggagccctg cccctggacg acgccgcccg 7020 cgtcgtcacc ctgcgcagca agtccatcgc cgcccacctc gccggcaagg gcggcatgct 7080 gtccctcgcg ctgaacgagg acgccgtcct ggagcgactg agtgacttcg acgggctgtc 7140 cgtcgccgcc gtcaacgggc ccaccgccac tgtcgtgtcg ggtgaccccg tacagatcga 7200 agagcttgct caggcgtgca aggcggacgg attccgcgcg cggatcattc ccgtcgacta 7260 cgcgtcccac agccggcagg tcgagatcat cgagagcgag ctcgcccagg tcctcgccgg 7320 tctcagcccg caggccccgc gcgtgccgtt cttctcgacg ctcgaaggca cctggatcac 7380 cgagcccgtc ctcgacggca cctactggta ccgcaacctc cgtcaccgcg tcggcttcgc 7440 ccccgccatc gagaccctgg ccgtcgacga gggcttcacg cacttcgtcg aggtcagcgc 7500 ccaccccgtc ctcaccatga ccctccccga gaccgtcacc ggcctcggca ccctccgtcg 7560 cgaacaggga ggccaagagc gtctggtcac ctcgctcgcc gaggcgtggg tcaacgggct 7620 tcccgtggca tggacttcgc tcctgcccgc cacggcctcc cgccccggtc tgcccaccta 7680 cgccttccag gccgagcgct actggctcga gaacactccc gccgccctgg ccaccggcga 7740 cgactggcgc taccgcatcg actggaagcg cctcccggcc gccgaggggt ccgagcgcac 7800 cggcctgtcc ggccgctggc tcgccgtcac gccggaggac cactccgcgc aggccgccgc 7860 cgtgctcacc gcgctggtcg acgccggggc gaaggtcgag gtgctgacgg ccggggcgga 7920 cgacgaccgt gaggccctcg ccgcccggct caccgcactg acgaccggtg acggcttcac 7980 cggcgtggtc tcgctcctcg acggactcgt accgcaggtc gcctgggtcc aggcgctcgg 8040 cgacgccgga atcaaggcgc ccctgtggtc cgtcacccag ggcgcggtct ccgtcggacg 8100 tctcgacacc cccgccgacc ccgaccgggc catgctctgg ggcctcggcc gcgtcgtcgc 8160 ccttgagcac cccgaacgct gggccggcct cgtcgacctc cccgcccagc ccgatgccgc 8220 cgccctcgcc cacctcgtca ccgcactctc cggcgccacc ggcgaggacc agatcgccat 8280 ccgcaccacc ggactccacg cccgccgcct cgcccgcgca cccctccacg gacgtcggcc 8340 cacccgcgac tggcagcccc acggcaccgt cctcatcacc ggcggcaccg gagccctcgg 8400 cagccacgcc gcacgctgga tggcccacca cggagccgaa cacctcctcc tcgtcagccg 8460 cagcggcgaa caagcccccg gagccaccca actcaccgcc gaactcaccg catcgggcgc 8520 ccgcgtcacc atcgccgcct gcgacgtcgc cgacccccac gccatgcgca ccctcctcga 8580 cgccatcccc gccgagacgc ccctcaccgc cgtcgtccac accgccggcg cgctcgacga 8640 cggcatcgtg gacacgctga ccgccgagca ggtccggcgg gcccaccgtg cgaaggccgt 8700 cggcgcctcg gtgctcgacg agctgacccg ggacctcgac ctcgacgcgt tcgtgctctt 8760 ctcgtccgtg tcgagcactc tgggcatccc cggtcagggc aactacgccc cgcacaacgc 8820 ctacctcgac gccctcgcgg ctcgccgccg ggccaccggc cggtccgccg tctcggtggc 8880 ctggggaccg tgggacggtg gcggcatggc cgccggtgac ggcgtggccg agcggctgcg 8940 caaccacggc gtgcccggca tggacccgga actcgccctg gccgcactgg agtccgcgct 9000 cggccgggac gagaccgcga tcaccgtcgc ggacatcgac tgggaccgct tctacctcgc 9060 gtactcctcc ggtcgcccgc agcccctcgt cgaggagctg cccgaggtgc ggcgcatcat 9120 cgacgcacgg gacagcgcca cgtccggaca gggcgggagc tccgcccagg gcgccaaccc 9180 cctggccgag cggctggccg ccgcggctcc cggcgagcgt acggagatcc tcctcggtct 9240 cgtacgggcg caggccgccg ccgtgctccg gatgcgttcg ccggaggacg tcgccgccga 9300 ccgcgccttc aaggacatcg gcttcgactc gctcgccggt gtcgagctgc gcaacaggct 9360 gacccgggcg accgggctcc agctgcccgc gacgctcgtc ttcgaccacc cgacgccgct 9420 ggccctcgtg tcgctgctcc gcagcgagtt cctcggtgac gaggagacgg cggacgcccg 9480 gcggtccgcg gcgctgcccg cgactgtcgg tgccggtgcc ggcgccggcg ccggcaccga 9540 tgccgacgac gatccgatcg cgatcgtcgc gatgagctgc cgctaccccg gtgacatccg 9600 cagcccggag gacctgtggc ggatgctgtc cgagggcggc gagggcatca cgccgttccc 9660 caccgaccgc ggctgggacc tcgacggcct gtacgacgcc gacccggacg cgctcggcag 9720 ggcgtacgtc cgcgagggcg ggttcctgca cgacgcggcc gagttcgacg cggagttctt 9780 cggcgtctcg ccgcgcgagg cgctggccat ggacccgcag cagcggatgc tcctgacgac 9840 gtcctgggag gccttcgagc gggccggcat cgagccggca tcgctgcgcg gcagcagcac 9900 cggtgtcttc atcggcctct cctaccagga ctacgcggcc cgcgtcccga acgccccgcg 9960 tggcgtggag ggttacctgc tgaccggcag cacgccgagc gtcgcgtcgg gccgtatcgc 10020 gtacaccttc ggtctcgaag ggcccgcgac gaccgtcgac accgcctgct cgtcgtcgct 10080 gaccgccctg cacctggcgg tgcgggcgct gcgcagcggc gagtgcacga tggcgctcgc 10140 cggtggcgtg gcgatgatgg cgaccccgca catgttcgtg gagttcagcc gtcagcgggc 10200 gctcgccccg gacggccgca gcaaggcctt ctcggcggac gccgacgggt tcggcgccgc 10260 ggagggcgtc ggcctgctgc tcgtggagcg gctctcggac gcgcggcgca acggtcaccc 10320 ggtgctcgcc gtggtccgcg gtaccgccgt caaccaggac ggcgccagca acgggctgac 10380 cgcgcccaac ggaccctcgc agcagcgggt gatccggcag gcgctcgccg acgcccggct 10440 ggcacccggc gacatcgacg ccgtcgagac gcacggcacg ggaacctcgc tgggcgaccc 10500 catcgaggcc cagggcctcc aggccacgta cggcaaggag cggcccgcgg aacggccgct 10560 cgccatcggc tccgtgaagt ccaacatcgg acacacccag gccgcggccg gtgcggcggg 10620 catcatcaag atggtcctcg cgatgcgcca cggcaccctg ccgaagaccc tccacgccga 10680 cgagccgagc ccgcacgtcg actgggcgaa cagcggcctg gccctcgtca ccgagccgat 10740 cgactggccg gccggcaccg gtccgcgccg cgccgccgtc tcctccttcg gcatcagcgg 10800 gacgaacgcg cacgtcgtgc tggagcaggc gccggatgct gctggtgagg tgcttggggc 10860 cgatgaggtg cctgaggtgt ctgagacggt agcgatggct gggacggctg ggacctccga 10920 ggtcgctgag ggctctgagg cctccgaggc ccccgcggcc cccggcagcc gtgaggcgtc 10980 cctccccggg cacctgccct gggtgctgtc cgccaaggac gagcagtcgc tgcgcggcca 11040 ggccgccgcc ctgcacgcgt ggctgtccga gcccgccgcc gacctgtcgg acgcggacgg 11100 accggcccgc ctgcgggacg tcgggtacac gctcgccacg agccgtaccg ccttcgcgca 11160 ccgcgccgcc gtgaccgccg ccgaccggga cgggttcctg gacgggctgg ccacgctggc 11220 ccagggcggc acctcggccc acgtccacct ggacaccgcc cgggacggca ccaccgcgtt 11280 cctcttcacc ggccagggca gtcagcgccc cggcgccggc cgtgagctgt acgaccggca 11340 ccccgtcttc gcccgggcgc tcgacgagat ctgcgcccac ctcgacggtc acctcgaact 11400 gcccctgctc gacgtgatgt tcgcggccga gggcagcgcg gaggccgcgc tgctcgacga 11460 gacgcggtac acgcagtgcg cgctgttcgc cctggaggtc gcgctcttcc ggctcgtcga 11520 gagctggggc atgcggccgg ccgcactgct cggtcactcg gtcggcgaga tcgccgccgc 11580 gcacgtcgcc ggtgtgttct cgctcgccga cgccgcccgc ctggtcgccg cgcgcggccg 11640 gctcatgcag gagctgcccg ccggtggcgc gatgctcgcc gtccaggccg cggaggacga 11700 gatccgcgtg tggctggaga cggaggagcg gtacgcggga cgtctggacg tcgccgccgt 11760 caacggcccc gaggccgccg tcctgtccgg cgacgcggac gcggcgcggg aggcggaggc 11820 gtactggtcc gggctcggcc gcaggacccg cgcgctgcgg gtcagccacg ccttccactc 11880 cgcgcacatg gacggcatgc tcgacgggtt ccgcgccgtc ctggagacgg tggagttccg 11940 gcgcccctcc ctgaccgtgg tctcgaacgt caccggcctg gccgccggcc cggacgacct 12000 gtgcgacccc gagtactggg tccggcacgt ccgcggcacc gtccgcttcc tcgacggcgt 12060 ccgtgtcctg cgcgacctcg gcgtgcggac ctgcctggag ctgggccccg acggggtcct 12120 caccgccatg gcggccgacg gcctcgcgga cacccccgcg gattccgctg ccggctcccc 12180 cgtcggctct cccgccggct ctcccgccga ctccgccgcc ggcgcgctcc ggccccggcc 12240 gctgctcgtg gcgctgctgc gccgcaagcg gtcggagacc gagaccgtcg cggacgccct 12300 cggcagggcg cacgcccacg gcaccggacc cgactggcac gcctggttcg ccggctccgg 12360 ggcgcaccgc gtggacctgc ccacgtactc cttccggcgc gaccgctact ggctggacgc 12420 cccggcggcc gacaccgcgg tggacaccgc cggcctcggt ctcggcaccg ccgaccaccc 12480 gctgctcggc gccgtggtca gccttccgga ccgggacggc ctgctgctca ccggccgcct 12540 ctccctgcgc acccacccgt ggctcgcgga ccacgccgtc ctggggagcg tcctgctccc 12600 cggcgccgcg atggtcgaac tcgccgcgca cgctgcggag tccgccggtc tgcgtgacgt 12660 gcgggagctg accctccttg aaccgctggt actgcccgag cacggtggcg tcgagctgcg 12720 cgtgacggtc ggggcgccgg ccggagagcc cggtggcgag tcggccgggg acggcgcacg 12780 gcccgtctcc ctccactcgc ggctcgccga cgcgcccgcc ggtaccgcct ggtcctgcca 12840 cgcgaccggt ctgctggcca ccgaccggcc cgagcttccc gtcgcgcccg accgtgcggc 12900 catgtggccg ccgcagggcg ccgaggaggt gccgctcgac ggtctctacg agcggctcga 12960 cgggaacggc ctcgccttcg gtccgctgtt ccaggggctg aacgcggtgt ggcggtacga 13020 gggtgaggtc ttcgccgaca tcgcgctccc cgccaccacg aatgcgaccg cgcccgcgac 13080 cgcgaacggc ggcgggagtg cggcggcggc cccctacggc atccaccccg ccctgctcga 13140 cgcttcgctg cacgccatcg cggtcggcgg tctcgtcgac gagcccgagc tcgtccgcgt 13200 ccccttccac tggagcggtg tcaccgtgca cgcggccggt gccgcggcgg cccgggtccg 13260 tctcgcctcc gcggggacgg acgccgtctc gctgtccctg acggacggcg agggacgccc 13320 gctggtctcc gtggaacggc tcacgctgcg cccggtcacc gccgatcagg cggcggcgag 13380 ccgcgtcggc gggctgatgc accgggtggc ctggcgtccg tacgccctcg cctcgtccgg 13440 cgaacaggac ccgcacgcca cttcgtacgg gccgaccgcc gtcctcggca aggacgagct 13500 gaaggtcgcc gccgccctgg agtccgcggg cgtcgaagtc gggctctacc ccgacctggc 13560 cgcgctgtcc caggacgtgg cggccggcgc cccggcgccc cgtaccgtcc ttgcgccgct 13620 gcccgcgggt cccgccgacg gcggcgcgga gggtgtacgg ggcacggtgg cccggacgct 13680 ggagctgctc caggcctggc tggccgacga gcacctcgcg ggcacccgcc tgctcctggt 13740 cacccgcggt gcggtgcggg accccgaggg gtccggcgcc gacgatggcg gcgaggacct 13800 gtcgcacgcg gccgcctggg gtctcgtacg gaccgcgcag accgagaacc ccggccgctt 13860 cggccttctc gacctggccg acgacgcctc gtcgtaccgg accctgccgt cggtgctctc 13920 cgacgcgggc ctgcgcgacg aaccgcagct cgccctgcac gacggcacca tcaggctggc 13980 ccgcctggcc tccgtccggc ccgagaccgg caccgccgca ccggcgctcg ccccggaggg 14040 cacggtcctg ctgaccggcg gcaccggcgg cctgggcgga ctggtcgccc ggcacgtggt 14100 gggcgagtgg ggcgtacgac gcctgctgct ggtgagccgg cggggcacgg acgccccggg 14160 cgccgacgag ctcgtgcacg agctggaggc cctgggagcc gacgtctcgg tggccgcgtg 14220 cgacgtcgcc gaccgcgaag ccctcaccgc cgtactcgac gccatccccg ccgaacaccc 14280 gctcaccgcg gtcgtccaca cggcaggcgt cctctccgac ggcaccctcc cgtccatgac 14340 gacggaggac gtggaacacg tactgcggcc caaggtcgac gccgcgttcc tcctcgacga 14400 actcacctcg acgcccgcat acgacctggc agcgttcgtc atgttctcct ccgccgccgc 14460 cgtcttcggt ggcgcggggc agggcgccta cgccgccgcc aacgccaccc tcgacgccct 14520 cgcctggcgc cgccgggcag ccggactccc cgccctctcc ctcggctggg gcctctgggc 14580 cgagaccagc ggcatgaccg gcgagctcgg ccaggcggac ctgcgccgga tgagccgcgc 14640 gggcatcggc gggatcagcg acgccgaggg catcgcgctc ctcgacgccg ccctccgcga 14700 cgaccgccac ccggtcctgc tgcccctgcg gctcgacgcc gccgggctgc gggacgcggc 14760 cgggaacgac ccggccggaa tcccggcgct cttccgggac gtcgtcggcg ccaggaccgt 14820 ccgggcccgg ccgtccgcgg cctccgcctc gacgacagcc gggacggccg gcacgccggg 14880 gacggcggac ggcgcggcgg aaacggcggc ggtcacgctc gccgaccggg ccgccaccgt 14940 ggacgggccc gcacggcagc gcctgctgct cgagttcgtc gtcggcgagg tcgccgaagt 15000 actcggccac gcccgcggtc accggatcga cgccgaacgg ggcttcctcg acctcggctt 15060 cgactccctg accgccgtcg aactccgcaa ccggctcaac tccgccggtg gcctcgccct 15120 cccggcgacc ctggtcttcg accacccaag cccggcggca ctcgcctccc acctggacgc 15180 cgagctgccg cgcggcgcct cggaccagga cggagccggg aaccggaacg ggaacgagaa 15240 cgggacgacg gcgtcccgga gcaccgccga gacggacgcg ctgctggcac aactgacccg 15300 cctggaaggc gccttggtgc tgacgggcct ctcggacgcc cccgggagcg aagaagtcct 15360 ggagcacctg cggtccctgc gctcgatggt cacgggcgag accgggaccg ggaccgcgtc 15420 cggagccccg gacggcgccg ggtccggcgc cgaggaccgg ccctgggcgg ccggggacgg 15480 agccgggggc gggagtgagg acggcgcggg agtgccggac ttcatgaacg cctcggccga 15540 ggaactcttc ggcctcctcg accaggaccc cagcacggac tgatccctgc cgcacggtcg 15600 cctcccgccc cggaccccgt cccgggcacc tcgactcgaa tcacttcatg cgcgcctcgg 15660 gcgcctccag gaactcaagg ggacagcgtg tccacggtga acgaagagaa gtacctcgac 15720 tacctgcgtc gtgccacggc ggacctccac gaggcccgtg gccgcctccg cgagctggag 15780 gcgaaggcgg gcgagccggt ggcgatcgtc ggcatggcct gccgcctgcc cggcggcgtc 15840 gcctcgcccg aggacctgtg gcggctggtg gccggcggcg aggacgcgat ctcggagttc 15900 ccccaggacc gcggctggga cgtggagggc ctgtacgacc cgaacccgga ggccacgggc 15960 aagagttacg cccgcgaggc cggattcctg tacgaggcgg gcgagttcga cgccgacttc 16020 ttcgggatct cgccgcgcga ggccctcgcc atggacccgc agcagcgtct cctcctggag 16080 gcctcctggg aggcgttcga gcacgccggg atcccggcgg ccaccgcgcg cggcacctcg 16140 gtcggcgtct tcaccggcgt gatgtaccac gactacgcca cccgtctcac cgatgtcccg 16200 gagggcatcg agggctacct gggcaccggc aactccggca gtgtcgcctc gggccgcgtc 16260 gcgtacacgc ttggcctgga ggggccggcc gtcacggtcg acaccgcctg ctcgtcctcg 16320 ctggtcgccc tgcacctcgc cgtgcaggcc ctgcgcaagg gcgaggtcga catggcgctc 16380 gccggcggcg tgacggtcat gtcgacgccc agcaccttcg tcgagttcag ccgtcagcgc 16440 gggctggcgc cggacggccg gtcgaagtcc ttctcgtcga cggccgacgg caccagctgg 16500 tccgagggcg tcggcgtcct cctcgtcgag cgcctgtccg acgcgcgtcg caagggccat 16560 cggatcctcg ccgtggtccg gggcaccgcc gtcaaccagg acggcgccag cagcggcctc 16620 acggctccga acgggccgtc gcagcagcgc gtcatccgac gtgccctggc ggacgcccgg 16680 ctcacgacct ccgacgtgga cgtcgtcgag gcccacggca cgggtacgcg actcggcgac 16740 ccgatcgagg cgcaggccgt catcgccacg tacgggcagg gccgtgacgg cgaacagccg 16800 ctgcgcctcg ggtcgttgaa gtccaacatc ggacacaccc aggccgccgc cggtgtctcc 16860 ggcgtgatca agatggtcca ggcgatgcgc cacggcgtcc tgccgaagac gctccacgtg 16920 gagaagccga cggaccaggt ggactggtcc gcgggcgcgg tcgagctgct caccgaggcc 16980 atggactggc cggacaaggg cgacggcgga ctgcgcaggg ccgcggtctc ctccttcggc 17040 gtcagcggga cgaacgcgca cgtcgtgctc gaagaggccc cggcggccga ggagacccct 17100 gcctccgagg cgaccccggc cgtcgagccg tcggtcggcg ccggcctggt gccgtggctg 17160 gtgtcggcga agactccggc cgcgctggac gcccagatcg gacgcctcgc cgcgttcgcc 17220 tcgcagggcc gtacggacgc cgccgatccg ggcgcggtcg ctcgcgtact ggccggcggg 17280 cgcgccgagt tcgagcaccg ggccgtcgtg ctcggcaccg gacaggacga tttcgcgcag 17340 gcgctgaccg ctccggaagg actgatacgc ggcacgccct cggacgtggg ccgggtggcg 17400 ttcgtgttcc ccggtcaggg cacgcagtgg gccgggatgg gcgccgaact cctcgacgtg 17460 tcgaaggagt tcgcggcggc catggccgag tgcgagagcg cgctctcccg ctatgtcgac 17520 tggtcgctgg aggccgtcgt ccggcaggcg ccgggcgcgc ccacgctgga gcgggtcgac 17580 gtcgtccagc ccgtgacctt cgctgtcatg gtttcgctgg cgaaggtctg gcagcaccac 17640 ggcgtgacgc cgcaggccgt cgtcggccac tcgcagggcg agatcgccgc cgcgtacgtc 17700 gccggtgccc tcaccctcga cgacgccgcc cgcgtcgtca ccctgcgcag caagtccatc 17760 gccgcccacc tcgccggcaa gggcggcatg atctccctcg ccctcagcga ggaagccacc 17820 cggcagcgca tcgagaacct ccacggactg tcgatcgccg ccgtcaacgg ccccaccgcc 17880 accgtggttt cgggcgaccc cacccagatc caagagctcg ctcaggcgtg tgaggccgac 17940 ggggtccgcg cacggatcat ccccgtcgac tacgcctccc acagcgccca cgtcgagacc 18000 atcgagagcg aactcgccga ggtcctcgcc gggctcagcc cgcggacacc tgaggtgccg 18060 ttcttctcga cactcgaagg cgcctggatc accgagccgg tgctcgacgg cacctactgg 18120 taccgcaacc tccgccaccg cgtcggcttc gcccccgccg tcgagaccct cgccaccgac 18180 gaaggcttca cccacttcat cgaggtcagc gcccaccccg tcctcaccat gaccctcccc 18240 gagaccgtca ccggcctcgg caccctccgc cgcgaacagg gaggccagga gcgtctggtc 18300 acctcactcg ccgaagcctg gaccaacggc ctcaccatcg actgggcgcc cgtcctcccc 18360 accgcaaccg gccaccaccc cgagctcccc acctacgcct tccagcgccg tcactactgg 18420 ctccacgact cccccgccgt ccagggctcc gtgcaggact cctggcgcta ccgcatcgac 18480 tggaagcgcc tcgcggtcgc cgacgcgtcc gagcgcgccg ggctgtccgg gcgctggctc 18540 gtcgtcgtcc ccgaggaccg ttccgccgag gccgccccgg tgctcgccgc gctgtccggc 18600 gccggcgccg accccgtaca gctggacgtg tccccgctgg gcgaccggca gcggctcgcc 18660 gcgacgctgg gcgaggccct ggcggcggcc ggtggagccg tcgacggcgt cctctcgctg 18720 ctcgcgtggg acgagagcgc gcaccccggc caccccgccc ccttcacccg gggcaccggc 18780 gccaccctca ccctggtgca ggcgctggag gacgccggcg tcgccgcccc gctgtggtgc 18840 gtgacccacg gcgcggtgtc cgtcggccgg gccgaccacg tcacctcccc cgcccaggcc 18900 atggtgtggg gcatgggccg ggtcgccgcc ctggagcacc ccgagcggtg gggcggcctg 18960 atcgacctgc cctcggacgc cgaccgggcg gccctggacc gcatgaccac ggtcctcgcc 19020 ggcggtacgg gtgaggacca ggtcgcggta cgcgcctccg ggctgctcgc ccgccgcctc 19080 gtccgcgcct ccctcccggc gcacggcacg gcttcgccgt ggtggcaggc cgacggcacg 19140 gtgctcgtca ccggtgccga ggagcctgcg gccgccgagg ccgcacgccg gctggcccgc 19200 gacggcgccg gacacctcct cctccacacc accccctccg gcagcgaagg cgccgaaggc 19260 acctccggtg ccgccgagga ctccggcctc gccgggctcg tcgccgaact cgcggacctg 19320 ggcgcgacgg ccaccgtcgt gacctgcgac ctcacggacg cggaggcggc cgcccggctg 19380 ctcgccggcg tctccgacgc gcacccgctc agcgccgtcc tccacctgcc gcccaccgtc 19440 gactccgagc cgctcgccgc gaccgacgcg gacgcgctcg cccgtgtcgt gaccgcgaag 19500 gccaccgccg cgctccacct ggaccgcctc ctgcgggagg ccgcggctgc cggaggccgt 19560 ccgcccgtcc tggtcctctt ctcctcggtc gccgcgatct ggggcggcgc cggtcagggc 19620 gcgtacgccg ccggtacggc cttcctcgac gccctcgccg gtcagcaccg ggccgacggc 19680 cccaccgtga cctcggtggc ctggagcccc tgggagggca gccgcgtcac cgagggtgcg 19740 accggggagc ggctgcgccg cctcggcctg cgccccctcg cccccgcgac ggcgctcacc 19800 gccctggaca ccgcgctcgg ccacggcgac accgccgtca cgatcgccga cgtcgactgg 19860 tcgagcttcg cccccggctt caccacggcc cggccgggca ccctcctcgc cgatctgccc 19920 gaggcgcgcc gcgcgctcga cgagcagcag tcgacgacgg ccgccgacga caccgtcctg 19980 agccgcgagc tcggtgcgct caccggcgcc gaacagcagc gccgtatgca ggagttggtc 20040 cgcgagcacc tcgccgtggt cctcaaccac ccctcccccg aggccgtcga cacggggcgg 20100 gccttccgtg acctcggatt cgactcgctg acggcggtcg agctccgcaa ccgcctcaag 20160 aacgccaccg gcctggccct cccggccact ctggtcttcg actacccgac cccccggacg 20220 ctggcggagt tcctcctcgc ggagatcctg ggcgagcagg ccggtgccgg cgagcagctt 20280 ccggtggacg gcggggtcga cgacgagccc gtcgcgatcg tcggcatggc gtgccgcctg 20340 ccgggcggtg tcgcctcgcc ggaggacctg tggcggctgg tggccggcgg cgaggacgcg 20400 atctccggct tcccgcagga ccgcggctgg gacgtggagg ggctgtacga cccggacccg 20460 gacgcgtccg ggcggacgta ctgccgtgcc ggtggcttcc tcgacgaggc gggcgagttc 20520 gacgccgact tcttcgggat ctcgccgcgc gaggccctcg ccatggaccc gcagcagcgg 20580 ctcctcctgg agacctcctg ggaggccgtc gaggacgccg ggatcgaccc gacctccctt 20640 caggggcagc aggtcggcgt gttcgcgggc accaacggcc cccactacga gccgctgctc 20700 cgcaacaccg ccgaggatct tgagggttac gtcgggacgg gcaacgccgc cagcatcatg 20760 tcgggccgtg tctcgtacac cctcggcctg gagggcccgg ccgtcacggt cgacaccgcc 20820 tgctcctcct cgctggtcgc cctgcacctc gccgtgcagg ccctgcgcaa gggcgaatgc 20880 ggactggcgc tcgcgggcgg tgtgacggtc atgtcgacgc ccacgacgtt cgtggagttc 20940 agccggcagc gcgggctcgc ggaggacggc cggtcgaagg cgttcgccgc gtcggcggac 21000 ggcttcggcc cggcggaggg cgtcggcatg ctcctcgtcg agcgcctgtc ggacgcccgc 21060 cgcaacggac accgtgtgct ggcggtcgtg cgcggcagcg cggtcaacca ggacggcgcg 21120 agcaacggcc tgaccgcccc gaacgggccc tcgcagcagc gcgtcatccg gcgcgcgctc 21180 gcggacgccc gactgacgac cgccgacgtg gacgtcgtcg aggcccacgg cacgggcacg 21240 cgactcggcg acccgatcga ggcacaggcc ctcatcgcca cctacggcca ggggcgcgac 21300 accgaacagc cgctgcgcct ggggtcgttg aagtccaaca tcggacacac ccaggccgcc 21360 gccggtgtct ccggcatcat caagatggtc caggcgatgc gccacggcgt cctgccgaag 21420 acgctccacg tggaccggcc gtcggaccag atcgactggt cggcgggcac ggtcgagctg 21480 ctcaccgagg ccatggactg gccgaggaag caggagggcg ggctgcgccg cgcggccgtc 21540 tcctccttcg gcatcagcgg cacgaacgcg cacatcgtgc tcgaagaagc cccggtcgac 21600 gaggacgccc cggcggacga gccgtcggtc ggcggtgtgg tgccgtggct cgtgtccgcg 21660 aagactccgg ccgcgctgga cgcccagatc ggacgcctcg ccgcgttcgc ctcgcagggc 21720 cgtacggacg ccgccgatcc gggcgcggtc gctcgcgtac tggccggcgg gcgtgcgcag 21780 ttcgagcacc gggccgtcgc gctcggcacc ggacaggacg acctggcggc cgcactggcc 21840 gcgcctgagg gtctggtccg gggtgtggcc tccggtgtgg gtcgagtggc gttcgtgttc 21900 ccgggacagg gcacgcagtg ggccgggatg ggtgccgaac tcctcgacgt gtcgaaggag 21960 ttcgcggcgg ccatggccga gtgcgaggcc gcgctcgctc cgtacgtgga ctggtcgctg 22020 gaggccgtcg tccgacaggc ccccggcgcg cccacgctgg agcgggtcga tgtcgtccag 22080 cccgtgacgt tcgccgtcat ggtctcgctg gcgaaggtct ggcagcacca cggggtgacc 22140 ccgcaagccg tcgtcggcca ctcgcagggc gagatcgccg ccgcgtacgt cgccggtgcc 22200 ctgagcctgg acgacgccgc tcgtgtcgtg accctgcgca gcaagtccat cggcgcccac 22260 ctcgcgggcc agggcggcat gctgtccctc gcgctgagcg aggcggccgt tgtggagcga 22320 ctggccgggt tcgacgggct gtccgtcgcc gccgtcaacg ggcctaccgc caccgtggtt 22380 tcgggcgacc cgacccagat ccaagagctc gctcaggcgt gtgaggccga cggggtccgc 22440 gcacggatca tccccgtcga ctacgcctcc cacagcgccc acgtcgagac catcgagagc 22500 gaactcgccg acgtcctggc ggggttgtcc ccccagacac cccaggtccc cttcttctcc 22560 accctcgaag gcgcctggat caccgaaccc gccctcgacg gcggctactg gtaccgcaac 22620 ctccgccatc gtgtgggctt cgccccggcc gtcgaaaccc tggccaccga cgaaggcttc 22680 acccacttcg tcgaggtcag cgcccacccc gtcctcacca tggcgctgcc cgagaccgtc 22740 accggactcg gcaccctccg ccgtgacaac ggcggacagc accgcctcac cacctccctc 22800 gccgaggcct gggccaacgg cctcaccgtc gactgggcct ctctcctccc caccacgacc 22860 acccaccccg atctgcccac ctacgccttc cagaccgagc gctactggcc gcagcccgac 22920 ctctccgccg ccggtgacat cacctccgcc ggtctcgggg cggccgagca cccgctgctc 22980 ggcgcggccg tggcgctcgc ggactccgac ggctgcctgc tcacggggag cctctccctc 23040 cgtacgcacc cctggctggc ggaccacgcg gtggccggca ccgtgctgct gccgggaacg 23100 gcgttcgtgg agctggcgtt ccgagccggg gaccaggtcg gttgcgatct ggtcgaggag 23160 ctcaccctcg acgcgccgct cgtgctgccc cgtcgtggcg cggtccgtgt gcagctgtcc 23220 gtcggcgcga gcgacgagtc cgggcgtcgt accttcgggc tctacgcgca cccggaggac 23280 gcgccgggcg aggcggagtg gacgcggcac gccaccggtg tgctggccgc ccgtgcggac 23340 cgcaccgccc ccgtcgccga cccggaggcc tggccgccgc cgggcgccga gccggtggac 23400 gtggacggtc tgtacgagcg cttcgcggcg aacggctacg gctacggccc cctcttccag 23460 ggcgtccgtg gtgtctggcg gcgtggcgac gaggtgttcg ccgacgtggc cctgccggcc 23520 gaggtcgccg gtgccgaggg cgcgcggttc ggccttcacc cggcgctgct cgacgccgcc 23580 gtgcaggcgg ccggtgcggg ccggggcgtt cggcgcgggc acgcggctgc cgttcgcctg 23640 gagcgggatc tcctgtacgc ggtcggcgcc accgccctcc gcgtgcggct ggcccccgcc 23700 ggcccggaca cggtgtccgt gagcgccgcc gactcctccg ggcagccggt gttcgccgcg 23760 gactccctca cggtgctgcc cgtcgacccc gcgcagctgg cggccttcag cgacccgact 23820 ctggacgcgc tgcacctgct ggagtggacc gcctgggacg gtgccgcgca ggccctgccc 23880 ggcgcggtcg tgctgggcgg cgacgccgac ggtctcgccg cggcgctgcg cgccggtggc 23940 accgaggtcc tgtccttccc ggaccttacg gacctggtgg aggccgtcga ccggggcgag 24000 accccggccc cggcgaccgt cctggtggcc tgccccgccg ccggccccga tgggccggag 24060 catgtccgcg aggccctgca cgggtcgctc gcgctgatgc aggcctggct ggccgacgag 24120 cggttcaccg atgggcgcct ggtgctcgtg acccgcgacg cggtcgccgc ccgttccggc 24180 gacggcctgc ggtccacggg acaggccgcc gtctggggcc tcggccggtc cgcgcagacg 24240 gagagcccgg gccggttcgt cctgctcgac ctcgccgggg aagcccggac ggccggggac 24300 gccaccgccg gggacggcct gacgaccggg gacgccaccg tcggcggcac ctctggagac 24360 gccgccctcg gcagcgccct cgcgaccgcc ctcggctcgg gcgagccgca gctcgccctc 24420 cgggacgggg cgctcctcgt accccgcctg gcgcgggccg ccgcgcccgc cgcggccgac 24480 ggcctcgccg cggccgacgg cctcgccgct ctgccgctgc ccgccgctcc ggccctctgg 24540 cgtctggagc ccggtacgga cggcagcctg gagagcctca cggcggcgcc cggcgacgcc 24600 gagaccctcg ccccggagcc gctcggcccg ggacaggtcc gcatcgcgat ccgggccacc 24660 ggtctcaact tccgcgacgt cctgatcgcc ctcggcatgt accccgatcc ggcgctgatg 24720 ggcaccgagg gagccggcgt ggtcaccgcg accggccccg gcgtcacgca cctcgccccc 24780 ggcgaccggg tcatgggcct gctctccggc gcgtacgccc cggtcgtcgt ggcggacgcg 24840 cggaccgtcg cgcggatgcc cgaggggtgg acgttcgccc agggcgcctc cgtgccggtg 24900 gtgttcctga cggccgtcta cgccctgcgc gacctggcgg acgtcaagcc cggcgagcgc 24960 ctcctggtcc actccgccgc cggtggcgtg ggcatggccg ccgtgcagct cgcccggcac 25020 tggggcgtgg aggtccacgg cacggcgagt cacgggaagt gggacgccct gcgcgcgctc 25080 ggcctggacg acgcgcacat cgcctcctcc cgcaccctgg acttcgagtc cgcgttccgt 25140 gccgcttccg gcggggcggg catggacgtc gtactgaact cgctcgcccg cgagttcgtc 25200 gacgcctcgc tgcgcctgct cgggccgggc ggccggttcg tggagatggg gaagaccgac 25260 gtccgcgacg cggagcgggt cgccgccgac caccccggtg tcggctaccg cgccttcgac 25320 ctgggcgagg ccgggccgga gcggatcggc gagatgctcg ccgaggtcat cgccctcttc 25380 gaggacgggg tgctccggca cctgcccgtc acgacctggg acgtgcgccg ggcccgcgac 25440 gccttccggc acgtcagcca ggcccgccac acgggcaagg tcgtcctcac gatgccgtcg 25500 ggcctcgacc cggagggtac ggtcctgctg accggcggca ccggtgcgct ggggggcatc 25560 gtggcccggc acgtggtggg cgagtggggc gtacgacgcc tgctgctcgt gagccggcgg 25620 ggcacggacg ccccgggcgc cggcgagctc gtgcacgagc tggaggccct gggagccgac 25680 gtctcggtgg ccgcgtgcga cgtcgccgac cgcgaagccc tcaccgccgt actcgactcg 25740 atccccgccg aacacccgct caccgcggtc gtccacacgg caggcgtcct ctccgacggc 25800 accctcccct cgatgacagc ggaggatgtg gaacacgtac tgcgtcccaa ggtcgacgcc 25860 gcgttcctcc tcgacgaact cacctcgacg cccggctacg acctggcagc gttcgtcatg 25920 ttctcctccg ccgccgccgt cttcggtggc gcggggcagg gcgcctacgc cgccgccaac 25980 gccaccctcg acgccctcgc ctggcgccgc cggacagccg gactccccgc cctctccctc 26040 ggctggggcc tctgggccga gaccagcggc atgaccggcg gactcagcga caccgaccgc 26100 tcgcggctgg cccgttccgg ggcgacgccc atggacagcg agctgaccct gtccctcctg 26160 gacgcggcca tgcgccgcga cgacccggcg ctcgtcccga tcgccctgga cgtcgccgcg 26220 ctccgcgccc agcagcgcga cggcatgctg gcgccgctgc tcagcgggct cacccgcgga 26280 tcgcgggtcg gcggcgcgcc ggtcaaccag cgcagggcag ccgccggagg cgcgggcgag 26340 gcggacacgg acctcggcgg gcggctcgcc gcgatgacac cggacgaccg ggtcgcgcac 26400 ctgcgggacc tcgtccgtac gcacgtggcg accgtcctgg gacacggcac cccgagccgg 26460 gtggacctgg agcgggcctt ccgcgacacc ggtttcgact cgctcaccgc cgtcgaactc 26520 cgcaaccgtc tcaacgccgc gaccgggctg cggctgccgg ccacgctggt cttcgaccac 26580 cccaccccgg gggagctcgc cgggcacctg ctcgacgaac tcgccacggc cgcgggcggg 26640 tcctgggcgg aaggcaccgg gtccggagac acggcctcgg cgaccgatcg gcagaccacg 26700 gcggccctcg ccgaactcga ccggctggaa ggcgtgctcg cctccctcgc gcccgccgcc 26760 ggcggccgtc cggagctcgc cgcccggctc agggcgctgg ccgcggccct gggggacgac 26820 ggcgacgacg ccaccgacct ggacgaggcg tccgacgacg acctcttctc cttcatcgac 26880 aaggagctgg gcgactccga cttctgacct gcccgacacc accggcacca ccggcaccac 26940 cagcccccct cacacacgga acacggaacg gacaggcgag aacgggagcc atggcgaaca 27000 acgaagacaa gctccgcgac tacctcaagc gcgtcaccgc cgagctgcag cagaacacca 27060 ggcgtctgcg cgagatcgag ggacgcacgc acgagccggt ggcgatcgtg ggcatggcct 27120 gccgcctgcc gggcggtgtc gcctcgcccg aggacctgtg gcagctggtg gccggggacg 27180 gggacgcgat ctcggagttc ccgcaggacc gcggctggga cgtggagggg ctgtacgacc 27240 ccgacccgga cgcgtccggc aggacgtact gccggtccgg cggattcctg cacgacgccg 27300 gcgagttcga cgccgacttc ttcgggatct cgccgcgcga ggccctcgcc atggacccgc 27360 agcagcgact gtccctcacc accgcgtggg aggcgatcga gagcgcgggc atcgacccga 27420 cggccctgaa gggcagcggc ctcggcgtct tcgtcggcgg ctggcacacc ggctacacct 27480 cggggcagac caccgccgtg cagtcgcccg agctggaggg ccacctggtc agcggcgcgg 27540 cgctgggctt cctgtccggc cgtatcgcgt acgtcctcgg tacggacgga ccggccctga 27600 ccgtggacac ggcctgctcg tcctcgctgg tcgccctgca cctcgccgtg caggccctcc 27660 gcaagggcga gtgcgacatg gccctcgccg gtggtgtcac ggtcatgccc aacgcggacc 27720 tgttcgtgca gttcagccgg cagcgcgggc tggccgcgga cggccggtcg aaggcgttcg 27780 ccacctcggc ggacggcttc ggccccgcgg agggcgccgg agtcctgctg gtggagcgcc 27840 tgtcggacgc ccgccgcaac ggacaccgga tcctcgcggt cgtccgcggc agcgcggtca 27900 accaggacgg cgccagcaac ggcctcacgg ctccgcacgg gccctcccag cagcgcgtca 27960 tccgacgggc cctggcggac gcccggctcg cgccgggtga cgtggacgtc gtcgaggcgc 28020 acggcacggg cacgcggctc ggcgacccga tcgaggcgca ggccctcatc gccacctacg 28080 gccaggagaa gagcagcgaa cagccgctga ggctgggcgc gttgaagtcg aacatcgggc 28140 acacgcaggc cgcggccggt gtcgcaggtg tcatcaagat ggtccaggcg atgcgccacg 28200 gactgctgcc gaagacgctg cacgtcgacg agccctcgga ccagatcgac tggtcggcgg 28260 gcacggtgga actcctcacc gaggccgtcg actggccgga gaagcaggac ggcgggctgc 28320 gccgcgcggc tgtctcctcc ttcggcatca gcgggacgaa cgcgcacgtc gtcctggagg 28380 aggccccggc ggtcgaggac tccccggccg tcgagccgcc ggccggtggc ggtgtggtgc 28440 cgtggccggt gtccgcgaag actccggccg cgctggacgc ccagatcggg cagctcgccg 28500 cgtacgcgga cggtcgtacg gacgtggatc cggcggtggc cgcccgcgcc ctggtcgaca 28560 gccgtacggc gatggagcac cgcgcggtcg cggtcggcga cagccgggag gcactgcggg 28620 acgccctgcg gatgccggaa ggactggtac gcggcacgtc ctcggacgtg ggccgggtgg 28680 cgttcgtctt ccccggccag ggcacgcagt gggccggcat gggcgccgaa ctccttgaca 28740 gctcaccgga gttcgctgcc tcgatggccg aatgcgagac cgcgctctcc cgctacgtcg 28800 actggtctct tgaagccgtc gtccgacagg aacccggcgc acccacgctc gaccgcgtcg 28860 acgtcgtcca gcccgtgacc ttcgctgtca tggtctcgct ggcgaaggtc tggcagcacc 28920 acggcatcac cccccaggcc gtcgtcggcc actcgcaggg cgagatcgcc gccgcgtacg 28980 tcgccggtgc actcaccctc gacgacgccg cccgcgtcgt caccctgcgc agcaagtcca 29040 tcgccgccca cctcgccggc aagggcggca tgatctccct cgccctcgac gaggcggccg 29100 tcctgaagcg actgagcgac ttcgacggac tctccgtcgc cgccgtcaac ggccccaccg 29160 ccaccgtcgt ctccggcgac ccgacccaga tcgaggaact cgcccgcacc tgcgaggccg 29220 acggcgtccg tgcgcggatc atcccggtcg actacgcctc ccacagccgg caggtcgaga 29280 tcatcgagaa ggagctggcc gaggtcctcg ccggactcgc cccgcaggct ccgcacgtgc 29340 cgttcttctc caccctcgaa ggcacctgga tcaccgagcc ggtgctcgac ggcacctact 29400 ggtaccgcaa cctgcgccat cgcgtgggct tcgcccccgc cgtggagacc ttggcggttg 29460 acggcttcac ccacttcatc gaggtcagcg cccaccccgt cctcaccatg accctccccg 29520 agaccgtcac cggcctcggc accctccgcc gcgaacaggg aggccaggag cgtctggtca 29580 cctcactcgc cgaagcctgg gccaacggcc tcaccatcga ctgggcgccc atcctcccca 29640 ccgcaaccgg ccaccacccc gagctcccca cctacgcctt ccagaccgag cgcttctggc 29700 tgcagagctc cgcgcccacc agcgccgccg acgactggcg ttaccgcgtc gagtggaagc 29760 cgctgacggc ctccggccag gcggacctgt ccgggcggtg gatcgtcgcc gtcgggagcg 29820 agccagaagc cgagctgctg ggcgcgctga aggccgcggg agcggaggtc gacgtactgg 29880 aagccggggc ggacgacgac cgtgaggccc tcgccgcccg gctcaccgca ctgacgaccg 29940 gcgacggctt caccggcgtg gtctcgctcc tcgacgacct cgtgccacag gtcgcctggg 30000 tgcaggcact cggcgacgcc ggaatcaagg cgcccctgtg gtccgtcacc cagggcgcgg 30060 tctccgtcgg acgtctcgac acccccgccg accccgaccg ggccatgctc tggggcctcg 30120 gccgcgtcgt cgcccttgag caccccgaac gctgggccgg cctcgtcgac ctccccgccc 30180 agcccgatgc cgccgccctc gcccacctcg tcaccgcact ctccggcgcc accggcgagg 30240 accagatcgc catccgcacc accggactcc acgcccgccg cctcgcccgc gcacccctcc 30300 acggacgtcg gcccacccgc gactggcagc cccacggcac cgtcctcatc accggcggca 30360 ccggagccct cggcagccac gccgcacgct ggatggccca ccacggagcc gaacacctcc 30420 tcctcgtcag ccgcagcggc gaacaagccc ccggagccac ccaactcacc gccgaactca 30480 ccgcatcggg cgcccgcgtc accatcgccg cctgcgacgt cgccgacccc cacgccatgc 30540 gcaccctcct cgacgccatc cccgccgaga cgcccctcac cgccgtcgtc cacaccgccg 30600 gcgcaccggg cggcgatccg ctggacgtca ccggcccgga ggacatcgcc cgcatcctgg 30660 gcgcgaagac gagcggcgcc gaggtcctcg acgacctgct ccgcggcact ccgctggacg 30720 ccttcgtcct ctactcctcg aacgccgggg tctggggcag cggcagccag ggcgtctacg 30780 cggcggccaa cgcccacctc gacgcgctcg ccgcccggcg ccgcgcccgg ggcgagacgg 30840 cgacctcggt cgcctggggc ctctgggccg gcgacggcat gggccggggc gccgacgacg 30900 cgtactggca gcgtcgcggc atccgtccga tgagccccga ccgcgccctg gacgaactgg 30960 ccaaggccct gagccacgac gagaccttcg tcgccgtggc cgatgtcgac tgggagcggt 31020 tcgcgcccgc gttcacggtg tcccgtccca gccttctgct cgacggcgtc ccggaggccc 31080 ggcaggcgct cgccgcaccc gtcggtgccc cggctcccgg cgacgccgcc gtggcgccga 31140 ccgggcagtc gtcggcgctg gccgcgatca ccgcgctccc cgagcccgag cgccggccgg 31200 cgctcctcac cctcgtccgt acccacgcgg cggccgtact cggccattcc tcccccgacc 31260 gggtggcccc cggccgtgcc ttcaccgagc tcggcttcga ctcgctgacg gccgtgcagc 31320 tccgcaacca gctctccacg gtggtcggca acaggctccc cgccaccacg gtcttcgacc 31380 acccgacgcc cgccgcactc gccgcgcacc tccacgaggc gtacctcgca ccggccgagc 31440 cggccccgac ggactgggag gggcgggtgc gccgggccct ggccgaactg cccctcgacc 31500 ggctgcggga cgcgggggtc ctcgacaccg tcctgcgcct caccggcatc gagcccgagc 31560 cgggttccgg cggttcggac ggcggcgccg ccgaccctgg tgcggagccg gaggcgtcga 31620 tcgacgacct ggacgccgag gccctgatcc ggatggctct cggcccccgt aacacctgac 31680 ccgaccgcgg tcctgcccca cgcgccgcac cccgcgcatc ccgcgcacca cccgccccca 31740 cacgcccaca accccatcca cgagcggaag accacaccca gatgacgagt tccaacgaac 31800 agttggtgga cgctctgcgc gcctctctca aggagaacga agaactccgg aaagagagcc 31860 gtcgccgggc cgaccgtcgg caggagccca tggcgatcgt cggcatgagc tgccggttcg 31920 cgggcggaat ccggtccccc gaggacctct gggacgccgt cgccgcgggc aaggacctgg 31980 tctccgaggt accggaggag cgcggctggg acatcgactc cctctacgac ccggtgcccg 32040 ggcgcaaggg cacgacgtac gtccgcaacg ccgcgttcct cgacgacgcc gccggattcg 32100 acgcggcctt cttcgggatc tcgccgcgcg aggccctcgc catggacccg cagcagcggc 32160 agctcctcga agcctcctgg gaggtcttcg agcgggccgg catcgacccc gcgtcggtcc 32220 gcggcaccga cgtcggcgtg tacgtgggct gtggctacca ggactacgcg ccggacatcc 32280 gggtcgcccc cgaaggcacc ggcggttacg tcgtcaccgg caactcctcc gccgtggcct 32340 ccgggcgcat cgcgtactcc ctcggcctgg agggacccgc cgtgaccgtg gacacggcgt 32400 gctcctcttc gctcgtcgcc ctgcacctcg ccctgaaggg cctgcggaac ggcgactgct 32460 cgacggcact cgtgggcggc gtggccgtcc tcgcgacgcc gggcgcgttc atcgagttca 32520 gcagccagca ggccatggcc gccgacggcc ggaccaaggg cttcgcctcg gcggcggacg 32580 gcctcgcctg gggcgagggc gtcgccgtac tcctcctcga acggctctcc gacgcgcggc 32640 gcaagggcca ccgggtcctg gccgtcgtgc gcggcagcgc catcaaccag gacggcgcga 32700 gcaacggcct cacggctccg cacgggccct cccagcagca cctgatccgc caggccctgg 32760 ccgacgcgcg gctcacgtcg agcgacgtgg acgtcgtgga gggccacggc acggggaccc 32820 gtctcggcga cccgatcgag gcgcaggcgc tgctcgccac gtacgggcag gggcgcgccc 32880 cggggcagcc gctgcggctg gggacgctga agtcgaacat cgggcacacg caggccgctt 32940 cgggtgtcgc cggtgtcatc aagatggtgc aggcgctgcg ccacggggtg ctgccgaaga 33000 ccctgcacgt ggacgagccg acggaccagg tcgactggtc ggccggttcg gtcgagctgc 33060 tcaccgaggc cgtggactgg ccggagcggc cgggccggct ccgccgggcg ggcgtctccg 33120 cgttcggcgt gggcgggacg aacgcgcacg tcgtcctgga ggaggccccg gcggtcgagg 33180 agtcccctgc cgtcgagccg ccggccggtg gcggcgtggt gccgtggccg gtgtccgcga 33240 agacctcggc cgcactggac gcccagatcg ggcagctcgc cgcatacgcg gaagaccgca 33300 cggacgtgga tccggcggtg gccgcccgcg ccctggtcga cagccgtacg gcgatggagc 33360 accgcgcggt cgcggtcggc gacagccggg aggcactgcg ggacgccctg cggatgccgg 33420 aaggactggt acggggcacg gtcaccgatc cgggccgggt ggcgttcgtc ttccccggcc 33480 agggcacgca gtgggccggc atgggcgccg aactcctcga cagctcaccc gaattcgccg 33540 ccgccatggc cgaatgcgag accgcactct ccccgtacgt cgactggtct ctcgaagccg 33600 tcgtccgaca ggctcccagc gcaccgacac tcgaccgcgt cgacgtcgtc cagcccgtca 33660 ccttcgccgt catggtctcc ctcgccaagg tctggcagca ccacggcatc acccccgagg 33720 ccgtcatcgg ccactcccag ggcgagatcg ccgccgcgta cgtcgccggt gccctcaccc 33780 tcgacgacgc cgctcgtgtc gtgaccctcc gcagcaagtc catcgccgcc cacctcgccg 33840 gcaagggcgg catgatctcc ctcgccctca gcgaggaagc cacccggcag cgcatcgaga 33900 acctccacgg actgtcgatc gccgccgtca acgggcctac cgccaccgtg gtttcgggcg 33960 accccaccca gatccaagaa cttgctcagg cgtgtgaggc cgacggcatc cgcgcacgga 34020 tcatccccgt cgactacgcc tcccacagcg cccacgtcga gaccatcgag aacgaactcg 34080 ccgacgtcct ggcggggttg tccccccaga caccccaggt ccccttcttc tccaccctcg 34140 aaggcacctg gatcaccgaa cccgccctcg acggcggcta ctggtaccgc aacctccgcc 34200 atcgtgtggg cttcgccccg gccgtcgaga ccctcgccac cgacgaaggc ttcacccact 34260 tcatcgaggt cagcgcccac cccgtcctca ccatgaccct ccccgacaag gtcaccggcc 34320 tggccaccct ccgacgcgag gacggcggac agcaccgcct caccacctcc cttgccgagg 34380 cctgggccaa cggcctcgcc ctcgactggg cctccctcct gcccgccacg ggcgccctca 34440 gccccgccgt ccccgacctc ccgacgtacg ccttccagca ccgctcgtac tggatcagcc 34500 ccgcgggtcc cggcgaggcg cccgcgcaca ccgcttccgg gcgcgaggcc gtcgccgaga 34560 cggggctcgc gtggggcccg ggtgccgagg acctcgacga ggagggccgg cgcagcgccg 34620 tactcgcgat ggtgatgcgg caggcggcct ccgtgctccg gtgcgactcg cccgaagagg 34680 tccccgtcga ccgcccgctg cgggagatcg gcttcgactc gctgaccgcc gtcgacttcc 34740 gcaaccgcgt caaccggctg accggtctcc agctgccgcc caccgtcgtg ttccagcacc 34800 cgacgcccgt cgcgctcgcc gagcgcatca gcgacgagct ggccgagcgg aactgggccg 34860 tcgccgagcc gtcggatcac gagcaggcgg aggaggagaa ggccgccgct ccggcggggg 34920 cccgctccgg ggccgacacc ggcgccggcg ccgggatgtt ccgcgccctg ttccggcagg 34980 ccgtggagga cgaccggtac ggcgagttcc tcgacgtcct cgccgaagcc tccgcgttcc 35040 gcccgcagtt cgcctcgccc gaggcctgct cggagcggct cgacccggtg ctgctcgccg 35100 gcggtccgac ggaccgggcg gaaggccgtg ccgttctcgt cggctgcacc ggcaccgcgg 35160 cgaacggcgg cccgcacgag ttcctgcggc tcagcacctc cttccaggag gagcgggact 35220 tcctcgccgt acctctcccc ggctacggca cgggtacggg caccggcacg gccctcctcc 35280 cggccgatct cgacaccgcg ctcgacgccc aggcccgggc gatcctccgg gccgccgggg 35340 acgccccggt cgtcctgctc gggcactccg gcggcgccct gctcgcgcac gagctggcct 35400 tccgcctgga gcgggcgcac ggcgcgccgc cggccgggat cgtcctggtc gacccctatc 35460 cgccgggcca tcaggagccc atcgaggtgt ggagcaggca gctgggcgag ggcctgttcg 35520 cgggcgagct ggagccgatg tccgatgcgc ggctgctggc catgggccgg tacgcgcggt 35580 tcctcgccgg cccgcggccg ggccgcagca gcgcgcccgt gcttctggtc cgtgcctccg 