AU2006201437A1 - Methods and compositions for making emamectin - Google Patents

Description

P/00/011 I Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT

(ORIGINAL)

Name of Applicant(s): Actual Inventor(s): Syngenta Participations AG, ACN of Schwarzwaldallee 215, CH-4058 Basel, SWITZERLAND James Madison Ligon, Philip Eugene Hammer, Thomas Gunter BUckel, Dwight Steven Hill, Johannes Paul Pachlatko, Ross Eric Zirkle, Istvan Molnar Address for Service: DAVIES COLLISON CAVE, Patent Trademark Attorneys, of 1 Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM "Methods and compositions for making emamectin" Invention Title: The following statement is a full description of this invention, including the best method of performing it known to us:- Case PB/5-60016A METHODS AND COMPOSITIONS FOR MAKING EMAMECTIN The invention relates to the field of agrochemicals, and in particular, to insecticides.

More specifically, this invention relates to the derivatization of avermectin, particularly for the synthesis of emamectin.

Emamectin is a potent insecticide and controls many pests such as thrips, leafminers, and worm pests including alfalfa caterpillar, beet armyworm, cabbage looper, corn earworm, cutworms, diamondback moth, tobacco budworm, tomato fruitworm, and tomato pinworm.

Emamectin 4 "-deoxy-4"-epi-N-methylamino avermectin Bla/BIb) is described in U.S. Patent No. 4,874,749 and in Cvetovich, R.J. et al., J. Organic Chem. 59:7704-7708, 1994 (as MK- 244).

U.S. Patent No. 5,288,710 describes salts of emamectin that are especially valuable agrochemically. These salts of emamectin are valuable pesticides, especially for combating insects and representatives of the order Acarina. Some pests for which emamectin is useful in combating are listed in European Patent Application EP-A 736,252.

One drawback to the use of emamectin is the difficulty of its synthesis from avermectin.

This is due to the first step of the process, which is the most costly and time-consuming step of producing emamectin, in which the 4"-carbinol group of avermectin must be oxidized to a ketone. The oxidation of the 4"-carbinol group is problematic due to the presence of two other hydroxyl groups on the molecule that must be chemically protected before oxidation and deprotected after oxidation. Thus, this first step, significantly increases the overall cost and time of producing emamectin from avermectin.

Because of the efficacy and potency of emamectin as an insecticide, there is a need to develop a cost and time effective method and/or reagent for regioselectively oxidizing the 4"carbinol group of avermectin to produce 4 "-keto-avermectin, which is a necessary intermediate for producing emamectin from avermectin.

The invention provides a novel family of P450 monooxygenases, each member of which is able to regioselectively oxidize the 4"-carbinol group of unprotected avermectin, thereby resulting in a cheap, effective method to produce 4 "-keto-avermectin, a necessary intermediate in the production of emamectin. The invention allows elimination of the costly, timeconsuming steps of chemically protecting the two other hydroxyl groups on the avermectin Case PB/5-60016A molecule prior to oxidation of the 4"-carbinol group that must be chemically protected before oxidation; and chemically deprotecting these two other hydroxyl groups after oxidation.

The invention thus provides reagents and methods for significantly reducing the overall cost of producing emamectin from avermectin.

Accordingly, in one aspect, the invention provides a purified nucleic acid molecule encoding a polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-keto-avermectin.

In a specific embodiment the invention relates to an purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-ketoavermectin, which polypeptide is substantially similar, and preferably has between at least and 99% amino acid sequence identity to the polypeptide of SEQ ID NO:2, with each individual number within this range of between 50% and 99% also being part of the invention.

S In a further specific embodiment the invention relates to an purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-keto-avermectin, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO:2.

The invention further provides a purified nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO:1; b) having substantial similarity to c) capable of hybridizing to or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 1, or the complement thereof; e) complementary to or 0 which is the reverse complement of or or -2- Case PB/5-60016A g) which is a functional part of or encoding a polypeptide that still exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-keto-avermectin.

In a specific embodiment the invention relates to a purified nucleic acid molecule comprsing a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-ketoavermectin, which polypeptide is substantially similar, and preferably has at least between and 99% amino acid sequence identity to the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID with each individual number within this range of between 60% and 99% also being part of the invention.