35640 aaccgctggg cgactggcag gaggagcggg gcgactggcg tgcccactgg gaccttccgc 35700 acaccgtcgc ggacgtgccg ggcgaccact tcacgatgat gcgggaccac gcgccggccg 35760 tcgccgaggc cgtcctctcc tggctcgacg ccatcgaggg catcgagggg gcgggcaagt 35820 gaccgacaga cctctgaacg tggacagcgg actgtggatc cggcgcttcc accccgcgcc 35880 gaacagcgcg gtgcggctgg tctgcctgcc gcacgccggc ggctccgcca gctacttctt 35940 ccgcttctcg gaggagctgc acccctccgt cgaggccctg tcggtgcagt atccgggccg 36000 ccaggaccgg cgtgccgagc cgtgtctgga gagcgtcgag gagctcgccg agcatgtggt 36060 cgcggccacc gaaccctggt ggcaggaggg ccggctggcc ttcttcgggc acagcctcgg 36120 cgcctccgtc gccttcgaga cggcccgcat cctggaacag cggcacgggg tacggcccga 36180 gggcctgtac gtctccggtc ggcgcgcccc gtcgctggcg ccggaccggc tcgtccacca 36240 gctggacgac cgggcgttcc tggccgagat ccggcggctc agcggcaccg acgagcggtt 36300 cctccaggac gacgagctgc tgcggctggt gctgcccgcg ctgcgcagcg actacaaggc 36360 ggcggagacg tacctgcacc ggccgtccgc caagctcacc tgcccggtga tggccctggc 36420 cggcgaccgt gacccgaagg cgccgctgaa cgaggtggcc gagtggcgtc ggcacaccag 36480 cgggccgttc tgcctccggg cgtactccgg cggccacttc tacctcaacg accagtggca 36540 cgagatctgc aacgacatct ccgaccacct gctcgtcacc cgcggcgcgc ccgatgcccg 36600 cgtcgtgcag cccccgacca gccttatcga aggagcggcg aagagatggc agaacccacg 36660 gtgaccgacg acctgacggg ggccctcacg cagcccccgc tgggccgcac cgtccgcgcg 36720 gtggccgacc gtgaactcgg cacccacctc ctggagaccc gcggcatcca ctggatcc 36778 6 11877 PRT Streptomyces venezuelae 6 Met Ala Met Arg Asp Ser Ile Pro Arg Arg Ala Asp Arg Asp Thr Leu 1 5 10 15 Arg Arg Glu Leu Gly Gln Asn Phe Leu Gln Asp Asp Arg Ala Val Arg 20 25 30 Asn Leu Val Thr His Val Glu Gly Asp Gly Arg Asn Val Leu Glu Ile 35 40 45 Gly Pro Gly Lys Gly Ala Ile Thr Glu Glu Leu Val Arg Ser Phe Asp 50 55 60 Thr Val Thr Val Val Glu Met Asp Pro His Trp Ala Ala His Val Arg 65 70 75 80 Arg Lys Phe Glu Gly Glu Arg Val Thr Val Phe Gln Gly Asp Phe Leu 85 90 95 Asp Phe Arg Ile Pro Arg Asp Ile Asp Thr Val Val Gly Asn Val Pro 100 105 110 Phe Gly Ile Thr Thr Gln Ile Leu Arg Ser Leu Leu Glu Ser Thr Asn 115 120 125 Trp Gln Ser Ala Ala Leu Ile Val Gln Trp Glu Val Ala Arg Lys Arg 130 135 140 Ala Gly Arg Ser Gly Gly Ser Leu Leu Thr Thr Ser Trp Ala Pro Trp 145 150 155 160 Tyr Glu Phe Ala Val His Asp Arg Val Arg Ala Ser Ser Phe Arg Pro 165 170 175 Met Pro Arg Val Asp Gly Gly Val Leu Thr Ile Arg Arg Arg Pro Gln 180 185 190 Pro Leu Leu Pro Glu Ser Ala Ser Arg Ala Phe Gln Asn Phe Ala Glu 195 200 205 Ala Val Phe Thr Gly Pro Gly Arg Gly Leu Ala Glu Ile Leu Arg Arg 210 215 220 His Ile Pro Lys Arg Thr Tyr Arg Ser Leu Ala Asp Arg His Gly Ile 225 230 235 240 Pro Asp Gly Gly Leu Pro Lys Asp Leu Thr Leu Thr Gln Trp Ile Ala 245 250 255 Leu Phe Gln Ala Ser Gln Pro Ser Tyr Ala Pro Gly Ala Pro Gly Thr 260 265 270 Arg Met Pro Gly Gln Gly Gly Gly Ala Gly Gly Arg Asp Tyr Asp Ser 275 280 285 Glu Thr Ser Arg Ala Ala Val Pro Gly Ser Arg Arg Tyr Gly Pro Thr 290 295 300 Arg Gly Gly Glu Pro Cys Ala Pro Arg Ala Gln Val Arg Gln Thr Lys 305 310 315 320 Gly Arg Gln Gly Ala Arg Gly Ser Ser Tyr Gly Arg Arg Thr Gly Arg 325 330 335 Met Ser Ser Ala Gly Ile Thr Arg Thr Gly Ala Arg Thr Pro Val Thr 340 345 350 Gly Arg Gly Ala Ala Ala Trp Asp Thr Gly Glu Val Arg Val Arg Arg 355 360 365 Gly Leu Pro Pro Ala Gly Pro Asp His Ala Glu His Ser Phe Ser Arg 370 375 380 Ala Pro Thr Gly Asp Val Arg Ala Glu Leu Ile Arg Gly Glu Met Ser 385 390 395 400 Thr Val Ser Lys Ser Glu Ser Glu Glu Phe Val Ser Val Ser Asn Asp 405 410 415 Ala Gly Ser Ala His Gly Thr Ala Glu Pro Val Ala Val Val Gly Ile 420 425 430 Ser Cys Arg Val Pro Gly Ala Arg Asp Pro Arg Glu Phe Trp Glu Leu 435 440 445 Leu Ala Ala Gly Gly Gln Ala Val Thr Asp Val Pro Ala Asp Arg Trp 450 455 460 Asn Ala Gly Asp Phe Tyr Asp Pro Asp Arg Ser Ala Pro Gly Arg Ser 465 470 475 480 Asn Ser Arg Trp Gly Gly Phe Ile Glu Asp Val Asp Arg Phe Asp Ala 485 490 495 Ala Phe Phe Gly Ile Ser Pro Arg Glu Ala Ala Glu Met Asp Pro Gln 500 505 510 Gln Arg Leu Ala Leu Glu Leu Gly Trp Glu Ala Leu Glu Arg Ala Gly 515 520 525 Ile Asp Pro Ser Ser Leu Thr Gly Thr Arg Thr Gly Val Phe Ala Gly 530 535 540 Ala Ile Trp Asp Asp Tyr Ala Thr Leu Lys His Arg Gln Gly Gly Ala 545 550 555 560 Ala Ile Thr Pro His Thr Val Thr Gly Leu His Arg Gly Ile Ile Ala 565 570 575 Asn Arg Leu Ser Tyr Thr Leu Gly Leu Arg Gly Pro Ser Met Val Val 580 585 590 Asp Ser Gly Gln Ser Ser Ser Leu Val Ala Val His Leu Ala Cys Glu 595 600 605 Ser Leu Arg Arg Gly Glu Ser Glu Leu Ala Leu Ala Gly Gly Val Ser 610 615 620 Leu Asn Leu Val Pro Asp Ser Ile Ile Gly Ala Ser Lys Phe Gly Gly 625 630 635 640 Leu Ser Pro Asp Gly Arg Ala Tyr Thr Phe Asp Ala Arg Ala Asn Gly 645 650 655 Tyr Val Arg Gly Glu Gly Gly Gly Phe Val Val Leu Lys Arg Leu Ser 660 665 670 Arg Ala Val Ala Asp Gly Asp Pro Val Leu Ala Val Ile Arg Gly Ser 675 680 685 Ala Val Asn Asn Gly Gly Ala Ala Gln Gly Met Thr Thr Pro Asp Ala 690 695 700 Gln Ala Gln Glu Ala Val Leu Arg Glu Ala His Glu Arg Ala Gly Thr 705 710 715 720 Ala Pro Ala Asp Val Arg Tyr Val Glu Leu His Gly Thr Gly Thr Pro 725 730 735 Val Gly Asp Pro Ile Glu Ala Ala Ala Leu Gly Ala Ala Leu Gly Thr 740 745 750 Gly Arg Pro Ala Gly Gln Pro Leu Leu Val Gly Ser Val Lys Thr Asn 755 760 765 Ile Gly His Leu Glu Gly Ala Ala Gly Ile Ala Gly Leu Ile Lys Ala 770 775 780 Val Leu Ala Val Arg Gly Arg Ala Leu Pro Ala Ser Leu Asn Tyr Glu 785 790 795 800 Thr Pro Asn Pro Ala Ile Pro Phe Glu Glu Leu Asn Leu Arg Val Asn 805 810 815 Thr Glu Tyr Leu Pro Trp Glu Pro Glu His Asp Gly Gln Arg Met Val 820 825 830 Val Gly Val Ser Ser Phe Gly Met Gly Gly Thr Asn Ala His Val Val 835 840 845 Leu Glu Glu Ala Pro Gly Gly Cys Arg Gly Ala Ser Val Val Glu Ser 850 855 860 Thr Val Gly Gly Ser Ala Val Gly Gly Gly Val Val Pro Trp Val Val 865 870 875 880 Ser Ala Lys Ser Ala Ala Ala Leu Asp Ala Gln Ile Glu Arg Leu Ala 885 890 895 Ala Phe Ala Ser Arg Asp Arg Thr Asp Gly Val Asp Ala Gly Ala Val 900 905 910 Asp Ala Gly Ala Val Asp Ala Gly Ala Val Ala Arg Val Leu Ala Gly 915 920 925 Gly Arg Ala Gln Phe Glu His Arg Ala Val Val Val Gly Ser Gly Pro 930 935 940 Asp Asp Leu Ala Ala Ala Leu Ala Ala Pro Glu Gly Leu Val Arg Gly 945 950 955 960 Val Ala Ser Gly Val Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly 965 970 975 Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Ser Ser Ala Val 980 985 990 Phe Ala Ala Ala Met Ala Glu Cys Glu Ala Ala Leu Ser Pro Tyr Val 995 1000 1005 Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Gly Ala Pro Thr 1010 1015 1020 Leu Glu Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val Met Val 1025 1030 1035 1040 Ser Leu Ala Arg Val Trp Gln His His Gly Val Thr Pro Gln Ala Val 1045 1050 1055 Val Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala 1060 1065 1070 Leu Ser Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser 1075 1080 1085 Ile Ala Ala His Leu Ala Gly Lys Gly Gly Met Leu Ser Leu Ala Leu 1090 1095 1100 Ser Glu Asp Ala Val Leu Glu Arg Leu Ala Gly Phe Asp Gly Leu Ser 1105 1110 1115 1120 Val Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro 1125 1130 1135 Val Gln Ile Glu Glu Leu Ala Arg Ala Cys Glu Ala Asp Gly Val Arg 1140 1145 1150 Ala Arg Val Ile Pro Val Asp Tyr Ala Ser His Ser Arg Gln Val Glu 1155 1160 1165 Ile Ile Glu Ser Glu Leu Ala Glu Val Leu Ala Gly Leu Ser Pro Gln 1170 1175 1180 Ala Pro Arg Val Pro Phe Phe Ser Thr Leu Glu Gly Ala Trp Ile Thr 1185 1190 1195 1200 Glu Pro Val Leu Asp Gly Gly Tyr Trp Tyr Arg Asn Leu Arg His Arg 1205 1210 1215 Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu Gly Phe 1220 1225 1230 Thr His Phe Val Glu Val Ser Ala His Pro Val Leu Thr Met Ala Leu 1235 1240 1245 Pro Gly Thr Val Thr Gly Leu Ala Thr Leu Arg Arg Asp Asn Gly Gly 1250 1255 1260 Gln Asp Arg Leu Val Ala Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu 1265 1270 1275 1280 Ala Val Asp Trp Ser Pro Leu Leu Pro Ser Ala Thr Gly His His Ser 1285 1290 1295 Asp Leu Pro Thr Tyr Ala Phe Gln Thr Glu Arg His Trp Leu Gly Glu 1300 1305 1310 Ile Glu Ala Leu Ala Pro Ala Gly Glu Pro Ala Val Gln Pro Ala Val 1315 1320 1325 Leu Arg Thr Glu Ala Ala Glu Pro Ala Glu Leu Asp Arg Asp Glu Gln 1330 1335 1340 Leu Arg Val Ile Leu Asp Lys Val Arg Ala Gln Thr Ala Gln Val Leu 1345 1350 1355 1360 Gly Tyr Ala Thr Gly Gly Gln Ile Glu Val Asp Arg Thr Phe Arg Glu 1365 1370 1375 Ala Gly Cys Thr Ser Leu Thr Gly Val Asp Leu Arg Asn Arg Ile Asn 1380 1385 1390 Ala Ala Phe Gly Val Arg Met Ala Pro Ser Met Ile Phe Asp Phe Pro 1395 1400 1405 Thr Pro Glu Ala Leu Ala Glu Gln Leu Leu Leu Val Val His Gly Glu 1410 1415 1420 Ala Ala Ala Asn Pro Ala Gly Ala Glu Pro Ala Pro Val Ala Ala Ala 1425 1430 1435 1440 Gly Ala Val Asp Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu 1445 1450 1455 Pro Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Gly 1460 1465 1470 Gly Gly Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val 1475 1480 1485 Glu Gly Leu Tyr His Pro Asp Pro Glu His Pro Gly Thr Ser Tyr Val 1490 1495 1500 Arg Gln Gly Gly Phe Ile Glu Asn Val Ala Gly Phe Asp Ala Ala Phe 1505 1510 1515 1520 Phe Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg 1525 1530 1535 Leu Leu Leu Glu Thr Ser Trp Glu Ala Val Glu Asp Ala Gly Ile Asp 1540 1545 1550 Pro Thr Ser Leu Arg Gly Arg Gln Val Gly Val Phe Thr Gly Ala Met 1555 1560 1565 Thr His Glu Tyr Gly Pro Ser Leu Arg Asp Gly Gly Glu Gly Leu Asp 1570 1575 1580 Gly Tyr Leu Leu Thr Gly Asn Thr Ala Ser Val Met Ser Gly Arg Val 1585 1590 1595 1600 Ser Tyr Thr Leu Gly Leu Glu Gly Pro Ala Leu Thr Val Asp Thr Ala 1605 1610 1615 Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg 1620 1625 1630 Lys Gly Glu Val Asp Met Ala Leu Ala Gly Gly Val Ala Val Met Pro 1635 1640 1645 Thr Pro Gly Met Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Gly 1650 1655 1660 Asp Gly Arg Ser Lys Ala Phe Ala Ala Ser Ala Asp Gly Thr Ser Trp 1665 1670 1675 1680 Ser Glu Gly Val Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg 1685 1690 1695 Arg Asn Gly His Gln Val Leu Ala Val Val Arg Gly Ser Ala Leu Asn 1700 1705 1710 Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln 1715 1720 1725 Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr Thr Ser 1730 1735 1740 Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp 1745 1750 1755 1760 Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Gly Arg Asp 1765 1770 1775 Asp Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile Gly His 1780 1785 1790 Thr Gln Ala Ala Ala Gly Val Ser Gly Val Ile Lys Met Val Gln Ala 1795 1800 1805 Met Arg His Gly Leu Leu Pro Lys Thr Leu His Val Asp Glu Pro Ser 1810 1815 1820 Asp Gln Ile Asp Trp Ser Ala Gly Ala Val Glu Leu Leu Thr Glu Ala 1825 1830 1835 1840 Val Asp Trp Pro Glu Lys Gln Asp Gly Gly Leu Arg Arg Ala Ala Val 1845 1850 1855 Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Val Val Leu Glu Glu 1860 1865 1870 Ala Pro Val Val Val Glu Gly Ala Ser Val Val Glu Pro Ser Val Gly 1875 1880 1885 Gly Ser Ala Val Gly Gly Gly Val Thr Pro Trp Val Val Ser Ala Lys 1890 1895 1900 Ser Ala Ala Ala Leu Asp Ala Gln Ile Glu Arg Leu Ala Ala Phe Ala 1905 1910 1915 1920 Ser Arg Asp Arg Thr Asp Asp Ala Asp Ala Gly Ala Val Asp Ala Gly 1925 1930 1935 Ala Val Ala His Val Leu Ala Asp Gly Arg Ala Gln Phe Glu His Arg 1940 1945 1950 Ala Val Ala Leu Gly Ala Gly Ala Asp Asp Leu Val Gln Ala Leu Ala 1955 1960 1965 Asp Pro Asp Gly Leu Ile Arg Gly Thr Ala Ser Gly Val Gly Arg Val 1970 1975 1980 Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly Met Gly Ala 1985 1990 1995 2000 Glu Leu Leu Asp Ser Ser Ala Val Phe Ala Ala Ala Met Ala Glu Cys 2005 2010 2015 Glu Ala Ala Leu Ser Pro Tyr Val Asp Trp Ser Leu Glu Ala Val Val 2020 2025 2030 Arg Gln Ala Pro Gly Ala Pro Thr Leu Glu Arg Val Asp Val Val Gln 2035 2040 2045 Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Arg Val Trp Gln His 2050 2055 2060 His Gly Val Thr Pro Gln Ala Val Val Gly His Ser Gln Gly Glu Ile 2065 2070 2075 2080 Ala Ala Ala Tyr Val Ala Gly Ala Leu Pro Leu Asp Asp Ala Ala Arg 2085 2090 2095 Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu Ala Gly Lys 2100 2105 2110 Gly Gly Met Leu Ser Leu Ala Leu Asn Glu Asp Ala Val Leu Glu Arg 2115 2120 2125 Leu Ser Asp Phe Asp Gly Leu Ser Val Ala Ala Val Asn Gly Pro Thr 2130 2135 2140 Ala Thr Val Val Ser Gly Asp Pro Val Gln Ile Glu Glu Leu Ala Gln 2145 2150 2155 2160 Ala Cys Lys Ala Asp Gly Phe Arg Ala Arg Ile Ile Pro Val Asp Tyr 2165 2170 2175 Ala Ser His Ser Arg Gln Val Glu Ile Ile Glu Ser Glu Leu Ala Gln 2180 2185 2190 Val Leu Ala Gly Leu Ser Pro Gln Ala Pro Arg Val Pro Phe Phe Ser 2195 2200 2205 Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Val Leu Asp Gly Thr Tyr 2210 2215 2220 Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro Ala Ile Glu 2225 2230 2235 2240 Thr Leu Ala Val Asp Glu Gly Phe Thr His Phe Val Glu Val Ser Ala 2245 2250 2255 His Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly Leu Gly 2260 2265 2270 Thr Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr Ser Leu 2275 2280 2285 Ala Glu Ala Trp Val Asn Gly Leu Pro Val Ala Trp Thr Ser Leu Leu 2290 2295 2300 Pro Ala Thr Ala Ser Arg Pro Gly Leu Pro Thr Tyr Ala Phe Gln Ala 2305 2310 2315 2320 Glu Arg Tyr Trp Leu Glu Asn Thr Pro Ala Ala Leu Ala Thr Gly Asp 2325 2330 2335 Asp Trp Arg Tyr Arg Ile Asp Trp Lys Arg Leu Pro Ala Ala Glu Gly 2340 2345 2350 Ser Glu Arg Thr Gly Leu Ser Gly Arg Trp Leu Ala Val Thr Pro Glu 2355 2360 2365 Asp His Ser Ala Gln Ala Ala Ala Val Leu Thr Ala Leu Val Asp Ala 2370 2375 2380 Gly Ala Lys Val Glu Val Leu Thr Ala Gly Ala Asp Asp Asp Arg Glu 2385 2390 2395 2400 Ala Leu Ala Ala Arg Leu Thr Ala Leu Thr Thr Gly Asp Gly Phe Thr 2405 2410 2415 Gly Val Val Ser Leu Leu Asp Gly Leu Val Pro Gln Val Ala Trp Val 2420 2425 2430 Gln Ala Leu Gly Asp Ala Gly Ile Lys Ala Pro Leu Trp Ser Val Thr 2435 2440 2445 Gln Gly Ala Val Ser Val Gly Arg Leu Asp Thr Pro Ala Asp Pro Asp 2450 2455 2460 Arg Ala Met Leu Trp Gly Leu Gly Arg Val Val Ala Leu Glu His Pro 2465 2470 2475 2480 Glu Arg Trp Ala Gly Leu Val Asp Leu Pro Ala Gln Pro Asp Ala Ala 2485 2490 2495 Ala Leu Ala His Leu Val Thr Ala Leu Ser Gly Ala Thr Gly Glu Asp 2500 2505 2510 Gln Ile Ala Ile Arg Thr Thr Gly Leu His Ala Arg Arg Leu Ala Arg 2515 2520 2525 Ala Pro Leu His Gly Arg Arg Pro Thr Arg Asp Trp Gln Pro His Gly 2530 2535 2540 Thr Val Leu Ile Thr Gly Gly Thr Gly Ala Leu Gly Ser His Ala Ala 2545 2550 2555 2560 Arg Trp Met Ala His His Gly Ala Glu His Leu Leu Leu Val Ser Arg 2565 2570 2575 Ser Gly Glu Gln Ala Pro Gly Ala Thr Gln Leu Thr Ala Glu Leu Thr 2580 2585 2590 Ala Ser Gly Ala Arg Val Thr Ile Ala Ala Cys Asp Val Ala Asp Pro 2595 2600 2605 His Ala Met Arg Thr Leu Leu Asp Ala Ile Pro Ala Glu Thr Pro Leu 2610 2615 2620 Thr Ala Val Val His Thr Ala Gly Ala Leu Asp Asp Gly Ile Val Asp 2625 2630 2635 2640 Thr Leu Thr Ala Glu Gln Val Arg Arg Ala His Arg Ala Lys Ala Val 2645 2650 2655 Gly Ala Ser Val Leu Asp Glu Leu Thr Arg Asp Leu Asp Leu Asp Ala 2660 2665 2670 Phe Val Leu Phe Ser Ser Val Ser Ser Thr Leu Gly Ile Pro Gly Gln 2675 2680 2685 Gly Asn Tyr Ala Pro His Asn Ala Tyr Leu Asp Ala Leu Ala Ala Arg 2690 2695 2700 Arg Arg Ala Thr Gly Arg Ser Ala Val Ser Val Ala Trp Gly Pro Trp 2705 2710 2715 2720 Asp Gly Gly Gly Met Ala Ala Gly Asp Gly Val Ala Glu Arg Leu Arg 2725 2730 2735 Asn His Gly Val Pro Gly Met Asp Pro Glu Leu Ala Leu Ala Ala Leu 2740 2745 2750 Glu Ser Ala Leu Gly Arg Asp Glu Thr Ala Ile Thr Val Ala Asp Ile 2755 2760 2765 Asp Trp Asp Arg Phe Tyr Leu Ala Tyr Ser Ser Gly Arg Pro Gln Pro 2770 2775 2780 Leu Val Glu Glu Leu Pro Glu Val Arg Arg Ile Ile Asp Ala Arg Asp 2785 2790 2795 2800 Ser Ala Thr Ser Gly Gln Gly Gly Ser Ser Ala Gln Gly Ala Asn Pro 2805 2810 2815 Leu Ala Glu Arg Leu Ala Ala Ala Ala Pro Gly Glu Arg Thr Glu Ile 2820 2825 2830 Leu Leu Gly Leu Val Arg Ala Gln Ala Ala Ala Val Leu Arg Met Arg 2835 2840 2845 Ser Pro Glu Asp Val Ala Ala Asp Arg Ala Phe Lys Asp Ile Gly Phe 2850 2855 2860 Asp Ser Leu Ala Gly Val Glu Leu Arg Asn Arg Leu Thr Arg Ala Thr 2865 2870 2875 2880 Gly Leu Gln Leu Pro Ala Thr Leu Val Phe Asp His Pro Thr Pro Leu 2885 2890 2895 Ala Leu Val Ser Leu Leu Arg Ser Glu Phe Leu Gly Asp Glu Glu Thr 2900 2905 2910 Ala Asp Ala Arg Arg Ser Ala Ala Leu Pro Ala Thr Val Gly Ala Gly 2915 2920 2925 Ala Gly Ala Gly Ala Gly Thr Asp Ala Asp Asp Asp Pro Ile Ala Ile 2930 2935 2940 Val Ala Met Ser Cys Arg Tyr Pro Gly Asp Ile Arg Ser Pro Glu Asp 2945 2950 2955 2960 Leu Trp Arg Met Leu Ser Glu Gly Gly Glu Gly Ile Thr Pro Phe Pro 2965 2970 2975 Thr Asp Arg Gly Trp Asp Leu Asp Gly Leu Tyr Asp Ala Asp Pro Asp 2980 2985 2990 Ala Leu Gly Arg Ala Tyr Val Arg Glu Gly Gly Phe Leu His Asp Ala 2995 3000 3005 Ala Glu Phe Asp Ala Glu Phe Phe Gly Val Ser Pro Arg Glu Ala Leu 3010 3015 3020 Ala Met Asp Pro Gln Gln Arg Met Leu Leu Thr Thr Ser Trp Glu Ala 3025 3030 3035 3040 Phe Glu Arg Ala Gly Ile Glu Pro Ala Ser Leu Arg Gly Ser Ser Thr 3045 3050 3055 Gly Val Phe Ile Gly Leu Ser Tyr Gln Asp Tyr Ala Ala Arg Val Pro 3060 3065 3070 Asn Ala Pro Arg Gly Val Glu Gly Tyr Leu Leu Thr Gly Ser Thr Pro 3075 3080 3085 Ser Val Ala Ser Gly Arg Ile Ala Tyr Thr Phe Gly Leu Glu Gly Pro 3090 3095 3100 Ala Thr Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Thr Ala Leu His 3105 3110 3115 3120 Leu Ala Val Arg Ala Leu Arg Ser Gly Glu Cys Thr Met Ala Leu Ala 3125 3130 3135 Gly Gly Val Ala Met Met Ala Thr Pro His Met Phe Val Glu Phe Ser 3140 3145 3150 Arg Gln Arg Ala Leu Ala Pro Asp Gly Arg Ser Lys Ala Phe Ser Ala 3155 3160 3165 Asp Ala Asp Gly Phe Gly Ala Ala Glu Gly Val Gly Leu Leu Leu Val 3170 3175 3180 Glu Arg Leu Ser Asp Ala Arg Arg Asn Gly His Pro Val Leu Ala Val 3185 3190 3195 3200 Val Arg Gly Thr Ala Val Asn Gln Asp Gly Ala Ser Asn Gly Leu Thr 3205 3210 3215 Ala Pro Asn Gly Pro Ser Gln Gln Arg Val Ile Arg Gln Ala Leu Ala 3220 3225 3230 Asp Ala Arg Leu Ala Pro Gly Asp Ile Asp Ala Val Glu Thr His Gly 3235 3240 3245 Thr Gly Thr Ser Leu Gly Asp Pro Ile Glu Ala Gln Gly Leu Gln Ala 3250 3255 3260 Thr Tyr Gly Lys Glu Arg Pro Ala Glu Arg Pro Leu Ala Ile Gly Ser 3265 3270 3275 3280 Val Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ala Gly Ala Ala Gly 3285 3290 3295 Ile Ile Lys Met Val Leu Ala Met Arg His Gly Thr Leu Pro Lys Thr 3300 3305 3310 Leu His Ala Asp Glu Pro Ser Pro His Val Asp Trp Ala Asn Ser Gly 3315 3320 3325 Leu Ala Leu Val Thr Glu Pro Ile Asp Trp Pro Ala Gly Thr Gly Pro 3330 3335 3340 Arg Arg Ala Ala Val Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His 3345 3350 3355 3360 Val Val Leu Glu Gln Ala Pro Asp Ala Ala Gly Glu Val Leu Gly Ala 3365 3370 3375 Asp Glu Val Pro Glu Val Ser Glu Thr Val Ala Met Ala Gly Thr Ala 3380 3385 3390 Gly Thr Ser Glu Val Ala Glu Gly Ser Glu Ala Ser Glu Ala Pro Ala 3395 3400 3405 Ala Pro Gly Ser Arg Glu Ala Ser Leu Pro Gly His Leu Pro Trp Val 3410 3415 3420 Leu Ser Ala Lys Asp Glu Gln Ser Leu Arg Gly Gln Ala Ala Ala Leu 3425 3430 3435 3440 His Ala Trp Leu Ser Glu Pro Ala Ala Asp Leu Ser Asp Ala Asp Gly 3445 3450 3455 Pro Ala Arg Leu Arg Asp Val Gly Tyr Thr Leu Ala Thr Ser Arg Thr 3460 3465 3470 Ala Phe Ala His Arg Ala Ala Val Thr Ala Ala Asp Arg Asp Gly Phe 3475 3480 3485 Leu Asp Gly Leu Ala Thr Leu Ala Gln Gly Gly Thr Ser Ala His Val 3490 3495 3500 His Leu Asp Thr Ala Arg Asp Gly Thr Thr Ala Phe Leu Phe Thr Gly 3505 3510 3515 3520 Gln Gly Ser Gln Arg Pro Gly Ala Gly Arg Glu Leu Tyr Asp Arg His 3525 3530 3535 Pro Val Phe Ala Arg Ala Leu Asp Glu Ile Cys Ala His Leu Asp Gly 3540 3545 3550 His Leu Glu Leu Pro Leu Leu Asp Val Met Phe Ala Ala Glu Gly Ser 3555 3560 3565 Ala Glu Ala Ala Leu Leu Asp Glu Thr Arg Tyr Thr Gln Cys Ala Leu 3570 3575 3580 Phe Ala Leu Glu Val Ala Leu Phe Arg Leu Val Glu Ser Trp Gly Met 3585 3590 3595 3600 Arg Pro Ala Ala Leu Leu Gly His Ser Val Gly Glu Ile Ala Ala Ala 3605 3610 3615 His Val Ala Gly Val Phe Ser Leu Ala Asp Ala Ala Arg Leu Val Ala 3620 3625 3630 Ala Arg Gly Arg Leu Met Gln Glu Leu Pro Ala Gly Gly Ala Met Leu 3635 3640 3645 Ala Val Gln Ala Ala Glu Asp Glu Ile Arg Val Trp Leu Glu Thr Glu 3650 3655 3660 Glu Arg Tyr Ala Gly Arg Leu Asp Val Ala Ala Val Asn Gly Pro Glu 3665 3670 3675 3680 Ala Ala Val Leu Ser Gly Asp Ala Asp Ala Ala Arg Glu Ala Glu Ala 3685 3690 3695 Tyr Trp Ser Gly Leu Gly Arg Arg Thr Arg Ala Leu Arg Val Ser His 3700 3705 3710 Ala Phe His Ser Ala His Met Asp Gly Met Leu Asp Gly Phe Arg Ala 3715 3720 3725 Val Leu Glu Thr Val Glu Phe Arg Arg Pro Ser Leu Thr Val Val Ser 3730 3735 3740 Asn Val Thr Gly Leu Ala Ala Gly Pro Asp Asp Leu Cys Asp Pro Glu 3745 3750 3755 3760 Tyr Trp Val Arg His Val Arg Gly Thr Val Arg Phe Leu Asp Gly Val 3765 3770 3775 Arg Val Leu Arg Asp Leu Gly Val Arg Thr Cys Leu Glu Leu Gly Pro 3780 3785 3790 Asp Gly Val Leu Thr Ala Met Ala Ala Asp Gly Leu Ala Asp Thr Pro 3795 3800 3805 Ala Asp Ser Ala Ala Gly Ser Pro Val Gly Ser Pro Ala Gly Ser Pro 3810 3815 3820 Ala Asp Ser Ala Ala Gly Ala Leu Arg Pro Arg Pro Leu Leu Val Ala 3825 3830 3835 3840 Leu Leu Arg Arg Lys Arg Ser Glu Thr Glu Thr Val Ala Asp Ala Leu 3845 3850 3855 Gly Arg Ala His Ala His Gly Thr Gly Pro Asp Trp His Ala Trp Phe 3860 3865 3870 Ala Gly Ser Gly Ala His Arg Val Asp Leu Pro Thr Tyr Ser Phe Arg 3875 3880 3885 Arg Asp Arg Tyr Trp Leu Asp Ala Pro Ala Ala Asp Thr Ala Val Asp 3890 3895 3900 Thr Ala Gly Leu Gly Leu Gly Thr Ala Asp His Pro Leu Leu Gly Ala 3905 3910 3915 3920 Val Val Ser Leu Pro Asp Arg Asp Gly Leu Leu Leu Thr Gly Arg Leu 3925 3930 3935 Ser Leu Arg Thr His Pro Trp Leu Ala Asp His Ala Val Leu Gly Ser 3940 3945 3950 Val Leu Leu Pro Gly Ala Ala Met Val Glu Leu Ala Ala His Ala Ala 3955 3960 3965 Glu Ser Ala Gly Leu Arg Asp Val Arg Glu Leu Thr Leu Leu Glu Pro 3970 3975 3980 Leu Val Leu Pro Glu His Gly Gly Val Glu Leu Arg Val Thr Val Gly 3985 3990 3995 4000 Ala Pro Ala Gly Glu Pro Gly Gly Glu Ser Ala Gly Asp Gly Ala Arg 4005 4010 4015 Pro Val Ser Leu His Ser Arg Leu Ala Asp Ala Pro Ala Gly Thr Ala 4020 4025 4030 Trp Ser Cys His Ala Thr Gly Leu Leu Ala Thr Asp Arg Pro Glu Leu 4035 4040 4045 Pro Val Ala Pro Asp Arg Ala Ala Met Trp Pro Pro Gln Gly Ala Glu 4050 4055 4060 Glu Val Pro Leu Asp Gly Leu Tyr Glu Arg Leu Asp Gly Asn Gly Leu 4065 4070 4075 4080 Ala Phe Gly Pro Leu Phe Gln Gly Leu Asn Ala Val Trp Arg Tyr Glu 4085 4090 4095 Gly Glu Val Phe Ala Asp Ile Ala Leu Pro Ala Thr Thr Asn Ala Thr 4100 4105 4110 Ala Pro Ala Thr Ala Asn Gly Gly Gly Ser Ala Ala Ala Ala Pro Tyr 4115 4120 4125 Gly Ile His Pro Ala Leu Leu Asp Ala Ser Leu His Ala Ile Ala Val 4130 4135 4140 Gly Gly Leu Val Asp Glu Pro Glu Leu Val Arg Val Pro Phe His Trp 4145 4150 4155 4160 Ser Gly Val Thr Val His Ala Ala Gly Ala Ala Ala Ala Arg Val Arg 4165 4170 4175 Leu Ala Ser Ala Gly Thr Asp Ala Val Ser Leu Ser Leu Thr Asp Gly 4180 4185 4190 Glu Gly Arg Pro Leu Val Ser Val Glu Arg Leu Thr Leu Arg Pro Val 4195 4200 4205 Thr Ala Asp Gln Ala Ala Ala Ser Arg Val Gly Gly Leu Met His Arg 4210 4215 4220 Val Ala Trp Arg Pro Tyr Ala Leu Ala Ser Ser Gly Glu Gln Asp Pro 4225 4230 4235 4240 His Ala Thr Ser Tyr Gly Pro Thr Ala Val Leu Gly Lys Asp Glu Leu 4245 4250 4255 Lys Val Ala Ala Ala Leu Glu Ser Ala Gly Val Glu Val Gly Leu Tyr 4260 4265 4270 Pro Asp Leu Ala Ala Leu Ser Gln Asp Val Ala Ala Gly Ala Pro Ala 4275 4280 4285 Pro Arg Thr Val Leu Ala Pro Leu Pro Ala Gly Pro Ala Asp Gly Gly 4290 4295 4300 Ala Glu Gly Val Arg Gly Thr Val Ala Arg Thr Leu Glu Leu Leu Gln 4305 4310 4315 4320 Ala Trp Leu Ala Asp Glu His Leu Ala Gly Thr Arg Leu Leu Leu Val 4325 4330 4335 Thr Arg Gly Ala Val Arg Asp Pro Glu Gly Ser Gly Ala Asp Asp Gly 4340 4345 4350 Gly Glu Asp Leu Ser His Ala Ala Ala Trp Gly Leu Val Arg Thr Ala 4355 4360 4365 Gln Thr Glu Asn Pro Gly Arg Phe Gly Leu Leu Asp Leu Ala Asp Asp 4370 4375 4380 Ala Ser Ser Tyr Arg Thr Leu Pro Ser Val Leu Ser Asp Ala Gly Leu 4385 4390 4395 4400 Arg Asp Glu Pro Gln Leu Ala Leu His Asp Gly Thr Ile Arg Leu Ala 4405 4410 4415 Arg Leu Ala Ser Val Arg Pro Glu Thr Gly Thr Ala Ala Pro Ala Leu 4420 4425 4430 Ala Pro Glu Gly Thr Val Leu Leu Thr Gly Gly Thr Gly Gly Leu Gly 4435 4440 4445 Gly Leu Val Ala Arg His Val Val Gly Glu Trp Gly Val Arg Arg Leu 4450 4455 4460 Leu Leu Val Ser Arg Arg Gly Thr Asp Ala Pro Gly Ala Asp Glu Leu 4465 4470 4475 4480 Val His Glu Leu Glu Ala Leu Gly Ala Asp Val Ser Val Ala Ala Cys 4485 4490 4495 Asp Val Ala Asp Arg Glu Ala Leu Thr Ala Val Leu Asp Ala Ile Pro 4500 4505 4510 Ala Glu His Pro Leu Thr Ala Val Val His Thr Ala Gly Val Leu Ser 4515 4520 4525 Asp Gly Thr Leu Pro Ser Met Thr Thr Glu Asp Val Glu His Val Leu 4530 4535 4540 Arg Pro Lys Val Asp Ala Ala Phe Leu Leu Asp Glu Leu Thr Ser Thr 4545 4550 4555 4560 Pro Ala Tyr Asp Leu Ala Ala Phe Val Met Phe Ser Ser Ala Ala Ala 4565 4570 4575 Val Phe Gly Gly Ala Gly Gln Gly Ala Tyr Ala Ala Ala Asn Ala Thr 4580 4585 4590 Leu Asp Ala Leu Ala Trp Arg Arg Arg Ala Ala Gly Leu Pro Ala Leu 4595 4600 4605 Ser Leu Gly Trp Gly Leu Trp Ala Glu Thr Ser Gly Met Thr Gly Glu 4610 4615 4620 Leu Gly Gln Ala Asp Leu Arg Arg Met Ser Arg Ala Gly Ile Gly Gly 4625 4630 4635 4640 Ile Ser Asp Ala Glu Gly Ile Ala Leu Leu Asp Ala Ala Leu Arg Asp 4645 4650 4655 Asp Arg His Pro Val Leu Leu Pro Leu Arg Leu Asp Ala Ala Gly Leu 4660 4665 4670 Arg Asp Ala Ala Gly Asn Asp Pro Ala Gly Ile Pro Ala Leu Phe Arg 4675 4680 4685 Asp Val Val Gly Ala Arg Thr Val Arg Ala Arg Pro Ser Ala Ala Ser 4690 4695 4700 Ala Ser Thr Thr Ala Gly Thr Ala Gly Thr Pro Gly Thr Ala Asp Gly 4705 4710 4715 4720 Ala Ala Glu Thr Ala Ala Val Thr Leu Ala Asp Arg Ala Ala Thr Val 4725 4730 4735 Asp Gly Pro Ala Arg Gln Arg Leu Leu Leu Glu Phe Val Val Gly Glu 4740 4745 4750 Val Ala Glu Val Leu Gly His Ala Arg Gly His Arg Ile Asp Ala Glu 4755 4760 4765 Arg Gly Phe Leu Asp Leu Gly Phe Asp Ser Leu Thr Ala Val Glu Leu 4770 4775 4780 Arg Asn Arg Leu Asn Ser Ala Gly Gly Leu Ala Leu Pro Ala Thr Leu 4785 4790 4795 4800 Val Phe Asp His Pro Ser Pro Ala Ala Leu Ala Ser His Leu Asp Ala 4805 4810 4815 Glu Leu Pro Arg Gly Ala Ser Asp Gln Asp Gly Ala Gly Asn Arg Asn 4820 4825 4830 Gly Asn Glu Asn Gly Thr Thr Ala Ser Arg Ser Thr Ala Glu Thr Asp 4835 4840 4845 Ala Leu Leu Ala Gln Leu Thr Arg Leu Glu Gly Ala Leu Val Leu Thr 4850 4855 4860 Gly Leu Ser Asp Ala Pro Gly Ser Glu Glu Val Leu Glu His Leu Arg 4865 4870 4875 4880 Ser Leu Arg Ser Met Val Thr Gly Glu Thr Gly Thr Gly Thr Ala Ser 4885 4890 4895 Gly Ala Pro Asp Gly Ala Gly Ser Gly Ala Glu Asp Arg Pro Trp Ala 4900 4905 4910 Ala Gly Asp Gly Ala Gly Gly Gly Ser Glu Asp Gly Ala Gly Val Pro 4915 4920 4925 Asp Phe Met Asn Ala Ser Ala Glu Glu Leu Phe Gly Leu Leu Asp Gln 4930 4935 4940 Asp Pro Ser Thr Asp Met Ser Thr Val Asn Glu Glu Lys Tyr Leu Asp 4945 4950 4955 4960 Tyr Leu Arg Arg Ala Thr Ala Asp Leu His Glu Ala Arg Gly Arg Leu 4965 4970 4975 Arg Glu Leu Glu Ala Lys Ala Gly Glu Pro Val Ala Ile Val Gly Met 4980 4985 4990 Ala Cys Arg Leu Pro Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg 4995 5000 5005 Leu Val Ala Gly Gly Glu Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg 5010 5015 5020 Gly Trp Asp Val Glu Gly Leu Tyr Asp Pro Asn Pro Glu Ala Thr Gly 5025 5030 5035 5040 Lys Ser Tyr Ala Arg Glu Ala Gly Phe Leu Tyr Glu Ala Gly Glu Phe 5045 5050 5055 Asp Ala Asp Phe Phe Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp 5060 5065 5070 Pro Gln Gln Arg Leu Leu Leu Glu Ala Ser Trp Glu Ala Phe Glu His 5075 5080 5085 Ala Gly Ile Pro Ala Ala Thr Ala Arg Gly Thr Ser Val Gly Val Phe 5090 5095 5100 Thr Gly Val Met Tyr His Asp Tyr Ala Thr Arg Leu Thr Asp Val Pro 5105 5110 5115 5120 Glu Gly Ile Glu Gly Tyr Leu Gly Thr Gly Asn Ser Gly Ser Val Ala 5125 5130 5135 Ser Gly Arg Val Ala Tyr Thr Leu Gly Leu Glu Gly Pro Ala Val Thr 5140 5145 5150 Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val 5155 5160 5165 Gln Ala Leu Arg Lys Gly Glu Val Asp Met Ala Leu Ala Gly Gly Val 5170 5175 5180 Thr Val Met Ser Thr Pro Ser Thr Phe Val Glu Phe Ser Arg Gln Arg 5185 5190 5195 5200 Gly Leu Ala Pro Asp Gly Arg Ser Lys Ser Phe Ser Ser Thr Ala Asp 5205 5210 5215 Gly Thr Ser Trp Ser Glu Gly Val Gly Val Leu Leu Val Glu Arg Leu 5220 5225 5230 Ser Asp Ala Arg Arg Lys Gly His Arg Ile Leu Ala Val Val Arg Gly 5235 5240 5245 Thr Ala Val Asn Gln Asp Gly Ala Ser Ser Gly Leu Thr Ala Pro Asn 5250 5255 5260 Gly Pro Ser Gln Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg 5265 5270 5275 5280 Leu Thr Thr Ser Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr 5285 5290 5295 Arg Leu Gly Asp Pro Ile Glu Ala Gln Ala Val Ile Ala Thr Tyr Gly 5300 5305 5310 Gln Gly Arg Asp Gly Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser 5315 5320 5325 Asn Ile Gly His Thr Gln Ala Ala Ala Gly Val Ser Gly Val Ile Lys 5330 5335 5340 Met Val Gln Ala Met Arg His Gly Val Leu Pro Lys Thr Leu His Val 5345 5350 5355 5360 Glu Lys Pro Thr Asp Gln Val Asp Trp Ser Ala Gly Ala Val Glu Leu 5365 5370 5375 Leu Thr Glu Ala Met Asp Trp Pro Asp Lys Gly Asp Gly Gly Leu Arg 5380 5385 5390 Arg Ala Ala Val Ser Ser Phe Gly Val Ser Gly Thr Asn Ala His Val 5395 5400 5405 Val Leu Glu Glu Ala Pro Ala Ala Glu Glu Thr Pro Ala Ser Glu Ala 5410 5415 5420 Thr Pro Ala Val Glu Pro Ser Val Gly Ala Gly Leu Val Pro Trp Leu 5425 5430 5435 5440 Val Ser Ala Lys Thr Pro Ala Ala Leu Asp Ala Gln Ile Gly Arg Leu 5445 5450 5455 Ala Ala Phe Ala Ser Gln Gly Arg Thr Asp Ala Ala Asp Pro Gly Ala 5460 5465 5470 Val Ala Arg Val Leu Ala Gly Gly Arg Ala Glu Phe Glu His Arg Ala 5475 5480 5485 Val Val Leu Gly Thr Gly Gln Asp Asp Phe Ala Gln Ala Leu Thr Ala 5490 5495 5500 Pro Glu Gly Leu Ile Arg Gly Thr Pro Ser Asp Val Gly Arg Val Ala 5505 5510 5515 5520 Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly Met Gly Ala Glu 5525 5530 5535 Leu Leu Asp Val Ser Lys Glu Phe Ala Ala Ala Met Ala Glu Cys Glu 5540 5545 5550 Ser Ala Leu Ser Arg Tyr Val Asp Trp Ser Leu Glu Ala Val Val Arg 5555 5560 5565 Gln Ala Pro Gly Ala Pro Thr Leu Glu Arg Val Asp Val Val Gln Pro 5570 5575 5580 Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val Trp Gln His His 5585 5590 5595 5600 Gly Val Thr Pro Gln Ala Val Val Gly His Ser Gln Gly Glu Ile Ala 5605 5610 5615 Ala Ala Tyr Val Ala Gly Ala Leu Thr Leu Asp Asp Ala Ala Arg Val 5620 5625 5630 Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu Ala Gly Lys Gly 5635 5640 5645 Gly Met Ile Ser Leu Ala Leu Ser Glu Glu Ala Thr Arg Gln Arg Ile 5650 5655 5660 Glu Asn Leu His Gly Leu Ser Ile Ala Ala Val Asn Gly Pro Thr Ala 5665 5670 5675 5680 Thr Val Val Ser Gly Asp Pro Thr Gln Ile Gln Glu Leu Ala Gln Ala 5685 5690 5695 Cys Glu Ala Asp Gly Val Arg Ala Arg Ile Ile Pro Val Asp Tyr Ala 5700 5705 5710 Ser His Ser Ala His Val Glu Thr Ile Glu Ser Glu Leu Ala Glu Val 5715 5720 5725 Leu Ala Gly Leu Ser Pro Arg Thr Pro Glu Val Pro Phe Phe Ser Thr 5730 5735 5740 Leu Glu Gly Ala Trp Ile Thr Glu Pro Val Leu Asp Gly Thr Tyr Trp 5745 5750 5755 5760 Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro Ala Val Glu Thr 5765 5770 5775 Leu Ala Thr Asp Glu Gly Phe Thr His Phe Ile Glu Val Ser Ala His 5780 5785 5790 Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly Leu Gly Thr 5795 5800 5805 Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr Ser Leu Ala 5810 5815 5820 Glu Ala Trp Thr Asn Gly Leu Thr Ile Asp Trp Ala Pro Val Leu Pro 5825 5830 5835 5840 Thr Ala Thr Gly His His Pro Glu Leu Pro Thr Tyr Ala Phe Gln Arg 5845 5850 5855 Arg His Tyr Trp Leu His Asp Ser Pro Ala Val Gln Gly Ser Val Gln 5860 5865 5870 Asp Ser Trp Arg Tyr Arg Ile Asp Trp Lys Arg Leu Ala Val Ala Asp 5875 5880 5885 Ala Ser Glu Arg Ala Gly Leu Ser Gly Arg Trp Leu Val Val Val Pro 5890 5895 5900 Glu Asp Arg Ser Ala Glu Ala Ala Pro Val Leu Ala Ala Leu Ser Gly 5905 5910 5915 5920 Ala Gly Ala Asp Pro Val Gln Leu Asp Val Ser Pro Leu Gly Asp Arg 5925 5930 5935 Gln Arg Leu Ala Ala Thr Leu Gly Glu Ala Leu Ala Ala Ala Gly Gly 5940 5945 5950 Ala Val Asp Gly Val Leu Ser Leu Leu Ala Trp Asp Glu Ser Ala His 5955 5960 5965 Pro Gly His Pro Ala Pro Phe Thr Arg Gly Thr Gly Ala Thr Leu Thr 5970 5975 5980 Leu Val Gln Ala Leu Glu Asp Ala Gly Val Ala Ala Pro Leu Trp Cys 5985 5990 5995 6000 Val Thr His Gly Ala Val Ser Val Gly Arg Ala Asp His Val Thr Ser 6005 6010 6015 Pro Ala Gln Ala Met Val Trp Gly Met Gly Arg Val Ala Ala Leu Glu 6020 6025 6030 His Pro Glu Arg Trp Gly Gly Leu Ile Asp Leu Pro Ser Asp Ala Asp 6035 6040 6045 Arg Ala Ala Leu Asp Arg Met Thr Thr Val Leu Ala Gly Gly Thr Gly 6050 6055 6060 Glu Asp Gln Val Ala Val Arg Ala Ser Gly Leu Leu Ala Arg Arg Leu 6065 6070 6075 6080 Val Arg Ala Ser Leu Pro Ala His Gly Thr Ala Ser Pro Trp Trp Gln 6085 6090 6095 Ala Asp Gly Thr Val Leu Val Thr Gly Ala Glu Glu Pro Ala Ala Ala 6100 6105 6110 Glu Ala Ala Arg Arg Leu Ala Arg Asp Gly Ala Gly His Leu Leu Leu 6115 6120 6125 His Thr Thr Pro Ser Gly Ser Glu Gly Ala Glu Gly Thr Ser Gly Ala 6130 6135 6140 Ala Glu Asp Ser Gly Leu Ala Gly Leu Val Ala Glu Leu Ala Asp Leu 6145 6150 6155 6160 Gly Ala Thr Ala Thr Val Val Thr Cys Asp Leu Thr Asp Ala Glu Ala 6165 6170 6175 Ala Ala Arg Leu Leu Ala Gly Val Ser Asp Ala His Pro Leu Ser Ala 6180 6185 6190 Val Leu His Leu Pro Pro Thr Val Asp Ser Glu Pro Leu Ala Ala Thr 6195 6200 6205 Asp Ala Asp Ala Leu Ala Arg Val Val Thr Ala Lys Ala Thr Ala Ala 6210 6215 6220 Leu His Leu Asp Arg Leu Leu Arg Glu Ala Ala Ala Ala Gly Gly Arg 6225 6230 6235 6240 Pro Pro Val Leu Val Leu Phe Ser Ser Val Ala Ala Ile Trp Gly Gly 6245 6250 6255 Ala Gly Gln Gly Ala Tyr Ala Ala Gly Thr Ala Phe Leu Asp Ala Leu 6260 6265 6270 Ala Gly Gln His Arg Ala Asp Gly Pro Thr Val Thr Ser Val Ala Trp 6275 6280 6285 Ser Pro Trp Glu Gly Ser Arg Val Thr Glu Gly Ala Thr Gly Glu Arg 6290 6295 6300 Leu Arg Arg Leu Gly Leu Arg Pro Leu Ala Pro Ala Thr Ala Leu Thr 6305 6310 6315 6320 Ala Leu Asp Thr Ala Leu Gly His Gly Asp Thr Ala Val Thr Ile Ala 6325 6330 6335 Asp Val Asp Trp Ser Ser Phe Ala Pro Gly Phe Thr Thr Ala Arg Pro 6340 6345 6350 Gly Thr Leu Leu Ala Asp Leu Pro Glu Ala Arg Arg Ala Leu Asp Glu 6355 6360 6365 Gln Gln Ser Thr Thr Ala Ala Asp Asp Thr Val Leu Ser Arg Glu Leu 6370 6375 6380 Gly Ala Leu Thr Gly Ala Glu Gln Gln Arg Arg Met Gln Glu Leu Val 6385 6390 6395 6400 Arg Glu His Leu Ala Val Val Leu Asn His Pro Ser Pro Glu Ala Val 6405 6410 6415 Asp Thr Gly Arg Ala Phe Arg Asp Leu Gly Phe Asp Ser Leu Thr Ala 6420 6425 6430 Val Glu Leu Arg Asn Arg Leu Lys Asn Ala Thr Gly Leu Ala Leu Pro 6435 6440 6445 Ala Thr Leu Val Phe Asp Tyr Pro Thr Pro Arg Thr Leu Ala Glu Phe 6450 6455 6460 Leu Leu Ala Glu Ile Leu Gly Glu Gln Ala Gly Ala Gly Glu Gln Leu 6465 6470 6475 6480 Pro Val Asp Gly Gly Val Asp Asp Glu Pro Val Ala Ile Val Gly Met 6485 6490 6495 Ala Cys Arg Leu Pro Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg 6500 6505 6510 Leu Val Ala Gly Gly Glu Asp Ala Ile Ser Gly Phe Pro Gln Asp Arg 6515 6520 6525 Gly Trp Asp Val Glu Gly Leu Tyr Asp Pro Asp Pro Asp Ala Ser Gly 6530 6535 6540 Arg Thr Tyr Cys Arg Ala Gly Gly Phe Leu Asp Glu Ala Gly Glu Phe 6545 6550 6555 6560 Asp Ala Asp Phe Phe Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp 6565 6570 6575 Pro Gln Gln Arg Leu Leu Leu Glu Thr