S In a further specific embodiment the invention relates to an purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4 "-keto-avermectin, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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 SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID SEQ ID NO:32, SEQ ID NO:34, or SEQ ID S The invention further provides a purified nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94; b) having substantial similarity to c) capable of hybridizing to or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 1, SEQ ID -3- Case PB/5-60016A NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: I1, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94 or the complement thereof; e) complementary to or 0 which is the reverse complement of or or g) which is a functional part of or encoding a polypeptide that still exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-keto-avermectin.

In certain embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least between 66%, and 99% identical to SEQ ID NO: 1, with each individual number within this range of between 66%, and 99% also being part of the invention..

In certain embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least between 70%, and 99% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94, with each individual number within this range of between 70%, and 99% also being part of the invention..

In some embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least 80% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94.

In certain embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least 90% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94.

Case PB/5-60016A S In certain embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:I, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94.

S In some embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, and SEQ ID NO:94.

S In particular embodiments, the nucleic acid molecule is isolated from a Streptomyces strain. In certain embodiments, the Streptomyces strain is selected from the group consisting of Streptomyces tubercidicus, Streptomyces lydicus, Streptomyces platensis, Streptomyces chattanoogensis, Streptomyces kasugaensis, and Streptomyces rimosus and Streptomyces albofaciens..

S In some embodiments of this aspect, the nucleic acid molecule further comprises a nucleic acid sequence encoding a tag which is linked to the P450 monooxygenase via a covalent bond. In certain embodiments, the tag is selected from the group consisting of a His tag, a GST tag, an HA tag, a HSV tag, a Myc-tag, and VSV-G-Tag.

S In another aspect, the invention provides a purified polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4 "-keto-avermectin.

In some embodiments, the polypeptide comprises or consists essentially of an amino acid sequence that is encoded by a nucleic acid molecule a) as given in SEQ ID NO:1 or the complement thereof; b) having substantial similarity to c) capable of hybridizing to or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: or the complement thereof; e) complementary to or Case PB/5-60016A f) which is the reverse complement of or or.

g) which is a functional part of or encoding a polypeptide that still exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-keto-avermectin.

In some embodiments, the polypeptide comprises or consists essentially of an amino acid sequence that is between at least 50%, and 99% identical to SEQ ID NO:2, with each individual number within this range of between 50% and 99% also being part of the invention..

In some embodiments, the polypeptide comprises or consists essentially of an amino acid sequence that is encoded by a nucleic acid molecule a) as given in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94 or the complement thereof; b) having substantial similarity to c) capable of hybridizing to or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94 or the complement thereof, or the complement thereof; e) complementary to or f) which is the reverse complement of or or g) which is a functional part of or encoding a polypeptide that still exhibits an enzymatic activity of a P450 monooxygenase and regioselectively oxidizes avermectin to 4"-keto-avermectin.

In some embodiments, the P450 monooxygenase comprises or consists essentially of an amino acid sequence that is between at least 60%, and 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, Case PB/5-60016A SEQ ID NO:26, SEQ ID NO:28, SEQ JD NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:95, with each individual number within this range of between 60% and 99% also being part of the invention..

In certain embodiments, the P450 monooxygenase comprises or consists essentially of an amino acid sequence that is at least 70% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In some embodiments, the P450 monooxygenase comprises or consists essentially of an amino acid sequence that is at least 80% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ D 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In some embodiments, the P450 monooxygenase comprises or consists essentially of an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In certain embodiments, the P450 monooxygenase comprises or consists essentially of an amino acid sequence that is at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In some embodiments of this aspect of the invention, the P450 monooxygenase comprises or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID In certain embodiments, the polypeptide according to the invention exhibiting an enzymatic activity of a P450 monooxygenase further comprises a tag. In some Case PB/5-60016A embodiments, the tag is selected from the group consisting of a His tag, a GST tag, an HA tag, a HSV tag, a Myc-tag, and VSV-G-Tag.

In another aspect, the invention provides a binding agent that specifically binds to a polypeptide according to the invention exhibiting an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin. In some embodiments, the binding agent is an antibody. In certain embodiments, the antibody is a polyclonal antibody or a monoclonal antibody.

In yet another aspect, the invention provides a family of P450 monooxygenase polypeptides, wherein each member of the family regioselectively oxidizes avermectin to 4"-keto-avermectin.

In certain embodiments, each member of the family comprises or consists essentially of an amino acid sequence that is between at least 50%, and 99% identical to SEQ ID NO:2, with each individual number within this range of between 50% and 99% also being part of the invention..

In certain embodiments, each member of the family comprises or consists essentially of an amino acid sequence that is between at least 60%, and 99% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:95, with each individual number within this range of between 60% and 99% also being part of the invention..

In some embodiments, each member of the family comprises or consists essentially of an amino acid sequence that is at least 70% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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 1D NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In certain embodiments, each member of the family comprises or consists essentially of an amino acid sequence that is at least 80% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ D NO:34, or SEQ ID NO:95. In Case PB/5-60016A some embodiments, each member of the family comprises or consists essentially of an amino acid sequence that is at least 90% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In certain embodiments, each member of the family comprises or consists essentially of an amino acid sequence that is at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ D In some embodiments of this aspect of the invention, each member of the family comprises or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID In still another aspect, the invention provides a cell genetically engineered to comprise a nucleic acid molecule encoding a polypeptide which exhibits an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin.

In some embodiments, the nucleic acid molecule is positioned for expression in the cell. In certain embodiments, the cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin protein.

In some embodiments, the cell further comprises a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin reductase protein.

In certain embodiments, the cell is a genetically engineered Streptomyces strain. In certain embodiments, the cell is a genetically engineered Streptomyces lividans strain. In particular embodiments, the genetically engineered Streptomyces lividans strain has NRRL Designation No. B-30478. In some embodiments, the cell is a genetically engineered Pseudomonas strain. In some embodiments, the cell is a genetically engineered Case PB/5-60016A Pseudomonas putida strain. In certain embodiments, the genetically engineered Pseudomonas putida strain has NRRL Designation No. B-30479. In some embodiments, the cell is a genetically engineered Escherichia coli strain.

In another aspect, the invention provides a purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin, wherein the nucleic acid molecule is isolated from a Streptomyces strain comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin.

In a specific embodiment the invention relates to an purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits the enzymatic activity of a ferredoxin, which polypeptide is substantially similar, and preferably has between at least 80%, and 99% amino acid sequence identity to the polypeptide of SEQ ID NO:36 or SEQ ID NO: 38, with each individual number within this range of between and 99% also being part of the invention.

S In still a further specific embodiment the invention relates to an purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits the enzymatic activity of a ferredoxin, which polypeptide is immunologically reactive with antibodies raised against a polypeptide of SEQ ID NO: 36 or SEQ ID NO: 38.

The invention further provides a purified nucleic acid molecule comprising a nucleotide sequence a) as given in SEQ ID NO:35 or SEQ ID NO: 37; b) having substantial similarity to c) capable of hybridizing to or the complement thereof; d) capable of hybridizing to a nucleic acid molecule comprising 50 to 200 or more consecutive nucleotides of a nucleotide sequence given in SEQ ID NO: 35 or SEQ ID NO: 37, or the complement thereof; e) complementary to or f which is the reverse complement of or or g) which is a functional part of or encoding a polypeptide that still exhibits an enzymatic activity of a ferredoxin and regioselectively oxidizes avermectin to 4"-keto-avermectin.

Case PB/5-60016A In certain embodiments, the nucleic acid molecule encoding a fen-edoxin of the invention comprises or consists essentially of a nucleic acid sequence that is at least 81% identical to SEQ ID NO:35 or SEQ ID NO:37. In some embodiments, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least or at least 90%, or at least 95%, or at least 99% identical to SEQ ID NO:35 or SEQ ID NO:37. In certain embodiments, the nucleic acid molecule encoding a ferredoxin of the invention comprises or consists essentially of the nucleic acid sequence of SEQ ID or SEQ ID NO:37.