Ser Trp Glu Ala Val Glu Asp 6580 6585 6590 Ala Gly Ile Asp Pro Thr Ser Leu Gln Gly Gln Gln Val Gly Val Phe 6595 6600 6605 Ala Gly Thr Asn Gly Pro His Tyr Glu Pro Leu Leu Arg Asn Thr Ala 6610 6615 6620 Glu Asp Leu Glu Gly Tyr Val Gly Thr Gly Asn Ala Ala Ser Ile Met 6625 6630 6635 6640 Ser Gly Arg Val Ser Tyr Thr Leu Gly Leu Glu Gly Pro Ala Val Thr 6645 6650 6655 Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val 6660 6665 6670 Gln Ala Leu Arg Lys Gly Glu Cys Gly Leu Ala Leu Ala Gly Gly Val 6675 6680 6685 Thr Val Met Ser Thr Pro Thr Thr Phe Val Glu Phe Ser Arg Gln Arg 6690 6695 6700 Gly Leu Ala Glu Asp Gly Arg Ser Lys Ala Phe Ala Ala Ser Ala Asp 6705 6710 6715 6720 Gly Phe Gly Pro Ala Glu Gly Val Gly Met Leu Leu Val Glu Arg Leu 6725 6730 6735 Ser Asp Ala Arg Arg Asn Gly His Arg Val Leu Ala Val Val Arg Gly 6740 6745 6750 Ser Ala Val Asn Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn 6755 6760 6765 Gly Pro Ser Gln Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg 6770 6775 6780 Leu Thr Thr Ala Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr 6785 6790 6795 6800 Arg Leu Gly Asp Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly 6805 6810 6815 Gln Gly Arg Asp Thr Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser 6820 6825 6830 Asn Ile Gly His Thr Gln Ala Ala Ala Gly Val Ser Gly Ile Ile Lys 6835 6840 6845 Met Val Gln Ala Met Arg His Gly Val Leu Pro Lys Thr Leu His Val 6850 6855 6860 Asp Arg Pro Ser Asp Gln Ile Asp Trp Ser Ala Gly Thr Val Glu Leu 6865 6870 6875 6880 Leu Thr Glu Ala Met Asp Trp Pro Arg Lys Gln Glu Gly Gly Leu Arg 6885 6890 6895 Arg Ala Ala Val Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Ile 6900 6905 6910 Val Leu Glu Glu Ala Pro Val Asp Glu Asp Ala Pro Ala Asp Glu Pro 6915 6920 6925 Ser Val Gly Gly Val Val Pro Trp Leu Val Ser Ala Lys Thr Pro Ala 6930 6935 6940 Ala Leu Asp Ala Gln Ile Gly Arg Leu Ala Ala Phe Ala Ser Gln Gly 6945 6950 6955 6960 Arg Thr Asp Ala Ala Asp Pro Gly Ala Val Ala Arg Val Leu Ala Gly 6965 6970 6975 Gly Arg Ala Gln Phe Glu His Arg Ala Val Ala Leu Gly Thr Gly Gln 6980 6985 6990 Asp Asp Leu Ala Ala Ala Leu Ala Ala Pro Glu Gly Leu Val Arg Gly 6995 7000 7005 Val Ala Ser Gly Val Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly 7010 7015 7020 Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Val Ser Lys Glu 7025 7030 7035 7040 Phe Ala Ala Ala Met Ala Glu Cys Glu Ala Ala Leu Ala Pro Tyr Val 7045 7050 7055 Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Gly Ala Pro Thr 7060 7065 7070 Leu Glu Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val Met Val 7075 7080 7085 Ser Leu Ala Lys Val Trp Gln His His Gly Val Thr Pro Gln Ala Val 7090 7095 7100 Val Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala 7105 7110 7115 7120 Leu Ser Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser 7125 7130 7135 Ile Gly Ala His Leu Ala Gly Gln Gly Gly Met Leu Ser Leu Ala Leu 7140 7145 7150 Ser Glu Ala Ala Val Val Glu Arg Leu Ala Gly Phe Asp Gly Leu Ser 7155 7160 7165 Val Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro 7170 7175 7180 Thr Gln Ile Gln Glu Leu Ala Gln Ala Cys Glu Ala Asp Gly Val Arg 7185 7190 7195 7200 Ala Arg Ile Ile Pro Val Asp Tyr Ala Ser His Ser Ala His Val Glu 7205 7210 7215 Thr Ile Glu Ser Glu Leu Ala Asp Val Leu Ala Gly Leu Ser Pro Gln 7220 7225 7230 Thr Pro Gln Val Pro Phe Phe Ser Thr Leu Glu Gly Ala Trp Ile Thr 7235 7240 7245 Glu Pro Ala Leu Asp Gly Gly Tyr Trp Tyr Arg Asn Leu Arg His Arg 7250 7255 7260 Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu Gly Phe 7265 7270 7275 7280 Thr His Phe Val Glu Val Ser Ala His Pro Val Leu Thr Met Ala Leu 7285 7290 7295 Pro Glu Thr Val Thr Gly Leu Gly Thr Leu Arg Arg Asp Asn Gly Gly 7300 7305 7310 Gln His Arg Leu Thr Thr Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu 7315 7320 7325 Thr Val Asp Trp Ala Ser Leu Leu Pro Thr Thr Thr Thr His Pro Asp 7330 7335 7340 Leu Pro Thr Tyr Ala Phe Gln Thr Glu Arg Tyr Trp Pro Gln Pro Asp 7345 7350 7355 7360 Leu Ser Ala Ala Gly Asp Ile Thr Ser Ala Gly Leu Gly Ala Ala Glu 7365 7370 7375 His Pro Leu Leu Gly Ala Ala Val Ala Leu Ala Asp Ser Asp Gly Cys 7380 7385 7390 Leu Leu Thr Gly Ser Leu Ser Leu Arg Thr His Pro Trp Leu Ala Asp 7395 7400 7405 His Ala Val Ala Gly Thr Val Leu Leu Pro Gly Thr Ala Phe Val Glu 7410 7415 7420 Leu Ala Phe Arg Ala Gly Asp Gln Val Gly Cys Asp Leu Val Glu Glu 7425 7430 7435 7440 Leu Thr Leu Asp Ala Pro Leu Val Leu Pro Arg Arg Gly Ala Val Arg 7445 7450 7455 Val Gln Leu Ser Val Gly Ala Ser Asp Glu Ser Gly Arg Arg Thr Phe 7460 7465 7470 Gly Leu Tyr Ala His Pro Glu Asp Ala Pro Gly Glu Ala Glu Trp Thr 7475 7480 7485 Arg His Ala Thr Gly Val Leu Ala Ala Arg Ala Asp Arg Thr Ala Pro 7490 7495 7500 Val Ala Asp Pro Glu Ala Trp Pro Pro Pro Gly Ala Glu Pro Val Asp 7505 7510 7515 7520 Val Asp Gly Leu Tyr Glu Arg Phe Ala Ala Asn Gly Tyr Gly Tyr Gly 7525 7530 7535 Pro Leu Phe Gln Gly Val Arg Gly Val Trp Arg Arg Gly Asp Glu Val 7540 7545 7550 Phe Ala Asp Val Ala Leu Pro Ala Glu Val Ala Gly Ala Glu Gly Ala 7555 7560 7565 Arg Phe Gly Leu His Pro Ala Leu Leu Asp Ala Ala Val Gln Ala Ala 7570 7575 7580 Gly Ala Gly Arg Gly Val Arg Arg Gly His Ala Ala Ala Val Arg Leu 7585 7590 7595 7600 Glu Arg Asp Leu Leu Tyr Ala Val Gly Ala Thr Ala Leu Arg Val Arg 7605 7610 7615 Leu Ala Pro Ala Gly Pro Asp Thr Val Ser Val Ser Ala Ala Asp Ser 7620 7625 7630 Ser Gly Gln Pro Val Phe Ala Ala Asp Ser Leu Thr Val Leu Pro Val 7635 7640 7645 Asp Pro Ala Gln Leu Ala Ala Phe Ser Asp Pro Thr Leu Asp Ala Leu 7650 7655 7660 His Leu Leu Glu Trp Thr Ala Trp Asp Gly Ala Ala Gln Ala Leu Pro 7665 7670 7675 7680 Gly Ala Val Val Leu Gly Gly Asp Ala Asp Gly Leu Ala Ala Ala Leu 7685 7690 7695 Arg Ala Gly Gly Thr Glu Val Leu Ser Phe Pro Asp Leu Thr Asp Leu 7700 7705 7710 Val Glu Ala Val Asp Arg Gly Glu Thr Pro Ala Pro Ala Thr Val Leu 7715 7720 7725 Val Ala Cys Pro Ala Ala Gly Pro Asp Gly Pro Glu His Val Arg Glu 7730 7735 7740 Ala Leu His Gly Ser Leu Ala Leu Met Gln Ala Trp Leu Ala Asp Glu 7745 7750 7755 7760 Arg Phe Thr Asp Gly Arg Leu Val Leu Val Thr Arg Asp Ala Val Ala 7765 7770 7775 Ala Arg Ser Gly Asp Gly Leu Arg Ser Thr Gly Gln Ala Ala Val Trp 7780 7785 7790 Gly Leu Gly Arg Ser Ala Gln Thr Glu Ser Pro Gly Arg Phe Val Leu 7795 7800 7805 Leu Asp Leu Ala Gly Glu Ala Arg Thr Ala Gly Asp Ala Thr Ala Gly 7810 7815 7820 Asp Gly Leu Thr Thr Gly Asp Ala Thr Val Gly Gly Thr Ser Gly Asp 7825 7830 7835 7840 Ala Ala Leu Gly Ser Ala Leu Ala Thr Ala Leu Gly Ser Gly Glu Pro 7845 7850 7855 Gln Leu Ala Leu Arg Asp Gly Ala Leu Leu Val Pro Arg Leu Ala Arg 7860 7865 7870 Ala Ala Ala Pro Ala Ala Ala Asp Gly Leu Ala Ala Ala Asp Gly Leu 7875 7880 7885 Ala Ala Leu Pro Leu Pro Ala Ala Pro Ala Leu Trp Arg Leu Glu Pro 7890 7895 7900 Gly Thr Asp Gly Ser Leu Glu Ser Leu Thr Ala Ala Pro Gly Asp Ala 7905 7910 7915 7920 Glu Thr Leu Ala Pro Glu Pro Leu Gly Pro Gly Gln Val Arg Ile Ala 7925 7930 7935 Ile Arg Ala Thr Gly Leu Asn Phe Arg Asp Val Leu Ile Ala Leu Gly 7940 7945 7950 Met Tyr Pro Asp Pro Ala Leu Met Gly Thr Glu Gly Ala Gly Val Val 7955 7960 7965 Thr Ala Thr Gly Pro Gly Val Thr His Leu Ala Pro Gly Asp Arg Val 7970 7975 7980 Met Gly Leu Leu Ser Gly Ala Tyr Ala Pro Val Val Val Ala Asp Ala 7985 7990 7995 8000 Arg Thr Val Ala Arg Met Pro Glu Gly Trp Thr Phe Ala Gln Gly Ala 8005 8010 8015 Ser Val Pro Val Val Phe Leu Thr Ala Val Tyr Ala Leu Arg Asp Leu 8020 8025 8030 Ala Asp Val Lys Pro Gly Glu Arg Leu Leu Val His Ser Ala Ala Gly 8035 8040 8045 Gly Val Gly Met Ala Ala Val Gln Leu Ala Arg His Trp Gly Val Glu 8050 8055 8060 Val His Gly Thr Ala Ser His Gly Lys Trp Asp Ala Leu Arg Ala Leu 8065 8070 8075 8080 Gly Leu Asp Asp Ala His Ile Ala Ser Ser Arg Thr Leu Asp Phe Glu 8085 8090 8095 Ser Ala Phe Arg Ala Ala Ser Gly Gly Ala Gly Met Asp Val Val Leu 8100 8105 8110 Asn Ser Leu Ala Arg Glu Phe Val Asp Ala Ser Leu Arg Leu Leu Gly 8115 8120 8125 Pro Gly Gly Arg Phe Val Glu Met Gly Lys Thr Asp Val Arg Asp Ala 8130 8135 8140 Glu Arg Val Ala Ala Asp His Pro Gly Val Gly Tyr Arg Ala Phe Asp 8145 8150 8155 8160 Leu Gly Glu Ala Gly Pro Glu Arg Ile Gly Glu Met Leu Ala Glu Val 8165 8170 8175 Ile Ala Leu Phe Glu Asp Gly Val Leu Arg His Leu Pro Val Thr Thr 8180 8185 8190 Trp Asp Val Arg Arg Ala Arg Asp Ala Phe Arg His Val Ser Gln Ala 8195 8200 8205 Arg His Thr Gly Lys Val Val Leu Thr Met Pro Ser Gly Leu Asp Pro 8210 8215 8220 Glu Gly Thr Val Leu Leu Thr Gly Gly Thr Gly Ala Leu Gly Gly Ile 8225 8230 8235 8240 Val Ala Arg His Val Val Gly Glu Trp Gly Val Arg Arg Leu Leu Leu 8245 8250 8255 Val Ser Arg Arg Gly Thr Asp Ala Pro Gly Ala Gly Glu Leu Val His 8260 8265 8270 Glu Leu Glu Ala Leu Gly Ala Asp Val Ser Val Ala Ala Cys Asp Val 8275 8280 8285 Ala Asp Arg Glu Ala Leu Thr Ala Val Leu Asp Ser Ile Pro Ala Glu 8290 8295 8300 His Pro Leu Thr Ala Val Val His Thr Ala Gly Val Leu Ser Asp Gly 8305 8310 8315 8320 Thr Leu Pro Ser Met Thr Ala Glu Asp Val Glu His Val Leu Arg Pro 8325 8330 8335 Lys Val Asp Ala Ala Phe Leu Leu Asp Glu Leu Thr Ser Thr Pro Gly 8340 8345 8350 Tyr Asp Leu Ala Ala Phe Val Met Phe Ser Ser Ala Ala Ala Val Phe 8355 8360 8365 Gly Gly Ala Gly Gln Gly Ala Tyr Ala Ala Ala Asn Ala Thr Leu Asp 8370 8375 8380 Ala Leu Ala Trp Arg Arg Arg Thr Ala Gly Leu Pro Ala Leu Ser Leu 8385 8390 8395 8400 Gly Trp Gly Leu Trp Ala Glu Thr Ser Gly Met Thr Gly Gly Leu Ser 8405 8410 8415 Asp Thr Asp Arg Ser Arg Leu Ala Arg Ser Gly Ala Thr Pro Met Asp 8420 8425 8430 Ser Glu Leu Thr Leu Ser Leu Leu Asp Ala Ala Met Arg Arg Asp Asp 8435 8440 8445 Pro Ala Leu Val Pro Ile Ala Leu Asp Val Ala Ala Leu Arg Ala Gln 8450 8455 8460 Gln Arg Asp Gly Met Leu Ala Pro Leu Leu Ser Gly Leu Thr Arg Gly 8465 8470 8475 8480 Ser Arg Val Gly Gly Ala Pro Val Asn Gln Arg Arg Ala Ala Ala Gly 8485 8490 8495 Gly Ala Gly Glu Ala Asp Thr Asp Leu Gly Gly Arg Leu Ala Ala Met 8500 8505 8510 Thr Pro Asp Asp Arg Val Ala His Leu Arg Asp Leu Val Arg Thr His 8515 8520 8525 Val Ala Thr Val Leu Gly His Gly Thr Pro Ser Arg Val Asp Leu Glu 8530 8535 8540 Arg Ala Phe Arg Asp Thr Gly Phe Asp Ser Leu Thr Ala Val Glu Leu 8545 8550 8555 8560 Arg Asn Arg Leu Asn Ala Ala Thr Gly Leu Arg Leu Pro Ala Thr Leu 8565 8570 8575 Val Phe Asp His Pro Thr Pro Gly Glu Leu Ala Gly His Leu Leu Asp 8580 8585 8590 Glu Leu Ala Thr Ala Ala Gly Gly Ser Trp Ala Glu Gly Thr Gly Ser 8595 8600 8605 Gly Asp Thr Ala Ser Ala Thr Asp Arg Gln Thr Thr Ala Ala Leu Ala 8610 8615 8620 Glu Leu Asp Arg Leu Glu Gly Val Leu Ala Ser Leu Ala Pro Ala Ala 8625 8630 8635 8640 Gly Gly Arg Pro Glu Leu Ala Ala Arg Leu Arg Ala Leu Ala Ala Ala 8645 8650 8655 Leu Gly Asp Asp Gly Asp Asp Ala Thr Asp Leu Asp Glu Ala Ser Asp 8660 8665 8670 Asp Asp Leu Phe Ser Phe Ile Asp Lys Glu Leu Gly Asp Ser Asp Phe 8675 8680 8685 Met Ala Asn Asn Glu Asp Lys Leu Arg Asp Tyr Leu Lys Arg Val Thr 8690 8695 8700 Ala Glu Leu Gln Gln Asn Thr Arg Arg Leu Arg Glu Ile Glu Gly Arg 8705 8710 8715 8720 Thr His Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro Gly 8725 8730 8735 Gly Val Ala Ser Pro Glu Asp Leu Trp Gln Leu Val Ala Gly Asp Gly 8740 8745 8750 Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val Glu Gly 8755 8760 8765 Leu Tyr Asp Pro Asp Pro Asp Ala Ser Gly Arg Thr Tyr Cys Arg Ser 8770 8775 8780 Gly Gly Phe Leu His Asp Ala Gly Glu Phe Asp Ala Asp Phe Phe Gly 8785 8790 8795 8800 Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu Ser 8805 8810 8815 Leu Thr Thr Ala Trp Glu Ala Ile Glu Ser Ala Gly Ile Asp Pro Thr 8820 8825 8830 Ala Leu Lys Gly Ser Gly Leu Gly Val Phe Val Gly Gly Trp His Thr 8835 8840 8845 Gly Tyr Thr Ser Gly Gln Thr Thr Ala Val Gln Ser Pro Glu Leu Glu 8850 8855 8860 Gly His Leu Val Ser Gly Ala Ala Leu Gly Phe Leu Ser Gly Arg Ile 8865 8870 8875 8880 Ala Tyr Val Leu Gly Thr Asp Gly Pro Ala Leu Thr Val Asp Thr Ala 8885 8890 8895 Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg 8900 8905 8910 Lys Gly Glu Cys Asp Met Ala Leu Ala Gly Gly Val Thr Val Met Pro 8915 8920 8925 Asn Ala Asp Leu Phe Val Gln Phe Ser Arg Gln Arg Gly Leu Ala Ala 8930 8935 8940 Asp Gly Arg Ser Lys Ala Phe Ala Thr Ser Ala Asp Gly Phe Gly Pro 8945 8950 8955 8960 Ala Glu Gly Ala Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg 8965 8970 8975 Arg Asn Gly His Arg Ile Leu Ala Val Val Arg Gly Ser Ala Val Asn 8980 8985 8990 Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro His Gly Pro Ser Gln 8995 9000 9005 Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Ala Pro Gly 9010 9015 9020 Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp 9025 9030 9035 9040 Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Glu Lys Ser 9045 9050 9055 Ser Glu Gln Pro Leu Arg Leu Gly Ala Leu Lys Ser Asn Ile Gly His 9060 9065 9070 Thr Gln Ala Ala Ala Gly Val Ala Gly Val Ile Lys Met Val Gln Ala 9075 9080 9085 Met Arg His Gly Leu Leu Pro Lys Thr Leu His Val Asp Glu Pro Ser 9090 9095 9100 Asp Gln Ile Asp Trp Ser Ala Gly Thr Val Glu Leu Leu Thr Glu Ala 9105 9110 9115 9120 Val Asp Trp Pro Glu Lys Gln Asp Gly Gly Leu Arg Arg Ala Ala Val 9125 9130 9135 Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Val Val Leu Glu Glu 9140 9145 9150 Ala Pro Ala Val Glu Asp Ser Pro Ala Val Glu Pro Pro Ala Gly Gly 9155 9160 9165 Gly Val Val Pro Trp Pro Val Ser Ala Lys Thr Pro Ala Ala Leu Asp 9170 9175 9180 Ala Gln Ile Gly Gln Leu Ala Ala Tyr Ala Asp Gly Arg Thr Asp Val 9185 9190 9195 9200 Asp Pro Ala Val Ala Ala Arg Ala Leu Val Asp Ser Arg Thr Ala Met 9205 9210 9215 Glu His Arg Ala Val Ala Val Gly Asp Ser Arg Glu Ala Leu Arg Asp 9220 9225 9230 Ala Leu Arg Met Pro Glu Gly Leu Val Arg Gly Thr Ser Ser Asp Val 9235 9240 9245 Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly 9250 9255 9260 Met Gly Ala Glu Leu Leu Asp Ser Ser Pro Glu Phe Ala Ala Ser Met 9265 9270 9275 9280 Ala Glu Cys Glu Thr Ala Leu Ser Arg Tyr Val Asp Trp Ser Leu Glu 9285 9290 9295 Ala Val Val Arg Gln Glu Pro Gly Ala Pro Thr Leu Asp Arg Val Asp 9300 9305 9310 Val Val Gln Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val 9315 9320 9325 Trp Gln His His Gly Ile Thr Pro Gln Ala Val Val Gly His Ser Gln 9330 9335 9340 Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala Leu Thr Leu Asp Asp 9345 9350 9355 9360 Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu 9365 9370 9375 Ala Gly Lys Gly Gly Met Ile Ser Leu Ala Leu Asp Glu Ala Ala Val 9380 9385 9390 Leu Lys Arg Leu Ser Asp Phe Asp Gly Leu Ser Val Ala Ala Val Asn 9395 9400 9405 Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Glu Glu 9410 9415 9420 Leu Ala Arg Thr Cys Glu Ala Asp Gly Val Arg Ala Arg Ile Ile Pro 9425 9430 9435 9440 Val Asp Tyr Ala Ser His Ser Arg Gln Val Glu Ile Ile Glu Lys Glu 9445 9450 9455 Leu Ala Glu Val Leu Ala Gly Leu Ala Pro Gln Ala Pro His Val Pro 9460 9465 9470 Phe Phe Ser Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Val Leu Asp 9475 9480 9485 Gly Thr Tyr Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro 9490 9495 9500 Ala Val Glu Thr Leu Ala Val Asp Gly Phe Thr His Phe Ile Glu Val 9505 9510 9515 9520 Ser Ala His Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly 9525 9530 9535 Leu Gly Thr Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr 9540 9545 9550 Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu Thr Ile Asp Trp Ala Pro 9555 9560 9565 Ile Leu Pro Thr Ala Thr Gly His His Pro Glu Leu Pro Thr Tyr Ala 9570 9575 9580 Phe Gln Thr Glu Arg Phe Trp Leu Gln Ser Ser Ala Pro Thr Ser Ala 9585 9590 9595 9600 Ala Asp Asp Trp Arg Tyr Arg Val Glu Trp Lys Pro Leu Thr Ala Ser 9605 9610 9615 Gly Gln Ala Asp Leu Ser Gly Arg Trp Ile Val Ala Val Gly Ser Glu 9620 9625 9630 Pro Glu Ala Glu Leu Leu Gly Ala Leu Lys Ala Ala Gly Ala Glu Val 9635 9640 9645 Asp Val Leu Glu Ala Gly Ala Asp Asp Asp Arg Glu Ala Leu Ala Ala 9650 9655 9660 Arg Leu Thr Ala Leu Thr Thr Gly Asp Gly Phe Thr Gly Val Val Ser 9665 9670 9675 9680 Leu Leu Asp Asp Leu Val Pro Gln Val Ala Trp Val Gln Ala Leu Gly 9685 9690 9695 Asp Ala Gly Ile Lys Ala Pro Leu Trp Ser Val Thr Gln Gly Ala Val 9700 9705 9710 Ser Val Gly Arg Leu Asp Thr Pro Ala Asp Pro Asp Arg Ala Met Leu 9715 9720 9725 Trp Gly Leu Gly Arg Val Val Ala Leu Glu His Pro Glu Arg Trp Ala 9730 9735 9740 Gly Leu Val Asp Leu Pro Ala Gln Pro Asp Ala Ala Ala Leu Ala His 9745 9750 9755 9760 Leu Val Thr Ala Leu Ser Gly Ala Thr Gly Glu Asp Gln Ile Ala Ile 9765 9770 9775 Arg Thr Thr Gly Leu His Ala Arg Arg Leu Ala Arg Ala Pro Leu His 9780 9785 9790 Gly Arg Arg Pro Thr Arg Asp Trp Gln Pro His Gly Thr Val Leu Ile 9795 9800 9805 Thr Gly Gly Thr Gly Ala Leu Gly Ser His Ala Ala Arg Trp Met Ala 9810 9815 9820 His His Gly Ala Glu His Leu Leu Leu Val Ser Arg Ser Gly Glu Gln 9825 9830 9835 9840 Ala Pro Gly Ala Thr Gln Leu Thr Ala Glu Leu Thr Ala Ser Gly Ala 9845 9850 9855 Arg Val Thr Ile Ala Ala Cys Asp Val Ala Asp Pro His Ala Met Arg 9860 9865 9870 Thr Leu Leu Asp Ala Ile Pro Ala Glu Thr Pro Leu Thr Ala Val Val 9875 9880 9885 His Thr Ala Gly Ala Pro Gly Gly Asp Pro Leu Asp Val Thr Gly Pro 9890 9895 9900 Glu Asp Ile Ala Arg Ile Leu Gly Ala Lys Thr Ser Gly Ala Glu Val 9905 9910 9915 9920 Leu Asp Asp Leu Leu Arg Gly Thr Pro Leu Asp Ala Phe Val Leu Tyr 9925 9930 9935 Ser Ser Asn Ala Gly Val Trp Gly Ser Gly Ser Gln Gly Val Tyr Ala 9940 9945 9950 Ala Ala Asn Ala His Leu Asp Ala Leu Ala Ala Arg Arg Arg Ala Arg 9955 9960 9965 Gly Glu Thr Ala Thr Ser Val Ala Trp Gly Leu Trp Ala Gly Asp Gly 9970 9975 9980 Met Gly Arg Gly Ala Asp Asp Ala Tyr Trp Gln Arg Arg Gly Ile Arg 9985 9990 9995 10000 Pro Met Ser Pro Asp Arg Ala Leu Asp Glu Leu Ala Lys Ala Leu Ser 10005 10010 10015 His Asp Glu Thr Phe Val Ala Val Ala Asp Val Asp Trp Glu Arg Phe 10020 10025 10030 Ala Pro Ala Phe Thr Val Ser Arg Pro Ser Leu Leu Leu Asp Gly Val 10035 10040 10045 Pro Glu Ala Arg Gln Ala Leu Ala Ala Pro Val Gly Ala Pro Ala Pro 10050 10055 10060 Gly Asp Ala Ala Val Ala Pro Thr Gly Gln Ser Ser Ala Leu Ala Ala 10065 10070 10075 10080 Ile Thr Ala Leu Pro Glu Pro Glu Arg Arg Pro Ala Leu Leu Thr Leu 10085 10090 10095 Val Arg Thr His Ala Ala Ala Val Leu Gly His Ser Ser Pro Asp Arg 10100 10105 10110 Val Ala Pro Gly Arg Ala Phe Thr Glu Leu Gly Phe Asp Ser Leu Thr 10115 10120 10125 Ala Val Gln Leu Arg Asn Gln Leu Ser Thr Val Val Gly Asn Arg Leu 10130 10135 10140 Pro Ala Thr Thr Val Phe Asp His Pro Thr Pro Ala Ala Leu Ala Ala 10145 10150 10155 10160 His Leu His Glu Ala Tyr Leu Ala Pro Ala Glu Pro Ala Pro Thr Asp 10165 10170 10175 Trp Glu Gly Arg Val Arg Arg Ala Leu Ala Glu Leu Pro Leu Asp Arg 10180 10185 10190 Leu Arg Asp Ala Gly Val Leu Asp Thr Val Leu Arg Leu Thr Gly Ile 10195 10200 10205 Glu Pro Glu Pro Gly Ser Gly Gly Ser Asp Gly Gly Ala Ala Asp Pro 10210 10215 10220 Gly Ala Glu Pro Glu Ala Ser Ile Asp Asp Leu Asp Ala Glu Ala Leu 10225 10230 10235 10240 Ile Arg Met Ala Leu Gly Pro Arg Asn Thr Met Thr Ser Ser Asn Glu 10245 10250 10255 Gln Leu Val Asp Ala Leu Arg Ala Ser Leu Lys Glu Asn Glu Glu Leu 10260 10265 10270 Arg Lys Glu Ser Arg Arg Arg Ala Asp Arg Arg Gln Glu Pro Met Ala 10275 10280 10285 Ile Val Gly Met Ser Cys Arg Phe Ala Gly Gly Ile Arg Ser Pro Glu 10290 10295 10300 Asp Leu Trp Asp Ala Val Ala Ala Gly Lys Asp Leu Val Ser Glu Val 10305 10310 10315 10320 Pro Glu Glu Arg Gly Trp Asp Ile Asp Ser Leu Tyr Asp Pro Val Pro 10325 10330 10335 Gly Arg Lys Gly Thr Thr Tyr Val Arg Asn Ala Ala Phe Leu Asp Asp 10340 10345 10350 Ala Ala Gly Phe Asp Ala Ala Phe Phe Gly Ile Ser Pro Arg Glu Ala 10355 10360 10365 Leu Ala Met Asp Pro Gln Gln Arg Gln Leu Leu Glu Ala Ser Trp Glu 10370 10375 10380 Val Phe Glu Arg Ala Gly Ile Asp Pro Ala Ser Val Arg Gly Thr Asp 10385 10390 10395 10400 Val Gly Val Tyr Val Gly Cys Gly Tyr Gln Asp Tyr Ala Pro Asp Ile 10405 10410 10415 Arg Val Ala Pro Glu Gly Thr Gly Gly Tyr Val Val Thr Gly Asn Ser 10420 10425 10430 Ser Ala Val Ala Ser Gly Arg Ile Ala Tyr Ser Leu Gly Leu Glu Gly 10435 10440 10445 Pro Ala Val Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Leu 10450 10455 10460 His Leu Ala Leu Lys Gly Leu Arg Asn Gly Asp Cys Ser Thr Ala Leu 10465 10470 10475 10480 Val Gly Gly Val Ala Val Leu Ala Thr Pro Gly Ala Phe Ile Glu Phe 10485 10490 10495 Ser Ser Gln Gln Ala Met Ala Ala Asp Gly Arg Thr Lys Gly Phe Ala 10500 10505 10510 Ser Ala Ala Asp Gly Leu Ala Trp Gly Glu Gly Val Ala Val Leu Leu 10515 10520 10525 Leu Glu Arg Leu Ser Asp Ala Arg Arg Lys Gly His Arg Val Leu Ala 10530 10535 10540 Val Val Arg Gly Ser Ala Ile Asn Gln Asp Gly Ala Ser Asn Gly Leu 10545 10550 10555 10560 Thr Ala Pro His Gly Pro Ser Gln Gln His Leu Ile Arg Gln Ala Leu 10565 10570 10575 Ala Asp Ala Arg Leu Thr Ser Ser Asp Val Asp Val Val Glu Gly His 10580 10585 10590 Gly Thr Gly Thr Arg Leu Gly Asp Pro Ile Glu Ala Gln Ala Leu Leu 10595 10600 10605 Ala Thr Tyr Gly Gln Gly Arg Ala Pro Gly Gln Pro Leu Arg Leu Gly 10610 10615 10620 Thr Leu Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ser Gly Val Ala 10625 10630 10635 10640 Gly Val Ile Lys Met Val Gln Ala Leu Arg His Gly Val Leu Pro Lys 10645 10650 10655 Thr Leu His Val Asp Glu Pro Thr Asp Gln Val Asp Trp Ser Ala Gly 10660 10665 10670 Ser Val Glu Leu Leu Thr Glu Ala Val Asp Trp Pro Glu Arg Pro Gly 10675 10680 10685 Arg Leu Arg Arg Ala Gly Val Ser Ala Phe Gly Val Gly Gly Thr Asn 10690 10695 10700 Ala His Val Val Leu Glu Glu Ala Pro Ala Val Glu Glu Ser Pro Ala 10705 10710 10715 10720 Val Glu Pro Pro Ala Gly Gly Gly Val Val Pro Trp Pro Val Ser Ala 10725 10730 10735 Lys Thr Ser Ala Ala Leu Asp Ala Gln Ile Gly Gln Leu Ala Ala Tyr 10740 10745 10750 Ala Glu Asp Arg Thr Asp Val Asp Pro Ala Val Ala Ala Arg Ala Leu 10755 10760 10765 Val Asp Ser Arg Thr Ala Met Glu His Arg Ala Val Ala Val Gly Asp 10770 10775 10780 Ser Arg Glu Ala Leu Arg Asp Ala Leu Arg Met Pro Glu Gly Leu Val 10785 10790 10795 10800 Arg Gly Thr Val Thr Asp Pro Gly Arg Val Ala Phe Val Phe Pro Gly 10805 10810 10815 Gln Gly Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Ser Ser 10820 10825 10830 Pro Glu Phe Ala Ala Ala Met Ala Glu Cys Glu Thr Ala Leu Ser Pro 10835 10840 10845 Tyr Val Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Ser Ala 10850 10855 10860 Pro Thr Leu Asp Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val 10865 10870 10875 10880 Met Val Ser Leu Ala Lys Val Trp Gln His His Gly Ile Thr Pro Glu 10885 10890 10895 Ala Val Ile Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala 10900 10905 10910 Gly Ala Leu Thr Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser 10915 10920 10925 Lys Ser Ile Ala Ala His Leu Ala Gly Lys Gly Gly Met Ile Ser Leu 10930 10935 10940 Ala Leu Ser Glu Glu Ala Thr Arg Gln Arg Ile Glu Asn Leu His Gly 10945 10950 10955 10960 Leu Ser Ile Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly 10965 10970 10975 Asp Pro Thr Gln Ile Gln Glu Leu Ala Gln Ala Cys Glu Ala Asp Gly 10980 10985 10990 Ile Arg Ala Arg Ile Ile Pro Val Asp Tyr Ala Ser His Ser Ala His 10995 11000 11005 Val Glu Thr Ile Glu Asn Glu Leu Ala Asp Val Leu Ala Gly Leu Ser 11010 11015 11020 Pro Gln Thr Pro Gln Val Pro Phe Phe Ser Thr Leu Glu Gly Thr Trp 11025 11030 11035 11040 Ile Thr Glu Pro Ala Leu Asp Gly Gly Tyr Trp Tyr Arg Asn Leu Arg 11045 11050 11055 His Arg Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu 11060 11065 11070 Gly Phe Thr His Phe Ile Glu Val Ser Ala His Pro Val Leu Thr Met 11075 11080 11085 Thr Leu Pro Asp Lys Val Thr Gly Leu Ala Thr Leu Arg Arg Glu Asp 11090 11095 11100 Gly Gly Gln His Arg Leu Thr Thr Ser Leu Ala Glu Ala Trp Ala Asn 11105 11110 11115 11120 Gly Leu Ala Leu Asp Trp Ala Ser Leu Leu Pro Ala Thr Gly Ala Leu 11125 11130 11135 Ser Pro Ala Val Pro Asp Leu Pro Thr Tyr Ala Phe Gln His Arg Ser 11140 11145 11150 Tyr Trp Ile Ser Pro Ala Gly Pro Gly Glu Ala Pro Ala His Thr Ala 11155 11160 11165 Ser Gly Arg Glu Ala Val Ala Glu Thr Gly Leu Ala Trp Gly Pro Gly 11170 11175 11180 Ala Glu Asp Leu Asp Glu Glu Gly Arg Arg Ser Ala Val Leu Ala Met 11185 11190 11195 11200 Val Met Arg Gln Ala Ala Ser Val Leu Arg Cys Asp Ser Pro Glu Glu 11205 11210 11215 Val Pro Val Asp Arg Pro Leu Arg Glu Ile Gly Phe Asp Ser Leu Thr 11220 11225 11230 Ala Val Asp Phe Arg Asn Arg Val Asn Arg Leu Thr Gly Leu Gln Leu 11235 11240 11245 Pro Pro Thr Val Val Phe Gln His Pro Thr Pro Val Ala Leu Ala Glu 11250 11255 11260 Arg Ile Ser Asp Glu Leu Ala Glu Arg Asn Trp Ala Val Ala Glu Pro 11265 11270 11275 11280 Ser Asp His Glu Gln Ala Glu Glu Glu Lys Ala Ala Ala Pro Ala Gly 11285 11290 11295 Ala Arg Ser Gly Ala Asp Thr Gly Ala Gly Ala Gly Met Phe Arg Ala 11300 11305 11310 Leu Phe Arg Gln Ala Val Glu Asp Asp Arg Tyr Gly Glu Phe Leu Asp 11315 11320 11325 Val Leu Ala Glu Ala Ser Ala Phe Arg Pro Gln Phe Ala Ser Pro Glu 11330 11335 11340 Ala Cys Ser Glu Arg Leu Asp Pro Val Leu Leu Ala Gly Gly Pro Thr 11345 11350 11355 11360 Asp Arg Ala Glu Gly Arg Ala Val Leu Val Gly Cys Thr Gly Thr Ala 11365 11370 11375 Ala Asn Gly Gly Pro His Glu Phe Leu Arg Leu Ser Thr Ser Phe Gln 11380 11385 11390 Glu Glu Arg Asp Phe Leu Ala Val Pro Leu Pro Gly Tyr Gly Thr Gly 11395 11400 11405 Thr Gly Thr Gly Thr Ala Leu Leu Pro Ala Asp Leu Asp Thr Ala Leu 11410 11415 11420 Asp Ala Gln Ala Arg Ala Ile Leu Arg Ala Ala Gly Asp Ala Pro Val 11425 11430 11435 11440 Val Leu Leu Gly His Ser Gly Gly Ala Leu Leu Ala His Glu Leu Ala 11445 11450 11455 Phe Arg Leu Glu Arg Ala His Gly Ala Pro Pro Ala Gly Ile Val Leu 11460 11465 11470 Val Asp Pro Tyr Pro Pro Gly His Gln Glu Pro Ile Glu Val Trp Ser 11475 11480 11485 Arg Gln Leu Gly Glu Gly Leu Phe Ala Gly Glu Leu Glu Pro Met Ser 11490 11495 11500 Asp Ala Arg Leu Leu Ala Met Gly Arg Tyr Ala Arg Phe Leu Ala Gly 11505 11510 11515 11520 Pro Arg Pro Gly Arg Ser Ser Ala Pro Val Leu Leu Val Arg Ala Ser 11525 11530 11535 Glu Pro Leu Gly Asp Trp Gln Glu Glu Arg Gly Asp Trp Arg Ala His 11540 11545 11550 Trp Asp Leu Pro His Thr Val Ala Asp Val Pro Gly Asp His Phe Thr 11555 11560 11565 Met Met Arg Asp His Ala Pro Ala Val Ala Glu Ala Val Leu Ser Trp 11570 11575 11580 Leu Asp Ala Ile Glu Gly Ile Glu Gly Ala Gly Lys Met Thr Asp Arg 11585 11590 11595 11600 Pro Leu Asn Val Asp Ser Gly Leu Trp Ile Arg Arg Phe His Pro Ala 11605 11610 11615 Pro Asn Ser Ala Val Arg Leu Val Cys Leu Pro His Ala Gly Gly Ser 11620 11625 11630 Ala Ser Tyr Phe Phe Arg Phe Ser Glu Glu Leu His Pro Ser Val Glu 11635 11640 11645 Ala Leu Ser Val Gln Tyr Pro Gly Arg Gln Asp Arg Arg Ala Glu Pro 11650 11655 11660 Cys Leu Glu Ser Val Glu Glu Leu Ala Glu His Val Val Ala Ala Thr 11665 11670 11675 11680 Glu Pro Trp Trp Gln Glu Gly Arg Leu Ala Phe Phe Gly His Ser Leu 11685 11690 11695 Gly Ala Ser Val Ala Phe Glu Thr Ala Arg Ile Leu Glu Gln Arg His 11700 11705 11710 Gly Val Arg Pro Glu Gly Leu Tyr Val Ser Gly Arg Arg Ala Pro Ser 11715 11720 11725 Leu Ala Pro Asp Arg Leu Val His Gln Leu Asp Asp Arg Ala Phe Leu 11730 11735 11740 Ala Glu Ile Arg Arg Leu Ser Gly Thr Asp Glu Arg Phe Leu Gln Asp 11745 11750 11755 11760 Asp Glu Leu Leu Arg Leu Val Leu Pro Ala Leu Arg Ser Asp Tyr Lys 11765 11770 11775 Ala Ala Glu Thr Tyr Leu His Arg Pro Ser Ala Lys Leu Thr Cys Pro 11780 11785 11790 Val Met Ala Leu Ala Gly Asp Arg Asp Pro Lys Ala Pro Leu Asn Glu 11795 11800 11805 Val Ala Glu Trp Arg Arg His Thr Ser Gly Pro Phe Cys Leu Arg Ala 11810 11815 11820 Tyr Ser Gly Gly His Phe Tyr Leu Asn Asp Gln Trp His Glu Ile Cys 11825 11830 11835 11840 Asn Asp Ile Ser Asp His Leu Leu Val Thr Arg Gly Ala Pro Asp Ala 11845 11850 11855 Arg Val Val Gln Pro Pro Thr Ser Leu Ile Glu Gly Ala Ala Lys Arg 11860 11865 11870 Trp Gln Asn Pro Arg 11875 7 1248 DNA Streptomyces venezuelae 7 gtgaaaagcg ccttatccga cctcgcattc ttcggcggcc ccgccgcttt cgaccagccg 60 ctcctcgtgg ggcggcccaa ccgcatcgac cgcgccaggc tgtacgagcg gctcgaccgg 120 gccctcgaca gccagtggct gtccaacggc ggcccgctcg tccgcgagtt cgaggagcgc 180 gtcgccgggc tcgccggggt ccggcatgcc gtggccacct gcaacgccac ggccgggctc 240 cagctcctcg cgcacgccgc cggcctcacc ggcgaagtga tcatgccgtc gatgacgttc 300 gccgccaccc cgcacgcact gcgctggatc ggcctcaccc cggtcttcgc cgacatcgac 360 ccggacaccg gcaacctcga cccggaccag gtggccgccg cggtcacacc ccgcacctcg 420 gccgtcgtcg gcgtccacct ctggggccgc ccctgcgccg ccgaccagct gcggaaggtc 480 gccgacgagc acggcctgcg gctgtacttc gacgccgcgc acgccctcgg ctgcgcggtc 540 gacggccggc ccgccggcag cctcggcgac gccgaggtct tcagcttcca cgccaccaag 600 gccgtcaacg ccttcgaggg cggcgccgtc gtcaccgacg acgccgacct cgccgcccgg 660 atccgcgccc tccacaactt cggcttcgac ctgcccggcg gcagccccgc cggcgggacc 720 aacgccaaga tgagcgaggc cgccgccgcc atgggcctca cctccctcga cgcgtttccc 780 gaggtcatcg accggaaccg gcgcaaccac gccgcctacc gcgagcacct cgcggacctc 840 cccggcgtcc tcgtcgccga ccacgaccgc cacggcctca acaaccacca gtacgtgatc 900 gtcgagatcg acgaggccac caccggcatc caccgcgacc tcgtcatgga ggtcctgaag 960 gccgaaggcg tgcacacccg cgcctacttc tcgccgggct gccacgagct ggagccgtac 1020 cgcgggcagc cgcacgcccc gctgccgcac accgaacgcc tcgccgcgcg cgtgctgtcc 1080 ctgccgaccg gcaccgccat cggcgacgac gacatccgcc gggtcgccga cctgctgcgt 1140 ctctgcgcga cccgcggccg cgaactgacc gcgcgccacc gcgacacggc ccccgccccg 1200 ctcgcggccc cccagacatc cacgcccacg attggacgct cccgatga 1248 8 415 PRT Streptomyces venezuelae 8 Met Lys Ser Ala Leu Ser Asp Leu Ala Phe Phe Gly Gly Pro Ala Ala 1 5 10 15 Phe Asp Gln Pro Leu Leu Val Gly Arg Pro Asn Arg Ile Asp Arg Ala 20 25 30 Arg Leu Tyr Glu Arg Leu Asp Arg Ala Leu Asp Ser Gln Trp Leu Ser 35 40 45 Asn Gly Gly Pro Leu Val Arg Glu Phe Glu Glu Arg Val Ala Gly Leu 50 55 60 Ala Gly Val Arg His Ala Val Ala Thr Cys Asn Ala Thr Ala Gly Leu 65 70 75 80 Gln Leu Leu Ala His Ala Ala Gly Leu Thr Gly Glu Val Ile Met Pro 85 90 95 Ser Met Thr Phe Ala Ala Thr Pro His Ala Leu Arg Trp Ile Gly Leu 100 105 110 Thr Pro Val Phe Ala Asp Ile Asp Pro Asp Thr Gly Asn Leu Asp Pro 115 120 125 Asp Gln Val Ala Ala Ala Val Thr Pro Arg Thr Ser Ala Val Val Gly 130 135 140 Val His Leu Trp Gly Arg Pro Cys Ala Ala Asp Gln Leu Arg Lys Val 145 150 155 160 Ala Asp Glu His Gly Leu Arg Leu Tyr Phe Asp Ala Ala His Ala Leu 165 170 175 Gly Cys Ala Val Asp Gly Arg Pro Ala Gly Ser Leu Gly Asp Ala Glu 180 185 190 Val Phe Ser Phe His Ala Thr Lys Ala Val Asn Ala Phe Glu Gly Gly 195 200 205 Ala Val Val Thr Asp Asp Ala Asp Leu Ala Ala Arg Ile Arg Ala Leu 210 215 220 His Asn Phe Gly Phe Asp Leu Pro Gly Gly Ser Pro Ala Gly Gly Thr 225 230 235 240 Asn Ala Lys Met Ser Glu Ala Ala Ala Ala Met Gly Leu Thr Ser Leu 245 250 255 Asp Ala Phe Pro Glu Val Ile Asp Arg Asn Arg Arg Asn His Ala Ala 260 265 270 Tyr Arg Glu His Leu Ala Asp Leu Pro Gly Val Leu Val Ala Asp His 275 280 285 Asp Arg His Gly Leu Asn Asn His Gln Tyr Val Ile Val Glu Ile Asp 290 295 300 Glu Ala Thr Thr Gly Ile His Arg Asp Leu Val Met Glu Val Leu Lys 305 310 315 320 Ala Glu Gly Val His Thr Arg Ala Tyr Phe Ser Pro Gly Cys His Glu 325 330 335 Leu Glu Pro Tyr Arg Gly Gln Pro His Ala Pro Leu Pro His Thr Glu 340 345 350 Arg Leu Ala Ala Arg Val Leu Ser Leu Pro Thr Gly Thr Ala Ile Gly 355 360 365 Asp Asp Asp Ile Arg Arg Val Ala Asp Leu Leu Arg Leu Cys Ala Thr 370 375 380 Arg Gly Arg Glu Leu Thr Ala Arg His Arg Asp Thr Ala Pro Ala Pro 385 390 395 400 Leu Ala Ala Pro Gln Thr Ser Thr Pro Thr Ile Gly Arg Ser Arg 405 410 415 9 1458 DNA Streptomyces venezuelae 9 atgaccgccc ccgccctttc cgccaccgcc ccggccgaac gctgcgcgca ccccggagcc 60 gatctggggg cggcggtcca cgccgtcggc cagaccctcg ccgccggcgg cctcgtgccg 120 cccgacgagg ccggaacgac cgcccgccac ctcgtccggc tcgccgtgcg ctacggcaac 180 agccccttca ccccgctgga ggaggcccgc cacgacctgg gcgtcgaccg ggacgccttc 240 cggcgcctcc tcgccctgtt cgggcaggtc ccggagctcc gcaccgcggt cgagaccggc 300 cccgccgggg cgtactggaa gaacaccctg ctcccgctcg aacagcgcgg cgtcttcgac 360 gcggcgctcg ccaggaagcc cgtcttcccg tacagcgtcg gcctctaccc cggcccgacc 420 tgcatgttcc gctgccactt ctgcgtccgt gtgaccggcg cccgctacga cccgtccgcc 480 ctcgacgccg gcaacgccat gttccggtcg gtcatcgacg agatacccgc gggcaacccc 540 tcggcgatgt acttctccgg cggcctggag ccgctcacca accccggcct cgggagcctg 600 gccgcgcacg ccaccgacca cggcctgcgg cccaccgtct acacgaactc cttcgcgctc 660 accgagcgca ccctggagcg ccagcccggc ctctggggcc tgcacgccat ccgcacctcg 720 ctctacggcc tcaacgacga ggagtacgag cagaccaccg gcaagaaggc cgccttccgc 780 cgcgtccgcg agaacctgcg ccgcttccag cagctgcgcg ccgagcgcga gtcgccgatc 840 aacctcggct tcgcctacat cgtgctcccg ggccgtgcct cccgcctgct cgacctggtc 900 gacttcatcg ccgacctcaa cgacgccggg cagggcagga cgatcgactt cgtcaacatt 960 cgcgaggact acagcggccg tgacgacggc aagctgccgc aggaggagcg ggccgagctc 1020 caggaggccc tcaacgcctt cgaggagcgg gtccgcgagc gcacccccgg actccacatc 1080 gactacggct acgccctgaa cagcctgcgc accggggccg acgccgaact gctgcggatc 1140 aagcccgcca ccatgcggcc caccgcgcac ccgcaggtcg cggtgcaggt cgatctcctc 1200 ggcgacgtgt acctgtaccg cgaggccggc ttccccgacc tggacggcgc gacccgctac 1260 atcgcgggcc gcgtgacccc cgacacctcc ctcaccgagg tcgtcaggga cttcgtcgag 1320 cgcggcggcg aggtggcggc cgtcgacggc gacgagtact tcatggacgg cttcgatcag 1380 gtcgtcaccg cccgcctgaa ccagctggag cgcgacgccg cggacggctg ggaggaggcc 1440 cgcggcttcc tgcgctga 1458 10 485 PRT Streptomyces venezuelae 10 Met Thr Ala Pro Ala Leu Ser Ala Thr Ala Pro Ala Glu Arg Cys Ala 1 5 10 15 His Pro Gly Ala Asp Leu Gly Ala Ala Val His Ala Val Gly Gln Thr 20 25 30 Leu Ala Ala Gly Gly Leu Val Pro Pro Asp Glu Ala Gly Thr Thr Ala 35 40 45 Arg His Leu Val Arg Leu Ala Val Arg Tyr Gly Asn Ser Pro Phe Thr 50 55 60 Pro Leu Glu Glu Ala Arg His Asp Leu Gly Val Asp Arg Asp Ala Phe 65 70 75 80 Arg Arg Leu Leu Ala Leu Phe Gly Gln Val Pro Glu Leu Arg Thr Ala 85 90 95 Val Glu Thr Gly Pro Ala Gly Ala Tyr Trp Lys Asn Thr Leu Leu Pro 100 105 110 Leu Glu Gln Arg Gly Val Phe Asp Ala Ala Leu Ala Arg Lys Pro Val 115 120 125 Phe Pro Tyr Ser Val Gly Leu Tyr Pro Gly Pro Thr Cys Met Phe Arg 130 135 140 Cys His Phe Cys Val Arg Val Thr Gly Ala Arg Tyr Asp Pro Ser Ala 145 150 155 160 Leu Asp Ala Gly Asn Ala Met Phe Arg Ser Val Ile Asp Glu Ile Pro 165 170 175 Ala Gly Asn Pro Ser Ala Met Tyr Phe Ser Gly Gly Leu Glu Pro Leu 180 185 190 Thr Asn Pro Gly Leu Gly Ser Leu Ala Ala His Ala Thr Asp His Gly 195 200 205 Leu Arg Pro Thr Val Tyr Thr Asn Ser Phe Ala Leu Thr Glu Arg Thr 210 215 220 Leu Glu Arg Gln Pro Gly Leu Trp Gly Leu His Ala Ile Arg Thr Ser 225 230 235 240 Leu Tyr Gly Leu Asn Asp Glu Glu Tyr Glu Gln Thr Thr Gly Lys Lys 245 250 255 Ala Ala Phe Arg Arg Val Arg Glu Asn Leu Arg Arg Phe Gln Gln Leu 260 265 270 Arg Ala Glu Arg Glu Ser Pro Ile Asn Leu Gly Phe Ala Tyr Ile Val 275 280 285 Leu Pro Gly Arg Ala Ser Arg Leu Leu Asp Leu Val Asp Phe Ile Ala 290 295 300 Asp Leu Asn Asp Ala Gly Gln Gly Arg Thr Ile Asp Phe Val Asn Ile 305 310 315 320 Arg Glu Asp Tyr Ser Gly Arg Asp Asp Gly Lys Leu Pro Gln Glu Glu 325 330 335 Arg Ala Glu Leu Gln Glu Ala Leu Asn Ala Phe Glu Glu Arg Val Arg 340 345 350 Glu Arg Thr Pro Gly Leu His Ile Asp Tyr Gly Tyr Ala Leu Asn Ser 355 360 365 Leu Arg Thr Gly Ala Asp Ala Glu Leu Leu Arg Ile Lys Pro Ala Thr 370 375 380 Met Arg Pro Thr Ala His Pro Gln Val Ala Val Gln Val Asp Leu Leu 385 390 395 400 Gly Asp Val Tyr Leu Tyr Arg Glu Ala Gly Phe Pro Asp Leu Asp Gly 405 410 415 Ala Thr Arg Tyr Ile Ala Gly Arg Val Thr Pro Asp Thr