In yet another aspect, the invention provides a purified ferredoxin protein, wherein the ferredoxin protein is isolated from a Streptomyces strain comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin. In certain embodiments, the ferredoxin of the invention comprises or consists essentially of an amino acid sequence that is at least 80% identical to SEQ ID NO:36 or SEQ ID NO:38. In some embodiments, the nucleic acid molecule comprises or consists essentially of an amino acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 99% identical to SEQ ID NO:36 or SEQ ID NO:38.

In particular embodiments, the ferredoxin of the invention comprises or consists essentially of the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:38.

In another aspect, the invention provides a purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin reductase, wherein the nucleic acid molecule is isolated from a Streptomyces strain comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin.

In certain embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin reductase comprises or consists essentially of the nucleic acid sequence of SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:104.

In yet another aspect, the invention provides a purified polypeptide exhibiting an enzymatic activity of a ferredoxin reductase protein, wherein the said polypeptide is isolated from a Streptomyces strain comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin. In certain embodiments, the 11 Case PB/5-60016A polypeptide of the invention comprises or consists essentially of the amino acid sequence of SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105.

In another aspect, the invention provides a process for the preparation a compound of the formula R8 R9N R 9 1N in which

RI-R

9 represent, independently of each other hydrogen or a substituent; m is 0, 1 or 2; n is 0, 2 or 3; and the bonds marked with A, B, C, D, E and F indicate, independently of each other, that two adjacent carbon atoms are connected by a double bond, a single bond, a single bond and a epoxide bridge of the formula H 0 H

H

2 H C H ,or a single bond and a methylene bridge of the formula 12- Case PB/5-60016A including, where applicable, an E/Z isomer, a mixture of E/Z isomers, and/or a tautomer thereof, in each case in free form or in salt form, which process comprises 1) bringing a compound of the formula

CH

2

R

6 wherein Ri-R 7 m, n, A, B, C, D, E and F have the same meanings as given for formula above, into contact with a polypeptide according to the invention that is capable of regioselectively oxidising the alcohol at position 4" in order to form a compound of the formula 13- Case PB/5-60016A 0- R5 DO 0 C OH

F

O

CH

2

R

6 R7 in which R 1

R

2

R

3

R

4

R

5

R

6

R

7 m, n, A, B, C, D, E and F have the meanings given for formula and 2) reacting the compound of the formula with an amine of the formula HN(R 8

)R

9 wherein R 8 and R 9 have the same meanings as given for formula and which is known, in the presence of a reducing agent; and, in each case, if desired, converting a compound of formula obtainable in accordance with the process or by another method, or an E/Z isomer or tautomer thereof, in each case in free form or in salt form, into a different compound of formula or an E/Z isomer or tautomer thereof, in each case in free form or in salt form, separating a mixture of E/Z isomers obtainable in accordance with the process and isolating the desired isomer, and/or converting a free compound of formula obtainable in accordance with the process or by another method, or an E/Z isomer or tautomer thereof, into a salt or converting a salt, obtainable in accordance with the process or by another method, of a compound of formula or of an E/Z isomer or tautomer thereof into the free compound of formula or an E/Z isomer or tautomer thereof or into a different salt.

In some embodiments, the compound of formula (II) is further brought into contact with a polypeptide according to the invention exhibiting an enzymatic activity of a 14- Case PB/5-60016A ferredoxin. In certain embodiments, the compound of formula is further brought into contact with a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin reductase. In some embodiments, the compound of formula (II) is further brought into contact with a reducing agent NADH or NADPH).

In still a further embodiment, the invention provides a process for the preparation of a compound of the formula 0 0

E

C OH S2

CHR,

R7 in which R 1

R

2

R

3

R

4

R

5

R

6

R

7 m, n, A, B, C, D, E and F have the meanings given for formula of claim 1, which process comprises 1) bringing a compound of the formula Case PB/5-60016A

HO

O0 0 0 R2 R1 B 3 R4 O 0 A O n m wherein Ri-R 7 m, n, A, B, C, D, E and F have the same meanings as given for formula above, into contact with a polypeptide according to the invention that is capable of regioselectively oxidising the alcohol at position maintaining said contact for a time sufficient for the oxidation reaction to occur and isolating and purifying the compound of formula In yet another embodiment, the invention provides a process according to the invention for the preparation of a compound of the formula in which n is 1;

E

mis 1; A is a double bond; B is single bond or a double bond, C is a double bond,

F

O CR6 R7 wherein D is m, n, A, B, C, D, E and F have the same meanings as given for formula above, into contact with a polypeptide according to the invention that is capable ofa double bond, regioselectively oxidising the alcohol at position maintaining said contact for a time sufficient for the bond; or a single bation reaction to occur and isolatinge; or and purifying the compoundand a of formula

(II).

In yet another embodiment, the invention provides a process according to the invention for the preparation of a compound of the formula in which n is 1; m is 1; methyA is a double bridge; B is single bond or a double bond, C is a double bond, D is a single bond, E is a double bond, F is a double bond; or a single bond and a epoxy bridge; or a single bond and a methylene bridge; RI, R2 and R3 are H; -16- Case PB/5-60016A

R

4 is methyl;

R

5 is Ci-Cio-alkyl, C 3 -C8-cycloalkyl or C 2 -Co1-alkenyl;

R

6 is H;

R

7 is OH;

R

8 and R 9 are independently of each other H; C 1 -Clo-alkyl or Ci-Clo-acyl; or together form and q is 4, 5 or 6.

S In still another embodiment, the invention provides a process according to the invention for the preparation of a compound of the formula in which n is 1; mis 1; A, B, C, E and F are double bonds; D is a single bond; Ri, R 2 and R 3 are H;

R

4 is methyl;

R

5 is s-butyl or isopropyl; R6 is H; R7 is OH; Rg is methyl

R

9 is H.

In still another embodiment, the invention provides a process according to the invention for the preparation of 4"-deoxy-4"-N-methylamino avermectin BIa/Bib benzoate salt.

In another aspect, the invention provides a method for making emamectin. The method comprises adding a polypeptide according to the invention exhibiting an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-ketoavermectin to a reaction mixture comprising avermectin and incubating the reaction mixture under conditions that allow the polypeptide to regioselectively oxidize avermectin to 4"-keto-avermectin. In some embodiments, the reaction mixture further comprises a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin. In 17- Case PB/5-60016A certain embodiments, the reaction mixture further comprises a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin reductase. In some embodiments, the reaction mixture further comprises a reducing agent NADH or

NADPH).

S In still another aspect, the invention provides a formulation for making a compound of formula comprising a polypeptide according to the invention exhibiting a P450 monooxygenase activity that is capable of regioselectively oxidising the alcohol at position 4" in order to form a compound of formula In some embodiments, the formulation further comprises a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin a ferredoxin from cell or strain from which the P450 monooxygenase was isolated or derived).

In still another aspect, the invention provides a formulation for making emamectin comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-ketoavermectin. In some embodiments, the formulation further comprises a ferredoxin a ferredoxin from cell or strain from which the P450 monooxygenase was isolated or derived).

In certain embodiments, the formulation further comprises a polypeptide according to the invention exhibiting an enzymatic activity of a ferredoxin reductase a ferredoxin from cell or strain from which the P450 monooxygenase was isolated or derived). In some embodiments, the formulation further comprises a reducing agent NADH or

NADPH).

Brief Description of the Drawings Figure 1 is a diagrammatic representation showing a map of plasmid pTBBKA.

Recognition sites by the indicated restriction endonucleases are shown, along with the location of the site in the nucleotide sequence of the plasmid. Also shown are genes kanamycin resistance "KanR"), and other functional aspects Tip promoter) contained in the plasmid.

Figure 2 is a diagrammatic representation showing a map of plasmid pTUAIA.

Recognition sites by the indicated restriction endonucleases are shown, along with the 18- Case PB/5-60016A location of the site in the nucleotide sequence of the plasmid. Also shown are genes ampicillin resistance "AmpR") and other functional aspects Tip promoter) contained in the plasmid.

Figure 3 is a diagrammatic representation showing a map of plasmid pRK-emal/fd233.