Ser Leu Thr 420 425 430 Glu Val Val Arg Asp Phe Val Glu Arg Gly Gly Glu Val Ala Ala Val 435 440 445 Asp Gly Asp Glu Tyr Phe Met Asp Gly Phe Asp Gln Val Val Thr Ala 450 455 460 Arg Leu Asn Gln Leu Glu Arg Asp Ala Ala Asp Gly Trp Glu Glu Ala 465 470 475 480 Arg Gly Phe Leu Arg 485 11 879 DNA Streptomyces venezuelae 11 atgaagggaa tagtcctggc cggcgggagc ggaactcggc tgcatccggc gacctcggtc 60 atttcgaagc agattcttcc ggtctacaac aaaccgatga tctactatcc gctgtcggtt 120 ctcatgctcg gcggtattcg cgagattcaa atcatctcga ccccccagca catcgaactc 180 ttccagtcgc ttctcggaaa cggcaggcac ctgggaatag aactcgacta tgcggtccag 240 aaagagcccg caggaatcgc ggacgcactt ctcgtcggag ccgagcacat cggcgacgac 300 acctgcgccc tgatcctggg cgacaacatc ttccacgggc ccggcctcta cacgctcctg 360 cgggacagca tcgcgcgcct cgacggctgc gtgctcttcg gctacccggt caaggacccc 420 gagcggtacg gcgtcgccga ggtggacgcg acgggccggc tgaccgacct cgtcgagaag 480 cccgtcaagc cgcgctccaa cctcgccgtc accggcctct acctctacga caacgacgtc 540 gtcgacatcg ccaagaacat ccggccctcg ccgcgcggcg agctggagat caccgacgtc 600 aaccgcgtct acctggagcg gggccgggcc gaactcgtca acctgggccg cggcttcgcc 660 tggctggaca ccggcaccca cgactcgctc ctgcgggccg cccagtacgt ccaggtcctg 720 gaggagcggc agggcgtctg gatcgcgggc cttgaggaga tcgccttccg catgggcttc 780 atcgacgccg aggcctgtca cggcctggga gaaggcctct cccgcaccga gtacggcagc 840 tatctgatgg agatcgccgg ccgcgaggga gccccgtga 879 12 292 PRT Streptomyces venezuelae 12 Met Lys Gly Ile Val Leu Ala Gly Gly Ser Gly Thr Arg Leu His Pro 1 5 10 15 Ala Thr Ser Val Ile Ser Lys Gln Ile Leu Pro Val Tyr Asn Lys Pro 20 25 30 Met Ile Tyr Tyr Pro Leu Ser Val Leu Met Leu Gly Gly Ile Arg Glu 35 40 45 Ile Gln Ile Ile Ser Thr Pro Gln His Ile Glu Leu Phe Gln Ser Leu 50 55 60 Leu Gly Asn Gly Arg His Leu Gly Ile Glu Leu Asp Tyr Ala Val Gln 65 70 75 80 Lys Glu Pro Ala Gly Ile Ala Asp Ala Leu Leu Val Gly Ala Glu His 85 90 95 Ile Gly Asp Asp Thr Cys Ala Leu Ile Leu Gly Asp Asn Ile Phe His 100 105 110 Gly Pro Gly Leu Tyr Thr Leu Leu Arg Asp Ser Ile Ala Arg Leu Asp 115 120 125 Gly Cys Val Leu Phe Gly Tyr Pro Val Lys Asp Pro Glu Arg Tyr Gly 130 135 140 Val Ala Glu Val Asp Ala Thr Gly Arg Leu Thr Asp Leu Val Glu Lys 145 150 155 160 Pro Val Lys Pro Arg Ser Asn Leu Ala Val Thr Gly Leu Tyr Leu Tyr 165 170 175 Asp Asn Asp Val Val Asp Ile Ala Lys Asn Ile Arg Pro Ser Pro Arg 180 185 190 Gly Glu Leu Glu Ile Thr Asp Val Asn Arg Val Tyr Leu Glu Arg Gly 195 200 205 Arg Ala Glu Leu Val Asn Leu Gly Arg Gly Phe Ala Trp Leu Asp Thr 210 215 220 Gly Thr His Asp Ser Leu Leu Arg Ala Ala Gln Tyr Val Gln Val Leu 225 230 235 240 Glu Glu Arg Gln Gly Val Trp Ile Ala Gly Leu Glu Glu Ile Ala Phe 245 250 255 Arg Met Gly Phe Ile Asp Ala Glu Ala Cys His Gly Leu Gly Glu Gly 260 265 270 Leu Ser Arg Thr Glu Tyr Gly Ser Tyr Leu Met Glu Ile Ala Gly Arg 275 280 285 Glu Gly Ala Pro 290 13 1014 DNA Streptomyces venezuelae 13 gtgcggcttc tggtgaccgg aggtgcgggc ttcatcggct cgcacttcgt gcggcagctc 60 ctcgccgggg cgtaccccga cgtgcccgcc gatgaggtga tcgtcctgga cagcctcacc 120 tacgcgggca accgcgccaa cctcgccccg gtggacgcgg acccgcgact gcgcttcgtc 180 cacggcgaca tccgcgacgc cggcctcctc gcccgggaac tgcgcggcgt ggacgccatc 240 gtccacttcg cggccgagag ccacgtggac cgctccatcg cgggcgcgtc cgtgttcacc 300 gagaccaacg tgcagggcac gcagacgctg ctccagtgcg ccgtcgacgc cggcgtcggc 360 cgggtcgtgc acgtctccac cgacgaggtg tacgggtcga tcgactccgg ctcctggacc 420 gagagcagcc cgctggagcc caactcgccc tacgcggcgt ccaaggccgg ctccgacctc 480 gttgcccgcg cctaccaccg gacgtacggc ctcgacgtac ggatcacccg ctgctgcaac 540 aactacgggc cgtaccagca ccccgagaag ctcatccccc tcttcgtgac gaacctcctc 600 gacggcggga cgctcccgct gtacggcgac ggcgcgaacg tccgcgagtg ggtgcacacc 660 gacgaccact gccggggcat cgcgctcgtc ctcgcgggcg gccgggccgg cgagatctac 720 cacatcggcg gcggcctgga gctgaccaac cgcgaactca ccggcatcct cctggactcg 780 ctcggcgccg actggtcctc ggtccggaag gtcgccgacc gcaagggcca cgacctgcgc 840 tactccctcg acggcggcga gatcgagcgc gagctcggct accgcccgca ggtctccttc 900 gcggacggcc tcgcgcggac cgtccgctgg taccgggaga accgcggctg gtgggagccg 960 ctcaaggcga ccgccccgca gctgcccgcc accgccgtgg aggtgtccgc gtga 1014 14 337 PRT Streptomyces venezuelae 14 Met Arg Leu Leu Val Thr Gly Gly Ala Gly Phe Ile Gly Ser His Phe 1 5 10 15 Val Arg Gln Leu Leu Ala Gly Ala Tyr Pro Asp Val Pro Ala Asp Glu 20 25 30 Val Ile Val Leu Asp Ser Leu Thr Tyr Ala Gly Asn Arg Ala Asn Leu 35 40 45 Ala Pro Val Asp Ala Asp Pro Arg Leu Arg Phe Val His Gly Asp Ile 50 55 60 Arg Asp Ala Gly Leu Leu Ala Arg Glu Leu Arg Gly Val Asp Ala Ile 65 70 75 80 Val His Phe Ala Ala Glu Ser His Val Asp Arg Ser Ile Ala Gly Ala 85 90 95 Ser Val Phe Thr Glu Thr Asn Val Gln Gly Thr Gln Thr Leu Leu Gln 100 105 110 Cys Ala Val Asp Ala Gly Val Gly Arg Val Val His Val Ser Thr Asp 115 120 125 Glu Val Tyr Gly Ser Ile Asp Ser Gly Ser Trp Thr Glu Ser Ser Pro 130 135 140 Leu Glu Pro Asn Ser Pro Tyr Ala Ala Ser Lys Ala Gly Ser Asp Leu 145 150 155 160 Val Ala Arg Ala Tyr His Arg Thr Tyr Gly Leu Asp Val Arg Ile Thr 165 170 175 Arg Cys Cys Asn Asn Tyr Gly Pro Tyr Gln His Pro Glu Lys Leu Ile 180 185 190 Pro Leu Phe Val Thr Asn Leu Leu Asp Gly Gly Thr Leu Pro Leu Tyr 195 200 205 Gly Asp Gly Ala Asn Val Arg Glu Trp Val His Thr Asp Asp His Cys 210 215 220 Arg Gly Ile Ala Leu Val Leu Ala Gly Gly Arg Ala Gly Glu Ile Tyr 225 230 235 240 His Ile Gly Gly Gly Leu Glu Leu Thr Asn Arg Glu Leu Thr Gly Ile 245 250 255 Leu Leu Asp Ser Leu Gly Ala Asp Trp Ser Ser Val Arg Lys Val Ala 260 265 270 Asp Arg Lys Gly His Asp Leu Arg Tyr Ser Leu Asp Gly Gly Glu Ile 275 280 285 Glu Arg Glu Leu Gly Tyr Arg Pro Gln Val Ser Phe Ala Asp Gly Leu 290 295 300 Ala Arg Thr Val Arg Trp Tyr Arg Glu Asn Arg Gly Trp Trp Glu Pro 305 310 315 320 Leu Lys Ala Thr Ala Pro Gln Leu Pro Ala Thr Ala Val Glu Val Ser 325 330 335 Ala 15 1140 DNA Streptomyces venezuelae 15 gtgagcagcc gcgccgagac cccccgcgtc cccttcctcg acctcaaggc cgcctacgag 60 gagctccgcg cggagaccga cgccgcgatc gcccgcgtcc tcgactcggg gcgctacctc 120 ctcggacccg aactcgaagg attcgaggcg gagttcgccg cgtactgcga gacggaccac 180 gccgtcggcg tgaacagcgg gatggacgcc ctccagctcg ccctccgcgg cctcggcatc 240 ggacccgggg acgaggtgat cgtcccctcg cacacgtaca tcgccagctg gctcgcggtg 300 tccgccaccg gcgcgacccc cgtgcccgtc gagccgcacg aggaccaccc caccctggac 360 ccgctgctcg tcgagaaggc gatcaccccc cgcacccggg cgctcctccc cgtccacctc 420 tacgggcacc ccgccgacat ggacgccctc cgcgagctcg cggaccggca cggcctgcac 480 atcgtcgagg acgccgcgca ggcccacggc gcccgctacc ggggccggcg gatcggcgcc 540 gggtcgtcgg tggccgcgtt cagcttctac ccgggcaaga acctcggctg cttcggcgac 600 ggcggcgccg tcgtcaccgg cgaccccgag ctcgccgaac ggctccggat gctccgcaac 660 tacggctcgc ggcagaagta cagccacgag acgaagggca ccaactcccg cctggacgag 720 atgcaggccg ccgtgctgcg gatccggctc gcccacctgg acagctggaa cggccgcagg 780 tcggcgctgg ccgcggagta cctctccggg ctcgccggac tgcccggcat cggcctgccg 840 gtgaccgcgc ccgacaccga cccggtctgg cacctcttca ccgtgcgcac cgagcgccgc 900 gacgagctgc gcagccacct cgacgcccgc ggcatcgaca ccctcacgca ctacccggta 960 cccgtgcacc tctcgcccgc ctacgcgggc gaggcaccgc cggaaggctc gctcccgcgg 1020 gccgagagct tcgcgcggca ggtcctcagc ctgccgatcg gcccgcacct ggagcgcccg 1080 caggcgctgc gggtgatcga cgccgtgcgc gaatgggccg agcgggtcga ccaggcctag 1140 16 379 PRT Streptomyces venezuelae 16 Met Ser Ser Arg Ala Glu Thr Pro Arg Val Pro Phe Leu Asp Leu Lys 1 5 10 15 Ala Ala Tyr Glu Glu Leu Arg Ala Glu Thr Asp Ala Ala Ile Ala Arg 20 25 30 Val Leu Asp Ser Gly Arg Tyr Leu Leu Gly Pro Glu Leu Glu Gly Phe 35 40 45 Glu Ala Glu Phe Ala Ala Tyr Cys Glu Thr Asp His Ala Val Gly Val 50 55 60 Asn Ser Gly Met Asp Ala Leu Gln Leu Ala Leu Arg Gly Leu Gly Ile 65 70 75 80 Gly Pro Gly Asp Glu Val Ile Val Pro Ser His Thr Tyr Ile Ala Ser 85 90 95 Trp Leu Ala Val Ser Ala Thr Gly Ala Thr Pro Val Pro Val Glu Pro 100 105 110 His Glu Asp His Pro Thr Leu Asp Pro Leu Leu Val Glu Lys Ala Ile 115 120 125 Thr Pro Arg Thr Arg Ala Leu Leu Pro Val His Leu Tyr Gly His Pro 130 135 140 Ala Asp Met Asp Ala Leu Arg Glu Leu Ala Asp Arg His Gly Leu His 145 150 155 160 Ile Val Glu Asp Ala Ala Gln Ala His Gly Ala Arg Tyr Arg Gly Arg 165 170 175 Arg Ile Gly Ala Gly Ser Ser Val Ala Ala Phe Ser Phe Tyr Pro Gly 180 185 190 Lys Asn Leu Gly Cys Phe Gly Asp Gly Gly Ala Val Val Thr Gly Asp 195 200 205 Pro Glu Leu Ala Glu Arg Leu Arg Met Leu Arg Asn Tyr Gly Ser Arg 210 215 220 Gln Lys Tyr Ser His Glu Thr Lys Gly Thr Asn Ser Arg Leu Asp Glu 225 230 235 240 Met Gln Ala Ala Val Leu Arg Ile Arg Leu Ala His Leu Asp Ser Trp 245 250 255 Asn Gly Arg Arg Ser Ala Leu Ala Ala Glu Tyr Leu Ser Gly Leu Ala 260 265 270 Gly Leu Pro Gly Ile Gly Leu Pro Val Thr Ala Pro Asp Thr Asp Pro 275 280 285 Val Trp His Leu Phe Thr Val Arg Thr Glu Arg Arg Asp Glu Leu Arg 290 295 300 Ser His Leu Asp Ala Arg Gly Ile Asp Thr Leu Thr His Tyr Pro Val 305 310 315 320 Pro Val His Leu Ser Pro Ala Tyr Ala Gly Glu Ala Pro Pro Glu Gly 325 330 335 Ser Leu Pro Arg Ala Glu Ser Phe Ala Arg Gln Val Leu Ser Leu Pro 340 345 350 Ile Gly Pro His Leu Glu Arg Pro Gln Ala Leu Arg Val Ile Asp Ala 355 360 365 Val Arg Glu Trp Ala Glu Arg Val Asp Gln Ala 370 375 17 714 DNA Streptomyces venezuelae 17 gtgtacgaag tcgaccacgc cgacgtctac gacctcttct acctgggtcg cggcaaggac 60 tacgccgccg aggcctccga catcgccgac ctggtgcgct cccgtacccc cgaggcctcc 120 tcgctcctgg acgtggcctg cggtacgggc acgcatctgg agcacttcac caaggagttc 180 ggcgacaccg ccggcctgga gctgtccgag gacatgctca cccacgcccg caagcggctg 240 cccgacgcca cgctccacca gggcgacatg cgggacttcc ggctcggccg gaagttctcc 300 gccgtggtca gcatgttcag ctccgtcggc tacctgaaga cgaccgagga actcggcgcg 360 gccgtcgcct cgttcgcgga gcacctggag cccggtggcg tcgtcgtcgt cgagccgtgg 420 tggttcccgg agaccttcgc cgacggctgg gtcagcgccg acgtcgtccg ccgtgacggg 480 cgcaccgtgg cccgtgtctc gcactcggtg cgggagggga acgcgacgcg catggaggtc 540 cacttcaccg tggccgaccc gggcaagggc gtgcggcact tctccgacgt ccatctcatc 600 accctgttcc accaggccga gtacgaggcc gcgttcacgg ccgccgggct gcgcgtcgag 660 tacctggagg gcggcccgtc gggccgtggc ctcttcgtcg gcgtccccgc ctga 714 18 237 PRT Streptomyces venezuelae 18 Met Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly 1 5 10 15 Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val 20 25 30 Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly 35 40 45 Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala 50 55 60 Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu 65 70 75 80 Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Arg Leu Gly 85 90 95 Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu 100 105 110 Lys Thr Thr Glu Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His 115 120 125 Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu 130 135 140 Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly 145 150 155 160 Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr 165 170 175 Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg 180 185 190 His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Ala Glu Tyr 195 200 205 Glu Ala Ala Phe Thr Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly 210 215 220 Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala 225 230 235 19 1281 DNA Streptomyces venezuelae 19 atgcgcgtcc tgctgacctc gttcgcacat cacacgcact actacggcct ggtgcccctg 60 gcctgggcgc tgctcgccgc cgggcacgag gtgcgggtcg ccagccagcc cgcgctcacg 120 gacaccatca ccgggtccgg gctcgccgcg gtgccggtcg gcaccgacca cctcatccac 180 gagtaccggg tgcggatggc gggcgagccg cgcccgaacc atccggcgat cgccttcgac 240 gaggcccgtc ccgagccgct ggactgggac cacgccctcg gcatcgaggc gatcctcgcc 300 ccgtacttcc atctgctcgc caacaacgac tcgatggtcg acgacctcgt cgacttcgcc 360 cggtcctggc agccggacct ggtgctgtgg gagccgacga cctacgcggg cgccgtcgcc 420 gcccaggtca ccggtgccgc gcacgcccgg gtcctgtggg ggcccgacgt gatgggcagc 480 gcccgccgca agttcgtcgc gctgcgggac cggcagccgc ccgagcaccg cgaggacccc 540 accgcggagt ggctgacgtg gacgctcgac cggtacggcg cctccttcga agaggagctg 600 ctcaccggcc agttcacgat cgacccgacc ccgccgagcc tgcgcctcga cacgggcctg 660 ccgaccgtcg ggatgcgtta tgttccgtac aacggcacgt cggtcgtgcc ggactggctg 720 agtgagccgc ccgcgcggcc ccgggtctgc ctgaccctcg gcgtctccgc gcgtgaggtc 780 ctcggcggcg acggcgtctc gcagggcgac atcctggagg cgctcgccga cctcgacatc 840 gagctcgtcg ccacgctcga cgcgagtcag cgcgccgaga tccgcaacta cccgaagcac 900 acccggttca cggacttcgt gccgatgcac gcgctcctgc cgagctgctc ggcgatcatc 960 caccacggcg gggcgggcac ctacgcgacc gccgtgatca acgcggtgcc gcaggtcatg 1020 ctcgccgagc tgtgggacgc gccggtcaag gcgcgggccg tcgccgagca gggggcgggg 1080 ttcttcctgc cgccggccga gctcacgccg caggccgtgc gggacgccgt cgtccgcatc 1140 ctcgacgacc cctcggtcgc caccgccgcg caccggctgc gcgaggagac cttcggcgac 1200 cccaccccgg ccgggatcgt ccccgagctg gagcggctcg ccgcgcagca ccgccgcccg 1260 ccggccgacg cccggcactg a 1281 20 426 PRT Streptomyces venezuelae 20 Met Arg Val Leu Leu Thr Ser Phe Ala His His Thr His Tyr Tyr Gly 1 5 10 15 Leu Val Pro Leu Ala Trp Ala Leu Leu Ala Ala Gly His Glu Val Arg 20 25 30 Val Ala Ser Gln Pro Ala Leu Thr Asp Thr Ile Thr Gly Ser Gly Leu 35 40 45 Ala Ala Val Pro Val Gly Thr Asp His Leu Ile His Glu Tyr Arg Val 50 55 60 Arg Met Ala Gly Glu Pro Arg Pro Asn His Pro Ala Ile Ala Phe Asp 65 70 75 80 Glu Ala Arg Pro Glu Pro Leu Asp Trp Asp His Ala Leu Gly Ile Glu 85 90 95 Ala Ile Leu Ala Pro Tyr Phe His Leu Leu Ala Asn Asn Asp Ser Met 100 105 110 Val Asp Asp Leu Val Asp Phe Ala Arg Ser Trp Gln Pro Asp Leu Val 115 120 125 Leu Trp Glu Pro Thr Thr Tyr Ala Gly Ala Val Ala Ala Gln Val Thr 130 135 140 Gly Ala Ala His Ala Arg Val Leu Trp Gly Pro Asp Val Met Gly Ser 145 150 155 160 Ala Arg Arg Lys Phe Val Ala Leu Arg Asp Arg Gln Pro Pro Glu His 165 170 175 Arg Glu Asp Pro Thr Ala Glu Trp Leu Thr Trp Thr Leu Asp Arg Tyr 180 185 190 Gly Ala Ser Phe Glu Glu Glu Leu Leu Thr Gly Gln Phe Thr Ile Asp 195 200 205 Pro Thr Pro Pro Ser Leu Arg Leu Asp Thr Gly Leu Pro Thr Val Gly 210 215 220 Met Arg Tyr Val Pro Tyr Asn Gly Thr Ser Val Val Pro Asp Trp Leu 225 230 235 240 Ser Glu Pro Pro Ala Arg Pro Arg Val Cys Leu Thr Leu Gly Val Ser 245 250 255 Ala Arg Glu Val Leu Gly Gly Asp Gly Val Ser Gln Gly Asp Ile Leu 260 265 270 Glu Ala Leu Ala Asp Leu Asp Ile Glu Leu Val Ala Thr Leu Asp Ala 275 280 285 Ser Gln Arg Ala Glu Ile Arg Asn Tyr Pro Lys His Thr Arg Phe Thr 290 295 300 Asp Phe Val Pro Met His Ala Leu Leu Pro Ser Cys Ser Ala Ile Ile 305 310 315 320 His His Gly Gly Ala Gly Thr Tyr Ala Thr Ala Val Ile Asn Ala Val 325 330 335 Pro Gln Val Met Leu Ala Glu Leu Trp Asp Ala Pro Val Lys Ala Arg 340 345 350 Ala Val Ala Glu Gln Gly Ala Gly Phe Phe Leu Pro Pro Ala Glu Leu 355 360 365 Thr Pro Gln Ala Val Arg Asp Ala Val Val Arg Ile Leu Asp Asp Pro 370 375 380 Ser Val Ala Thr Ala Ala His Arg Leu Arg Glu Glu Thr Phe Gly Asp 385 390 395 400 Pro Thr Pro Ala Gly Ile Val Pro Glu Leu Glu Arg Leu Ala Ala Gln 405 410 415 His Arg Arg Pro Pro Ala Asp Ala Arg His 420 425 21 1209 DNA Streptomyces venezuelae 21 gtgaccgacg acctgacggg ggccctcacg cagcccccgc tgggccgcac cgtccgcgcg 60 gtggccgacc gtgaactcgg cacccacctc ctggagaccc gcggcatcca ctggatccac 120 gccgcgaacg gcgacccgta cgccaccgtg ctgcgcggcc aggcggacga cccgtatccc 180 gcgtacgagc gggtgcgtgc ccgcggcgcg ctctccttca gcccgacggg cagctgggtc 240 accgccgatc acgccctggc ggcgagcatc ctctgctcga cggacttcgg ggtctccggc 300 gccgacggcg tcccggtgcc gcagcaggtc ctctcgtacg gggagggctg tccgctggag 360 cgcgagcagg tgctgccggc ggccggtgac gtgccggagg gcgggcagcg tgccgtggtc 420 gaggggatcc accgggagac gctggagggt ctcgcgccgg acccgtcggc gtcgtacgcc 480 ttcgagctgc tgggcggttt cgtccgcccg gcggtgacgg ccgctgccgc cgccgtgctg 540 ggtgttcccg cggaccggcg cgcggacttc gcggatctgc tggagcggct ccggccgctg 600 tccgacagcc tgctggcccc gcagtccctg cggacggtac gggcggcgga cggcgcgctg 660 gccgagctca cggcgctgct cgccgattcg gacgactccc ccggggccct gctgtcggcg 720 ctcggggtca ccgcagccgt ccagctcacc gggaacgcgg tgctcgcgct cctcgcgcat 780 cccgagcagt ggcgggagct gtgcgaccgg cccgggctcg cggcggccgc ggtggaggag 840 accctccgct acgacccgcc ggtgcagctc gacgcccggg tggtccgcgg ggagacggag 900 ctggcgggcc ggcggctgcc ggccggggcg catgtcgtcg tcctgaccgc cgcgaccggc 960 cgggacccgg aggtcttcac ggacccggag cgcttcgacc tcgcgcgccc cgacgccgcc 1020 gcgcacctcg cgctgcaccc cgccggtccg tacggcccgg tggcgtccct ggtccggctt 1080 caggcggagg tcgcgctgcg gaccctggcc gggcgtttcc ccgggctgcg gcaggcgggg 1140 gacgtgctcc gcccccgccg cgcgcctgtc ggccgcgggc cgctgagcgt cccggtcagc 1200 agctcctga 1209 22 402 PRT Streptomyces venezuelae 22 Met Thr Asp Asp Leu Thr Gly Ala Leu Thr Gln Pro Pro Leu Gly Arg 1 5 10 15 Thr Val Arg Ala Val Ala Asp Arg Glu Leu Gly Thr His Leu Leu Glu 20 25 30 Thr Arg Gly Ile His Trp Ile His Ala Ala Asn Gly Asp Pro Tyr Ala 35 40 45 Thr Val Leu Arg Gly Gln Ala Asp Asp Pro Tyr Pro Ala Tyr Glu Arg 50 55 60 Val Arg Ala Arg Gly Ala Leu Ser Phe Ser Pro Thr Gly Ser Trp Val 65 70 75 80 Thr Ala Asp His Ala Leu Ala Ala Ser Ile Leu Cys Ser Thr Asp Phe 85 90 95 Gly Val Ser Gly Ala Asp Gly Val Pro Val Pro Gln Gln Val Leu Ser 100 105 110 Tyr Gly Glu Gly Cys Pro Leu Glu Arg Glu Gln Val Leu Pro Ala Ala 115 120 125 Gly Asp Val Pro Glu Gly Gly Gln Arg Ala Val Val Glu Gly Ile His 130 135 140 Arg Glu Thr Leu Glu Gly Leu Ala Pro Asp Pro Ser Ala Ser Tyr Ala 145 150 155 160 Phe Glu Leu Leu Gly Gly Phe Val Arg Pro Ala Val Thr Ala Ala Ala 165 170 175 Ala Ala Val Leu Gly Val Pro Ala Asp Arg Arg Ala Asp Phe Ala Asp 180 185 190 Leu Leu Glu Arg Leu Arg Pro Leu Ser Asp Ser Leu Leu Ala Pro Gln 195 200 205 Ser Leu Arg Thr Val Arg Ala Ala Asp Gly Ala Leu Ala Glu Leu Thr 210 215 220 Ala Leu Leu Ala Asp Ser Asp Asp Ser Pro Gly Ala Leu Leu Ser Ala 225 230 235 240 Leu Gly Val Thr Ala Ala Val Gln Leu Thr Gly Asn Ala Val Leu Ala 245 250 255 Leu Leu Ala His Pro Glu Gln Trp Arg Glu Leu Cys Asp Arg Pro Gly 260 265 270 Leu Ala Ala Ala Ala Val Glu Glu Thr Leu Arg Tyr Asp Pro Pro Val 275 280 285 Gln Leu Asp Ala Arg Val Val Arg Gly Glu Thr Glu Leu Ala Gly Arg 290 295 300 Arg Leu Pro Ala Gly Ala His Val Val Val Leu Thr Ala Ala Thr Gly 305 310 315 320 Arg Asp Pro Glu Val Phe Thr Asp Pro Glu Arg Phe Asp Leu Ala Arg 325 330 335 Pro Asp Ala Ala Ala His Leu Ala Leu His Pro Ala Gly Pro Tyr Gly 340 345 350 Pro Val Ala Ser Leu Val Arg Leu Gln Ala Glu Val Ala Leu Arg Thr 355 360 365 Leu Ala Gly Arg Phe Pro Gly Leu Arg Gln Ala Gly Asp Val Leu Arg 370 375 380 Pro Arg Arg Ala Pro Val Gly Arg Gly Pro Leu Ser Val Pro Val Ser 385 390 395 400 Ser Ser 23 2430 DNA Streptomyces venezuelae 23 gtgacaggta agacccgaat accgcgtgtc cgccgcggcc gcaccacgcc cagggccttc 60 accctggccg tcgtcggcac cctgctggcg ggcaccaccg tggcggccgc cgctcccggc 120 gccgccgaca cggccaatgt tcagtacacg agccgggcgg cggagctcgt cgcccagatg 180 acgctcgacg agaagatcag cttcgtccac tgggcgctgg accccgaccg gcagaacgtc 240 ggctaccttc ccggcgtgcc gcgtctgggc atcccggagc tgcgtgccgc cgacggcccg 300 aacggcatcc gcctggtggg gcagaccgcc accgcgctgc ccgcgccggt cgccctggcc 360 agcaccttcg acgacaccat ggccgacagc tacggcaagg tcatgggccg cgacggtcgc 420 gcgctcaacc aggacatggt cctgggcccg atgatgaaca acatccgggt gccgcacggc 480 ggccggaact acgagacctt cagcgaggac cccctggtct cctcgcgcac cgcggtcgcc 540 cagatcaagg gcatccaggg tgcgggtctg atgaccacgg ccaagcactt cgcggccaac 600 aaccaggaga acaaccgctt ctccgtgaac gccaatgtcg acgagcagac gctccgcgag 660 atcgagttcc cggcgttcga ggcgtcctcc aaggccggcg cggcctcctt catgtgtgcc 720 tacaacggcc tcaacgggaa gccgtcctgc ggcaacgacg agctcctcaa caacgtgctg 780 cgcacgcagt ggggcttcca gggctgggtg atgtccgact ggctcgccac cccgggcacc 840 gacgccatca ccaagggcct cgaccaggag atgggcgtcg agctccccgg cgacgtcccg 900 aagggcgagc cctcgccgcc ggccaagttc ttcggcgagg cgctgaagac ggccgtcctg 960 aacggcacgg tccccgaggc ggccgtgacg cggtcggcgg agcggatcgt cggccagatg 1020 gagaagttcg gtctgctcct cgccactccg gcgccgcggc ccgagcgcga caaggcgggt 1080 gcccaggcgg tgtcccgcaa ggtcgccgag aacggcgcgg tgctcctgcg caacgagggc 1140 caggccctgc cgctcgccgg tgacgccggc aagagcatcg cggtcatcgg cccgacggcc 1200 gtcgacccca aggtcaccgg cctgggcagc gcccacgtcg tcccggactc ggcggcggcg 1260 ccactcgaca ccatcaaggc ccgcgcgggt gcgggtgcga cggtgacgta cgagacgggt 1320 gaggagacct tcgggacgca gatcccggcg gggaacctca gcccggcgtt caaccagggc 1380 caccagctcg agccgggcaa ggcgggggcg ctgtacgacg gcacgctgac cgtgcccgcc 1440 gacggcgagt accgcatcgc ggtccgtgcc accggtggtt acgccacggt gcagctcggc 1500 agccacacca tcgaggccgg tcaggtctac ggcaaggtga gcagcccgct cctcaagctg 1560 accaagggca cgcacaagct cacgatctcg ggcttcgcga tgagtgccac cccgctctcc 1620 ctggagctgg gctgggtgac gccggcggcg gccgacgcga cgatcgcgaa ggccgtggag 1680 tcggcgcgga aggcccgtac ggcggtcgtc ttcgcctacg acgacggcac cgagggcgtc 1740 gaccgtccga acctgtcgct gccgggtacg caggacaagc tgatctcggc tgtcgcggac 1800 gccaacccga acacgatcgt ggtcctcaac accggttcgt cggtgctgat gccgtggctg 1860 tccaagaccc gcgcggtcct ggacatgtgg tacccgggcc aggcgggcgc cgaggccacc 1920 gccgcgctgc tctacggtga cgtcaacccg agcggcaagc tcacgcagag cttcccggcc 1980 gccgagaacc agcacgcggt cgccggcgac ccgacaagct acccgggcgt cgacaaccag 2040 cagacgtacc gcgagggcat ccacgtcggg taccgctggt tcgacaagga gaacgtcaag 2100 ccgctgttcc cgttcgggca cggcctgtcg tacacctcgt tcacgcagag cgccccgacc 2160 gtcgtgcgta cgtccacggg tggtctgaag gtcacggtca cggtccgcaa cagcgggaag 2220 cgcgccggcc aggaggtcgt ccaggcgtac ctcggtgcca gcccgaacgt gacggctccg 2280 caggcgaaga agaagctcgt gggctacacg aaggtctcgc tcgccgcggg cgaggcgaag 2340 acggtgacgg tgaacgtcga ccgccgtcag ctgcagaccg gttcgtcctc cgccgacctg 2400 cggggcagcg ccacggtcaa cgtctggtga 2430 24 809 PRT Streptomyces venezuelae 24 Met Thr Gly Lys Thr Arg Ile Pro Arg Val Arg Arg Gly Arg Thr Thr 1 5 10 15 Pro Arg Ala Phe Thr Leu Ala Val Val Gly Thr Leu Leu Ala Gly Thr 20 25 30 Thr Val Ala Ala Ala Ala Pro Gly Ala Ala Asp Thr Ala Asn Val Gln 35 40 45 Tyr Thr Ser Arg Ala Ala Glu Leu Val Ala Gln Met Thr Leu Asp Glu 50 55 60 Lys Ile Ser Phe Val His Trp Ala Leu Asp Pro Asp Arg Gln Asn Val 65 70 75 80 Gly Tyr Leu Pro Gly Val Pro Arg Leu Gly Ile Pro Glu Leu Arg Ala 85 90 95 Ala Asp Gly Pro Asn Gly Ile Arg Leu Val Gly Gln Thr Ala Thr Ala 100 105 110 Leu Pro Ala Pro Val Ala Leu Ala Ser Thr Phe Asp Asp Thr Met Ala 115 120 125 Asp Ser Tyr Gly Lys Val Met Gly Arg Asp Gly Arg Ala Leu Asn Gln 130 135 140 Asp Met Val Leu Gly Pro Met Met Asn Asn Ile Arg Val Pro His Gly 145 150 155 160 Gly Arg Asn Tyr Glu Thr Phe Ser Glu Asp Pro Leu Val Ser Ser Arg 165 170 175 Thr Ala Val Ala Gln Ile Lys Gly Ile Gln Gly Ala Gly Leu Met Thr 180 185 190 Thr Ala Lys His Phe Ala Ala Asn Asn Gln Glu Asn Asn Arg Phe Ser 195 200 205 Val Asn Ala Asn Val Asp Glu Gln Thr Leu Arg Glu Ile Glu Phe Pro 210 215 220 Ala Phe Glu Ala Ser Ser Lys Ala Gly Ala Ala Ser Phe Met Cys Ala 225 230 235 240 Tyr Asn Gly Leu Asn Gly Lys Pro Ser Cys Gly Asn Asp Glu Leu Leu 245 250 255 Asn Asn Val Leu Arg Thr Gln Trp Gly Phe Gln Gly Trp Val Met Ser 260 265 270 Asp Trp Leu Ala Thr Pro Gly Thr Asp Ala Ile Thr Lys Gly Leu Asp 275 280 285 Gln Glu Met Gly Val Glu Leu Pro Gly Asp Val Pro Lys Gly Glu Pro 290 295 300 Ser Pro Pro Ala Lys Phe Phe Gly Glu Ala Leu Lys Thr Ala Val Leu 305 310 315 320 Asn Gly Thr Val Pro Glu Ala Ala Val Thr Arg Ser Ala Glu Arg Ile 325 330 335 Val Gly Gln Met Glu Lys Phe Gly Leu Leu Leu Ala Thr Pro Ala Pro 340 345 350 Arg Pro Glu Arg Asp Lys Ala Gly Ala Gln Ala Val Ser Arg Lys Val 355 360 365 Ala Glu Asn Gly Ala Val Leu Leu Arg Asn Glu Gly Gln Ala Leu Pro 370 375 380 Leu Ala Gly Asp Ala Gly Lys Ser Ile Ala Val Ile Gly Pro Thr Ala 385 390 395 400 Val Asp Pro Lys Val Thr Gly Leu Gly Ser Ala His Val Val Pro Asp 405 410 415 Ser Ala Ala Ala Pro Leu Asp Thr Ile Lys Ala Arg Ala Gly Ala Gly 420 425 430 Ala Thr Val Thr Tyr Glu Thr Gly Glu Glu Thr Phe Gly Thr Gln Ile 435 440 445 Pro Ala Gly Asn Leu Ser Pro Ala Phe Asn Gln Gly His Gln Leu Glu 450 455 460 Pro Gly Lys Ala Gly Ala Leu Tyr Asp Gly Thr Leu Thr Val Pro Ala 465 470 475 480 Asp Gly Glu Tyr Arg Ile Ala Val Arg Ala Thr Gly Gly Tyr Ala Thr 485 490 495 Val Gln Leu Gly Ser His Thr Ile Glu Ala Gly Gln Val Tyr Gly Lys 500 505 510 Val Ser Ser Pro Leu Leu Lys Leu Thr Lys Gly Thr His Lys Leu Thr 515 520 525 Ile Ser Gly Phe Ala Met Ser Ala Thr Pro Leu Ser Leu Glu Leu Gly 530 535 540 Trp Val Thr Pro Ala Ala Ala Asp Ala Thr Ile Ala Lys Ala Val Glu 545 550 555 560 Ser Ala Arg Lys Ala Arg Thr Ala Val Val Phe Ala Tyr Asp Asp Gly 565 570 575 Thr Glu Gly Val Asp Arg Pro Asn Leu Ser Leu Pro Gly Thr Gln Asp 580 585 590 Lys Leu Ile Ser Ala Val Ala Asp Ala Asn Pro Asn Thr Ile Val Val 595 600 605 Leu Asn Thr Gly Ser Ser Val Leu Met Pro Trp Leu Ser Lys Thr Arg 610 615 620 Ala Val Leu Asp Met Trp Tyr Pro Gly Gln Ala Gly Ala Glu Ala Thr 625 630 635 640 Ala Ala Leu Leu Tyr Gly Asp Val Asn Pro Ser Gly Lys Leu Thr Gln 645 650 655 Ser Phe Pro Ala Ala Glu Asn Gln His Ala Val Ala Gly Asp Pro Thr 660 665 670 Ser Tyr Pro Gly Val Asp Asn Gln Gln Thr Tyr Arg Glu Gly Ile His 675 680 685 Val Gly Tyr Arg Trp Phe Asp Lys Glu Asn Val Lys Pro Leu Phe Pro 690 695 700 Phe Gly His Gly Leu Ser Tyr Thr Ser Phe Thr Gln Ser Ala Pro Thr 705 710 715 720 Val Val Arg Thr Ser Thr Gly Gly Leu Lys Val Thr Val Thr Val Arg 725 730 735 Asn Ser Gly Lys Arg Ala Gly Gln Glu Val Val Gln Ala Tyr Leu Gly 740 745 750 Ala Ser Pro Asn Val Thr Ala Pro Gln Ala Lys Lys Lys Leu Val Gly 755 760 765 Tyr Thr Lys Val Ser Leu Ala Ala Gly Glu Ala Lys Thr Val Thr Val 770 775 780 Asn Val Asp Arg Arg Gln Leu Gln Thr Gly Ser Ser Ser Ala Asp Leu 785 790 795 800 Arg Gly Ser Ala Thr Val Asn Val Trp 805 25 9 PRT Artificial Sequence A consensus sequence. 25 Leu Leu Asp Val Ala Cys Gly Thr Gly 1 5 26 1011 DNA Streptomyces venezuelae 26 atggcaatgc gcgactccat accgaggcga gcggaccgcg acacccttcg ccgcgaatta 60 ggccagaact tccttcagga cgacagagcc gtgcgcaatc tcgtcacgca tgtcgagggg 120 gacggtagga acgttctcga aatcggcccc ggaaagggcg cgataaccga ggagttggtg 180 cgctccttcg acaccgtgac ggtcgtggag atggacccgc actgggccgc gcatgtgcgg 240 cggaaattcg aaggggagag ggtcaccgta ttccagggtg atttcctcga cttccgcatt 300 ccgcgcgata tcgacaccgt cgtcggaaac gttcccttcg gcatcacgac ccagattctc 360 cggagtctcc tggaatcgac gaactggcag tcggcggccc tgatagtgca gtgggaggtc 420 gcccgcaaac gcgccggtcg cagcggcgga tcgctcctca cgacctcctg ggccccctgg 480 tacgagttcg cggtccacga ccgcgtccgc gcctcgtcgt tccgtccgat gccccgcgtc 540 gacggcggcg tcctgacgat caggcgacgc ccccagcccc tgctgcccga gagcgcgagc 600 cgcgccttcc agaacttcgc cgaagccgtc ttcaccggcc ccggacgggg cctcgcggag 660 atcctccggc gccacatccc caagcggacc taccgttccc tcgccgaccg ccacggaatt 720 ccggacggcg gactgccgaa ggacctcacg ctcacccaat ggatcgccct tttccaggcc 780 tcccagccga gttacgcgcc gggggcgccc ggcacgcgca tgccgggcca gggcggtggc 840 gccggcggca gggactatga ctcggagacg agcagggccg ccgtgcccgg gagccgcaga 900 tacggcccca cgcgcggcgg cgaaccctgc gcaccccgcg cacaggtccg gcagaccaag 960 ggccgccagg gcgcgcgagg ctcgtcgtac ggacgccgca cgggccgtta g 1011 27 336 PRT Streptomyces venezuelae 27 Met Ala Met Arg Asp Ser Ile Pro Arg Arg Ala Asp Arg Asp Thr Leu 1 5 10 15 Arg Arg Glu Leu Gly Gln Asn Phe Leu Gln Asp Asp Arg Ala Val Arg 20 25 30 Asn Leu Val Thr His Val Glu Gly Asp Gly Arg Asn Val Leu Glu Ile 35 40 45 Gly Pro Gly Lys Gly Ala Ile Thr Glu Glu Leu Val Arg Ser Phe Asp 50 55 60 Thr Val Thr Val Val Glu Met Asp Pro His Trp Ala Ala His Val Arg 65 70 75 80 Arg Lys Phe Glu Gly Glu Arg Val Thr Val Phe Gln Gly Asp Phe Leu 85 90 95 Asp Phe Arg Ile Pro Arg Asp Ile Asp Thr Val Val Gly Asn Val Pro 100 105 110 Phe Gly Ile Thr Thr Gln Ile Leu Arg Ser Leu Leu Glu Ser Thr Asn 115 120 125 Trp Gln Ser Ala Ala Leu Ile Val Gln Trp Glu Val Ala Arg Lys Arg 130 135 140 Ala Gly Arg Ser Gly Gly Ser Leu Leu Thr Thr Ser Trp Ala Pro Trp 145 150 155 160 Tyr Glu Phe Ala Val His Asp Arg Val Arg Ala Ser Ser Phe Arg Pro 165 170 175 Met Pro Arg Val Asp Gly Gly Val Leu Thr Ile Arg Arg Arg Pro Gln 180 185 190 Pro Leu Leu Pro Glu Ser Ala Ser Arg Ala Phe Gln Asn Phe Ala Glu 195 200 205 Ala Val Phe Thr Gly Pro Gly Arg Gly Leu Ala Glu Ile Leu Arg Arg 210 215 220 His Ile Pro Lys Arg Thr Tyr Arg Ser Leu Ala Asp Arg His Gly Ile 225 230 235 240 Pro Asp Gly Gly Leu Pro Lys Asp Leu Thr Leu Thr Gln Trp Ile Ala 245 250 255 Leu Phe Gln Ala Ser Gln Pro Ser Tyr Ala Pro Gly Ala Pro Gly Thr 260 265 270 Arg Met Pro Gly Gln Gly Gly Gly Ala Gly Gly Arg Asp Tyr Asp Ser 275 280 285 Glu Thr Ser Arg Ala Ala Val Pro Gly Ser Arg Arg Tyr Gly Pro Thr 290 295 300 Arg Gly Gly Glu Pro Cys Ala Pro Arg Ala Gln Val Arg Gln Thr Lys 305 310 315 320 Gly Arg Gln Gly Ala Arg Gly Ser Ser Tyr Gly Arg Arg Thr Gly Arg 325 330 335 28 28 This Sequence is intentionally skipped 29 29 This Sequence is intentionally skipped 30 13842 DNA Streptomyces venezuelae 30 atgtcttcag ccggaattac caggaccggt gcgagaacac cggtgacagg gcgtggggcg 60 gcagcgtggg acacggggga agtgcgggtc cgacgggggt tgccccctgc cggccccgat 120 catgcggagc actccttctc tcgtgctcct accggtgatg tgcgcgccga attgattcgt 180 ggagagatgt cgacagtgtc caagagtgag tccgaggaat tcgtgtccgt gtcgaacgac 240 gccggttccg cgcacggcac agcggaaccc gtcgccgtcg tcggcatctc ctgccgggtg 300 cccggcgccc gggacccgag agagttctgg gaactcctgg cggcaggcgg ccaggccgtc 360 accgacgtcc ccgcggaccg ctggaacgcc ggcgacttct acgacccgga ccgctccgcc 420 cccggccgct cgaacagccg gtggggcggg ttcatcgagg acgtcgaccg gttcgacgcc 480 gccttcttcg gcatctcgcc ccgcgaggcc gcggagatgg acccgcagca gcggctcgcc 540 ctggagctgg gctgggaggc cctggagcgc gccgggatcg acccgtcctc gctcaccggc 600 acccgcaccg gcgtcttcgc cggcgccatc tgggacgact acgccaccct gaagcaccgc 660 cagggcggcg ccgcgatcac cccgcacacc gtcaccggcc tccaccgcgg catcatcgcg 720 aaccgactct cgtacacgct cgggctccgc ggccccagca tggtcgtcga ctccggccag 780 tcctcgtcgc tcgtcgccgt ccacctcgcg tgcgagagcc tgcggcgcgg cgagtccgag 840 ctcgccctcg ccggcggcgt ctcgctcaac ctggtgccgg acagcatcat cggggcgagc 900 aagttcggcg gcctctcccc cgacggccgc gcctacacct tcgacgcgcg cgccaacggc 960 tacgtacgcg gcgagggcgg cggtttcgtc gtcctgaagc gcctctcccg ggccgtcgcc 1020 gacggcgacc cggtgctcgc cgtgatccgg ggcagcgccg tcaacaacgg cggcgccgcc 1080 cagggcatga cgacccccga cgcgcaggcg caggaggccg tgctccgcga ggcccacgag 1140 cgggccggga ccgcgccggc cgacgtgcgg tacgtcgagc tgcacggcac cggcaccccc 1200 gtgggcgacc cgatcgaggc cgctgcgctc ggcgccgccc tcggcaccgg ccgcccggcc 1260 ggacagccgc tcctggtcgg ctcggtcaag acgaacatcg gccacctgga gggcgcggcc 1320 ggcatcgccg gcctcatcaa ggccgtcctg gcggtccgcg gtcgcgcgct gcccgccagc 1380 ctgaactacg agaccccgaa cccggcgatc ccgttcgagg aactgaacct ccgggtgaac 1440 acggagtacc tgccgtggga gccggagcac gacgggcagc ggatggtcgt cggcgtgtcc 1500 tcgttcggca tgggcggcac gaacgcgcat gtcgtgctcg aagaggcccc cgggggttgt 1560 cgaggtgctt cggtcgtgga gtcgacggtc ggcgggtcgg cggtcggcgg cggtgtggtg 1620 ccgtgggtgg tgtcggcgaa gtccgctgcc gcgctggacg cgcagatcga gcggcttgcc 1680 gcgttcgcct cgcgggatcg tacggatggt gtcgacgcgg gcgctgtcga tgcgggtgct 1740 gtcgatgcgg gtgctgtcgc tcgcgtactg gccggcgggc gtgctcagtt cgagcaccgg 1800 gccgtcgtcg tcggcagcgg gccggacgat ctggcggcag cgctggccgc gcctgagggt 1860 ctggtccggg gcgtggcttc cggtgtcggg cgagtggcgt tcgtgttccc cgggcagggc 1920 acgcagtggg ccggcatggg tgccgaactg ctggactctt ccgcggtgtt cgcggcggcc 1980 atggccgaat gcgaggccgc actctccccg tacgtcgact ggtcgctgga ggccgtcgta 2040 cggcaggccc ccggtgcgcc cacgctggag cgggtcgatg tcgtgcagcc tgtgacgttc 2100 gccgtcatgg tctcgctggc tcgcgtgtgg cagcaccacg gggtgacgcc ccaggcggtc 2160 gtcggccact cgcagggcga gatcgccgcc gcgtacgtcg ccggtgccct gagcctggac 2220 gacgccgctc gtgtcgtgac cctgcgcagc aagtccatcg ccgcccacct cgccggcaag 2280 ggcggcatgc tgtccctcgc gctgagcgag gacgccgtcc tggagcgact ggccgggttc 2340 gacgggctgt ccgtcgccgc tgtgaacggg cccaccgcca ccgtggtctc cggtgacccc 2400 gtacagatcg aagagcttgc tcgggcgtgt gaggccgatg gggtccgtgc gcgggtcatt 2460 cccgtcgact acgcgtccca cagccggcag gtcgagatca tcgagagcga gctcgccgag 2520 gtcctcgccg ggctcagccc gcaggctccg cgcgtgccgt tcttctcgac actcgaaggc 2580 gcctggatca ccgagcccgt gctcgacggc ggctactggt accgcaacct gcgccatcgt 2640 gtgggcttcg ccccggccgt cgagaccctg gccaccgacg agggcttcac ccacttcgtc 2700 gaggtcagcg cccaccccgt cctcaccatg gccctccccg ggaccgtcac cggtctggcg 2760 accctgcgtc gcgacaacgg cggtcaggac cgcctagtcg cctccctcgc cgaagcatgg 2820 gccaacggac tcgcggtcga ctggagcccg ctcctcccct ccgcgaccgg ccaccactcc 2880 gacctcccca cctacgcgtt ccagaccgag cgccactggc tgggcgagat cgaggcgctc 2940 gccccggcgg gcgagccggc ggtgcagccc gccgtcctcc gcacggaggc ggccgagccg 3000 gcggagctcg accgggacga gcagctgcgc gtgatcctgg acaaggtccg ggcgcagacg 3060 gcccaggtgc tggggtacgc gacaggcggg cagatcgagg tcgaccggac cttccgtgag 3120 gccggttgca cctccctgac cggcgtggac ctgcgcaacc ggatcaacgc cgccttcggc 3180 gtacggatgg cgccgtccat gatcttcgac ttccccaccc ccgaggctct cgcggagcag 3240 ctgctcctcg tcgtgcacgg ggaggcggcg gcgaacccgg ccggtgcgga gccggctccg 3300 gtggcggcgg ccggtgccgt cgacgagccg gtggcgatcg tcggcatggc ctgccgcctg 3360 cccggtgggg tcgcctcgcc ggaggacctg tggcggctgg tggccggcgg cggggacgcg 3420 atctcggagt tcccgcagga ccgcggctgg gacgtggagg ggctgtacca cccggatccg 3480 gagcaccccg gcacgtcgta cgtccgccag ggcggtttca tcgagaacgt cgccggcttc 3540 gacgcggcct tcttcgggat ctcgccgcgc gaggccctcg ccatggaccc gcagcagcgg 3600 ctcctcctcg aaacctcctg ggaggccgtc gaggacgccg ggatcgaccc gacctccctg 3660 cggggacggc aggtcggcgt cttcactggg gcgatgaccc acgagtacgg gccgagcctg 3720 cgggacggcg gggaaggcct cgacggctac ctgctgaccg gcaacacggc cagcgtgatg 3780 tcgggccgcg tctcgtacac actcggcctt gagggccccg ccctgacggt ggacacggcc 3840 tgctcgtcgt cgctggtcgc cctgcacctc gccgtgcagg ccctgcgcaa gggcgaggtc 3900 gacatggcgc tcgccggcgg cgtggccgtg atgcccacgc ccgggatgtt cgtcgagttc 3960 agccggcagc gcgggctggc cggggacggc cggtcgaagg cgttcgccgc gtcggcggac 4020 ggcaccagct ggtccgaggg cgtcggcgtc ctcctcgtcg agcgcctgtc ggacgcccgc 4080 cgcaacggac accaggtcct cgcggtcgtc cgcggcagcg ccttgaacca ggacggcgcg 4140 agcaacggcc tcacggctcc gaacgggccc tcgcagcagc gcgtcatccg gcgcgcgctg 4200 gcggacgccc ggctgacgac ctccgacgtg gacgtcgtcg aggcacacgg cacgggcacg 4260 cgactcggcg acccgatcga ggcgcaggcc ctgatcgcca cctacggcca gggccgtgac 4320 gacgaacagc cgctgcgcct cgggtcgttg aagtccaaca tcgggcacac ccaggccgcg 4380 gccggcgtct ccggtgtcat caagatggtc caggcgatgc gccacggact gctgccgaag 4440 acgctgcacg tcgacgagcc ctcggaccag atcgactggt cggctggcgc cgtggaactc 4500 ctcaccgagg ccgtcgactg gccggagaag caggacggcg ggctgcgccg ggccgccgtc 4560 tcctccttcg ggatcagcgg caccaatgcg catgtggtgc tcgaagaggc cccggtggtt 4620 gtcgagggtg cttcggtcgt cgagccgtcg gttggcgggt cggcggtcgg cggcggtgtg 4680 acgccttggg tggtgtcggc gaagtccgct gccgcgctcg acgcgcagat cgagcggctt 4740 gccgcattcg cctcgcggga tcgtacggat gacgccgacg ccggtgctgt cgacgcgggc 4800 gctgtcgctc acgtactggc tgacgggcgt gctcagttcg agcaccgggc cgtcgcgctc 4860 ggcgccgggg cggacgacct cgtacaggcg ctggccgatc cggacgggct gatacgcgga 4920 acggcttccg gtgtcgggcg agtggcgttc gtgttccccg gtcagggcac gcagtgggct 4980 ggcatgggtg ccgaactgct ggactcttcc gcggtgttcg cggcggccat ggccgagtgt 5040 gaggccgcgc tgtccccgta cgtcgactgg tcgctggagg ccgtcgtacg gcaggccccc 5100 ggtgcgccca cgctggagcg ggtcgatgtc gtgcagcctg tgacgttcgc cgtcatggtc 5160 tcgctggctc gcgtgtggca gcaccacggt gtgacgcccc aggcggtcgt cggccactcg 5220 cagggcgaga tcgccgccgc gtacgtcgcc ggagccctgc ccctggacga cgccgcccgc 5280 gtcgtcaccc tgcgcagcaa gtccatcgcc gcccacctcg ccggcaaggg cggcatgctg 5340 tccctcgcgc tgaacgagga cgccgtcctg gagcgactga gtgacttcga cgggctgtcc 5400 gtcgccgccg tcaacgggcc caccgccact gtcgtgtcgg gtgaccccgt acagatcgaa 5460 gagcttgctc aggcgtgcaa ggcggacgga ttccgcgcgc ggatcattcc cgtcgactac 5520 gcgtcccaca gccggcaggt cgagatcatc gagagcgagc tcgcccaggt cctcgccggt 5580 ctcagcccgc aggccccgcg cgtgccgttc ttctcgacgc