This plasmid was derived by ligating a Bgll fragment containing the emal andfd233 genes organized on a single transcriptional unit into the BglUl site of the broad host-range plasmid pRK290. The emal/fd233 genes are expressed by the tac promoter (Ptac), and they are followed by the tac terminator (Ttac). Restriction endonuclease recognition sites shown are Bgll EcoRI Pad Pmel and Sail The present invention provides a family of polypeptides which exhibit an enzymatic activity of a P450 monooxygenases and are capable of regioselectively oxidizing the alcohol at position 4" of a compound of formular such as avermectin in order to produce a compound of the formula but especially 4"-keto-avermectin.

More particularly, the family of polypeptides according to the invention may be used in a process for the preparation a compound of the formula R8 0 R9 4" O O R2 R1 B 3 R4 4O 0 A 0 n O0 R5 (1)

E

D O 0 C OH

F

O

CHR,

R7 in which

RI-R

9 represent, independently of each other hydrogen or a substituent; 19- Case PB/5-60016A m is0, 1 or 2; n is 0, 1,2 or 3; and the bonds marked with A, B, C, D, E and F indicate, independently of each other, that two adjacent carbon atoms are connected by a double bond, a single bond, a single bond and a epoxide bridge of the formula H 0 H )L1(.

,or a single bond and a methylene bridge of the formula

H

2 H C H including, where applicable, an E/Z isomer, a mixture of E/Z isomers, and/or a tautomer thereof, in each case in free form or in salt form, which process comprises 1) bringing a compound of the formula 0

HO

O 0 O R2 R1 B 3 R4 M O AO nR 0 R5 (11);

F

2CH2R wherein

RI-R

7 m, n, A, B, C, D, E and F have the same meanings as given for formula above, Case PB/5-60016A into contact with a polypeptide according to the invention which exhibits an enzymatic activity of a P450 monooxygenases and is capable of regioselectively oxidizing the alcohol at position 4" of fonnular (11) in order to produce a compound of the formula (lIn) 0O 0 O O. R2 R1 B 3 R4 O A O n

E

D o ,o C OH

F

R7 in which RI, R2, R3, R4, R5, R6, R7, m, n, A, B, C, D, E and F have the meanings given for formula and 2) reacting the compound of the formula (LI) with an amine of the formula HN(Rs)R 9 wherein R 8 and R 9 have the same meanings as given for formula and which is known, in the presence of a reducing agent; and, in each case, if desired, converting a compound of formula obtainable in accordance with the process or by another method, or an E/Z isomer or tautomer thereof, in each case in free form or in salt form, into a different compound of formula or an E/Z isomer or tautomer thereof, in each case in free form or in salt form, separating a mixture of E/Z isomers obtainable in accordance with the process and isolating the desired isomer, and/or converting a free compound of formula obtainable in accordance with the process or by another method, or an E/Z isomer or tautomer thereof, into a salt or converting a salt, obtainable in accordance with the process or by another method, of a compound of formula or of an E/Z isomer or tautomer thereof into the free compound of formula or an E/Z isomer or tautomer thereof or into a different salt.

-21 Case PB/5-60016A Methods of synthesis for the compounds of formula are described in the literature. It has been found, however, that the processes known in the literature cause considerable problems during production basically on account of the low yields and the tedious procedures which have to be used. Accordingly, the known processes are not satisfactory in that respect, giving rise to the need to make available improved preparation processes for those compounds.

The compounds (II) and (IU) may be in the form of tautomers. Accordingly, hereinbefore and hereinafter, where appropriate the compounds (II) and (111) are to be understood to include corresponding tautomers, even if the latter are not specifically mentioned in each case.