tcgaaggcac ctggatcacc 5640 gagcccgtcc tcgacggcac ctactggtac cgcaacctcc gtcaccgcgt cggcttcgcc 5700 cccgccatcg agaccctggc cgtcgacgag ggcttcacgc acttcgtcga ggtcagcgcc 5760 caccccgtcc tcaccatgac cctccccgag accgtcaccg gcctcggcac cctccgtcgc 5820 gaacagggag gccaagagcg tctggtcacc tcgctcgccg aggcgtgggt caacgggctt 5880 cccgtggcat ggacttcgct cctgcccgcc acggcctccc gccccggtct gcccacctac 5940 gccttccagg ccgagcgcta ctggctcgag aacactcccg ccgccctggc caccggcgac 6000 gactggcgct accgcatcga ctggaagcgc ctcccggccg ccgaggggtc cgagcgcacc 6060 ggcctgtccg gccgctggct cgccgtcacg ccggaggacc actccgcgca ggccgccgcc 6120 gtgctcaccg cgctggtcga cgccggggcg aaggtcgagg tgctgacggc cggggcggac 6180 gacgaccgtg aggccctcgc cgcccggctc accgcactga cgaccggtga cggcttcacc 6240 ggcgtggtct cgctcctcga cggactcgta ccgcaggtcg cctgggtcca ggcgctcggc 6300 gacgccggaa tcaaggcgcc cctgtggtcc gtcacccagg gcgcggtctc cgtcggacgt 6360 ctcgacaccc ccgccgaccc cgaccgggcc atgctctggg gcctcggccg cgtcgtcgcc 6420 cttgagcacc ccgaacgctg ggccggcctc gtcgacctcc ccgcccagcc cgatgccgcc 6480 gccctcgccc acctcgtcac cgcactctcc ggcgccaccg gcgaggacca gatcgccatc 6540 cgcaccaccg gactccacgc ccgccgcctc gcccgcgcac ccctccacgg acgtcggccc 6600 acccgcgact ggcagcccca cggcaccgtc ctcatcaccg gcggcaccgg agccctcggc 6660 agccacgccg cacgctggat ggcccaccac ggagccgaac acctcctcct cgtcagccgc 6720 agcggcgaac aagcccccgg agccacccaa ctcaccgccg aactcaccgc atcgggcgcc 6780 cgcgtcacca tcgccgcctg cgacgtcgcc gacccccacg ccatgcgcac cctcctcgac 6840 gccatccccg ccgagacgcc cctcaccgcc gtcgtccaca ccgccggcgc gctcgacgac 6900 ggcatcgtgg acacgctgac cgccgagcag gtccggcggg cccaccgtgc gaaggccgtc 6960 ggcgcctcgg tgctcgacga gctgacccgg gacctcgacc tcgacgcgtt cgtgctcttc 7020 tcgtccgtgt cgagcactct gggcatcccc ggtcagggca actacgcccc gcacaacgcc 7080 tacctcgacg ccctcgcggc tcgccgccgg gccaccggcc ggtccgccgt ctcggtggcc 7140 tggggaccgt gggacggtgg cggcatggcc gccggtgacg gcgtggccga gcggctgcgc 7200 aaccacggcg tgcccggcat ggacccggaa ctcgccctgg ccgcactgga gtccgcgctc 7260 ggccgggacg agaccgcgat caccgtcgcg gacatcgact gggaccgctt ctacctcgcg 7320 tactcctccg gtcgcccgca gcccctcgtc gaggagctgc ccgaggtgcg gcgcatcatc 7380 gacgcacggg acagcgccac gtccggacag ggcgggagct ccgcccaggg cgccaacccc 7440 ctggccgagc ggctggccgc cgcggctccc ggcgagcgta cggagatcct cctcggtctc 7500 gtacgggcgc aggccgccgc cgtgctccgg atgcgttcgc cggaggacgt cgccgccgac 7560 cgcgccttca aggacatcgg cttcgactcg ctcgccggtg tcgagctgcg caacaggctg 7620 acccgggcga ccgggctcca gctgcccgcg acgctcgtct tcgaccaccc gacgccgctg 7680 gccctcgtgt cgctgctccg cagcgagttc ctcggtgacg aggagacggc ggacgcccgg 7740 cggtccgcgg cgctgcccgc gactgtcggt gccggtgccg gcgccggcgc cggcaccgat 7800 gccgacgacg atccgatcgc gatcgtcgcg atgagctgcc gctaccccgg tgacatccgc 7860 agcccggagg acctgtggcg gatgctgtcc gagggcggcg agggcatcac gccgttcccc 7920 accgaccgcg gctgggacct cgacggcctg tacgacgccg acccggacgc gctcggcagg 7980 gcgtacgtcc gcgagggcgg gttcctgcac gacgcggccg agttcgacgc ggagttcttc 8040 ggcgtctcgc cgcgcgaggc gctggccatg gacccgcagc agcggatgct cctgacgacg 8100 tcctgggagg ccttcgagcg ggccggcatc gagccggcat cgctgcgcgg cagcagcacc 8160 ggtgtcttca tcggcctctc ctaccaggac tacgcggccc gcgtcccgaa cgccccgcgt 8220 ggcgtggagg gttacctgct gaccggcagc acgccgagcg tcgcgtcggg ccgtatcgcg 8280 tacaccttcg gtctcgaagg gcccgcgacg accgtcgaca ccgcctgctc gtcgtcgctg 8340 accgccctgc acctggcggt gcgggcgctg cgcagcggcg agtgcacgat ggcgctcgcc 8400 ggtggcgtgg cgatgatggc gaccccgcac atgttcgtgg agttcagccg tcagcgggcg 8460 ctcgccccgg acggccgcag caaggccttc tcggcggacg ccgacgggtt cggcgccgcg 8520 gagggcgtcg gcctgctgct cgtggagcgg ctctcggacg cgcggcgcaa cggtcacccg 8580 gtgctcgccg tggtccgcgg taccgccgtc aaccaggacg gcgccagcaa cgggctgacc 8640 gcgcccaacg gaccctcgca gcagcgggtg atccggcagg cgctcgccga cgcccggctg 8700 gcacccggcg acatcgacgc cgtcgagacg cacggcacgg gaacctcgct gggcgacccc 8760 atcgaggccc agggcctcca ggccacgtac ggcaaggagc ggcccgcgga acggccgctc 8820 gccatcggct ccgtgaagtc caacatcgga cacacccagg ccgcggccgg tgcggcgggc 8880 atcatcaaga tggtcctcgc gatgcgccac ggcaccctgc cgaagaccct ccacgccgac 8940 gagccgagcc cgcacgtcga ctgggcgaac agcggcctgg ccctcgtcac cgagccgatc 9000 gactggccgg ccggcaccgg tccgcgccgc gccgccgtct cctccttcgg catcagcggg 9060 acgaacgcgc acgtcgtgct ggagcaggcg ccggatgctg ctggtgaggt gcttggggcc 9120 gatgaggtgc ctgaggtgtc tgagacggta gcgatggctg ggacggctgg gacctccgag 9180 gtcgctgagg gctctgaggc ctccgaggcc cccgcggccc ccggcagccg tgaggcgtcc 9240 ctccccgggc acctgccctg ggtgctgtcc gccaaggacg agcagtcgct gcgcggccag 9300 gccgccgccc tgcacgcgtg gctgtccgag cccgccgccg acctgtcgga cgcggacgga 9360 ccggcccgcc tgcgggacgt cgggtacacg ctcgccacga gccgtaccgc cttcgcgcac 9420 cgcgccgccg tgaccgccgc cgaccgggac gggttcctgg acgggctggc cacgctggcc 9480 cagggcggca cctcggccca cgtccacctg gacaccgccc gggacggcac caccgcgttc 9540 ctcttcaccg gccagggcag tcagcgcccc ggcgccggcc gtgagctgta cgaccggcac 9600 cccgtcttcg cccgggcgct cgacgagatc tgcgcccacc tcgacggtca cctcgaactg 9660 cccctgctcg acgtgatgtt cgcggccgag ggcagcgcgg aggccgcgct gctcgacgag 9720 acgcggtaca cgcagtgcgc gctgttcgcc ctggaggtcg cgctcttccg gctcgtcgag 9780 agctggggca tgcggccggc cgcactgctc ggtcactcgg tcggcgagat cgccgccgcg 9840 cacgtcgccg gtgtgttctc gctcgccgac gccgcccgcc tggtcgccgc gcgcggccgg 9900 ctcatgcagg agctgcccgc cggtggcgcg atgctcgccg tccaggccgc ggaggacgag 9960 atccgcgtgt ggctggagac ggaggagcgg tacgcgggac gtctggacgt cgccgccgtc 10020 aacggccccg aggccgccgt cctgtccggc gacgcggacg cggcgcggga ggcggaggcg 10080 tactggtccg ggctcggccg caggacccgc gcgctgcggg tcagccacgc cttccactcc 10140 gcgcacatgg acggcatgct cgacgggttc cgcgccgtcc tggagacggt ggagttccgg 10200 cgcccctccc tgaccgtggt ctcgaacgtc accggcctgg ccgccggccc ggacgacctg 10260 tgcgaccccg agtactgggt ccggcacgtc cgcggcaccg tccgcttcct cgacggcgtc 10320 cgtgtcctgc gcgacctcgg cgtgcggacc tgcctggagc tgggccccga cggggtcctc 10380 accgccatgg cggccgacgg cctcgcggac acccccgcgg attccgctgc cggctccccc 10440 gtcggctctc ccgccggctc tcccgccgac tccgccgccg gcgcgctccg gccccggccg 10500 ctgctcgtgg cgctgctgcg ccgcaagcgg tcggagaccg agaccgtcgc ggacgccctc 10560 ggcagggcgc acgcccacgg caccggaccc gactggcacg cctggttcgc cggctccggg 10620 gcgcaccgcg tggacctgcc cacgtactcc ttccggcgcg accgctactg gctggacgcc 10680 ccggcggccg acaccgcggt ggacaccgcc ggcctcggtc tcggcaccgc cgaccacccg 10740 ctgctcggcg ccgtggtcag ccttccggac cgggacggcc tgctgctcac cggccgcctc 10800 tccctgcgca cccacccgtg gctcgcggac cacgccgtcc tggggagcgt cctgctcccc 10860 ggcgccgcga tggtcgaact cgccgcgcac gctgcggagt ccgccggtct gcgtgacgtg 10920 cgggagctga ccctccttga accgctggta ctgcccgagc acggtggcgt cgagctgcgc 10980 gtgacggtcg gggcgccggc cggagagccc ggtggcgagt cggccgggga cggcgcacgg 11040 cccgtctccc tccactcgcg gctcgccgac gcgcccgccg gtaccgcctg gtcctgccac 11100 gcgaccggtc tgctggccac cgaccggccc gagcttcccg tcgcgcccga ccgtgcggcc 11160 atgtggccgc cgcagggcgc cgaggaggtg ccgctcgacg gtctctacga gcggctcgac 11220 gggaacggcc tcgccttcgg tccgctgttc caggggctga acgcggtgtg gcggtacgag 11280 ggtgaggtct tcgccgacat cgcgctcccc gccaccacga atgcgaccgc gcccgcgacc 11340 gcgaacggcg gcgggagtgc ggcggcggcc ccctacggca tccaccccgc cctgctcgac 11400 gcttcgctgc acgccatcgc ggtcggcggt ctcgtcgacg agcccgagct cgtccgcgtc 11460 cccttccact ggagcggtgt caccgtgcac gcggccggtg ccgcggcggc ccgggtccgt 11520 ctcgcctccg cggggacgga cgccgtctcg ctgtccctga cggacggcga gggacgcccg 11580 ctggtctccg tggaacggct cacgctgcgc ccggtcaccg ccgatcaggc ggcggcgagc 11640 cgcgtcggcg ggctgatgca ccgggtggcc tggcgtccgt acgccctcgc ctcgtccggc 11700 gaacaggacc cgcacgccac ttcgtacggg ccgaccgccg tcctcggcaa ggacgagctg 11760 aaggtcgccg ccgccctgga gtccgcgggc gtcgaagtcg ggctctaccc cgacctggcc 11820 gcgctgtccc aggacgtggc ggccggcgcc ccggcgcccc gtaccgtcct tgcgccgctg 11880 cccgcgggtc ccgccgacgg cggcgcggag ggtgtacggg gcacggtggc ccggacgctg 11940 gagctgctcc aggcctggct ggccgacgag cacctcgcgg gcacccgcct gctcctggtc 12000 acccgcggtg cggtgcggga ccccgagggg tccggcgccg acgatggcgg cgaggacctg 12060 tcgcacgcgg ccgcctgggg tctcgtacgg accgcgcaga ccgagaaccc cggccgcttc 12120 ggccttctcg acctggccga cgacgcctcg tcgtaccgga ccctgccgtc ggtgctctcc 12180 gacgcgggcc tgcgcgacga accgcagctc gccctgcacg acggcaccat caggctggcc 12240 cgcctggcct ccgtccggcc cgagaccggc accgccgcac cggcgctcgc cccggagggc 12300 acggtcctgc tgaccggcgg caccggcggc ctgggcggac tggtcgcccg gcacgtggtg 12360 ggcgagtggg gcgtacgacg cctgctgctg gtgagccggc ggggcacgga cgccccgggc 12420 gccgacgagc tcgtgcacga gctggaggcc ctgggagccg acgtctcggt ggccgcgtgc 12480 gacgtcgccg accgcgaagc cctcaccgcc gtactcgacg ccatccccgc cgaacacccg 12540 ctcaccgcgg tcgtccacac ggcaggcgtc ctctccgacg gcaccctccc gtccatgacg 12600 acggaggacg tggaacacgt actgcggccc aaggtcgacg ccgcgttcct cctcgacgaa 12660 ctcacctcga cgcccgcata cgacctggca gcgttcgtca tgttctcctc cgccgccgcc 12720 gtcttcggtg gcgcggggca gggcgcctac gccgccgcca acgccaccct cgacgccctc 12780 gcctggcgcc gccgggcagc cggactcccc gccctctccc tcggctgggg cctctgggcc 12840 gagaccagcg gcatgaccgg cgagctcggc caggcggacc tgcgccggat gagccgcgcg 12900 ggcatcggcg ggatcagcga cgccgagggc atcgcgctcc tcgacgccgc cctccgcgac 12960 gaccgccacc cggtcctgct gcccctgcgg ctcgacgccg ccgggctgcg ggacgcggcc 13020 gggaacgacc cggccggaat cccggcgctc ttccgggacg tcgtcggcgc caggaccgtc 13080 cgggcccggc cgtccgcggc ctccgcctcg acgacagccg ggacggccgg cacgccgggg 13140 acggcggacg gcgcggcgga aacggcggcg gtcacgctcg ccgaccgggc cgccaccgtg 13200 gacgggcccg cacggcagcg cctgctgctc gagttcgtcg tcggcgaggt cgccgaagta 13260 ctcggccacg cccgcggtca ccggatcgac gccgaacggg gcttcctcga cctcggcttc 13320 gactccctga ccgccgtcga actccgcaac cggctcaact ccgccggtgg cctcgccctc 13380 ccggcgaccc tggtcttcga ccacccaagc ccggcggcac tcgcctccca cctggacgcc 13440 gagctgccgc gcggcgcctc ggaccaggac ggagccggga accggaacgg gaacgagaac 13500 gggacgacgg cgtcccggag caccgccgag acggacgcgc tgctggcaca actgacccgc 13560 ctggaaggcg ccttggtgct gacgggcctc tcggacgccc ccgggagcga agaagtcctg 13620 gagcacctgc ggtccctgcg ctcgatggtc acgggcgaga ccgggaccgg gaccgcgtcc 13680 ggagccccgg acggcgccgg gtccggcgcc gaggaccggc cctgggcggc cggggacgga 13740 gccgggggcg ggagtgagga cggcgcggga gtgccggact tcatgaacgc ctcggccgag 13800 gaactcttcg gcctcctcga ccaggacccc agcacggact ga 13842 31 4613 PRT Streptomyces venezuelae 31 Met Ser Ser Ala Gly Ile Thr Arg Thr Gly Ala Arg Thr Pro Val Thr 1 5 10 15 Gly Arg Gly Ala Ala Ala Trp Asp Thr Gly Glu Val Arg Val Arg Arg 20 25 30 Gly Leu Pro Pro Ala Gly Pro Asp His Ala Glu His Ser Phe Ser Arg 35 40 45 Ala Pro Thr Gly Asp Val Arg Ala Glu Leu Ile Arg Gly Glu Met Ser 50 55 60 Thr Val Ser Lys Ser Glu Ser Glu Glu Phe Val Ser Val Ser Asn Asp 65 70 75 80 Ala Gly Ser Ala His Gly Thr Ala Glu Pro Val Ala Val Val Gly Ile 85 90 95 Ser Cys Arg Val Pro Gly Ala Arg Asp Pro Arg Glu Phe Trp Glu Leu 100 105 110 Leu Ala Ala Gly Gly Gln Ala Val Thr Asp Val Pro Ala Asp Arg Trp 115 120 125 Asn Ala Gly Asp Phe Tyr Asp Pro Asp Arg Ser Ala Pro Gly Arg Ser 130 135 140 Asn Ser Arg Trp Gly Gly Phe Ile Glu Asp Val Asp Arg Phe Asp Ala 145 150 155 160 Ala Phe Phe Gly Ile Ser Pro Arg Glu Ala Ala Glu Met Asp Pro Gln 165 170 175 Gln Arg Leu Ala Leu Glu Leu Gly Trp Glu Ala Leu Glu Arg Ala Gly 180 185 190 Ile Asp Pro Ser Ser Leu Thr Gly Thr Arg Thr Gly Val Phe Ala Gly 195 200 205 Ala Ile Trp Asp Asp Tyr Ala Thr Leu Lys His Arg Gln Gly Gly Ala 210 215 220 Ala Ile Thr Pro His Thr Val Thr Gly Leu His Arg Gly Ile Ile Ala 225 230 235 240 Asn Arg Leu Ser Tyr Thr Leu Gly Leu Arg Gly Pro Ser Met Val Val 245 250 255 Asp Ser Gly Gln Ser Ser Ser Leu Val Ala Val His Leu Ala Cys Glu 260 265 270 Ser Leu Arg Arg Gly Glu Ser Glu Leu Ala Leu Ala Gly Gly Val Ser 275 280 285 Leu Asn Leu Val Pro Asp Ser Ile Ile Gly Ala Ser Lys Phe Gly Gly 290 295 300 Leu Ser Pro Asp Gly Arg Ala Tyr Thr Phe Asp Ala Arg Ala Asn Gly 305 310 315 320 Tyr Val Arg Gly Glu Gly Gly Gly Phe Val Val Leu Lys Arg Leu Ser 325 330 335 Arg Ala Val Ala Asp Gly Asp Pro Val Leu Ala Val Ile Arg Gly Ser 340 345 350 Ala Val Asn Asn Gly Gly Ala Ala Gln Gly Met Thr Thr Pro Asp Ala 355 360 365 Gln Ala Gln Glu Ala Val Leu Arg Glu Ala His Glu Arg Ala Gly Thr 370 375 380 Ala Pro Ala Asp Val Arg Tyr Val Glu Leu His Gly Thr Gly Thr Pro 385 390 395 400 Val Gly Asp Pro Ile Glu Ala Ala Ala Leu Gly Ala Ala Leu Gly Thr 405 410 415 Gly Arg Pro Ala Gly Gln Pro Leu Leu Val Gly Ser Val Lys Thr Asn 420 425 430 Ile Gly His Leu Glu Gly Ala Ala Gly Ile Ala Gly Leu Ile Lys Ala 435 440 445 Val Leu Ala Val Arg Gly Arg Ala Leu Pro Ala Ser Leu Asn Tyr Glu 450 455 460 Thr Pro Asn Pro Ala Ile Pro Phe Glu Glu Leu Asn Leu Arg Val Asn 465 470 475 480 Thr Glu Tyr Leu Pro Trp Glu Pro Glu His Asp Gly Gln Arg Met Val 485 490 495 Val Gly Val Ser Ser Phe Gly Met Gly Gly Thr Asn Ala His Val Val 500 505 510 Leu Glu Glu Ala Pro Gly Gly Cys Arg Gly Ala Ser Val Val Glu Ser 515 520 525 Thr Val Gly Gly Ser Ala Val Gly Gly Gly Val Val Pro Trp Val Val 530 535 540 Ser Ala Lys Ser Ala Ala Ala Leu Asp Ala Gln Ile Glu Arg Leu Ala 545 550 555 560 Ala Phe Ala Ser Arg Asp Arg Thr Asp Gly Val Asp Ala Gly Ala Val 565 570 575 Asp Ala Gly Ala Val Asp Ala Gly Ala Val Ala Arg Val Leu Ala Gly 580 585 590 Gly Arg Ala Gln Phe Glu His Arg Ala Val Val Val Gly Ser Gly Pro 595 600 605 Asp Asp Leu Ala Ala Ala Leu Ala Ala Pro Glu Gly Leu Val Arg Gly 610 615 620 Val Ala Ser Gly Val Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly 625 630 635 640 Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Ser Ser Ala Val 645 650 655 Phe Ala Ala Ala Met Ala Glu Cys Glu Ala Ala Leu Ser Pro Tyr Val 660 665 670 Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Gly Ala Pro Thr 675 680 685 Leu Glu Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val Met Val 690 695 700 Ser Leu Ala Arg Val Trp Gln His His Gly Val Thr Pro Gln Ala Val 705 710 715 720 Val Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala 725 730 735 Leu Ser Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser 740 745 750 Ile Ala Ala His Leu Ala Gly Lys Gly Gly Met Leu Ser Leu Ala Leu 755 760 765 Ser Glu Asp Ala Val Leu Glu Arg Leu Ala Gly Phe Asp Gly Leu Ser 770 775 780 Val Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro 785 790 795 800 Val Gln Ile Glu Glu Leu Ala Arg Ala Cys Glu Ala Asp Gly Val Arg 805 810 815 Ala Arg Val Ile Pro Val Asp Tyr Ala Ser His Ser Arg Gln Val Glu 820 825 830 Ile Ile Glu Ser Glu Leu Ala Glu Val Leu Ala Gly Leu Ser Pro Gln 835 840 845 Ala Pro Arg Val Pro Phe Phe Ser Thr Leu Glu Gly Ala Trp Ile Thr 850 855 860 Glu Pro Val Leu Asp Gly Gly Tyr Trp Tyr Arg Asn Leu Arg His Arg 865 870 875 880 Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu Gly Phe 885 890 895 Thr His Phe Val Glu Val Ser Ala His Pro Val Leu Thr Met Ala Leu 900 905 910 Pro Gly Thr Val Thr Gly Leu Ala Thr Leu Arg Arg Asp Asn Gly Gly 915 920 925 Gln Asp Arg Leu Val Ala Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu 930 935 940 Ala Val Asp Trp Ser Pro Leu Leu Pro Ser Ala Thr Gly His His Ser 945 950 955 960 Asp Leu Pro Thr Tyr Ala Phe Gln Thr Glu Arg His Trp Leu Gly Glu 965 970 975 Ile Glu Ala Leu Ala Pro Ala Gly Glu Pro Ala Val Gln Pro Ala Val 980 985 990 Leu Arg Thr Glu Ala Ala Glu Pro Ala Glu Leu Asp Arg Asp Glu Gln 995 1000 1005 Leu Arg Val Ile Leu Asp Lys Val Arg Ala Gln Thr Ala Gln Val Leu 1010 1015 1020 Gly Tyr Ala Thr Gly Gly Gln Ile Glu Val Asp Arg Thr Phe Arg Glu 1025 1030 1035 1040 Ala Gly Cys Thr Ser Leu Thr Gly Val Asp Leu Arg Asn Arg Ile Asn 1045 1050 1055 Ala Ala Phe Gly Val Arg Met Ala Pro Ser Met Ile Phe Asp Phe Pro 1060 1065 1070 Thr Pro Glu Ala Leu Ala Glu Gln Leu Leu Leu Val Val His Gly Glu 1075 1080 1085 Ala Ala Ala Asn Pro Ala Gly Ala Glu Pro Ala Pro Val Ala Ala Ala 1090 1095 1100 Gly Ala Val Asp Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu 1105 1110 1115 1120 Pro Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Gly 1125 1130 1135 Gly Gly Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val 1140 1145 1150 Glu Gly Leu Tyr His Pro Asp Pro Glu His Pro Gly Thr Ser Tyr Val 1155 1160 1165 Arg Gln Gly Gly Phe Ile Glu Asn Val Ala Gly Phe Asp Ala Ala Phe 1170 1175 1180 Phe Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg 1185 1190 1195 1200 Leu Leu Leu Glu Thr Ser Trp Glu Ala Val Glu Asp Ala Gly Ile Asp 1205 1210 1215 Pro Thr Ser Leu Arg Gly Arg Gln Val Gly Val Phe Thr Gly Ala Met 1220 1225 1230 Thr His Glu Tyr Gly Pro Ser Leu Arg Asp Gly Gly Glu Gly Leu Asp 1235 1240 1245 Gly Tyr Leu Leu Thr Gly Asn Thr Ala Ser Val Met Ser Gly Arg Val 1250 1255 1260 Ser Tyr Thr Leu Gly Leu Glu Gly Pro Ala Leu Thr Val Asp Thr Ala 1265 1270 1275 1280 Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg 1285 1290 1295 Lys Gly Glu Val Asp Met Ala Leu Ala Gly Gly Val Ala Val Met Pro 1300 1305 1310 Thr Pro Gly Met Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Gly 1315 1320 1325 Asp Gly Arg Ser Lys Ala Phe Ala Ala Ser Ala Asp Gly Thr Ser Trp 1330 1335 1340 Ser Glu Gly Val Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg 1345 1350 1355 1360 Arg Asn Gly His Gln Val Leu Ala Val Val Arg Gly Ser Ala Leu Asn 1365 1370 1375 Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln 1380 1385 1390 Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr Thr Ser 1395 1400 1405 Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp 1410 1415 1420 Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Gly Arg Asp 1425 1430 1435 1440 Asp Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile Gly His 1445 1450 1455 Thr Gln Ala Ala Ala Gly Val Ser Gly Val Ile Lys Met Val Gln Ala 1460 1465 1470 Met Arg His Gly Leu Leu Pro Lys Thr Leu His Val Asp Glu Pro Ser 1475 1480 1485 Asp Gln Ile Asp Trp Ser Ala Gly Ala Val Glu Leu Leu Thr Glu Ala 1490 1495 1500 Val Asp Trp Pro Glu Lys Gln Asp Gly Gly Leu Arg Arg Ala Ala Val 1505 1510 1515 1520 Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Val Val Leu Glu Glu 1525 1530 1535 Ala Pro Val Val Val Glu Gly Ala Ser Val Val Glu Pro Ser Val Gly 1540 1545 1550 Gly Ser Ala Val Gly Gly Gly Val Thr Pro Trp Val Val Ser Ala Lys 1555 1560 1565 Ser Ala Ala Ala Leu Asp Ala Gln Ile Glu Arg Leu Ala Ala Phe Ala 1570 1575 1580 Ser Arg Asp Arg Thr Asp Asp Ala Asp Ala Gly Ala Val Asp Ala Gly 1585 1590 1595 1600 Ala Val Ala His Val Leu Ala Asp Gly Arg Ala Gln Phe Glu His Arg 1605 1610 1615 Ala Val Ala Leu Gly Ala Gly Ala Asp Asp Leu Val Gln Ala Leu Ala 1620 1625 1630 Asp Pro Asp Gly Leu Ile Arg Gly Thr Ala Ser Gly Val Gly Arg Val 1635 1640 1645 Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly Met Gly Ala 1650 1655 1660 Glu Leu Leu Asp Ser Ser Ala Val Phe Ala Ala Ala Met Ala Glu Cys 1665 1670 1675 1680 Glu Ala Ala Leu Ser Pro Tyr Val Asp Trp Ser Leu Glu Ala Val Val 1685 1690 1695 Arg Gln Ala Pro Gly Ala Pro Thr Leu Glu Arg Val Asp Val Val Gln 1700 1705 1710 Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Arg Val Trp Gln His 1715 1720 1725 His Gly Val Thr Pro Gln Ala Val Val Gly His Ser Gln Gly Glu Ile 1730 1735 1740 Ala Ala Ala Tyr Val Ala Gly Ala Leu Pro Leu Asp Asp Ala Ala Arg 1745 1750 1755 1760 Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu Ala Gly Lys 1765 1770 1775 Gly Gly Met Leu Ser Leu Ala Leu Asn Glu Asp Ala Val Leu Glu Arg 1780 1785 1790 Leu Ser Asp Phe Asp Gly Leu Ser Val Ala Ala Val Asn Gly Pro Thr 1795 1800 1805 Ala Thr Val Val Ser Gly Asp Pro Val Gln Ile Glu Glu Leu Ala Gln 1810 1815 1820 Ala Cys Lys Ala Asp Gly Phe Arg Ala Arg Ile Ile Pro Val Asp Tyr 1825 1830 1835 1840 Ala Ser His Ser Arg Gln Val Glu Ile Ile Glu Ser Glu Leu Ala Gln 1845 1850 1855 Val Leu Ala Gly Leu Ser Pro Gln Ala Pro Arg Val Pro Phe Phe Ser 1860 1865 1870 Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Val Leu Asp Gly Thr Tyr 1875 1880 1885 Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro Ala Ile Glu 1890 1895 1900 Thr Leu Ala Val Asp Glu Gly Phe Thr His Phe Val Glu Val Ser Ala 1905 1910 1915 1920 His Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly Leu Gly 1925 1930 1935 Thr Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr Ser Leu 1940 1945 1950 Ala Glu Ala Trp Val Asn Gly Leu Pro Val Ala Trp Thr Ser Leu Leu 1955 1960 1965 Pro Ala Thr Ala Ser Arg Pro Gly Leu Pro Thr Tyr Ala Phe Gln Ala 1970 1975 1980 Glu Arg Tyr Trp Leu Glu Asn Thr Pro Ala Ala Leu Ala Thr Gly Asp 1985 1990 1995 2000 Asp Trp Arg Tyr Arg Ile Asp Trp Lys Arg Leu Pro Ala Ala Glu Gly 2005 2010 2015 Ser Glu Arg Thr Gly Leu Ser Gly Arg Trp Leu Ala Val Thr Pro Glu 2020 2025 2030 Asp His Ser Ala Gln Ala Ala Ala Val Leu Thr Ala Leu Val Asp Ala 2035 2040 2045 Gly Ala Lys Val Glu Val Leu Thr Ala Gly Ala Asp Asp Asp Arg Glu 2050 2055 2060 Ala Leu Ala Ala Arg Leu Thr Ala Leu Thr Thr Gly Asp Gly Phe Thr 2065 2070 2075 2080 Gly Val Val Ser Leu Leu Asp Gly Leu Val Pro Gln Val Ala Trp Val 2085 2090 2095 Gln Ala Leu Gly Asp Ala Gly Ile Lys Ala Pro Leu Trp Ser Val Thr 2100 2105 2110 Gln Gly Ala Val Ser Val Gly Arg Leu Asp Thr Pro Ala Asp Pro Asp 2115 2120 2125 Arg Ala Met Leu Trp Gly Leu Gly Arg Val Val Ala Leu Glu His Pro 2130 2135 2140 Glu Arg Trp Ala Gly Leu Val Asp Leu Pro Ala Gln Pro Asp Ala Ala 2145 2150 2155 2160 Ala Leu Ala His Leu Val Thr Ala Leu Ser Gly Ala Thr Gly Glu Asp 2165 2170 2175 Gln Ile Ala Ile Arg Thr Thr Gly Leu His Ala Arg Arg Leu Ala Arg 2180 2185 2190 Ala Pro Leu His Gly Arg Arg Pro Thr Arg Asp Trp Gln Pro His Gly 2195 2200 2205 Thr Val Leu Ile Thr Gly Gly Thr Gly Ala Leu Gly Ser His Ala Ala 2210 2215 2220 Arg Trp Met Ala His His Gly Ala Glu His Leu Leu Leu Val Ser Arg 2225 2230 2235 2240 Ser Gly Glu Gln Ala Pro Gly Ala Thr Gln Leu Thr Ala Glu Leu Thr 2245 2250 2255 Ala Ser Gly Ala Arg Val Thr Ile Ala Ala Cys Asp Val Ala Asp Pro 2260 2265 2270 His Ala Met Arg Thr Leu Leu Asp Ala Ile Pro Ala Glu Thr Pro Leu 2275 2280 2285 Thr Ala Val Val His Thr Ala Gly Ala Leu Asp Asp Gly Ile Val Asp 2290 2295 2300 Thr Leu Thr Ala Glu Gln Val Arg Arg Ala His Arg Ala Lys Ala Val 2305 2310 2315 2320 Gly Ala Ser Val Leu Asp Glu Leu Thr Arg Asp Leu Asp Leu Asp Ala 2325 2330 2335 Phe Val Leu Phe Ser Ser Val Ser Ser Thr Leu Gly Ile Pro Gly Gln 2340 2345 2350 Gly Asn Tyr Ala Pro His Asn Ala Tyr Leu Asp Ala Leu Ala Ala Arg 2355 2360 2365 Arg Arg Ala Thr Gly Arg Ser Ala Val Ser Val Ala Trp Gly Pro Trp 2370 2375 2380 Asp Gly Gly Gly Met Ala Ala Gly Asp Gly Val Ala Glu Arg Leu Arg 2385 2390 2395 2400 Asn His Gly Val Pro Gly Met Asp Pro Glu Leu Ala Leu Ala Ala Leu 2405 2410 2415 Glu Ser Ala Leu Gly Arg Asp Glu Thr Ala Ile Thr Val Ala Asp Ile 2420 2425 2430 Asp Trp Asp Arg Phe Tyr Leu Ala Tyr Ser Ser Gly Arg Pro Gln Pro 2435 2440 2445 Leu Val Glu Glu Leu Pro Glu Val Arg Arg Ile Ile Asp Ala Arg Asp 2450 2455 2460 Ser Ala Thr Ser Gly Gln Gly Gly Ser Ser Ala Gln Gly Ala Asn Pro 2465 2470 2475 2480 Leu Ala Glu Arg Leu Ala Ala Ala Ala Pro Gly Glu Arg Thr Glu Ile 2485 2490 2495 Leu Leu Gly Leu Val Arg Ala Gln Ala Ala Ala Val Leu Arg Met Arg 2500 2505 2510 Ser Pro Glu Asp Val Ala Ala Asp Arg Ala Phe Lys Asp Ile Gly Phe 2515 2520 2525 Asp Ser Leu Ala Gly Val Glu Leu Arg Asn Arg Leu Thr Arg Ala Thr 2530 2535 2540 Gly Leu Gln Leu Pro Ala Thr Leu Val Phe Asp His Pro Thr Pro Leu 2545 2550 2555 2560 Ala Leu Val Ser Leu Leu Arg Ser Glu Phe Leu Gly Asp Glu Glu Thr 2565 2570 2575 Ala Asp Ala Arg Arg Ser Ala Ala Leu Pro Ala Thr Val Gly Ala Gly 2580 2585 2590 Ala Gly Ala Gly Ala Gly Thr Asp Ala Asp Asp Asp Pro Ile Ala Ile 2595 2600 2605 Val Ala Met Ser Cys Arg Tyr Pro Gly Asp Ile Arg Ser Pro Glu Asp 2610 2615 2620 Leu Trp Arg Met Leu Ser Glu Gly Gly Glu Gly Ile Thr Pro Phe Pro 2625 2630 2635 2640 Thr Asp Arg Gly Trp Asp Leu Asp Gly Leu Tyr Asp Ala Asp Pro Asp 2645 2650 2655 Ala Leu Gly Arg Ala Tyr Val Arg Glu Gly Gly Phe Leu His Asp Ala 2660 2665 2670 Ala Glu Phe Asp Ala Glu Phe Phe Gly Val Ser Pro Arg Glu Ala Leu 2675 2680 2685 Ala Met Asp Pro Gln Gln Arg Met Leu Leu Thr Thr Ser Trp Glu Ala 2690 2695 2700 Phe Glu Arg Ala Gly Ile Glu Pro Ala Ser Leu Arg Gly Ser Ser Thr 2705 2710 2715 2720 Gly Val Phe Ile Gly Leu Ser Tyr Gln Asp Tyr Ala Ala Arg Val Pro 2725 2730 2735 Asn Ala Pro Arg Gly Val Glu Gly Tyr Leu Leu Thr Gly Ser Thr Pro 2740 2745 2750 Ser Val Ala Ser Gly Arg Ile Ala Tyr Thr Phe Gly Leu Glu Gly Pro 2755 2760 2765 Ala Thr Thr Val Asp Thr Ala Cys Ser Ser Ser Leu Thr Ala Leu His 2770 2775 2780 Leu Ala Val Arg Ala Leu Arg Ser Gly Glu Cys Thr Met Ala Leu Ala 2785 2790 2795 2800 Gly Gly Val Ala Met Met Ala Thr Pro His Met Phe Val Glu Phe Ser 2805 2810 2815 Arg Gln Arg Ala Leu Ala Pro Asp Gly Arg Ser Lys Ala Phe Ser Ala 2820 2825 2830 Asp Ala Asp Gly Phe Gly Ala Ala Glu Gly Val Gly Leu Leu Leu Val 2835 2840 2845 Glu Arg Leu Ser Asp Ala Arg Arg Asn Gly His Pro Val Leu Ala Val 2850 2855 2860 Val Arg Gly Thr Ala Val Asn Gln Asp Gly Ala Ser Asn Gly Leu Thr 2865 2870 2875 2880 Ala Pro Asn Gly Pro Ser Gln Gln Arg Val Ile Arg Gln Ala Leu Ala 2885 2890 2895 Asp Ala Arg Leu Ala Pro Gly Asp Ile Asp Ala Val Glu Thr His Gly 2900 2905 2910 Thr Gly Thr Ser Leu Gly Asp Pro Ile Glu Ala Gln Gly Leu Gln Ala 2915 2920 2925 Thr Tyr Gly Lys Glu Arg Pro Ala Glu Arg Pro Leu Ala Ile Gly Ser 2930 2935 2940 Val Lys Ser Asn Ile Gly His Thr Gln Ala Ala Ala Gly Ala Ala Gly 2945 2950 2955 2960 Ile Ile Lys Met Val Leu Ala Met Arg His Gly Thr Leu Pro Lys Thr 2965 2970 2975 Leu His Ala Asp Glu Pro Ser Pro His Val Asp Trp Ala Asn Ser Gly 2980 2985 2990 Leu Ala Leu Val Thr Glu Pro Ile Asp Trp Pro Ala Gly Thr Gly Pro 2995 3000 3005 Arg Arg Ala Ala Val Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His 3010 3015 3020 Val Val Leu Glu Gln Ala Pro Asp Ala Ala Gly Glu Val Leu Gly Ala 3025 3030 3035 3040 Asp Glu Val Pro Glu Val Ser Glu Thr Val Ala Met Ala Gly Thr Ala 3045 3050 3055 Gly Thr Ser Glu Val Ala Glu Gly Ser Glu Ala Ser Glu Ala Pro Ala 3060 3065 3070 Ala Pro Gly Ser Arg Glu Ala Ser Leu Pro Gly His Leu Pro Trp Val 3075 3080 3085 Leu Ser Ala Lys Asp Glu Gln Ser Leu Arg Gly Gln Ala Ala Ala Leu 3090 3095 3100 His Ala Trp Leu Ser Glu Pro Ala Ala Asp Leu Ser Asp Ala Asp Gly 3105 3110 3115 3120 Pro Ala Arg Leu Arg Asp Val Gly Tyr Thr Leu Ala Thr Ser Arg Thr 3125 3130 3135 Ala Phe Ala His Arg Ala Ala Val Thr Ala Ala Asp Arg Asp Gly Phe 3140 3145 3150 Leu Asp Gly Leu Ala Thr Leu Ala Gln Gly Gly Thr Ser Ala His Val 3155 3160 3165 His Leu Asp Thr Ala Arg Asp Gly Thr Thr Ala Phe Leu Phe Thr Gly 3170 3175 3180 Gln Gly Ser Gln Arg Pro Gly Ala Gly Arg Glu Leu Tyr Asp Arg His 3185 3190 3195 3200 Pro Val Phe Ala Arg Ala Leu Asp Glu Ile Cys Ala His Leu Asp Gly 3205 3210 3215 His Leu Glu Leu Pro Leu Leu Asp Val Met Phe Ala Ala Glu Gly Ser 3220 3225 3230 Ala Glu Ala Ala Leu Leu Asp Glu Thr Arg Tyr Thr Gln Cys Ala Leu 3235 3240 3245 Phe Ala Leu Glu Val Ala Leu Phe Arg Leu Val Glu Ser Trp Gly Met 3250 3255 3260 Arg Pro Ala Ala Leu Leu Gly His Ser Val Gly Glu Ile Ala Ala Ala 3265 3270 3275 3280 His Val Ala Gly Val Phe Ser Leu Ala Asp Ala Ala Arg Leu Val Ala 3285 3290 3295 Ala Arg Gly Arg Leu Met Gln Glu Leu Pro Ala Gly Gly Ala Met Leu 3300 3305 3310 Ala Val Gln Ala Ala Glu Asp Glu Ile Arg Val Trp Leu Glu Thr Glu 3315 3320 3325 Glu Arg Tyr Ala Gly Arg Leu Asp Val Ala Ala Val Asn Gly Pro Glu 3330 3335 3340 Ala Ala Val Leu Ser Gly Asp Ala Asp Ala Ala Arg Glu Ala Glu Ala 3345 3350 3355 3360 Tyr Trp Ser Gly Leu Gly Arg Arg Thr Arg Ala Leu Arg Val Ser His 3365 3370 3375 Ala Phe His Ser Ala His Met Asp Gly Met Leu Asp Gly Phe Arg Ala 3380 3385 3390 Val Leu Glu Thr Val Glu Phe Arg Arg Pro Ser Leu Thr Val Val Ser 3395 3400 3405 Asn Val Thr Gly Leu Ala Ala Gly Pro Asp Asp Leu Cys Asp Pro Glu 3410 3415 3420 Tyr Trp Val Arg His Val Arg Gly Thr Val Arg Phe Leu Asp Gly Val 3425 3430 3435 3440 Arg Val Leu Arg Asp Leu Gly Val Arg Thr Cys Leu Glu Leu Gly Pro 3445 3450 3455 Asp Gly Val Leu Thr Ala Met Ala Ala Asp Gly Leu Ala Asp Thr Pro 3460 3465 3470 Ala Asp Ser Ala Ala Gly Ser Pro Val Gly Ser Pro Ala Gly Ser Pro 3475 3480 3485 Ala Asp Ser Ala Ala Gly Ala Leu Arg Pro Arg Pro Leu Leu Val Ala 3490 3495 3500 Leu Leu Arg Arg Lys Arg Ser Glu Thr Glu Thr Val Ala Asp Ala Leu 3505 3510 3515 3520 Gly Arg Ala His Ala His Gly Thr Gly Pro Asp Trp His Ala Trp Phe 3525 3530 3535 Ala Gly Ser Gly Ala His Arg Val Asp Leu Pro Thr Tyr Ser Phe Arg 3540 3545 3550 Arg Asp Arg Tyr Trp Leu Asp Ala Pro Ala Ala Asp Thr Ala Val Asp 3555 3560 3565 Thr Ala Gly Leu Gly Leu Gly Thr Ala Asp His Pro Leu Leu Gly Ala 3570 3575 3580 Val Val Ser Leu Pro Asp Arg Asp Gly Leu Leu Leu Thr Gly Arg Leu 3585 3590 3595 3600 Ser Leu Arg Thr His Pro Trp Leu Ala Asp His Ala Val Leu Gly Ser 3605 3610 3615 Val Leu Leu Pro Gly Ala Ala Met Val Glu Leu Ala Ala His Ala Ala 3620 3625 3630 Glu Ser Ala Gly Leu Arg Asp Val Arg Glu Leu Thr Leu Leu Glu Pro 3635 3640 3645 Leu Val Leu Pro Glu His Gly Gly Val Glu Leu Arg Val Thr Val Gly 3650 3655 3660 Ala Pro Ala Gly Glu Pro Gly Gly Glu Ser Ala Gly Asp Gly Ala Arg 3665 3670 3675 3680 Pro Val Ser Leu His Ser Arg Leu Ala Asp Ala Pro Ala Gly Thr Ala 3685 3690 3695 Trp Ser Cys His Ala Thr Gly Leu Leu Ala Thr Asp Arg Pro Glu Leu 3700 3705 3710 Pro Val Ala Pro Asp Arg Ala Ala Met Trp Pro Pro Gln Gly Ala Glu 3715 3720 3725 Glu Val Pro Leu Asp Gly Leu Tyr Glu Arg Leu Asp Gly Asn Gly Leu 3730 3735 3740 Ala Phe Gly Pro Leu Phe Gln Gly Leu Asn Ala Val Trp Arg Tyr Glu 3745 3750 3755 3760 Gly Glu Val Phe Ala Asp Ile Ala Leu Pro Ala Thr Thr Asn Ala Thr 3765 3770 3775 Ala Pro Ala Thr Ala Asn Gly Gly Gly Ser Ala Ala Ala Ala Pro Tyr 3780 3785 3790 Gly Ile His Pro Ala Leu Leu Asp Ala Ser Leu His Ala Ile Ala Val 3795 3800 3805 Gly Gly Leu Val Asp Glu Pro Glu Leu Val Arg Val Pro Phe His Trp 3810 3815 3820 Ser Gly Val Thr Val His Ala Ala Gly Ala Ala Ala Ala Arg Val Arg 3825 3830 3835 3840 Leu Ala Ser Ala Gly Thr Asp Ala Val Ser Leu Ser Leu Thr Asp Gly 3845 3850 3855 Glu Gly Arg Pro Leu Val Ser Val Glu Arg Leu Thr Leu Arg Pro Val 3860 3865 3870 Thr Ala Asp Gln Ala Ala Ala Ser Arg Val Gly Gly Leu Met His Arg 3875 3880 3885 Val Ala Trp Arg Pro Tyr Ala Leu Ala Ser Ser Gly Glu Gln Asp Pro 3890 3895 3900 His Ala Thr Ser Tyr Gly Pro Thr Ala Val Leu Gly Lys Asp Glu Leu 3905 3910 3915 3920 Lys Val Ala Ala Ala Leu Glu Ser Ala Gly Val Glu Val Gly Leu Tyr 3925 3930 3935 Pro Asp Leu Ala Ala Leu Ser Gln Asp Val Ala Ala Gly Ala Pro Ala 3940 3945 3950 Pro Arg Thr Val Leu Ala Pro Leu Pro Ala Gly Pro Ala Asp Gly Gly 3955 3960 3965 Ala Glu Gly Val Arg Gly Thr Val Ala Arg Thr Leu Glu Leu Leu Gln 3970 3975 3980 Ala Trp Leu Ala Asp Glu His Leu Ala Gly Thr Arg Leu Leu Leu Val 3985 3990 3995 4000 Thr Arg Gly Ala Val Arg Asp Pro Glu Gly Ser Gly Ala Asp Asp Gly 4005 4010 4015 Gly Glu Asp Leu Ser His Ala Ala Ala Trp Gly Leu Val Arg Thr Ala 4020 4025 4030 Gln Thr Glu Asn Pro Gly Arg Phe Gly Leu Leu Asp Leu Ala Asp Asp 4035 4040 4045 Ala Ser Ser Tyr Arg Thr Leu Pro Ser Val Leu Ser Asp Ala Gly Leu 4050 4055 4060 Arg Asp Glu Pro Gln Leu Ala Leu His Asp Gly Thr Ile Arg Leu Ala 4065 4070 4075 4080 Arg Leu Ala Ser Val Arg Pro Glu Thr Gly Thr Ala Ala Pro Ala Leu 4085 4090 4095 Ala Pro Glu Gly Thr Val Leu Leu Thr Gly Gly Thr Gly Gly Leu Gly 4100 4105 4110 Gly Leu Val Ala Arg His Val Val Gly Glu Trp Gly Val Arg Arg Leu 4115 4120 4125 Leu Leu Val Ser Arg Arg Gly Thr Asp Ala Pro Gly Ala Asp Glu Leu 4130 4135 4140 Val His Glu Leu Glu Ala Leu Gly Ala Asp Val Ser Val Ala Ala Cys 4145 4150 4155 4160 Asp Val Ala Asp Arg Glu Ala Leu Thr Ala Val Leu Asp Ala Ile Pro 4165 4170 4175 Ala Glu His Pro Leu Thr Ala Val Val His Thr Ala Gly Val Leu Ser 4180 4185 4190 Asp Gly Thr Leu Pro Ser Met Thr Thr Glu Asp Val Glu His Val Leu 4195 4200 4205 Arg Pro Lys Val Asp Ala Ala Phe Leu Leu Asp Glu Leu Thr Ser Thr 4210 4215 4220 Pro Ala Tyr Asp Leu Ala Ala Phe Val Met Phe Ser Ser Ala Ala Ala 4225 4230 4235 4240 Val Phe Gly Gly Ala Gly Gln Gly Ala Tyr Ala Ala Ala Asn Ala Thr 4245 4250 4255 Leu Asp Ala Leu Ala Trp Arg Arg Arg Ala Ala Gly Leu Pro Ala Leu 4260 4265 4270 Ser Leu Gly Trp Gly Leu Trp Ala Glu Thr Ser Gly Met Thr Gly Glu 4275 4280 4285 Leu Gly Gln Ala Asp Leu Arg Arg Met Ser Arg Ala Gly Ile Gly Gly 4290 4295 4300 Ile Ser Asp Ala Glu Gly Ile Ala Leu Leu Asp Ala Ala Leu Arg Asp 4305 4310 4315 4320 Asp Arg His Pro Val Leu Leu Pro Leu Arg Leu Asp Ala Ala Gly Leu 4325 4330 4335 Arg Asp Ala Ala Gly Asn Asp Pro Ala Gly Ile Pro Ala Leu Phe Arg 4340 4345 4350 Asp Val Val Gly Ala Arg Thr Val Arg Ala Arg Pro Ser Ala Ala Ser 4355 4360 4365 Ala Ser Thr Thr Ala Gly Thr Ala Gly Thr Pro Gly Thr Ala Asp Gly 4370 4375 4380 Ala Ala Glu Thr Ala Ala Val Thr Leu Ala Asp Arg Ala Ala Thr Val 4385 4390 4395 4400 Asp Gly Pro Ala Arg Gln Arg Leu Leu Leu Glu Phe Val Val Gly Glu 4405 4410 4415 Val Ala Glu Val Leu Gly His Ala Arg Gly His Arg Ile Asp Ala Glu 4420 4425 4430 Arg Gly Phe Leu Asp Leu Gly Phe Asp Ser Leu Thr Ala Val Glu Leu 4435 4440 4445 Arg Asn Arg Leu Asn Ser Ala Gly Gly Leu Ala Leu Pro Ala Thr Leu 4450 4455 4460 Val Phe Asp His Pro Ser Pro Ala Ala Leu Ala Ser His Leu Asp Ala 4465 4470 4475 4480 Glu Leu Pro Arg Gly Ala Ser Asp Gln Asp Gly Ala Gly Asn Arg Asn 4485 4490 4495 Gly Asn Glu Asn Gly Thr Thr Ala Ser Arg Ser Thr Ala Glu Thr Asp 4500 4505 4510 Ala Leu Leu Ala Gln Leu Thr Arg Leu Glu Gly Ala Leu Val Leu Thr 4515 4520 4525 Gly Leu Ser Asp Ala Pro Gly Ser Glu Glu Val Leu Glu His Leu Arg 4530 4535 4540 Ser Leu Arg Ser Met Val Thr Gly Glu Thr Gly Thr Gly Thr Ala Ser 4545 4550 4555 4560 Gly Ala Pro Asp Gly Ala Gly Ser Gly Ala Glu Asp Arg Pro Trp Ala 4565 4570 4575 Ala Gly Asp Gly Ala Gly Gly Gly Ser Glu Asp Gly Ala Gly Val Pro 4580 4585 4590 Asp Phe Met Asn Ala Ser Ala Glu Glu Leu Phe Gly Leu Leu Asp Gln 4595 4600 4605 Asp Pro Ser Thr Asp 4610 32 11220 DNA Streptomyces venezuelae 32 gtgtccacgg tgaacgaaga gaagtacctc gactacctgc gtcgtgccac ggcggacctc 60 cacgaggccc gtggccgcct ccgcgagctg gaggcgaagg cgggcgagcc ggtggcgatc 120 gtcggcatgg cctgccgcct gcccggcggc gtcgcctcgc ccgaggacct gtggcggctg 180 gtggccggcg gcgaggacgc gatctcggag ttcccccagg accgcggctg ggacgtggag 240 ggcctgtacg acccgaaccc ggaggccacg ggcaagagtt acgcccgcga ggccggattc 300 ctgtacgagg cgggcgagtt cgacgccgac ttcttcggga tctcgccgcg cgaggccctc 360 gccatggacc cgcagcagcg tctcctcctg gaggcctcct gggaggcgtt cgagcacgcc 420 gggatcccgg cggccaccgc gcgcggcacc tcggtcggcg tcttcaccgg cgtgatgtac 480 cacgactacg ccacccgtct caccgatgtc ccggagggca tcgagggcta cctgggcacc 540 ggcaactccg gcagtgtcgc ctcgggccgc gtcgcgtaca cgcttggcct ggaggggccg 600 gccgtcacgg tcgacaccgc ctgctcgtcc tcgctggtcg ccctgcacct cgccgtgcag 660 gccctgcgca agggcgaggt cgacatggcg ctcgccggcg gcgtgacggt catgtcgacg 720 cccagcacct tcgtcgagtt cagccgtcag cgcgggctgg cgccggacgg ccggtcgaag 780 tccttctcgt cgacggccga cggcaccagc tggtccgagg gcgtcggcgt cctcctcgtc 840 gagcgcctgt ccgacgcgcg tcgcaagggc catcggatcc tcgccgtggt ccggggcacc 900 gccgtcaacc aggacggcgc cagcagcggc ctcacggctc cgaacgggcc gtcgcagcag 960 cgcgtcatcc gacgtgccct ggcggacgcc cggctcacga cctccgacgt ggacgtcgtc 1020 gaggcccacg gcacgggtac gcgactcggc gacccgatcg aggcgcaggc cgtcatcgcc 1080 acgtacgggc agggccgtga cggcgaacag ccgctgcgcc tcgggtcgtt gaagtccaac 1140 atcggacaca cccaggccgc cgccggtgtc tccggcgtga tcaagatggt ccaggcgatg 1200 cgccacggcg tcctgccgaa gacgctccac gtggagaagc cgacggacca ggtggactgg 1260 tccgcgggcg cggtcgagct gctcaccgag gccatggact ggccggacaa gggcgacggc 1320 ggactgcgca gggccgcggt ctcctccttc ggcgtcagcg ggacgaacgc gcacgtcgtg 1380 ctcgaagagg ccccggcggc cgaggagacc cctgcctccg aggcgacccc ggccgtcgag 1440 ccgtcggtcg gcgccggcct ggtgccgtgg ctggtgtcgg cgaagactcc ggccgcgctg 1500 gacgcccaga tcggacgcct cgccgcgttc gcctcgcagg gccgtacgga cgccgccgat 1560 ccgggcgcgg tcgctcgcgt actggccggc gggcgcgccg agttcgagca ccgggccgtc 1620 gtgctcggca ccggacagga cgatttcgcg caggcgctga ccgctccgga aggactgata 1680 cgcggcacgc cctcggacgt gggccgggtg gcgttcgtgt tccccggtca gggcacgcag 1740 tgggccggga tgggcgccga actcctcgac gtgtcgaagg agttcgcggc ggccatggcc 1800 gagtgcgaga gcgcgctctc ccgctatgtc gactggtcgc tggaggccgt cgtccggcag 1860 gcgccgggcg cgcccacgct ggagcgggtc gacgtcgtcc agcccgtgac cttcgctgtc 1920 atggtttcgc tggcgaaggt ctggcagcac cacggcgtga cgccgcaggc cgtcgtcggc 1980 cactcgcagg gcgagatcgc cgccgcgtac gtcgccggtg ccctcaccct cgacgacgcc 2040 gcccgcgtcg tcaccctgcg cagcaagtcc atcgccgccc acctcgccgg caagggcggc 2100 atgatctccc tcgccctcag cgaggaagcc acccggcagc gcatcgagaa cctccacgga 2160 ctgtcgatcg ccgccgtcaa cggccccacc gccaccgtgg tttcgggcga ccccacccag 2220 atccaagagc tcgctcaggc gtgtgaggcc gacggggtcc gcgcacggat catccccgtc 2280 gactacgcct cccacagcgc ccacgtcgag accatcgaga gcgaactcgc cgaggtcctc 2340 gccgggctca gcccgcggac acctgaggtg ccgttcttct cgacactcga aggcgcctgg 2400 atcaccgagc cggtgctcga cggcacctac tggtaccgca acctccgcca ccgcgtcggc 2460 ttcgcccccg ccgtcgagac cctcgccacc gacgaaggct tcacccactt catcgaggtc 2520 agcgcccacc ccgtcctcac catgaccctc cccgagaccg tcaccggcct cggcaccctc 2580 cgccgcgaac agggaggcca ggagcgtctg gtcacctcac tcgccgaagc ctggaccaac 2640 ggcctcacca tcgactgggc gcccgtcctc cccaccgcaa ccggccacca ccccgagctc 2700 cccacctacg ccttccagcg ccgtcactac tggctccacg actcccccgc cgtccagggc 2760 tccgtgcagg actcctggcg ctaccgcatc gactggaagc gcctcgcggt cgccgacgcg 2820 tccgagcgcg ccgggctgtc cgggcgctgg ctcgtcgtcg tccccgagga ccgttccgcc 2880 gaggccgccc cggtgctcgc cgcgctgtcc ggcgccggcg ccgaccccgt acagctggac 2940 gtgtccccgc tgggcgaccg gcagcggctc gccgcgacgc tgggcgaggc cctggcggcg 3000 gccggtggag ccgtcgacgg cgtcctctcg ctgctcgcgt gggacgagag cgcgcacccc 3060 ggccaccccg cccccttcac ccggggcacc ggcgccaccc tcaccctggt gcaggcgctg 3120 gaggacgccg gcgtcgccgc cccgctgtgg tgcgtgaccc acggcgcggt gtccgtcggc 3180 cgggccgacc acgtcacctc ccccgcccag gccatggtgt ggggcatggg ccgggtcgcc 3240 gccctggagc accccgagcg gtggggcggc ctgatcgacc tgccctcgga cgccgaccgg 3300 gcggccctgg accgcatgac cacggtcctc gccggcggta cgggtgagga ccaggtcgcg 3360 gtacgcgcct ccgggctgct cgcccgccgc ctcgtccgcg cctccctccc ggcgcacggc 3420 acggcttcgc cgtggtggca ggccgacggc acggtgctcg tcaccggtgc cgaggagcct 3480 gcggccgccg aggccgcacg ccggctggcc cgcgacggcg ccggacacct cctcctccac 3540 accaccccct ccggcagcga aggcgccgaa ggcacctccg gtgccgccga ggactccggc 3600 ctcgccgggc tcgtcgccga actcgcggac ctgggcgcga cggccaccgt cgtgacctgc 3660 gacctcacgg acgcggaggc ggccgcccgg ctgctcgccg gcgtctccga cgcgcacccg 3720 ctcagcgccg tcctccacct gccgcccacc gtcgactccg agccgctcgc cgcgaccgac 3780 gcggacgcgc tcgcccgtgt cgtgaccgcg aaggccaccg ccgcgctcca cctggaccgc 3840 ctcctgcggg aggccgcggc tgccggaggc cgtccgcccg tcctggtcct cttctcctcg 3900 gtcgccgcga tctggggcgg cgccggtcag ggcgcgtacg ccgccggtac ggccttcctc 3960 gacgccctcg ccggtcagca ccgggccgac ggccccaccg tgacctcggt ggcctggagc 4020 ccctgggagg gcagccgcgt caccgagggt gcgaccgggg agcggctgcg ccgcctcggc 4080 ctgcgccccc tcgcccccgc gacggcgctc accgccctgg acaccgcgct cggccacggc 4140 gacaccgccg tcacgatcgc cgacgtcgac tggtcgagct tcgcccccgg cttcaccacg 4200 gcccggccgg gcaccctcct cgccgatctg cccgaggcgc gccgcgcgct cgacgagcag 4260 cagtcgacga cggccgccga cgacaccgtc ctgagccgcg agctcggtgc gctcaccggc 4320 gccgaacagc agcgccgtat gcaggagttg gtccgcgagc acctcgccgt ggtcctcaac 4380 cacccctccc ccgaggccgt cgacacgggg cgggccttcc gtgacctcgg attcgactcg 4440 ctgacggcgg tcgagctccg caaccgcctc aagaacgcca ccggcctggc cctcccggcc 4500 actctggtct tcgactaccc gaccccccgg acgctggcgg agttcctcct cgcggagatc 4560 ctgggcgagc aggccggtgc cggcgagcag cttccggtgg acggcggggt cgacgacgag 4620 cccgtcgcga tcgtcggcat ggcgtgccgc ctgccgggcg gtgtcgcctc gccggaggac 4680 ctgtggcggc tggtggccgg cggcgaggac gcgatctccg gcttcccgca ggaccgcggc 4740 tgggacgtgg aggggctgta cgacccggac ccggacgcgt ccgggcggac gtactgccgt 4800 gccggtggct tcctcgacga ggcgggcgag ttcgacgccg acttcttcgg gatctcgccg 4860 cgcgaggccc tcgccatgga cccgcagcag cggctcctcc tggagacctc ctgggaggcc 4920 gtcgaggacg ccgggatcga cccgacctcc cttcaggggc agcaggtcgg cgtgttcgcg 4980 ggcaccaacg gcccccacta cgagccgctg ctccgcaaca ccgccgagga tcttgagggt 5040 tacgtcggga cgggcaacgc cgccagcatc atgtcgggcc gtgtctcgta caccctcggc 5100 ctggagggcc cggccgtcac ggtcgacacc gcctgctcct cctcgctggt cgccctgcac 5160 ctcgccgtgc aggccctgcg caagggcgaa tgcggactgg cgctcgcggg cggtgtgacg 5220 gtcatgtcga cgcccacgac gttcgtggag ttcagccggc agcgcgggct cgcggaggac 5280 ggccggtcga aggcgttcgc cgcgtcggcg gacggcttcg gcccggcgga gggcgtcggc 5340 atgctcctcg tcgagcgcct gtcggacgcc cgccgcaacg gacaccgtgt gctggcggtc 5400 gtgcgcggca gcgcggtcaa ccaggacggc gcgagcaacg gcctgaccgc cccgaacggg 5460 ccctcgcagc agcgcgtcat ccggcgcgcg ctcgcggacg cccgactgac gaccgccgac 5520 gtggacgtcg tcgaggccca cggcacgggc acgcgactcg gcgacccgat cgaggcacag 5580 gccctcatcg ccacctacgg ccaggggcgc gacaccgaac agccgctgcg cctggggtcg 5640 ttgaagtcca acatcggaca cacccaggcc gccgccggtg tctccggcat catcaagatg 5700 gtccaggcga tgcgccacgg cgtcctgccg aagacgctcc acgtggaccg gccgtcggac 5760 cagatcgact ggtcggcggg cacggtcgag ctgctcaccg aggccatgga ctggccgagg 5820 aagcaggagg gcgggctgcg ccgcgcggcc gtctcctcct tcggcatcag cggcacgaac 5880 gcgcacatcg tgctcgaaga agccccggtc gacgaggacg ccccggcgga cgagccgtcg 5940 gtcggcggtg tggtgccgtg gctcgtgtcc gcgaagactc cggccgcgct ggacgcccag 6000 atcggacgcc tcgccgcgtt cgcctcgcag ggccgtacgg acgccgccga tccgggcgcg 6060 gtcgctcgcg tactggccgg cgggcgtgcg cagttcgagc accgggccgt cgcgctcggc 6120 accggacagg acgacctggc ggccgcactg gccgcgcctg agggtctggt ccggggtgtg 6180 gcctccggtg tgggtcgagt ggcgttcgtg ttcccgggac agggcacgca gtgggccggg 6240 atgggtgccg aactcctcga cgtgtcgaag gagttcgcgg cggccatggc cgagtgcgag 6300 gccgcgctcg ctccgtacgt ggactggtcg ctggaggccg tcgtccgaca ggcccccggc 6360 gcgcccacgc tggagcgggt cgatgtcgtc cagcccgtga cgttcgccgt catggtctcg 6420 ctggcgaagg tctggcagca ccacggggtg accccgcaag ccgtcgtcgg ccactcgcag 6480 ggcgagatcg ccgccgcgta cgtcgccggt gccctgagcc tggacgacgc cgctcgtgtc 6540 gtgaccctgc gcagcaagtc catcggcgcc cacctcgcgg gccagggcgg catgctgtcc 6600 ctcgcgctga gcgaggcggc cgttgtggag cgactggccg ggttcgacgg gctgtccgtc 6660 gccgccgtca acgggcctac cgccaccgtg gtttcgggcg acccgaccca gatccaagag 6720 ctcgctcagg cgtgtgaggc cgacggggtc cgcgcacgga tcatccccgt cgactacgcc 6780 tcccacagcg cccacgtcga gaccatcgag agcgaactcg ccgacgtcct ggcggggttg 6840 tccccccaga caccccaggt ccccttcttc tccaccctcg aaggcgcctg gatcaccgaa 6900 cccgccctcg acggcggcta ctggtaccgc aacctccgcc atcgtgtggg cttcgccccg 6960 gccgtcgaaa ccctggccac cgacgaaggc ttcacccact tcgtcgaggt cagcgcccac 7020 cccgtcctca ccatggcgct gcccgagacc gtcaccggac tcggcaccct ccgccgtgac 7080 aacggcggac agcaccgcct caccacctcc ctcgccgagg cctgggccaa cggcctcacc 7140 gtcgactggg cctctctcct ccccaccacg accacccacc ccgatctgcc cacctacgcc 7200 ttccagaccg agcgctactg gccgcagccc gacctctccg ccgccggtga catcacctcc 7260 gccggtctcg gggcggccga gcacccgctg ctcggcgcgg ccgtggcgct cgcggactcc 7320 gacggctgcc tgctcacggg gagcctctcc ctccgtacgc acccctggct ggcggaccac 7380 gcggtggccg gcaccgtgct gctgccggga acggcgttcg tggagctggc gttccgagcc 7440 ggggaccagg tcggttgcga tctggtcgag gagctcaccc tcgacgcgcc gctcgtgctg 7500 ccccgtcgtg gcgcggtccg tgtgcagctg tccgtcggcg cgagcgacga gtccgggcgt 7560 cgtaccttcg ggctctacgc gcacccggag gacgcgccgg gcgaggcgga gtggacgcgg 7620 cacgccaccg gtgtgctggc cgcccgtgcg gaccgcaccg cccccgtcgc cgacccggag 7680 gcctggccgc cgccgggcgc cgagccggtg gacgtggacg gtctgtacga gcgcttcgcg 7740 gcgaacggct acggctacgg ccccctcttc cagggcgtcc gtggtgtctg gcggcgtggc 7800 gacgaggtgt tcgccgacgt ggccctgccg gccgaggtcg ccggtgccga gggcgcgcgg 7860 ttcggccttc acccggcgct gctcgacgcc gccgtgcagg cggccggtgc gggccggggc 7920 gttcggcgcg ggcacgcggc tgccgttcgc ctggagcggg atctcctgta cgcggtcggc 7980 gccaccgccc tccgcgtgcg gctggccccc gccggcccgg acacggtgtc cgtgagcgcc 8040 gccgactcct ccgggcagcc ggtgttcgcc gcggactccc tcacggtgct gcccgtcgac 8100 cccgcgcagc tggcggcctt cagcgacccg actctggacg cgctgcacct gctggagtgg 8160 accgcctggg acggtgccgc gcaggccctg cccggcgcgg tcgtgctggg cggcgacgcc 8220 gacggtctcg ccgcggcgct gcgcgccggt ggcaccgagg tcctgtcctt cccggacctt 8280 acggacctgg tggaggccgt cgaccggggc gagaccccgg ccccggcgac cgtcctggtg 8340 gcctgccccg ccgccggccc cgatgggccg gagcatgtcc gcgaggccct gcacgggtcg 8400 ctcgcgctga tgcaggcctg gctggccgac gagcggttca ccgatgggcg cctggtgctc 8460 gtgacccgcg acgcggtcgc cgcccgttcc ggcgacggcc tgcggtccac gggacaggcc 8520 gccgtctggg gcctcggccg gtccgcgcag acggagagcc cgggccggtt cgtcctgctc 8580 gacctcgccg gggaagcccg gacggccggg gacgccaccg ccggggacgg cctgacgacc 8640 ggggacgcca ccgtcggcgg cacctctgga gacgccgccc tcggcagcgc cctcgcgacc 8700 gccctcggct cgggcgagcc gcagctcgcc ctccgggacg gggcgctcct cgtaccccgc 8760 ctggcgcggg ccgccgcgcc cgccgcggcc gacggcctcg ccgcggccga cggcctcgcc 8820 gctctgccgc tgcccgccgc tccggccctc tggcgtctgg agcccggtac ggacggcagc 8880 ctggagagcc tcacggcggc gcccggcgac gccgagaccc tcgccccgga gccgctcggc 8940 ccgggacagg tccgcatcgc gatccgggcc accggtctca acttccgcga cgtcctgatc 9000 gccctcggca tgtaccccga tccggcgctg atgggcaccg agggagccgg cgtggtcacc 9060 gcgaccggcc ccggcgtcac gcacctcgcc cccggcgacc gggtcatggg cctgctctcc 9120 ggcgcgtacg ccccggtcgt cgtggcggac gcgcggaccg tcgcgcggat gcccgagggg 9180 tggacgttcg cccagggcgc ctccgtgccg gtggtgttcc tgacggccgt ctacgccctg 9240 cgcgacctgg cggacgtcaa gcccggcgag cgcctcctgg tccactccgc cgccggtggc 9300 gtgggcatgg ccgccgtgca gctcgcccgg cactggggcg tggaggtcca cggcacggcg 9360 agtcacggga agtgggacgc cctgcgcgcg ctcggcctgg acgacgcgca catcgcctcc 9420 tcccgcaccc tggacttcga gtccgcgttc cgtgccgctt ccggcggggc gggcatggac 9480 gtcgtactga actcgctcgc ccgcgagttc gtcgacgcct cgctgcgcct gctcgggccg 9540 ggcggccggt tcgtggagat ggggaagacc gacgtccgcg acgcggagcg ggtcgccgcc 9600 gaccaccccg gtgtcggcta ccgcgccttc gacctgggcg aggccgggcc ggagcggatc 9660 ggcgagatgc tcgccgaggt catcgccctc ttcgaggacg gggtgctccg gcacctgccc 9720 gtcacgacct gggacgtgcg ccgggcccgc gacgccttcc ggcacgtcag ccaggcccgc 9780 cacacgggca aggtcgtcct cacgatgccg tcgggcctcg acccggaggg tacggtcctg 9840 ctgaccggcg gcaccggtgc gctggggggc atcgtggccc ggcacgtggt gggcgagtgg 9900 ggcgtacgac gcctgctgct cgtgagccgg cggggcacgg acgccccggg cgccggcgag 9960 ctcgtgcacg agctggaggc cctgggagcc gacgtctcgg tggccgcgtg cgacgtcgcc 10020 gaccgcgaag ccctcaccgc cgtactcgac tcgatccccg ccgaacaccc gctcaccgcg 10080 gtcgtccaca cggcaggcgt cctctccgac ggcaccctcc cctcgatgac agcggaggat 10140 gtggaacacg tactgcgtcc caaggtcgac gccgcgttcc tcctcgacga actcacctcg 10200 acgcccggct acgacctggc agcgttcgtc atgttctcct ccgccgccgc cgtcttcggt 10260 ggcgcggggc agggcgccta cgccgccgcc aacgccaccc tcgacgccct cgcctggcgc 10320 cgccggacag ccggactccc cgccctctcc ctcggctggg gcctctgggc cgagaccagc 10380 ggcatgaccg gcggactcag cgacaccgac cgctcgcggc tggcccgttc cggggcgacg 10440 cccatggaca gcgagctgac cctgtccctc ctggacgcgg ccatgcgccg cgacgacccg 10500 gcgctcgtcc cgatcgccct ggacgtcgcc gcgctccgcg cccagcagcg cgacggcatg 10560 ctggcgccgc tgctcagcgg gctcacccgc ggatcgcggg tcggcggcgc gccggtcaac 10620 cagcgcaggg cagccgccgg aggcgcgggc gaggcggaca cggacctcgg cgggcggctc 10680 gccgcgatga caccggacga ccgggtcgcg cacctgcggg acctcgtccg tacgcacgtg 10740 gcgaccgtcc tgggacacgg caccccgagc cgggtggacc tggagcgggc cttccgcgac 10800 accggtttcg actcgctcac cgccgtcgaa ctccgcaacc gtctcaacgc cgcgaccggg 10860 ctgcggctgc cggccacgct ggtcttcgac caccccaccc cgggggagct cgccgggcac 10920 ctgctcgacg aactcgccac ggccgcgggc gggtcctggg cggaaggcac cgggtccgga 10980 gacacggcct cggcgaccga tcggcagacc acggcggccc tcgccgaact cgaccggctg 11040 gaaggcgtgc tcgcctccct cgcgcccgcc gccggcggcc gtccggagct cgccgcccgg 11100 ctcagggcgc tggccgcggc cctgggggac gacggcgacg acgccaccga cctggacgag 11160 gcgtccgacg acgacctctt ctccttcatc gacaaggagc tgggcgactc cgacttctga 11220 33 3739 PRT Streptomyces venezuelae 33 Met Ser Thr Val Asn Glu Glu Lys Tyr Leu Asp Tyr Leu Arg Arg Ala 1 5 10 15 Thr Ala Asp Leu His Glu Ala Arg Gly Arg Leu Arg Glu Leu Glu Ala 20 25 30 Lys Ala Gly Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro 35 40 45 Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Gly Gly 50 55 60 Glu Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val Glu 65 70 75 80 Gly Leu Tyr Asp Pro Asn Pro Glu Ala Thr Gly Lys Ser Tyr Ala Arg 85 90 95 Glu Ala Gly Phe Leu Tyr Glu Ala Gly Glu Phe Asp Ala Asp Phe Phe 100 105 110 Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu 115 120 125 Leu Leu Glu Ala Ser Trp Glu Ala Phe Glu His Ala Gly Ile Pro Ala 130 135 140 Ala Thr Ala Arg Gly Thr Ser Val Gly Val Phe Thr Gly Val Met Tyr 145 150 155 160 His Asp Tyr Ala Thr Arg Leu Thr Asp Val Pro Glu Gly Ile Glu Gly 165 170 175 Tyr Leu Gly Thr Gly Asn Ser Gly Ser Val Ala Ser Gly Arg Val Ala 180 185 190 Tyr Thr Leu Gly Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys 195 200 205 Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg Lys 210 215 220 Gly Glu Val Asp Met Ala Leu Ala Gly Gly Val Thr Val Met Ser Thr 225 230 235 240 Pro Ser Thr Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Pro Asp 245 250 255 Gly Arg Ser Lys Ser Phe Ser Ser Thr Ala Asp Gly Thr Ser Trp Ser 260 265 270 Glu Gly Val Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg Arg 275 280 285 Lys Gly His Arg Ile Leu Ala Val Val Arg Gly Thr Ala Val Asn Gln 290 295 300 Asp Gly Ala Ser Ser Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln 305 310 315 320 Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr Thr Ser Asp 325 330 335 Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro 340 345 350 Ile Glu Ala Gln Ala Val Ile Ala Thr Tyr Gly Gln Gly Arg Asp Gly 355 360 365 Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile Gly His Thr 370 375 380 Gln Ala Ala Ala Gly Val Ser Gly Val Ile Lys Met Val Gln Ala Met 385 390 395 400 Arg His Gly Val Leu Pro Lys Thr Leu His Val Glu Lys Pro Thr Asp 405 410 415 Gln Val Asp Trp Ser Ala Gly Ala Val Glu Leu Leu Thr Glu Ala Met 420 425 430 Asp Trp Pro Asp Lys Gly Asp Gly Gly Leu Arg Arg Ala Ala Val Ser 435 440 445 Ser Phe Gly Val Ser Gly Thr Asn Ala His Val Val Leu Glu Glu Ala 450 455 460 Pro Ala Ala Glu Glu Thr Pro Ala Ser Glu Ala Thr Pro Ala Val Glu 465 470 475 480 Pro Ser Val Gly Ala Gly Leu Val Pro Trp Leu Val Ser Ala Lys Thr 485 490 495 Pro Ala Ala Leu Asp Ala Gln Ile Gly Arg Leu Ala Ala Phe Ala Ser 500 505 510 Gln Gly Arg Thr Asp Ala Ala Asp Pro Gly Ala Val Ala Arg Val Leu 515 520 525 Ala Gly Gly Arg Ala Glu Phe Glu His Arg Ala Val Val Leu Gly Thr 530 535 540 Gly Gln Asp Asp Phe Ala Gln Ala Leu Thr Ala Pro Glu Gly Leu Ile 545 550 555 560 Arg Gly Thr Pro Ser Asp Val Gly Arg Val Ala Phe Val Phe Pro Gly 565 570 575 Gln Gly Thr Gln Trp Ala Gly Met Gly Ala Glu Leu Leu Asp Val Ser 580 585 590 Lys Glu Phe Ala Ala Ala Met Ala Glu Cys Glu Ser Ala Leu Ser Arg 595 600 605 Tyr Val Asp Trp Ser Leu Glu Ala Val Val Arg Gln Ala Pro Gly Ala 610 615 620 Pro Thr Leu Glu Arg Val Asp Val Val Gln Pro Val Thr Phe Ala Val 625 630 635 640 Met Val Ser Leu Ala Lys Val Trp Gln His His Gly Val Thr Pro Gln 645 650 655 Ala Val Val Gly His Ser Gln Gly Glu Ile Ala Ala Ala Tyr Val Ala 660 665 670 Gly Ala Leu Thr Leu Asp Asp Ala Ala Arg Val Val Thr Leu Arg Ser 675 680 685 Lys Ser Ile Ala Ala His Leu Ala Gly Lys Gly Gly Met Ile Ser Leu 690 695 700 Ala Leu Ser Glu Glu Ala Thr Arg Gln Arg Ile Glu Asn Leu His Gly 705 710 715 720 Leu Ser Ile Ala Ala Val Asn Gly Pro Thr Ala Thr Val Val Ser Gly 725 730 735 Asp Pro Thr Gln Ile Gln Glu Leu Ala Gln Ala Cys Glu Ala Asp Gly 740 745 750 Val Arg Ala Arg Ile Ile Pro Val Asp Tyr Ala Ser His Ser Ala His 755 760 765 Val Glu Thr Ile Glu Ser Glu Leu Ala Glu Val Leu Ala Gly Leu Ser 770 775 780 Pro Arg Thr Pro Glu Val Pro Phe Phe Ser Thr Leu Glu Gly Ala Trp 785 790 795 800 Ile Thr Glu Pro Val Leu Asp Gly Thr Tyr Trp Tyr Arg Asn Leu Arg 805 810 815 His Arg Val Gly Phe Ala Pro Ala Val Glu Thr Leu Ala Thr Asp Glu 820 825 830 Gly Phe Thr His Phe Ile Glu Val Ser Ala His Pro Val Leu Thr Met 835 840 845 Thr Leu Pro Glu Thr Val Thr Gly Leu Gly Thr Leu Arg Arg Glu Gln 850 855 860 Gly Gly Gln Glu Arg Leu Val Thr Ser Leu Ala Glu Ala Trp Thr Asn 865 870 875 880 Gly Leu Thr Ile Asp Trp Ala Pro Val Leu Pro Thr Ala Thr Gly His 885 890 895 His Pro Glu Leu Pro Thr Tyr Ala Phe Gln Arg Arg His Tyr Trp Leu 900 905 910 His Asp Ser Pro Ala Val Gln Gly Ser Val Gln Asp Ser Trp Arg Tyr 915 920 925 Arg Ile Asp Trp Lys Arg Leu Ala Val Ala Asp Ala Ser Glu Arg Ala 930 935 940 Gly Leu Ser Gly Arg Trp Leu Val Val Val Pro Glu Asp Arg Ser Ala 945 950 955 960 Glu Ala Ala Pro Val Leu Ala Ala Leu Ser Gly Ala Gly Ala Asp Pro 965 970 975 Val Gln Leu Asp Val Ser Pro Leu Gly Asp Arg Gln Arg Leu Ala Ala 980 985 990 Thr Leu Gly Glu Ala Leu Ala Ala Ala Gly Gly Ala Val Asp Gly Val 995 1000 1005 Leu Ser Leu Leu Ala Trp Asp Glu Ser Ala His Pro Gly His Pro Ala 1010 1015 1020 Pro Phe Thr Arg Gly Thr Gly Ala Thr Leu Thr Leu Val Gln Ala Leu 1025 1030 1035 1040 Glu Asp Ala Gly Val Ala Ala Pro Leu Trp Cys Val Thr His Gly Ala 1045 1050 1055 Val Ser Val Gly Arg Ala Asp His Val Thr Ser Pro Ala Gln Ala Met 1060 1065 1070 Val Trp Gly Met Gly Arg Val Ala Ala Leu Glu His Pro Glu Arg Trp 1075 1080 1085 Gly Gly Leu Ile Asp Leu Pro Ser Asp Ala Asp Arg Ala Ala Leu Asp 1090 1095 1100 Arg Met Thr Thr Val Leu Ala Gly Gly Thr Gly Glu Asp Gln Val Ala 1105 1110 1115 1120 Val Arg Ala Ser Gly Leu Leu Ala Arg Arg Leu Val Arg Ala Ser Leu 1125 1130 1135 Pro Ala His Gly Thr Ala Ser Pro Trp Trp Gln Ala Asp Gly Thr Val 1140 1145 1150 Leu Val Thr Gly Ala Glu Glu Pro Ala Ala Ala Glu Ala Ala Arg Arg 1155 1160 1165 Leu Ala Arg Asp Gly Ala Gly His Leu Leu Leu His Thr Thr Pro Ser 1170 1175 1180 Gly Ser Glu Gly Ala Glu Gly Thr Ser Gly Ala Ala Glu Asp Ser Gly 1185 1190 1195 1200 Leu Ala Gly Leu Val Ala Glu Leu Ala Asp Leu Gly Ala Thr Ala Thr 1205 1210 1215 Val Val Thr Cys Asp Leu Thr Asp Ala Glu Ala Ala Ala Arg Leu Leu 1220 1225 1230 Ala Gly Val Ser Asp Ala His Pro Leu Ser Ala Val Leu His Leu Pro 1235 1240 1245 Pro Thr Val Asp Ser Glu Pro Leu Ala Ala Thr Asp Ala Asp Ala Leu 1250 1255 1260 Ala Arg Val Val Thr Ala Lys Ala Thr Ala Ala Leu His Leu Asp Arg 1265 1270 1275 1280 Leu Leu Arg Glu Ala Ala Ala Ala Gly Gly Arg Pro Pro Val Leu Val 1285 1290 1295 Leu Phe Ser Ser Val Ala Ala Ile Trp Gly Gly Ala Gly Gln Gly Ala 1300 1305 1310 Tyr Ala Ala Gly Thr Ala Phe Leu Asp Ala Leu Ala Gly Gln His Arg 1315 1320 1325 Ala Asp Gly Pro Thr Val Thr Ser Val Ala Trp Ser Pro Trp Glu Gly 1330 1335 1340 Ser Arg Val Thr Glu Gly Ala Thr Gly Glu Arg Leu Arg Arg Leu Gly 1345 1350 1355 1360 Leu Arg Pro Leu Ala Pro Ala Thr Ala Leu Thr Ala Leu Asp Thr Ala 1365 1370 1375 Leu Gly His Gly Asp Thr Ala Val Thr Ile Ala Asp Val Asp Trp Ser 1380 1385 1390 Ser Phe Ala Pro Gly Phe Thr Thr Ala Arg Pro Gly Thr Leu Leu Ala 1395 1400 1405 Asp Leu Pro Glu Ala Arg Arg Ala Leu Asp Glu Gln Gln Ser Thr Thr 1410 1415 1420 Ala Ala Asp Asp Thr Val Leu Ser Arg Glu Leu Gly Ala Leu Thr Gly 1425 1430 1435 1440 Ala Glu Gln Gln Arg Arg Met Gln Glu Leu Val Arg Glu His Leu Ala 1445 1450 1455 Val Val Leu Asn His Pro Ser Pro Glu Ala Val Asp Thr Gly Arg Ala 1460 1465 1470 Phe Arg Asp Leu Gly Phe Asp Ser Leu Thr Ala Val Glu Leu Arg Asn 1475 1480 1485 Arg Leu Lys Asn Ala Thr Gly Leu Ala Leu Pro Ala Thr Leu Val Phe 1490 1495 1500 Asp Tyr Pro Thr Pro Arg Thr Leu Ala Glu Phe Leu Leu Ala Glu Ile 1505 1510 1515 1520 Leu Gly Glu Gln Ala Gly Ala Gly Glu Gln Leu Pro Val Asp Gly Gly 1525 1530 1535 Val Asp Asp Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro 1540 1545 1550 Gly Gly Val Ala Ser Pro Glu Asp Leu Trp Arg Leu Val Ala Gly Gly 1555 1560 1565 Glu Asp Ala Ile Ser Gly Phe Pro Gln Asp Arg Gly Trp Asp Val Glu 1570 1575 1580 Gly Leu Tyr Asp Pro Asp Pro Asp Ala Ser Gly Arg Thr Tyr Cys Arg 1585 1590 1595 1600 Ala Gly Gly Phe Leu Asp Glu Ala Gly Glu Phe Asp Ala Asp Phe Phe 1605 1610 1615 Gly Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu 1620 1625 1630 Leu Leu Glu Thr Ser Trp Glu Ala Val Glu Asp Ala Gly Ile Asp Pro 1635 1640 1645 Thr Ser Leu Gln Gly Gln Gln Val Gly Val Phe Ala Gly Thr Asn Gly 1650 1655 1660 Pro His Tyr Glu Pro Leu Leu Arg Asn Thr Ala Glu Asp Leu Glu Gly 1665 1670 1675 1680 Tyr Val Gly Thr Gly Asn Ala Ala Ser Ile Met Ser Gly Arg Val Ser 1685 1690 1695 Tyr Thr Leu Gly Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys 1700 1705 1710 Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg Lys 1715 1720 1725 Gly Glu Cys Gly Leu Ala Leu Ala Gly Gly Val Thr Val Met Ser Thr 1730 1735 1740 Pro Thr Thr Phe Val Glu Phe Ser Arg Gln Arg Gly Leu Ala Glu Asp 1745 1750 1755 1760 Gly Arg Ser Lys Ala Phe Ala Ala Ser Ala Asp Gly Phe Gly Pro Ala 1765 1770 1775 Glu Gly Val Gly Met Leu Leu Val Glu Arg Leu Ser Asp Ala Arg Arg 1780 1785 1790 Asn Gly His Arg Val Leu Ala Val Val Arg Gly Ser Ala Val Asn Gln 1795 1800 1805 Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro Asn Gly Pro Ser Gln Gln 1810 1815 1820 Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Thr Thr Ala Asp 1825 1830 1835 1840 Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp Pro 1845 1850 1855 Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Gly Arg Asp Thr 1860 1865 1870 Glu Gln Pro Leu Arg Leu Gly Ser Leu Lys Ser Asn Ile Gly His Thr 1875 1880 1885 Gln Ala Ala Ala Gly Val Ser Gly Ile Ile Lys Met Val Gln Ala Met 1890 1895 1900 Arg His Gly Val Leu Pro Lys Thr Leu His Val Asp Arg Pro Ser Asp 1905 1910 1915 1920 Gln Ile Asp Trp Ser Ala Gly Thr Val Glu Leu Leu Thr Glu Ala Met 1925 1930 1935 Asp Trp Pro Arg Lys Gln Glu Gly Gly Leu Arg Arg Ala Ala Val Ser 1940 1945 1950 Ser Phe Gly Ile Ser Gly Thr Asn Ala His Ile Val Leu Glu Glu Ala 1955 1960 1965 Pro Val Asp Glu Asp Ala Pro Ala Asp Glu Pro Ser Val Gly Gly Val 1970 1975 1980 Val Pro Trp Leu Val Ser Ala Lys Thr Pro Ala Ala Leu Asp Ala Gln 1985 1990 1995 2000 Ile Gly Arg Leu Ala Ala Phe Ala Ser Gln Gly Arg Thr Asp Ala Ala 2005 2010 2015 Asp Pro Gly Ala Val Ala Arg Val Leu Ala Gly Gly Arg Ala Gln Phe 2020 2025 2030 Glu His Arg Ala Val Ala Leu Gly Thr Gly Gln Asp Asp Leu Ala Ala 2035 2040 2045 Ala Leu Ala Ala Pro Glu Gly Leu Val Arg Gly Val Ala Ser Gly Val 2050 2055 2060 Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly 2065 2070 2075 2080 Met Gly Ala Glu Leu Leu Asp Val Ser Lys Glu Phe Ala Ala Ala Met 2085 2090 2095 Ala Glu Cys Glu Ala Ala Leu Ala Pro Tyr Val Asp Trp Ser Leu Glu 2100 2105 2110 Ala Val Val Arg Gln Ala Pro Gly Ala Pro Thr Leu Glu Arg Val Asp 2115 2120 2125 Val Val Gln Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val 2130 2135 2140 Trp Gln His His Gly Val Thr Pro Gln Ala Val Val Gly His Ser Gln 2145 2150 2155 2160 Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala Leu Ser Leu Asp Asp 2165 2170 2175 Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser Ile Gly Ala His Leu 2180 2185 2190 Ala Gly Gln Gly Gly Met Leu Ser Leu Ala Leu Ser Glu Ala Ala Val 2195 2200 2205 Val Glu Arg Leu Ala Gly Phe Asp Gly Leu Ser Val Ala Ala Val Asn 2210 2215 2220 Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Gln Glu 2225 2230 2235 2240 Leu Ala Gln Ala Cys Glu Ala Asp Gly Val Arg Ala Arg Ile Ile Pro 2245 2250 2255 Val Asp Tyr Ala Ser His Ser Ala His Val Glu Thr Ile Glu Ser Glu 2260 2265 2270 Leu Ala Asp Val Leu Ala Gly Leu Ser Pro Gln Thr Pro Gln Val Pro 2275 2280 2285 Phe Phe Ser Thr Leu Glu Gly Ala Trp Ile Thr Glu Pro Ala Leu Asp 2290 2295 2300 Gly Gly Tyr Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro 2305 2310 2315 2320 Ala Val Glu Thr Leu Ala Thr Asp Glu Gly Phe Thr His Phe Val Glu 2325 2330 2335 Val Ser Ala His Pro Val Leu Thr Met Ala Leu Pro Glu Thr Val Thr 2340 2345 2350 Gly Leu Gly Thr Leu Arg Arg Asp Asn Gly Gly Gln His Arg Leu Thr 2355 2360 2365 Thr Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu Thr Val Asp Trp Ala 2370 2375 2380 Ser Leu Leu Pro Thr Thr Thr Thr His Pro Asp Leu Pro Thr Tyr Ala 2385 2390 2395 2400 Phe Gln Thr Glu Arg Tyr Trp Pro Gln Pro Asp Leu Ser Ala Ala Gly 2405 2410 2415 Asp Ile Thr Ser Ala Gly Leu Gly Ala Ala Glu His Pro Leu Leu Gly 2420 2425 2430 Ala Ala Val Ala Leu Ala Asp Ser Asp Gly Cys Leu Leu Thr Gly Ser 2435 2440 2445 Leu Ser Leu Arg Thr His Pro Trp Leu Ala Asp His Ala Val Ala Gly 2450 2455 2460 Thr Val Leu Leu Pro Gly Thr Ala Phe Val Glu Leu Ala Phe Arg Ala 2465 2470 2475 2480 Gly Asp Gln Val Gly Cys Asp Leu Val Glu Glu Leu Thr Leu Asp Ala 2485 2490 2495 Pro Leu Val Leu Pro Arg Arg Gly Ala Val Arg Val Gln Leu Ser Val 2500 2505 2510 Gly Ala Ser Asp Glu Ser Gly Arg Arg Thr Phe Gly Leu Tyr Ala His 2515 2520 2525 Pro Glu Asp Ala Pro Gly Glu Ala Glu Trp Thr Arg His Ala Thr Gly 2530 2535 2540 Val Leu Ala Ala Arg Ala Asp Arg Thr Ala Pro Val Ala Asp Pro Glu 2545 2550 2555 2560 Ala Trp Pro Pro Pro Gly Ala Glu Pro Val Asp Val Asp Gly Leu Tyr 2565 2570 2575 Glu Arg Phe Ala Ala Asn Gly Tyr Gly Tyr Gly Pro Leu Phe Gln Gly 2580 2585 2590 Val Arg Gly Val Trp Arg Arg Gly Asp Glu Val Phe Ala Asp Val Ala 2595 2600 2605 Leu Pro Ala Glu Val Ala Gly Ala Glu Gly Ala Arg Phe Gly Leu His 2610 2615 2620 Pro Ala Leu Leu Asp Ala Ala Val Gln Ala Ala Gly Ala Gly Arg Gly 2625 2630 2635 2640 Val Arg Arg Gly His Ala Ala Ala Val Arg Leu Glu Arg Asp Leu Leu 2645 2650 2655 Tyr Ala Val Gly Ala Thr Ala Leu Arg Val Arg Leu Ala Pro Ala Gly 2660 2665 2670 Pro Asp Thr Val Ser Val Ser Ala Ala Asp Ser Ser Gly Gln Pro Val 2675 2680 2685 Phe Ala Ala Asp Ser Leu Thr Val Leu Pro Val Asp Pro Ala Gln Leu 2690 2695 2700 Ala Ala Phe Ser Asp Pro Thr Leu Asp Ala Leu His Leu Leu Glu Trp 2705 2710 2715 2720 Thr Ala Trp Asp Gly Ala Ala Gln Ala Leu Pro Gly Ala Val Val Leu 2725 2730 2735 Gly Gly Asp Ala Asp Gly Leu Ala Ala Ala Leu Arg Ala Gly Gly Thr 2740 2745 2750 Glu Val Leu Ser Phe Pro Asp Leu Thr Asp Leu Val Glu Ala Val Asp 2755 2760 2765 Arg Gly Glu Thr Pro Ala Pro Ala Thr Val Leu Val Ala Cys Pro Ala 2770 2775 2780 Ala Gly Pro Asp Gly Pro Glu His Val Arg Glu Ala Leu His Gly Ser 2785 2790 2795 2800 Leu Ala Leu Met Gln Ala Trp Leu Ala Asp Glu Arg Phe Thr Asp Gly 2805 2810 2815 Arg Leu Val Leu Val Thr Arg Asp Ala Val Ala Ala Arg Ser Gly Asp 2820 2825 2830 Gly Leu Arg Ser Thr Gly Gln Ala Ala Val Trp Gly Leu Gly Arg Ser 2835 2840 2845 Ala Gln Thr Glu Ser Pro Gly Arg Phe Val Leu Leu Asp Leu Ala Gly 2850 2855 2860 Glu Ala Arg Thr Ala Gly Asp Ala Thr Ala Gly Asp Gly Leu Thr Thr 2865 2870 2875 2880 Gly Asp Ala Thr Val Gly Gly Thr Ser Gly Asp Ala Ala Leu Gly Ser 2885 2890 2895 Ala Leu Ala Thr Ala Leu Gly Ser Gly Glu Pro Gln Leu Ala Leu Arg 2900 2905 2910 Asp Gly Ala Leu Leu Val Pro Arg Leu Ala Arg Ala Ala Ala Pro Ala 2915 2920 2925 Ala Ala Asp Gly Leu Ala Ala Ala Asp Gly Leu Ala Ala Leu Pro Leu 2930 2935 2940 Pro Ala Ala Pro Ala Leu Trp Arg Leu Glu Pro Gly Thr Asp Gly Ser 2945 2950 2955 2960 Leu Glu Ser Leu Thr Ala Ala Pro Gly Asp Ala Glu Thr Leu Ala Pro 2965 2970 2975 Glu Pro Leu Gly Pro Gly Gln Val Arg Ile Ala Ile Arg Ala Thr Gly 2980 2985 2990 Leu Asn Phe Arg Asp Val Leu Ile Ala Leu Gly Met Tyr Pro Asp Pro 2995 3000 3005 Ala Leu Met Gly Thr Glu Gly Ala Gly Val Val Thr Ala Thr Gly Pro 3010 3015 3020 Gly Val Thr His Leu Ala Pro Gly Asp Arg Val Met Gly Leu Leu Ser 3025 3030 3035 3040 Gly Ala Tyr Ala Pro Val Val Val Ala Asp Ala Arg Thr Val Ala Arg 3045 3050 3055 Met Pro Glu Gly Trp Thr Phe Ala Gln Gly Ala Ser Val Pro Val Val 3060 3065 3070 Phe Leu Thr Ala Val Tyr Ala Leu Arg Asp Leu Ala Asp Val Lys Pro 3075 3080 3085 Gly Glu Arg Leu Leu Val His Ser Ala Ala Gly Gly Val Gly Met Ala 3090 3095 3100 Ala Val Gln Leu Ala Arg His Trp Gly Val Glu Val His Gly Thr Ala 3105 3110 3115 3120 Ser His Gly Lys Trp Asp Ala Leu Arg Ala Leu Gly Leu Asp Asp Ala 3125 3130 3135 His Ile Ala Ser Ser Arg Thr Leu Asp Phe Glu Ser Ala Phe Arg Ala 3140 3145 3150 Ala Ser Gly Gly Ala Gly Met Asp Val Val Leu Asn Ser Leu Ala Arg 3155 3160 3165 Glu Phe Val Asp Ala Ser Leu Arg Leu Leu Gly Pro Gly Gly Arg Phe 3170 3175 3180 Val Glu Met Gly Lys Thr Asp Val Arg Asp Ala Glu Arg Val Ala Ala 3185 3190 3195 3200 Asp His Pro Gly Val Gly Tyr Arg Ala Phe Asp Leu Gly Glu Ala Gly 3205 3210 3215 Pro Glu Arg Ile Gly Glu Met Leu Ala Glu Val Ile Ala Leu Phe Glu 3220 3225 3230 Asp Gly Val Leu Arg His Leu Pro Val Thr Thr Trp Asp Val Arg Arg 3235 3240 3245 Ala Arg Asp Ala Phe Arg His Val Ser Gln Ala Arg His Thr Gly Lys 3250 3255 3260 Val Val Leu Thr Met Pro Ser Gly Leu Asp Pro Glu Gly Thr Val Leu 3265 3270 3275 3280 Leu Thr Gly Gly Thr Gly Ala Leu Gly Gly Ile Val Ala Arg His Val 3285 3290 3295 Val Gly Glu Trp Gly Val Arg Arg Leu Leu Leu Val Ser Arg Arg Gly 3300 3305 3310 Thr Asp Ala Pro Gly Ala Gly Glu Leu Val His Glu Leu Glu Ala Leu 3315 3320 3325 Gly Ala Asp Val Ser Val Ala Ala Cys Asp Val Ala Asp Arg Glu Ala 3330 3335 3340 Leu Thr Ala Val Leu Asp Ser Ile Pro Ala Glu His Pro Leu Thr Ala 3345 3350 3355 3360 Val Val His Thr Ala Gly Val Leu Ser Asp Gly Thr Leu Pro Ser Met 3365 3370 3375 Thr Ala Glu Asp Val Glu His Val Leu Arg Pro Lys Val Asp Ala Ala 3380 3385 3390 Phe Leu Leu Asp Glu Leu Thr Ser Thr Pro Gly Tyr Asp Leu Ala Ala 3395 3400 3405 Phe Val Met Phe Ser Ser Ala Ala Ala Val Phe Gly Gly Ala Gly Gln 3410 3415 3420 Gly Ala Tyr Ala Ala Ala Asn Ala Thr Leu Asp Ala Leu Ala Trp Arg 3425 3430 3435 3440 Arg Arg Thr Ala Gly Leu Pro Ala Leu Ser Leu Gly Trp Gly Leu Trp 3445 3450 3455 Ala Glu Thr Ser Gly Met Thr Gly Gly Leu Ser Asp Thr Asp Arg Ser 3460 3465 3470 Arg Leu Ala Arg Ser Gly Ala Thr Pro Met Asp Ser Glu Leu Thr Leu 3475 3480 3485 Ser Leu Leu Asp Ala Ala Met Arg Arg Asp Asp Pro Ala Leu Val Pro 3490 3495 3500 Ile Ala Leu Asp Val Ala Ala Leu Arg Ala Gln Gln Arg Asp Gly Met 3505 3510 3515 3520 Leu Ala Pro Leu Leu Ser Gly Leu Thr Arg Gly Ser Arg Val Gly Gly 3525 3530 3535 Ala Pro Val Asn Gln Arg Arg Ala Ala Ala Gly Gly Ala Gly Glu Ala 3540 3545 3550 Asp Thr Asp Leu Gly Gly Arg Leu Ala Ala Met Thr Pro Asp Asp Arg 3555 3560 3565 Val Ala His Leu Arg Asp Leu Val Arg Thr His Val Ala Thr Val Leu 3570 3575 3580 Gly His Gly Thr Pro Ser Arg Val Asp Leu Glu Arg Ala Phe Arg Asp 3585 3590 3595 3600 Thr Gly Phe Asp Ser Leu Thr Ala Val Glu Leu Arg Asn Arg Leu Asn 3605 3610 3615 Ala Ala Thr Gly Leu Arg Leu Pro Ala Thr Leu Val Phe Asp His Pro 3620 3625 3630 Thr Pro Gly Glu Leu Ala Gly His Leu Leu Asp Glu Leu Ala Thr Ala 3635 3640 3645 Ala Gly Gly Ser Trp Ala Glu Gly Thr Gly Ser Gly Asp Thr Ala Ser 3650 3655 3660 Ala Thr Asp Arg Gln Thr Thr Ala Ala Leu Ala Glu Leu Asp Arg Leu 3665 3670 3675 3680 Glu Gly Val Leu Ala Ser Leu Ala Pro Ala Ala Gly Gly Arg Pro Glu 3685 3690 3695 Leu Ala Ala Arg Leu Arg Ala Leu Ala Ala Ala Leu Gly Asp Asp Gly 3700 3705 3710 Asp Asp Ala Thr Asp Leu Asp Glu Ala Ser Asp Asp Asp Leu Phe Ser 3715 3720 3725 Phe Ile Asp Lys Glu Leu Gly Asp Ser Asp Phe 3730 3735 34 4689 DNA Streptomyces venezuelae 34 atggcgaaca acgaagacaa gctccgcgac tacctcaagc gcgtcaccgc cgagctgcag 60 cagaacacca ggcgtctgcg cgagatcgag ggacgcacgc acgagccggt ggcgatcgtg 120 ggcatggcct gccgcctgcc gggcggtgtc gcctcgcccg aggacctgtg gcagctggtg 180 gccggggacg gggacgcgat ctcggagttc ccgcaggacc gcggctggga cgtggagggg 240 ctgtacgacc ccgacccgga cgcgtccggc aggacgtact gccggtccgg cggattcctg 300 cacgacgccg gcgagttcga cgccgacttc ttcgggatct cgccgcgcga ggccctcgcc 360 atggacccgc agcagcgact gtccctcacc accgcgtggg aggcgatcga gagcgcgggc 420 atcgacccga cggccctgaa gggcagcggc ctcggcgtct tcgtcggcgg ctggcacacc 480 ggctacacct cggggcagac caccgccgtg cagtcgcccg agctggaggg ccacctggtc 540 agcggcgcgg cgctgggctt cctgtccggc cgtatcgcgt acgtcctcgg tacggacgga 600 ccggccctga ccgtggacac ggcctgctcg tcctcgctgg tcgccctgca cctcgccgtg 660 caggccctcc gcaagggcga gtgcgacatg gccctcgccg gtggtgtcac ggtcatgccc 720 aacgcggacc tgttcgtgca gttcagccgg cagcgcgggc tggccgcgga cggccggtcg 780 aaggcgttcg ccacctcggc ggacggcttc ggccccgcgg agggcgccgg agtcctgctg 840 gtggagcgcc tgtcggacgc ccgccgcaac ggacaccgga tcctcgcggt cgtccgcggc 900 agcgcggtca accaggacgg cgccagcaac ggcctcacgg ctccgcacgg gccctcccag 960 cagcgcgtca tccgacgggc cctggcggac gcccggctcg cgccgggtga cgtggacgtc 1020 gtcgaggcgc acggcacggg cacgcggctc ggcgacccga tcgaggcgca ggccctcatc 1080 gccacctacg gccaggagaa gagcagcgaa cagccgctga ggctgggcgc gttgaagtcg 1140 aacatcgggc acacgcaggc cgcggccggt gtcgcaggtg tcatcaagat ggtccaggcg 1200 atgcgccacg gactgctgcc gaagacgctg cacgtcgacg agccctcgga ccagatcgac 1260 tggtcggcgg gcacggtgga actcctcacc gaggccgtcg actggccgga gaagcaggac 1320 ggcgggctgc gccgcgcggc tgtctcctcc ttcggcatca gcgggacgaa cgcgcacgtc 1380 gtcctggagg aggccccggc ggtcgaggac tccccggccg tcgagccgcc ggccggtggc 1440 ggtgtggtgc cgtggccggt gtccgcgaag actccggccg cgctggacgc ccagatcggg 1500 cagctcgccg cgtacgcgga cggtcgtacg gacgtggatc cggcggtggc cgcccgcgcc 1560 ctggtcgaca gccgtacggc gatggagcac cgcgcggtcg cggtcggcga cagccgggag 1620 gcactgcggg acgccctgcg gatgccggaa ggactggtac gcggcacgtc ctcggacgtg 1680 ggccgggtgg cgttcgtctt ccccggccag ggcacgcagt gggccggcat gggcgccgaa 1740 ctccttgaca gctcaccgga gttcgctgcc tcgatggccg aatgcgagac cgcgctctcc 1800 cgctacgtcg actggtctct tgaagccgtc gtccgacagg aacccggcgc acccacgctc 1860 gaccgcgtcg acgtcgtcca gcccgtgacc ttcgctgtca tggtctcgct ggcgaaggtc 1920 tggcagcacc acggcatcac cccccaggcc gtcgtcggcc actcgcaggg cgagatcgcc 1980 gccgcgtacg tcgccggtgc actcaccctc gacgacgccg cccgcgtcgt caccctgcgc 2040 agcaagtcca tcgccgccca cctcgccggc aagggcggca tgatctccct cgccctcgac 2100 gaggcggccg tcctgaagcg actgagcgac ttcgacggac tctccgtcgc cgccgtcaac 2160 ggccccaccg ccaccgtcgt ctccggcgac ccgacccaga tcgaggaact cgcccgcacc 2220 tgcgaggccg acggcgtccg tgcgcggatc atcccggtcg actacgcctc ccacagccgg 2280 caggtcgaga tcatcgagaa ggagctggcc gaggtcctcg ccggactcgc cccgcaggct 2340 ccgcacgtgc cgttcttctc caccctcgaa ggcacctgga tcaccgagcc ggtgctcgac 2400 ggcacctact ggtaccgcaa cctgcgccat cgcgtgggct tcgcccccgc cgtggagacc 2460 ttggcggttg acggcttcac ccacttcatc gaggtcagcg cccaccccgt cctcaccatg 2520 accctccccg agaccgtcac cggcctcggc accctccgcc gcgaacaggg aggccaggag 2580 cgtctggtca cctcactcgc cgaagcctgg gccaacggcc tcaccatcga ctgggcgccc 2640 atcctcccca ccgcaaccgg ccaccacccc gagctcccca cctacgcctt ccagaccgag 2700 cgcttctggc tgcagagctc cgcgcccacc agcgccgccg acgactggcg ttaccgcgtc 2760 gagtggaagc cgctgacggc ctccggccag gcggacctgt ccgggcggtg gatcgtcgcc 2820 gtcgggagcg agccagaagc cgagctgctg ggcgcgctga aggccgcggg agcggaggtc 2880 gacgtactgg aagccggggc ggacgacgac cgtgaggccc tcgccgcccg gctcaccgca 2940 ctgacgaccg gcgacggctt caccggcgtg gtctcgctcc tcgacgacct cgtgccacag 3000 gtcgcctggg tgcaggcact cggcgacgcc ggaatcaagg cgcccctgtg gtccgtcacc 3060 cagggcgcgg tctccgtcgg acgtctcgac acccccgccg accccgaccg ggccatgctc 3120 tggggcctcg gccgcgtcgt cgcccttgag caccccgaac gctgggccgg cctcgtcgac 3180 ctccccgccc agcccgatgc cgccgccctc gcccacctcg tcaccgcact ctccggcgcc 3240 accggcgagg accagatcgc catccgcacc accggactcc acgcccgccg cctcgcccgc 3300 gcacccctcc acggacgtcg gcccacccgc gactggcagc cccacggcac