The compounds (II) and (III) are capable of forming acid addition salts. Those salts are formed, for example, with strong inorganic acids, such as mineral acids, for example perchloric acid, sulfuric acid, nitric acid, nitrous acid, a phosphoric acid or a hydrohalic acid, with strong organic carboxylic acids, such as unsubstituted or substituted, for example halosubstituted, Ci-C 4 alkanecarboxylic acids, for example acetic acid, saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric or phthalic acid, hydroxycarboxylic acids, for example ascorbic, lactic, malic, tartaric or citric acid, or benzoic acid, or with organic sulfonic acids, such as unsubstituted or substituted, for example halosubstituted, C 1

-C

4 alkane- or aryl-sulfonic acids, for example methane- or p-toluene-sulfonic acid. Furthermore, compounds of formula (II) and (111) having at least one acidic group are capable of forming salts with bases. Suitable salts with bases are, for example, metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium salts, or salts with ammonia or an organic amine, such as morpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, diethyl-, triethyl- or dimethyl-propyl-amine, or a mono-, di- or tri-hydroxy-lower alkylamine, for example mono-, di- or tri-ethanolamine. In addition, corresponding internal salts may also be formed. Preference is given within the scope of the invention to agrochemically advantageous salts. In view of the close relationship between the compounds of formula (II) and in free form and in the form of their salts, any reference hereinbefore or hereinafter to the free compounds of formula and (11I) or to their respective salts is to be understood as including also the corresponding salts or the free compounds of formula (II) and where appropriate and -22- Case PB/5-60016A expedient. The same applies in the case of tautomers of compounds of formula and and the salts thereof. The free form is generally preferred in each case.

Preferred within the scope of this invention is a process for the preparation of compounds of the formula in which n is 1; m is 1: A is a double bond; B is single bond or a double bond, C is a double bond, D is a single bond, E is a double bond, F is a double bond; or a single bond and a epoxiy bridge; or a single bond and a methylene bridge; Ri, R 2 and R 3 are H;

R

4 is methyl; Rs is Cj-Clo-alkyl, C 3 -Cs-cycloalkyl or C 2 -Co 1 -alkenyl; R6 is H;

R

7 is OH; Rg and R 9 are independently of each other H; Ci-Clo-alkyl or Ci-Clo-acyl; or together form and q is 4, 5 or 6.

Especially preferred within the scope of this invention is a process for the preparation of a compound of the formula in which n is 1; m is 1; A, B, C, E and F are double bonds; D is a single bond;

R

1

R

2 and R 3 are H;

R

4 is methyl;

R

5 is s-butyl or isopropyl; -23- Case PB/5-60016A

R

6 is H; R7 is OH; Rs is methyl

R

9 is H.

Very especially preferred is a process for the preparation of emamectin, more particularly the benzoate salt of emamectin. Emamectin is a mixture of 4"-deoxy-4"-Nmethylamino avermectin Bla/Bib and is described in US-P-4,4874,749 and as MK-244 in Journal of Organic Chemistry, Vol. 59 (1994), 7704-7708. Salts of emamectin that are especially valuable agrochemically are described in US-P-5,288,710. Each member of this family of peptides exhibiting an enzymatic activity of a P450 monooxygenases as described hereinbefore is able to oxidize unprotected avermectin regioselectively at position thus opening a new and more economical route for the production of emamectin.

The family members each catalyze the following reaction: 0o- o- o r 0 O Oo- H O- g 0 0 0 0 H H 0 0 0 H- I 0 S0 OZ, Y

S

R

0 5 51 'H family member chemical conversion 0o OH by reductive amination OH avermectin 4 "-keto-avermectin emamectin Sla and Blb Bla (R=CH and Blb

R=CHH

Accordingly, the invention provides a purified nucleic acid molecule encoding a polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and is capable of regioselectively oxidizing the alcohol at position 4" of a compound of formular (II) such as avermectin in order to produce a compound of formula (Ill), but especially 4"-ketoavermectin.

In particular, the invention provides a purified nucleic acid molecule encoding a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin. A "nucleic -24- Case PB/5-60016A acid molecule" refers to single-stranded or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogs of either DNA or RNA.

The invention also provides a purified polypeptide that exhibits an enzymatic activity of a P450 monooxygenase and is capable of regioselectively oxidizing the alcohol at position 4" of a compound of formular such as avermectin in order to produce a compound of formula but especially 4"-keto-avermectin.

In particular, the invention also provides a purified P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin.

As used herein, by "purified" is meant a nucleic acid molecule or polypeptide an enzyme or antibody) that has been separated from components which naturally accompany it.