cgtcctcatc 3360 accggcggca ccggagccct cggcagccac gccgcacgct ggatggccca ccacggagcc 3420 gaacacctcc tcctcgtcag ccgcagcggc gaacaagccc ccggagccac ccaactcacc 3480 gccgaactca ccgcatcggg cgcccgcgtc accatcgccg cctgcgacgt cgccgacccc 3540 cacgccatgc gcaccctcct cgacgccatc cccgccgaga cgcccctcac cgccgtcgtc 3600 cacaccgccg gcgcaccggg cggcgatccg ctggacgtca ccggcccgga ggacatcgcc 3660 cgcatcctgg gcgcgaagac gagcggcgcc gaggtcctcg acgacctgct ccgcggcact 3720 ccgctggacg ccttcgtcct ctactcctcg aacgccgggg tctggggcag cggcagccag 3780 ggcgtctacg cggcggccaa cgcccacctc gacgcgctcg ccgcccggcg ccgcgcccgg 3840 ggcgagacgg cgacctcggt cgcctggggc ctctgggccg gcgacggcat gggccggggc 3900 gccgacgacg cgtactggca gcgtcgcggc atccgtccga tgagccccga ccgcgccctg 3960 gacgaactgg ccaaggccct gagccacgac gagaccttcg tcgccgtggc cgatgtcgac 4020 tgggagcggt tcgcgcccgc gttcacggtg tcccgtccca gccttctgct cgacggcgtc 4080 ccggaggccc ggcaggcgct cgccgcaccc gtcggtgccc cggctcccgg cgacgccgcc 4140 gtggcgccga ccgggcagtc gtcggcgctg gccgcgatca ccgcgctccc cgagcccgag 4200 cgccggccgg cgctcctcac cctcgtccgt acccacgcgg cggccgtact cggccattcc 4260 tcccccgacc gggtggcccc cggccgtgcc ttcaccgagc tcggcttcga ctcgctgacg 4320 gccgtgcagc tccgcaacca gctctccacg gtggtcggca acaggctccc cgccaccacg 4380 gtcttcgacc acccgacgcc cgccgcactc gccgcgcacc tccacgaggc gtacctcgca 4440 ccggccgagc cggccccgac ggactgggag gggcgggtgc gccgggccct ggccgaactg 4500 cccctcgacc ggctgcggga cgcgggggtc ctcgacaccg tcctgcgcct caccggcatc 4560 gagcccgagc cgggttccgg cggttcggac ggcggcgccg ccgaccctgg tgcggagccg 4620 gaggcgtcga tcgacgacct ggacgccgag gccctgatcc ggatggctct cggcccccgt 4680 aacacctga 4689 35 1562 PRT Streptomyces venezuelae 35 Met Ala Asn Asn Glu Asp Lys Leu Arg Asp Tyr Leu Lys Arg Val Thr 1 5 10 15 Ala Glu Leu Gln Gln Asn Thr Arg Arg Leu Arg Glu Ile Glu Gly Arg 20 25 30 Thr His Glu Pro Val Ala Ile Val Gly Met Ala Cys Arg Leu Pro Gly 35 40 45 Gly Val Ala Ser Pro Glu Asp Leu Trp Gln Leu Val Ala Gly Asp Gly 50 55 60 Asp Ala Ile Ser Glu Phe Pro Gln Asp Arg Gly Trp Asp Val Glu Gly 65 70 75 80 Leu Tyr Asp Pro Asp Pro Asp Ala Ser Gly Arg Thr Tyr Cys Arg Ser 85 90 95 Gly Gly Phe Leu His Asp Ala Gly Glu Phe Asp Ala Asp Phe Phe Gly 100 105 110 Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Leu Ser 115 120 125 Leu Thr Thr Ala Trp Glu Ala Ile Glu Ser Ala Gly Ile Asp Pro Thr 130 135 140 Ala Leu Lys Gly Ser Gly Leu Gly Val Phe Val Gly Gly Trp His Thr 145 150 155 160 Gly Tyr Thr Ser Gly Gln Thr Thr Ala Val Gln Ser Pro Glu Leu Glu 165 170 175 Gly His Leu Val Ser Gly Ala Ala Leu Gly Phe Leu Ser Gly Arg Ile 180 185 190 Ala Tyr Val Leu Gly Thr Asp Gly Pro Ala Leu Thr Val Asp Thr Ala 195 200 205 Cys Ser Ser Ser Leu Val Ala Leu His Leu Ala Val Gln Ala Leu Arg 210 215 220 Lys Gly Glu Cys Asp Met Ala Leu Ala Gly Gly Val Thr Val Met Pro 225 230 235 240 Asn Ala Asp Leu Phe Val Gln Phe Ser Arg Gln Arg Gly Leu Ala Ala 245 250 255 Asp Gly Arg Ser Lys Ala Phe Ala Thr Ser Ala Asp Gly Phe Gly Pro 260 265 270 Ala Glu Gly Ala Gly Val Leu Leu Val Glu Arg Leu Ser Asp Ala Arg 275 280 285 Arg Asn Gly His Arg Ile Leu Ala Val Val Arg Gly Ser Ala Val Asn 290 295 300 Gln Asp Gly Ala Ser Asn Gly Leu Thr Ala Pro His Gly Pro Ser Gln 305 310 315 320 Gln Arg Val Ile Arg Arg Ala Leu Ala Asp Ala Arg Leu Ala Pro Gly 325 330 335 Asp Val Asp Val Val Glu Ala His Gly Thr Gly Thr Arg Leu Gly Asp 340 345 350 Pro Ile Glu Ala Gln Ala Leu Ile Ala Thr Tyr Gly Gln Glu Lys Ser 355 360 365 Ser Glu Gln Pro Leu Arg Leu Gly Ala Leu Lys Ser Asn Ile Gly His 370 375 380 Thr Gln Ala Ala Ala Gly Val Ala Gly Val Ile Lys Met Val Gln Ala 385 390 395 400 Met Arg His Gly Leu Leu Pro Lys Thr Leu His Val Asp Glu Pro Ser 405 410 415 Asp Gln Ile Asp Trp Ser Ala Gly Thr Val Glu Leu Leu Thr Glu Ala 420 425 430 Val Asp Trp Pro Glu Lys Gln Asp Gly Gly Leu Arg Arg Ala Ala Val 435 440 445 Ser Ser Phe Gly Ile Ser Gly Thr Asn Ala His Val Val Leu Glu Glu 450 455 460 Ala Pro Ala Val Glu Asp Ser Pro Ala Val Glu Pro Pro Ala Gly Gly 465 470 475 480 Gly Val Val Pro Trp Pro Val Ser Ala Lys Thr Pro Ala Ala Leu Asp 485 490 495 Ala Gln Ile Gly Gln Leu Ala Ala Tyr Ala Asp Gly Arg Thr Asp Val 500 505 510 Asp Pro Ala Val Ala Ala Arg Ala Leu Val Asp Ser Arg Thr Ala Met 515 520 525 Glu His Arg Ala Val Ala Val Gly Asp Ser Arg Glu Ala Leu Arg Asp 530 535 540 Ala Leu Arg Met Pro Glu Gly Leu Val Arg Gly Thr Ser Ser Asp Val 545 550 555 560 Gly Arg Val Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly 565 570 575 Met Gly Ala Glu Leu Leu Asp Ser Ser Pro Glu Phe Ala Ala Ser Met 580 585 590 Ala Glu Cys Glu Thr Ala Leu Ser Arg Tyr Val Asp Trp Ser Leu Glu 595 600 605 Ala Val Val Arg Gln Glu Pro Gly Ala Pro Thr Leu Asp Arg Val Asp 610 615 620 Val Val Gln Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val 625 630 635 640 Trp Gln His His Gly Ile Thr Pro Gln Ala Val Val Gly His Ser Gln 645 650 655 Gly Glu Ile Ala Ala Ala Tyr Val Ala Gly Ala Leu Thr Leu Asp Asp 660 665 670 Ala Ala Arg Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu 675 680 685 Ala Gly Lys Gly Gly Met Ile Ser Leu Ala Leu Asp Glu Ala Ala Val 690 695 700 Leu Lys Arg Leu Ser Asp Phe Asp Gly Leu Ser Val Ala Ala Val Asn 705 710 715 720 Gly Pro Thr Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Glu Glu 725 730 735 Leu Ala Arg Thr Cys Glu Ala Asp Gly Val Arg Ala Arg Ile Ile Pro 740 745 750 Val Asp Tyr Ala Ser His Ser Arg Gln Val Glu Ile Ile Glu Lys Glu 755 760 765 Leu Ala Glu Val Leu Ala Gly Leu Ala Pro Gln Ala Pro His Val Pro 770 775 780 Phe Phe Ser Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Val Leu Asp 785 790 795 800 Gly Thr Tyr Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro 805 810 815 Ala Val Glu Thr Leu Ala Val Asp Gly Phe Thr His Phe Ile Glu Val 820 825 830 Ser Ala His Pro Val Leu Thr Met Thr Leu Pro Glu Thr Val Thr Gly 835 840 845 Leu Gly Thr Leu Arg Arg Glu Gln Gly Gly Gln Glu Arg Leu Val Thr 850 855 860 Ser Leu Ala Glu Ala Trp Ala Asn Gly Leu Thr Ile Asp Trp Ala Pro 865 870 875 880 Ile Leu Pro Thr Ala Thr Gly His His Pro Glu Leu Pro Thr Tyr Ala 885 890 895 Phe Gln Thr Glu Arg Phe Trp Leu Gln Ser Ser Ala Pro Thr Ser Ala 900 905 910 Ala Asp Asp Trp Arg Tyr Arg Val Glu Trp Lys Pro Leu Thr Ala Ser 915 920 925 Gly Gln Ala Asp Leu Ser Gly Arg Trp Ile Val Ala Val Gly Ser Glu 930 935 940 Pro Glu Ala Glu Leu Leu Gly Ala Leu Lys Ala Ala Gly Ala Glu Val 945 950 955 960 Asp Val Leu Glu Ala Gly Ala Asp Asp Asp Arg Glu Ala Leu Ala Ala 965 970 975 Arg Leu Thr Ala Leu Thr Thr Gly Asp Gly Phe Thr Gly Val Val Ser 980 985 990 Leu Leu Asp Asp Leu Val Pro Gln Val Ala Trp Val Gln Ala Leu Gly 995 1000 1005 Asp Ala Gly Ile Lys Ala Pro Leu Trp Ser Val Thr Gln Gly Ala Val 1010 1015 1020 Ser Val Gly Arg Leu Asp Thr Pro Ala Asp Pro Asp Arg Ala Met Leu 1025 1030 1035 1040 Trp Gly Leu Gly Arg Val Val Ala Leu Glu His Pro Glu Arg Trp Ala 1045 1050 1055 Gly Leu Val Asp Leu Pro Ala Gln Pro Asp Ala Ala Ala Leu Ala His 1060 1065 1070 Leu Val Thr Ala Leu Ser Gly Ala Thr Gly Glu Asp Gln Ile Ala Ile 1075 1080 1085 Arg Thr Thr Gly Leu His Ala Arg Arg Leu Ala Arg Ala Pro Leu His 1090 1095 1100 Gly Arg Arg Pro Thr Arg Asp Trp Gln Pro His Gly Thr Val Leu Ile 1105 1110 1115 1120 Thr Gly Gly Thr Gly Ala Leu Gly Ser His Ala Ala Arg Trp Met Ala 1125 1130 1135 His His Gly Ala Glu His Leu Leu Leu Val Ser Arg Ser Gly Glu Gln 1140 1145 1150 Ala Pro Gly Ala Thr Gln Leu Thr Ala Glu Leu Thr Ala Ser Gly Ala 1155 1160 1165 Arg Val Thr Ile Ala Ala Cys Asp Val Ala Asp Pro His Ala Met Arg 1170 1175 1180 Thr Leu Leu Asp Ala Ile Pro Ala Glu Thr Pro Leu Thr Ala Val Val 1185 1190 1195 1200 His Thr Ala Gly Ala Pro Gly Gly Asp Pro Leu Asp Val Thr Gly Pro 1205 1210 1215 Glu Asp Ile Ala Arg Ile Leu Gly Ala Lys Thr Ser Gly Ala Glu Val 1220 1225 1230 Leu Asp Asp Leu Leu Arg Gly Thr Pro Leu Asp Ala Phe Val Leu Tyr 1235 1240 1245 Ser Ser Asn Ala Gly Val Trp Gly Ser Gly Ser Gln Gly Val Tyr Ala 1250 1255 1260 Ala Ala Asn Ala His Leu Asp Ala Leu Ala Ala Arg Arg Arg Ala Arg 1265 1270 1275 1280 Gly Glu Thr Ala Thr Ser Val Ala Trp Gly Leu Trp Ala Gly Asp Gly 1285 1290 1295 Met Gly Arg Gly Ala Asp Asp Ala Tyr Trp Gln Arg Arg Gly Ile Arg 1300 1305 1310 Pro Met Ser Pro Asp Arg Ala Leu Asp Glu Leu Ala Lys Ala Leu Ser 1315 1320 1325 His Asp Glu Thr Phe Val Ala Val Ala Asp Val Asp Trp Glu Arg Phe 1330 1335 1340 Ala Pro Ala Phe Thr Val Ser Arg Pro Ser Leu Leu Leu Asp Gly Val 1345 1350 1355 1360 Pro Glu Ala Arg Gln Ala Leu Ala Ala Pro Val Gly Ala Pro Ala Pro 1365 1370 1375 Gly Asp Ala Ala Val Ala Pro Thr Gly Gln Ser Ser Ala Leu Ala Ala 1380 1385 1390 Ile Thr Ala Leu Pro Glu Pro Glu Arg Arg Pro Ala Leu Leu Thr Leu 1395 1400 1405 Val Arg Thr His Ala Ala Ala Val Leu Gly His Ser Ser Pro Asp Arg 1410 1415 1420 Val Ala Pro Gly Arg Ala Phe Thr Glu Leu Gly Phe Asp Ser Leu Thr 1425 1430 1435 1440 Ala Val Gln Leu Arg Asn Gln Leu Ser Thr Val Val Gly Asn Arg Leu 1445 1450 1455 Pro Ala Thr Thr Val Phe Asp His Pro Thr Pro Ala Ala Leu Ala Ala 1460 1465 1470 His Leu His Glu Ala Tyr Leu Ala Pro Ala Glu Pro Ala Pro Thr Asp 1475 1480 1485 Trp Glu Gly Arg Val Arg Arg Ala Leu Ala Glu Leu Pro Leu Asp Arg 1490 1495 1500 Leu Arg Asp Ala Gly Val Leu Asp Thr Val Leu Arg Leu Thr Gly Ile 1505 1510 1515 1520 Glu Pro Glu Pro Gly Ser Gly Gly Ser Asp Gly Gly Ala Ala Asp Pro 1525 1530 1535 Gly Ala Glu Pro Glu Ala Ser Ile Asp Asp Leu Asp Ala Glu Ala Leu 1540 1545 1550 Ile Arg Met Ala Leu Gly Pro Arg Asn Thr 1555 1560 36 4041 DNA Streptomyces venezuelae 36 atgacgagtt ccaacgaaca gttggtggac gctctgcgcg cctctctcaa ggagaacgaa 60 gaactccgga aagagagccg tcgccgggcc gaccgtcggc aggagcccat ggcgatcgtc 120 ggcatgagct gccggttcgc gggcggaatc cggtcccccg aggacctctg ggacgccgtc 180 gccgcgggca aggacctggt ctccgaggta ccggaggagc gcggctggga catcgactcc 240 ctctacgacc cggtgcccgg gcgcaagggc acgacgtacg tccgcaacgc cgcgttcctc 300 gacgacgccg ccggattcga cgcggccttc ttcgggatct cgccgcgcga ggccctcgcc 360 atggacccgc agcagcggca gctcctcgaa gcctcctggg aggtcttcga gcgggccggc 420 atcgaccccg cgtcggtccg cggcaccgac gtcggcgtgt acgtgggctg tggctaccag 480 gactacgcgc cggacatccg ggtcgccccc gaaggcaccg gcggttacgt cgtcaccggc 540 aactcctccg ccgtggcctc cgggcgcatc gcgtactccc tcggcctgga gggacccgcc 600 gtgaccgtgg acacggcgtg ctcctcttcg ctcgtcgccc tgcacctcgc cctgaagggc 660 ctgcggaacg gcgactgctc gacggcactc gtgggcggcg tggccgtcct cgcgacgccg 720 ggcgcgttca tcgagttcag cagccagcag gccatggccg ccgacggccg gaccaagggc 780 ttcgcctcgg cggcggacgg cctcgcctgg ggcgagggcg tcgccgtact cctcctcgaa 840 cggctctccg acgcgcggcg caagggccac cgggtcctgg ccgtcgtgcg cggcagcgcc 900 atcaaccagg acggcgcgag caacggcctc acggctccgc acgggccctc ccagcagcac 960 ctgatccgcc aggccctggc cgacgcgcgg ctcacgtcga gcgacgtgga cgtcgtggag 1020 ggccacggca cggggacccg tctcggcgac ccgatcgagg cgcaggcgct gctcgccacg 1080 tacgggcagg ggcgcgcccc ggggcagccg ctgcggctgg ggacgctgaa gtcgaacatc 1140 gggcacacgc aggccgcttc gggtgtcgcc ggtgtcatca agatggtgca ggcgctgcgc 1200 cacggggtgc tgccgaagac cctgcacgtg gacgagccga cggaccaggt cgactggtcg 1260 gccggttcgg tcgagctgct caccgaggcc gtggactggc cggagcggcc gggccggctc 1320 cgccgggcgg gcgtctccgc gttcggcgtg ggcgggacga acgcgcacgt cgtcctggag 1380 gaggccccgg cggtcgagga gtcccctgcc gtcgagccgc cggccggtgg cggcgtggtg 1440 ccgtggccgg tgtccgcgaa gacctcggcc gcactggacg cccagatcgg gcagctcgcc 1500 gcatacgcgg aagaccgcac ggacgtggat ccggcggtgg ccgcccgcgc cctggtcgac 1560 agccgtacgg cgatggagca ccgcgcggtc gcggtcggcg acagccggga ggcactgcgg 1620 gacgccctgc ggatgccgga aggactggta cggggcacgg tcaccgatcc gggccgggtg 1680 gcgttcgtct tccccggcca gggcacgcag tgggccggca tgggcgccga actcctcgac 1740 agctcacccg aattcgccgc cgccatggcc gaatgcgaga ccgcactctc cccgtacgtc 1800 gactggtctc tcgaagccgt cgtccgacag gctcccagcg caccgacact cgaccgcgtc 1860 gacgtcgtcc agcccgtcac cttcgccgtc atggtctccc tcgccaaggt ctggcagcac 1920 cacggcatca cccccgaggc cgtcatcggc cactcccagg gcgagatcgc cgccgcgtac 1980 gtcgccggtg ccctcaccct cgacgacgcc gctcgtgtcg tgaccctccg cagcaagtcc 2040 atcgccgccc acctcgccgg caagggcggc atgatctccc tcgccctcag cgaggaagcc 2100 acccggcagc gcatcgagaa cctccacgga ctgtcgatcg ccgccgtcaa cgggcctacc 2160 gccaccgtgg tttcgggcga ccccacccag atccaagaac ttgctcaggc gtgtgaggcc 2220 gacggcatcc gcgcacggat catccccgtc gactacgcct cccacagcgc ccacgtcgag 2280 accatcgaga acgaactcgc cgacgtcctg gcggggttgt ccccccagac accccaggtc 2340 cccttcttct ccaccctcga aggcacctgg atcaccgaac ccgccctcga cggcggctac 2400 tggtaccgca acctccgcca tcgtgtgggc ttcgccccgg ccgtcgagac cctcgccacc 2460 gacgaaggct tcacccactt catcgaggtc agcgcccacc ccgtcctcac catgaccctc 2520 cccgacaagg tcaccggcct ggccaccctc cgacgcgagg acggcggaca gcaccgcctc 2580 accacctccc ttgccgaggc ctgggccaac ggcctcgccc tcgactgggc ctccctcctg 2640 cccgccacgg gcgccctcag ccccgccgtc cccgacctcc cgacgtacgc cttccagcac 2700 cgctcgtact ggatcagccc cgcgggtccc ggcgaggcgc ccgcgcacac cgcttccggg 2760 cgcgaggccg tcgccgagac ggggctcgcg tggggcccgg gtgccgagga cctcgacgag 2820 gagggccggc gcagcgccgt actcgcgatg gtgatgcggc aggcggcctc cgtgctccgg 2880 tgcgactcgc ccgaagaggt ccccgtcgac cgcccgctgc gggagatcgg cttcgactcg 2940 ctgaccgccg tcgacttccg caaccgcgtc aaccggctga ccggtctcca gctgccgccc 3000 accgtcgtgt tccagcaccc gacgcccgtc gcgctcgccg agcgcatcag cgacgagctg 3060 gccgagcgga actgggccgt cgccgagccg tcggatcacg agcaggcgga ggaggagaag 3120 gccgccgctc cggcgggggc ccgctccggg gccgacaccg gcgccggcgc cgggatgttc 3180 cgcgccctgt tccggcaggc cgtggaggac gaccggtacg gcgagttcct cgacgtcctc 3240 gccgaagcct ccgcgttccg cccgcagttc gcctcgcccg aggcctgctc ggagcggctc 3300 gacccggtgc tgctcgccgg cggtccgacg gaccgggcgg aaggccgtgc cgttctcgtc 3360 ggctgcaccg gcaccgcggc gaacggcggc ccgcacgagt tcctgcggct cagcacctcc 3420 ttccaggagg agcgggactt cctcgccgta cctctccccg gctacggcac gggtacgggc 3480 accggcacgg ccctcctccc ggccgatctc gacaccgcgc tcgacgccca ggcccgggcg 3540 atcctccggg ccgccgggga cgccccggtc gtcctgctcg ggcactccgg cggcgccctg 3600 ctcgcgcacg agctggcctt ccgcctggag cgggcgcacg gcgcgccgcc ggccgggatc 3660 gtcctggtcg acccctatcc gccgggccat caggagccca tcgaggtgtg gagcaggcag 3720 ctgggcgagg gcctgttcgc gggcgagctg gagccgatgt ccgatgcgcg gctgctggcc 3780 atgggccggt acgcgcggtt cctcgccggc ccgcggccgg gccgcagcag cgcgcccgtg 3840 cttctggtcc gtgcctccga accgctgggc gactggcagg aggagcgggg cgactggcgt 3900 gcccactggg accttccgca caccgtcgcg gacgtgccgg gcgaccactt cacgatgatg 3960 cgggaccacg cgccggccgt cgccgaggcc gtcctctcct ggctcgacgc catcgagggc 4020 atcgaggggg cgggcaagtg a 4041 37 1346 PRT Streptomyces venezuelae 37 Met Thr Ser Ser Asn Glu Gln Leu Val Asp Ala Leu Arg Ala Ser Leu 1 5 10 15 Lys Glu Asn Glu Glu Leu Arg Lys Glu Ser Arg Arg Arg Ala Asp Arg 20 25 30 Arg Gln Glu Pro Met Ala Ile Val Gly Met Ser Cys Arg Phe Ala Gly 35 40 45 Gly Ile Arg Ser Pro Glu Asp Leu Trp Asp Ala Val Ala Ala Gly Lys 50 55 60 Asp Leu Val Ser Glu Val Pro Glu Glu Arg Gly Trp Asp Ile Asp Ser 65 70 75 80 Leu Tyr Asp Pro Val Pro Gly Arg Lys Gly Thr Thr Tyr Val Arg Asn 85 90 95 Ala Ala Phe Leu Asp Asp Ala Ala Gly Phe Asp Ala Ala Phe Phe Gly 100 105 110 Ile Ser Pro Arg Glu Ala Leu Ala Met Asp Pro Gln Gln Arg Gln Leu 115 120 125 Leu Glu Ala Ser Trp Glu Val Phe Glu Arg Ala Gly Ile Asp Pro Ala 130 135 140 Ser Val Arg Gly Thr Asp Val Gly Val Tyr Val Gly Cys Gly Tyr Gln 145 150 155 160 Asp Tyr Ala Pro Asp Ile Arg Val Ala Pro Glu Gly Thr Gly Gly Tyr 165 170 175 Val Val Thr Gly Asn Ser Ser Ala Val Ala Ser Gly Arg Ile Ala Tyr 180 185 190 Ser Leu Gly Leu Glu Gly Pro Ala Val Thr Val Asp Thr Ala Cys Ser 195 200 205 Ser Ser Leu Val Ala Leu His Leu Ala Leu Lys Gly Leu Arg Asn Gly 210 215 220 Asp Cys Ser Thr Ala Leu Val Gly Gly Val Ala Val Leu Ala Thr Pro 225 230 235 240 Gly Ala Phe Ile Glu Phe Ser Ser Gln Gln Ala Met Ala Ala Asp Gly 245 250 255 Arg Thr Lys Gly Phe Ala Ser Ala Ala Asp Gly Leu Ala Trp Gly Glu 260 265 270 Gly Val Ala Val Leu Leu Leu Glu Arg Leu Ser Asp Ala Arg Arg Lys 275 280 285 Gly His Arg Val Leu Ala Val Val Arg Gly Ser Ala Ile Asn Gln Asp 290 295 300 Gly Ala Ser Asn Gly Leu Thr Ala Pro His Gly Pro Ser Gln Gln His 305 310 315 320 Leu Ile Arg Gln Ala Leu Ala Asp Ala Arg Leu Thr Ser Ser Asp Val 325 330 335 Asp Val Val Glu Gly His Gly Thr Gly Thr Arg Leu Gly Asp Pro Ile 340 345 350 Glu Ala Gln Ala Leu Leu Ala Thr Tyr Gly Gln Gly Arg Ala Pro Gly 355 360 365 Gln Pro Leu Arg Leu Gly Thr Leu Lys Ser Asn Ile Gly His Thr Gln 370 375 380 Ala Ala Ser Gly Val Ala Gly Val Ile Lys Met Val Gln Ala Leu Arg 385 390 395 400 His Gly Val Leu Pro Lys Thr Leu His Val Asp Glu Pro Thr Asp Gln 405 410 415 Val Asp Trp Ser Ala Gly Ser Val Glu Leu Leu Thr Glu Ala Val Asp 420 425 430 Trp Pro Glu Arg Pro Gly Arg Leu Arg Arg Ala Gly Val Ser Ala Phe 435 440 445 Gly Val Gly Gly Thr Asn Ala His Val Val Leu Glu Glu Ala Pro Ala 450 455 460 Val Glu Glu Ser Pro Ala Val Glu Pro Pro Ala Gly Gly Gly Val Val 465 470 475 480 Pro Trp Pro Val Ser Ala Lys Thr Ser Ala Ala Leu Asp Ala Gln Ile 485 490 495 Gly Gln Leu Ala Ala Tyr Ala Glu Asp Arg Thr Asp Val Asp Pro Ala 500 505 510 Val Ala Ala Arg Ala Leu Val Asp Ser Arg Thr Ala Met Glu His Arg 515 520 525 Ala Val Ala Val Gly Asp Ser Arg Glu Ala Leu Arg Asp Ala Leu Arg 530 535 540 Met Pro Glu Gly Leu Val Arg Gly Thr Val Thr Asp Pro Gly Arg Val 545 550 555 560 Ala Phe Val Phe Pro Gly Gln Gly Thr Gln Trp Ala Gly Met Gly Ala 565 570 575 Glu Leu Leu Asp Ser Ser Pro Glu Phe Ala Ala Ala Met Ala Glu Cys 580 585 590 Glu Thr Ala Leu Ser Pro Tyr Val Asp Trp Ser Leu Glu Ala Val Val 595 600 605 Arg Gln Ala Pro Ser Ala Pro Thr Leu Asp Arg Val Asp Val Val Gln 610 615 620 Pro Val Thr Phe Ala Val Met Val Ser Leu Ala Lys Val Trp Gln His 625 630 635 640 His Gly Ile Thr Pro Glu Ala Val Ile Gly His Ser Gln Gly Glu Ile 645 650 655 Ala Ala Ala Tyr Val Ala Gly Ala Leu Thr Leu Asp Asp Ala Ala Arg 660 665 670 Val Val Thr Leu Arg Ser Lys Ser Ile Ala Ala His Leu Ala Gly Lys 675 680 685 Gly Gly Met Ile Ser Leu Ala Leu Ser Glu Glu Ala Thr Arg Gln Arg 690 695 700 Ile Glu Asn Leu His Gly Leu Ser Ile Ala Ala Val Asn Gly Pro Thr 705 710 715 720 Ala Thr Val Val Ser Gly Asp Pro Thr Gln Ile Gln Glu Leu Ala Gln 725 730 735 Ala Cys Glu Ala Asp Gly Ile Arg Ala Arg Ile Ile Pro Val Asp Tyr 740 745 750 Ala Ser His Ser Ala His Val Glu Thr Ile Glu Asn Glu Leu Ala Asp 755 760 765 Val Leu Ala Gly Leu Ser Pro Gln Thr Pro Gln Val Pro Phe Phe Ser 770 775 780 Thr Leu Glu Gly Thr Trp Ile Thr Glu Pro Ala Leu Asp Gly Gly Tyr 785 790 795 800 Trp Tyr Arg Asn Leu Arg His Arg Val Gly Phe Ala Pro Ala Val Glu 805 810 815 Thr Leu Ala Thr Asp Glu Gly Phe Thr His Phe Ile Glu Val Ser Ala 820 825 830 His Pro Val Leu Thr Met Thr Leu Pro Asp Lys Val Thr Gly Leu Ala 835 840 845 Thr Leu Arg Arg Glu Asp Gly Gly Gln His Arg Leu Thr Thr Ser Leu 850 855 860 Ala Glu Ala Trp Ala Asn Gly Leu Ala Leu Asp Trp Ala Ser Leu Leu 865 870 875 880 Pro Ala Thr Gly Ala Leu Ser Pro Ala Val Pro Asp Leu Pro Thr Tyr 885 890 895 Ala Phe Gln His Arg Ser Tyr Trp Ile Ser Pro Ala Gly Pro Gly Glu 900 905 910 Ala Pro Ala His Thr Ala Ser Gly Arg Glu Ala Val Ala Glu Thr Gly 915 920 925 Leu Ala Trp Gly Pro Gly Ala Glu Asp Leu Asp Glu Glu Gly Arg Arg 930 935 940 Ser Ala Val Leu Ala Met Val Met Arg Gln Ala Ala Ser Val Leu Arg 945 950 955 960 Cys Asp Ser Pro Glu Glu Val Pro Val Asp Arg Pro Leu Arg Glu Ile 965 970 975 Gly Phe Asp Ser Leu Thr Ala Val Asp Phe Arg Asn Arg Val Asn Arg 980 985 990 Leu Thr Gly Leu Gln Leu Pro Pro Thr Val Val Phe Gln His Pro Thr 995 1000 1005 Pro Val Ala Leu Ala Glu Arg Ile Ser Asp Glu Leu Ala Glu Arg Asn 1010 1015 1020 Trp Ala Val Ala Glu Pro Ser Asp His Glu Gln Ala Glu Glu Glu Lys 1025 1030 1035 1040 Ala Ala Ala Pro Ala Gly Ala Arg Ser Gly Ala Asp Thr Gly Ala Gly 1045 1050 1055 Ala Gly Met Phe Arg Ala Leu Phe Arg Gln Ala Val Glu Asp Asp Arg 1060 1065 1070 Tyr Gly Glu Phe Leu Asp Val Leu Ala Glu Ala Ser Ala Phe Arg Pro 1075 1080 1085 Gln Phe Ala Ser Pro Glu Ala Cys Ser Glu Arg Leu Asp Pro Val Leu 1090 1095 1100 Leu Ala Gly Gly Pro Thr Asp Arg Ala Glu Gly Arg Ala Val Leu Val 1105 1110 1115 1120 Gly Cys Thr Gly Thr Ala Ala Asn Gly Gly Pro His Glu Phe Leu Arg 1125 1130 1135 Leu Ser Thr Ser Phe Gln Glu Glu Arg Asp Phe Leu Ala Val Pro Leu 1140 1145 1150 Pro Gly Tyr Gly Thr Gly Thr Gly Thr Gly Thr Ala Leu Leu Pro Ala 1155 1160 1165 Asp Leu Asp Thr Ala Leu Asp Ala Gln Ala Arg Ala Ile Leu Arg Ala 1170 1175 1180 Ala Gly Asp Ala Pro Val Val Leu Leu Gly His Ser Gly Gly Ala Leu 1185 1190 1195 1200 Leu Ala His Glu Leu Ala Phe Arg Leu Glu Arg Ala His Gly Ala Pro 1205 1210 1215 Pro Ala Gly Ile Val Leu Val Asp Pro Tyr Pro Pro Gly His Gln Glu 1220 1225 1230 Pro Ile Glu Val Trp Ser Arg Gln Leu Gly Glu Gly Leu Phe Ala Gly 1235 1240 1245 Glu Leu Glu Pro Met Ser Asp Ala Arg Leu Leu Ala Met Gly Arg Tyr 1250 1255 1260 Ala Arg Phe Leu Ala Gly Pro Arg Pro Gly Arg Ser Ser Ala Pro Val 1265 1270 1275 1280 Leu Leu Val Arg Ala Ser Glu Pro Leu Gly Asp Trp Gln Glu Glu Arg 1285 1290 1295 Gly Asp Trp Arg Ala His Trp Asp Leu Pro His Thr Val Ala Asp Val 1300 1305 1310 Pro Gly Asp His Phe Thr Met Met Arg Asp His Ala Pro Ala Val Ala 1315 1320 1325 Glu Ala Val Leu Ser Trp Leu Asp Ala Ile Glu Gly Ile Glu Gly Ala 1330 1335 1340 Gly Lys 1345 38 1251 DNA Streptomyces venezuelae 38 gtgcgccgta cccagcaggg aacgaccgct tctcccccgg tactcgacct cggggccctg 60 gggcaggatt tcgcggccga tccgtatccg acgtacgcga gactgcgtgc cgagggtccg 120 gcccaccggg tgcgcacccc cgagggggac gaggtgtggc tggtcgtcgg ctacgaccgg 180 gcgcgggcgg tcctcgccga tccccggttc agcaaggact ggcgcaactc cacgactccc 240 ctgaccgagg ccgaggccgc gctcaaccac aacatgctgg agtccgaccc gccgcggcac 300 acccggctgc gcaagctggt ggcccgtgag ttcaccatgc gccgggtcga gttgctgcgg 360 ccccgggtcc aggagatcgt cgacgggctc gtggacgcca tgctggcggc gcccgacggc 420 cgcgccgatc tgatggagtc cctggcctgg ccgctgccga tcaccgtgat ctccgaactc 480 ctcggcgtgc ccgagccgga ccgcgccgcc ttccgcgtct ggaccgacgc cttcgtcttc 540 ccggacgatc ccgcccaggc ccagaccgcc atggccgaga tgagcggcta tctctcccgg 600 ctcatcgact ccaagcgcgg gcaggacggc gaggacctgc tcagcgcgct cgtgcggacc 660 agcgacgagg acggctcccg gctgacctcc gaggagctgc tcggtatggc ccacatcctg 720 ctcgtcgcgg ggcacgagac cacggtcaat ctgatcgcca acggcatgta cgcgctgctc 780 tcgcaccccg accagctggc cgccctgcgg gccgacatga cgctcttgga cggcgcggtg 840 gaggagatgt tgcgctacga gggcccggtg gaatccgcga cctaccgctt cccggtcgag 900 cccgtcgacc tggacggcac ggtcatcccg gccggtgaca cggtcctcgt cgtcctggcc 960 gacgcccacc gcacccccga gcgcttcccg gacccgcacc gcttcgacat ccgccgggac 1020 accgccggcc atctcgcctt cggccacggc atccacttct gcatcggcgc ccccttggcc 1080 cggttggagg cccggatcgc cgtccgcgcc cttctcgaac gctgcccgga cctcgccctg 1140 gacgtctccc ccggcgaact cgtgtggtat ccgaacccga tgattcgcgg gctcaaggcc 1200 ctgccgatcc gctggcggcg aggacgggag gcgggccgcc gtaccggttg a 1251 39 416 PRT Streptomyces venezuelae 39 Met Arg Arg Thr Gln Gln Gly Thr Thr Ala Ser Pro Pro Val Leu Asp 1 5 10 15 Leu Gly Ala Leu Gly Gln Asp Phe Ala Ala Asp Pro Tyr Pro Thr Tyr 20 25 30 Ala Arg Leu Arg Ala Glu Gly Pro Ala His Arg Val Arg Thr Pro Glu 35 40 45 Gly Asp Glu Val Trp Leu Val Val Gly Tyr Asp Arg Ala Arg Ala Val 50 55 60 Leu Ala Asp Pro Arg Phe Ser Lys Asp Trp Arg Asn Ser Thr Thr Pro 65 70 75 80 Leu Thr Glu Ala Glu Ala Ala Leu Asn His Asn Met Leu Glu Ser Asp 85 90 95 Pro Pro Arg His Thr Arg Leu Arg Lys Leu Val Ala Arg Glu Phe Thr 100 105 110 Met Arg Arg Val Glu Leu Leu Arg Pro Arg Val Gln Glu Ile Val Asp 115 120 125 Gly Leu Val Asp Ala Met Leu Ala Ala Pro Asp Gly Arg Ala Asp Leu 130 135 140 Met Glu Ser Leu Ala Trp Pro Leu Pro Ile Thr Val Ile Ser Glu Leu 145 150 155 160 Leu Gly Val Pro Glu Pro Asp Arg Ala Ala Phe Arg Val Trp Thr Asp 165 170 175 Ala Phe Val Phe Pro Asp Asp Pro Ala Gln Ala Gln Thr Ala Met Ala 180 185 190 Glu Met Ser Gly Tyr Leu Ser Arg Leu Ile Asp Ser Lys Arg Gly Gln 195 200 205 Asp Gly Glu Asp Leu Leu Ser Ala Leu Val Arg Thr Ser Asp Glu Asp 210 215 220 Gly Ser Arg Leu Thr Ser Glu Glu Leu Leu Gly Met Ala His Ile Leu 225 230 235 240 Leu Val Ala Gly His Glu Thr Thr Val Asn Leu Ile Ala Asn Gly Met 245 250 255 Tyr Ala Leu Leu Ser His Pro Asp Gln Leu Ala Ala Leu Arg Ala Asp 260 265 270 Met Thr Leu Leu Asp Gly Ala Val Glu Glu Met Leu Arg Tyr Glu Gly 275 280 285 Pro Val Glu Ser Ala Thr Tyr Arg Phe Pro Val Glu Pro Val Asp Leu 290 295 300 Asp Gly Thr Val Ile Pro Ala Gly Asp Thr Val Leu Val Val Leu Ala 305 310 315 320 Asp Ala His Arg Thr Pro Glu Arg Phe Pro Asp Pro His Arg Phe Asp 325 330 335 Ile Arg Arg Asp Thr Ala Gly His Leu Ala Phe Gly His Gly Ile His 340 345 350 Phe Cys Ile Gly Ala Pro Leu Ala Arg Leu Glu Ala Arg Ile Ala Val 355 360 365 Arg Ala Leu Leu Glu Arg Cys Pro Asp Leu Ala Leu Asp Val Ser Pro 370 375 380 Gly Glu Leu Val Trp Tyr Pro Asn Pro Met Ile Arg Gly Leu Lys Ala 385 390 395 400 Leu Pro Ile Arg Trp Arg Arg Gly Arg Glu Ala Gly Arg Arg Thr Gly 405 410 415 40 2787 DNA Streptomyces venezuelae 40 atgaatctgg tggaacgcga cggggagata gcccatctca gggccgttct tgacgcatcc 60 gccgcaggtg acgggacgct cttactcgtc tccggaccgg ccggcagcgg gaagacggag 120 ctgctgcggt cgctccgccg gctggccgcc gagcgggaga cccccgtctg gtcggtccgg 180 gcgctgccgg gtgaccgcga catccccctg ggcgtcctct gccagttact ccgcagcgcc 240 gaacaacacg gtgccgacac ctccgccgtc cgcgacctgc tggacgccgc ctcgcggcgg 300 gccggaaacc tcacctcccc cgccgacgcg ccgctccgcg tcgacgagac acaccgcctg 360 cacgactggc tgctctccgt ctcccgccgc accccgttcc tcgtcgccgt cgacgacctg 420 acccacgccg acaccgcgtc cctgaggttc ctcctgtact gcgccgccca ccacgaccag 480 ggcggcatcg gcttcgtcat gaccgagcgg gcctcgcagc gcgccggata ccgggtgttc 540 cgcgccgagc tgctccgcca gccgcactgc cgcaacatgt ggctctccgg gcttcccccc 600 agcggggtac gccagttact cgcccactac tacggccccg aggccgccga gcggcgggcc 660 cccgcgtacc acgcgacgac cggcgggaac ccgctgctcc tgcgggcgct gacccaggac 720 cggcaggcct cccacaccac cctcggcgcg gccggcggcg acgagcccgt ccacggcgac 780 gccttcgccc aggccgtcct cgactgcctg caccgcagcg ccgagggcac actggagacc 840 gcccgctggc tcgcggtcct cgaacagtcc gacccgctcc tggtggagcg gctcacggga 900 acgaccgccg ccgccgtcga gcgccacatc caggagctcg ccgccatcgg cctcctggac 960 gaggacggca ccctgggaca gcccgcgatc cgcgaggccg ccctccagga cctgccggcc 1020 ggcgagcgca ccgaactgca ccggcgcgcc gcggagcagc tgcaccggga cggcgccgac 1080 gaggacaccg tggcccgcca cctgctggtc ggcggcgccc ccgacgctcc ctgggcgctg 1140 cccctgctcg aacggggcgc gcagcaggcc ctgttcgacg accgactcga cgacgccttc 1200 cggatcctcg agttcgccgt gcggtcgagc accgacaaca cccagctggc ccgcctcgcc 1260 ccacacctgg tcgcggcctc ctggcggatg aacccgcaca tgacgacccg ggccctcgca 1320 ctcttcgacc ggctcctgag cggtgaactg ccgcccagcc acccggtcat ggccctgatc 1380 cgctgcctcg tctggtacgg gcggctgccc gaggccgccg acgcgctgtc ccggctgcgg 1440 cccagctccg acaacgatgc cttggagctg tcgctcaccc ggatgtggct cgcggcgctg 1500 tgcccgccgc tcctggagtc cctgccggcc acgccggagc cggagcgggg tcccgtcccc 1560 gtacggctcg cgccgcggac gaccgcgctc caggcccagg ccggcgtctt ccagcggggc 1620 ccggacaacg cctcggtcgc gcaggccgaa cagatcctgc agggctgccg gctgtcggag 1680 gagacgtacg aggccctgga gacggccctc ttggtcctcg tccacgccga ccggctcgac 1740 cgggcgctgt tctggtcgga cgccctgctc gccgaggccg tggagcggcg gtcgctcggc 1800 tgggaggcgg tcttcgccgc gacccgggcg atgatcgcga tccgctgcgg cgacctcccg 1860 acggcgcggg agcgggccga gctggcgctc tcccacgcgg cgccggagag ctggggcctc 1920 gccgtgggca tgcccctctc cgcgctgctg ctcgcctgca cggaggccgg cgagtacgaa 1980 caggcggagc gggtcctgcg gcagccggtg ccggacgcga tgttcgactc gcggcacggc 2040 atggagtaca tgcacgcccg gggccgctac tggctggcga cgggccggct gcacgcggcg 2100 ctgggcgagt tcatgctctg cggggagatc ctgggcagct ggaacctcga ccagccctcg 2160 atcgtgccct ggcggacctc cgccgccgag gtgtacctgc ggctcggcaa ccgccagaag 2220 gccagggcgc tggccgaggc ccagctcgcc ctggtgcggc ccgggcgctc ccgcacccgg 2280 ggtctcaccc tgcgggtcct ggcggcggcg gtggacggcc agcaggcgga gcggctgcac 2340 gccgaggcgg tcgacatgct gcacgacagc ggcgaccggc tcgaacacgc ccgcgcgctc 2400 gccgggatga gccgccacca gcaggcccag ggggacaact accgggcgag gatgacggcg 2460 cggctcgccg gcgacatggc gtgggcctgc ggcgcgtacc cgctggccga ggagatcgtg 2520 ccgggccgcg gcggccgccg ggcgaaggcg gtgagcacgg agctggaact gccgggcggc 2580 ccggacgtcg gcctgctctc ggaggccgaa cgccgggtgg cggccctggc agcccgagga 2640 ttgacgaacc gccagatagc gcgccggctc tgcgtcaccg cgagcacggt cgaacagcac 2700 ctgacgcgcg tctaccgcaa actgaacgtg acccgccgag cagacctccc gatcagcctc 2760 gcccaggaca agtccgtcac ggcctga 2787 41 928 PRT Streptomyces venezuelae 41 Met Asn Leu Val Glu Arg Asp Gly Glu Ile Ala His Leu Arg Ala Val 1 5 10 15 Leu Asp Ala Ser Ala Ala Gly Asp Gly Thr Leu Leu Leu Val Ser Gly 20 25 30 Pro Ala Gly Ser Gly Lys Thr Glu Leu Leu Arg Ser Leu Arg Arg Leu 35 40 45 Ala Ala Glu Arg Glu Thr Pro Val Trp Ser Val Arg Ala Leu Pro Gly 50 55 60 Asp Arg Asp Ile Pro Leu Gly Val Leu Cys Gln Leu Leu Arg Ser Ala 65 70 75 80 Glu Gln His Gly Ala Asp Thr Ser Ala Val Arg Asp Leu Leu Asp Ala 85 90 95 Ala Ser Arg Arg Ala Gly Asn Leu Thr Ser Pro Ala Asp Ala Pro Leu 100 105 110 Arg Val Asp Glu Thr His Arg Leu His Asp Trp Leu Leu Ser Val Ser 115 120 125 Arg Arg Thr Pro Phe Leu Val Ala Val Asp Asp Leu Thr His Ala Asp 130 135 140 Thr Ala Ser Leu Arg Phe Leu Leu Tyr Cys Ala Ala His His Asp Gln 145 150 155 160 Gly Gly Ile Gly Phe Val Met Thr Glu Arg Ala Ser Gln Arg Ala Gly 165 170 175 Tyr Arg Val Phe Arg Ala Glu Leu Leu Arg Gln Pro His Cys Arg Asn 180 185 190 Met Trp Leu Ser Gly Leu Pro Pro Ser Gly Val Arg Gln Leu Leu Ala 195 200 205 His Tyr Tyr Gly Pro Glu Ala Ala Glu Arg Arg Ala Pro Ala Tyr His 210 215 220 Ala Thr Thr Gly Gly Asn Pro Leu Leu Leu Arg Ala Leu Thr Gln Asp 225 230 235 240 Arg Gln Ala Ser His Thr Thr Leu Gly Ala Ala Gly Gly Asp Glu Pro 245 250 255 Val His Gly Asp Ala Phe Ala Gln Ala Val Leu Asp Cys Leu His Arg 260 265 270 Ser Ala Glu Gly Thr Leu Glu Thr Ala Arg Trp Leu Ala Val Leu Glu 275 280 285 Gln Ser Asp Pro Leu Leu Val Glu Arg Leu Thr Gly Thr Thr Ala Ala 290 295 300 Ala Val Glu Arg His Ile Gln Glu Leu Ala Ala Ile Gly Leu Leu Asp 305 310 315 320 Glu Asp Gly Thr Leu Gly Gln Pro Ala Ile Arg Glu Ala Ala Leu Gln 325 330 335 Asp Leu Pro Ala Gly Glu Arg Thr Glu Leu His Arg Arg Ala Ala Glu 340 345 350 Gln Leu His Arg Asp Gly Ala Asp Glu Asp Thr Val Ala Arg His Leu 355 360 365 Leu Val Gly Gly Ala Pro Asp Ala Pro Trp Ala Leu Pro Leu Leu Glu 370 375 380 Arg Gly Ala Gln Gln Ala Leu Phe Asp Asp Arg Leu Asp Asp Ala Phe 385 390 395 400 Arg Ile Leu Glu Phe Ala Val Arg Ser Ser Thr Asp Asn Thr Gln Leu 405 410 415 Ala Arg Leu Ala Pro His Leu Val Ala Ala Ser Trp Arg Met Asn Pro 420 425 430 His Met Thr Thr Arg Ala Leu Ala Leu Phe Asp Arg Leu Leu Ser Gly 435 440 445 Glu Leu Pro Pro Ser His Pro Val Met Ala Leu Ile Arg Cys Leu Val 450 455 460 Trp Tyr Gly Arg Leu Pro Glu Ala Ala Asp Ala Leu Ser Arg Leu Arg 465 470 475 480 Pro Ser Ser Asp Asn Asp Ala Leu Glu Leu Ser Leu Thr Arg Met Trp 485 490 495 Leu Ala Ala Leu Cys Pro Pro Leu Leu Glu Ser Leu Pro Ala Thr Pro 500 505 510 Glu Pro Glu Arg Gly Pro Val Pro Val Arg Leu Ala Pro Arg Thr Thr 515 520 525 Ala Leu Gln Ala Gln Ala Gly Val Phe Gln Arg Gly Pro Asp Asn Ala 530 535 540 Ser Val Ala Gln Ala Glu Gln Ile Leu Gln Gly Cys Arg Leu Ser Glu 545 550 555 560 Glu Thr Tyr Glu Ala Leu Glu Thr Ala Leu Leu Val Leu Val His Ala 565 570 575 Asp Arg Leu Asp Arg Ala Leu Phe Trp Ser Asp Ala Leu Leu Ala Glu 580 585 590 Ala Val Glu Arg Arg Ser Leu Gly Trp Glu Ala Val Phe Ala Ala Thr 595 600 605 Arg Ala Met Ile Ala Ile Arg Cys Gly Asp Leu Pro Thr Ala Arg Glu 610 615 620 Arg Ala Glu Leu Ala Leu Ser His Ala Ala Pro Glu Ser Trp Gly Leu 625 630 635 640 Ala Val Gly Met Pro Leu Ser Ala Leu Leu Leu Ala Cys Thr Glu Ala 645 650 655 Gly Glu Tyr Glu Gln Ala Glu Arg Val Leu Arg Gln Pro Val Pro Asp 660 665 670 Ala Met Phe Asp Ser Arg His Gly Met Glu Tyr Met His Ala Arg Gly 675 680 685 Arg Tyr Trp Leu Ala Thr Gly Arg Leu His Ala Ala Leu Gly Glu Phe 690 695 700 Met Leu Cys Gly Glu Ile Leu Gly Ser Trp Asn Leu Asp Gln Pro Ser 705 710 715 720 Ile Val Pro Trp Arg Thr Ser Ala Ala Glu Val Tyr Leu Arg Leu Gly 725 730 735 Asn Arg Gln Lys Ala Arg Ala Leu Ala Glu Ala Gln Leu Ala Leu Val 740 745 750 Arg Pro Gly Arg Ser Arg Thr Arg Gly Leu Thr Leu Arg Val Leu Ala 755 760 765 Ala Ala Val Asp Gly Gln Gln Ala Glu Arg Leu His Ala Glu Ala Val 770 775 780 Asp Met Leu His Asp Ser Gly Asp Arg Leu Glu His Ala Arg Ala Leu 785 790 795 800 Ala Gly Met Ser Arg His Gln Gln Ala Gln Gly Asp Asn Tyr Arg Ala 805 810 815 Arg Met Thr Ala Arg Leu Ala Gly Asp Met Ala Trp Ala Cys Gly Ala 820 825 830 Tyr Pro Leu Ala Glu Glu Ile Val Pro Gly Arg Gly Gly Arg Arg Ala 835 840 845 Lys Ala Val Ser Thr Glu Leu Glu Leu Pro Gly Gly Pro Asp Val Gly 850 855 860 Leu Leu Ser Glu Ala Glu Arg Arg Val Ala Ala Leu Ala Ala Arg Gly 865 870 875 880 Leu Thr Asn Arg Gln Ile Ala Arg Arg Leu Cys Val Thr Ala Ser Thr 885 890 895 Val Glu Gln His Leu Thr Arg Val Tyr Arg Lys Leu Asn Val Thr Arg 900 905 910 Arg Ala Asp Leu Pro Ile Ser Leu Ala Gln Asp Lys Ser Val Thr Ala 915 920 925 42 846 DNA Streptomyces venezuelae 42 gtgaccgaca gacctctgaa cgtggacagc ggactgtgga tccggcgctt ccaccccgcg 60 ccgaacagcg cggtgcggct ggtctgcctg ccgcacgccg gcggctccgc cagctacttc 120 ttccgcttct cggaggagct gcacccctcc gtcgaggccc tgtcggtgca gtatccgggc 180 cgccaggacc ggcgtgccga gccgtgtctg gagagcgtcg aggagctcgc cgagcatgtg 240 gtcgcggcca ccgaaccctg gtggcaggag ggccggctgg ccttcttcgg gcacagcctc 300 ggcgcctccg tcgccttcga gacggcccgc atcctggaac agcggcacgg ggtacggccc 360 gagggcctgt acgtctccgg tcggcgcgcc ccgtcgctgg cgccggaccg gctcgtccac 420 cagctggacg accgggcgtt cctggccgag atccggcggc tcagcggcac cgacgagcgg 480 ttcctccagg acgacgagct gctgcggctg gtgctgcccg cgctgcgcag cgactacaag 540 gcggcggaga cgtacctgca ccggccgtcc gccaagctca cctgcccggt gatggccctg 600 gccggcgacc gtgacccgaa ggcgccgctg aacgaggtgg ccgagtggcg tcggcacacc 660 agcgggccgt tctgcctccg ggcgtactcc ggcggccact tctacctcaa cgaccagtgg 720 cacgagatct gcaacgacat ctccgaccac ctgctcgtca cccgcggcgc gcccgatgcc 780 cgcgtcgtgc agcccccgac cagccttatc gaaggagcgg cgaagagatg gcagaaccca 840 cggtga 846 43 281 PRT Streptomyces venezuelae 43 Met Thr Asp Arg Pro Leu Asn Val Asp Ser Gly Leu Trp Ile Arg Arg 1 5 10 15 Phe His Pro Ala Pro Asn Ser Ala Val Arg Leu Val Cys Leu Pro His 20 25 30 Ala Gly Gly Ser Ala Ser Tyr Phe Phe Arg Phe Ser Glu Glu Leu His 35 40 45 Pro Ser Val Glu Ala Leu Ser Val Gln Tyr Pro Gly Arg Gln Asp Arg 50 55 60 Arg Ala Glu Pro Cys Leu Glu Ser Val Glu Glu Leu Ala Glu His Val 65 70 75 80 Val Ala Ala Thr Glu Pro Trp Trp Gln Glu Gly Arg Leu Ala Phe Phe 85 90 95 Gly His Ser Leu Gly Ala Ser Val Ala Phe Glu Thr Ala Arg Ile Leu 100 105 110 Glu Gln Arg His Gly Val Arg Pro Glu Gly Leu Tyr Val Ser Gly Arg 115 120 125 Arg Ala Pro Ser Leu Ala Pro Asp Arg Leu Val His Gln Leu Asp Asp 130 135 140 Arg Ala Phe Leu Ala Glu Ile Arg Arg Leu Ser Gly Thr Asp Glu Arg 145 150 155 160 Phe Leu Gln Asp Asp Glu Leu Leu Arg Leu Val Leu Pro Ala Leu Arg 165 170 175 Ser Asp Tyr Lys Ala Ala Glu Thr Tyr Leu His Arg Pro Ser Ala Lys 180 185 190 Leu Thr Cys Pro Val Met Ala Leu Ala Gly Asp Arg Asp Pro Lys Ala 195 200 205 Pro Leu Asn Glu Val Ala Glu Trp Arg Arg His Thr Ser Gly Pro Phe 210 215 220 Cys Leu Arg Ala Tyr Ser Gly Gly His Phe Tyr Leu Asn Asp Gln Trp 225 230 235 240 His Glu Ile Cys Asn Asp Ile Ser Asp His Leu Leu Val Thr Arg Gly 245 250 255 Ala Pro Asp Ala Arg Val Val Gln Pro Pro Thr Ser Leu Ile Glu Gly 260 265 270 Ala Ala Lys Arg Trp Gln Asn Pro Arg 275 280

Claims

What is claimed is:

1. An isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a desosamine biosynthetic gene cluster, a fragment or a biologically active variant thereof, wherein the nucleic acid sequence is not derived from the eryC gene cluster of Saccharopolyspora erythraea or Streptomyces antibioticus.

2. The isolated and purified nucleic acid segment of claim 1 comprising SEQ ID NO:3.

3. The isolated and purified nucleic acid segment of claim 1 which encodes DesI, DesII, DesIII, DesIV, DesV, DesVI, DesVII, DesVIII or DesR.

4. The isolated and purified nucleic acid segment of claim 1 which is from Streptomyces venezuelae.

5. An expression cassette comprising the nucleic acid segment of claim 1 operably linked to a promoter functional in a host cell.

6. A recombinant bacterial host cell in which at least a portion of a nucleic acid sequence encoding desosamine is disrupted so as to result in a decrease or lack of desosamine synthesis, wherein the nucleic acid sequence which is disrupted is not derived from the eryC gene cluster of Saccharopolyspora erythraea.

7. The host cell of claim 6 wherein the nucleic acid sequence which is disrupted encodes DesI, DesII, DesIII, DesIV, DesV, DesVI, DesVII, DesVIII or DesR.

8. A host cell, the genome of which is augmented with the expression cassette of claim 5.

9. A product produced by the host cell of claim 6 which is not produced by the corresponding non-recombinant host cell.

10. The product of claim 9 which comprises a macrolide.

11. An isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a macrolide biosynthetic gene cluster encoding methymycin, pikomycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof.

12. The isolated and purified nucleic acid segment of claim 11 comprising SEQ ID NO:5.

13. The isolated and purified nucleic acid segment of claim 11 comprising a biologically active variant or fragment of SEQ ID NO:5.

14. The isolated and purified nucleic acid segment of claim 11 which encodes PikR1, PikR2, PikAI, PikAII, PikAIII, PikAIV, PikAV, PikC or PikD.

15. The isolated and purified nucleic acid segment of claim 11 which is from Streptomyces venezuelae.

16. A host cell, the genome of which is augmented with the nucleic acid segment of claim 11.

17. An isolated and purified nucleic acid sequence comprising SEQ ID NO:3, SEQ ID NO:5, a fragment thereof, the complement thereto, or which hybridizes thereto.

18. An isolated polypeptide encoded by the nucleic acid segment of claim 1 or 11.

19. A recombinant host cell in which a pikAI gene, a pikAII gene, a pikAIII gene, a pikAIV gene, a pikB gene cluster, a pikAV gene cluster, a pikC gene, a pikR1 gene, apikR2 gene, or a combination thereof, is disrupted so as to reduce or eliminate production of methymycin, neomethymycin, pikromycin, narbomycin, or a combination thereof.

20. A macrolide or polyketide produced by the host cell of claim 19 which is not produced by the corresponding non-recombinant host cell.

21. An isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae.

22. A method of providing a polyhydroxyalkanoate monomer, comprising:

(a) introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell, wherein the recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, and wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae; and

(b) expressing the DNA encoding the recombinant polyhydroxyalkanoate monomer synthase in the host cell so as to generate a polyhydroxyalkanoate monomer.

23. A recombinant vector comprising one or more modules of a polyketide synthase wherein at least one module is from Streptomyces venezuelae.

24. The method of claim 22 wherein the first module encodes a fatty acid synthase.

25. A method of providing a polyhydroxyalkanoate monomer, comprising:

(a) introducing into a host cell a DNA molecule encoding a fusion polypeptide, wherein the DNA molecule comprises a first DNA segment operably linked to a promoter functional in the host cell and a second DNA segment, wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae; and

(b) expressing the DNA in the host cell so as to generate the fusion polypeptide.

26. The host cell of claim 16 the native genome of which does not comprise an intact macrolide biosynthetic gene cluster encoding methymycin, pikomycin, neomethymycin, or narbomycin.