An example of such a nucleotide sequence or segment of interest "purified" from a source, would be nucleotide sequence or segment that is excised or removed from said source by chemical means, by the use of restriction endonucleases, so that it can be further manipulated, amplified, for use in the invention, by the methodology of genetic engineering. Such a nucleotide sequence or segment is commonly referred to as "recombinant.". In one specific aspect, the purified nucleic acid molecule may be separated from nucleotide sequences, such as promoter or enhancer sequences, that flank the nucleic acid molecule as it naturally occurs in the chromosome.

In the case of a protein or a polypeptide, the purified protein and polypeptide, respectively, is separated from components, such as other proteins or fragments of cell membrane, that accompany it in the cell. Of course, those of ordinary skill in molecular biology will understand that water, buffers, and other small molecules may additionally be present in a purified nucleic acid molecule or purified protein preparation. A purified nucleic acid molecule or purified polypeptide enzyme) of the invention that is at least 95% by weight, or at least 98% by weight, or at least 99% by weight, or 100% by weight free of components which naturally accompany the nucleic acid molecule or polypeptide.

According to the invention, a purified nucleic acid molecule may be generated, for example, by excising the nucleic acid molecule from the chromosome. It may then be ligated into an expression plasmid. Other methods for generating a purified nucleic acid molecule encoding a P450 monooxygenase of the invention are available and include, without limitation, artificial synthesis of the nucleic acid molecule on a nucleic acid synthesizer.

Case PB/5-60016A Similarly, a purified P450 monooxygenase of the invention may be generated, for example, by recombinant expression of a nucleic acid molecule encoding the P450 monooxygenase in a cell in which the P450 monooxygenase does not naturally occur. Of course, other methods for obtaining a purified P450 monooxygenase of the invention include, without limitation, artificial synthesis of the P450 monooxygenase on a polypeptide synthesizer and isolation of the P450 monooxygenase from a cell in which it naturally occurs using, an antibody that specifically binds the P450 monooxygenase.

Biotransformations of secondary alcohols to ketones by Streptomyces bacteria are known to be catalyzed by dehydrogenases or oxidases. However, prior to the present discovery of the cytochrome P450 monooxygenase from Streptomyces lubercidicus strain R- 922 responsible for the regioselective oxidation of avermectin to 4"-keto-avermectin, no experimental data of another cytochrome P450 monooxygenase from Streptomyces to oxidize a secondary alcohol to a ketone had been reported.

According to some embodiments of the invention, the nucleic acid molecule and/or the polypeptide encoded by the nucleic acid molecule are isolated from a Streptomyces strain.

Thus, the nucleic acid molecule (or polypeptide encoded thereby) may be isolated from, without limitation, Streptomyces tubercidicus, Streptomyces lydicus, Streptomyces platensis, Streptomyces chattanoogensis, Streptomyces kasugaensis, Streptomyces rimosus, and Streptomyces albofaciens.

As mentioned above and described in more detail below, an entire family of polypeptides exhibiting an enzymatic activity of P450 monooxygenases capable of regioselectively oxidizing avermectin to 4"-keto-avermectin are provided herein. All of these family members are related by at least 60% identity at the amino acid level. A useful nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase comprises or consists essentially of a nucleic acid sequence that is at least 70% identical to SEQ ID NO: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94. In certain embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase -26 Case PB/5-60016A comprises or consists essentially of a nucleic acid sequence that is at least 80% identical; or at least 85% identical; or at least 90% identical; or at least 95% identical; or at least 98% identical to SEQ ID NO: I, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ 1ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ FD NO:94.

Similarly, the invention provides a purified polypeptide exhibiting an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin which, in some embodiments, comprises or consists essentially of an amino acid sequence that is at least 60% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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 SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:95. In certain embodiments, the purified polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase comprises or consists essentially of an amino acid sequence that is at least 70% identical; or at least identical; or at least 90% identical; or at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In some embodiments, the nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase comprises or consists essentially of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33. or SEQ ID NO:94. Similarly, the purified polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase, in some embodiments, comprises or consists essentially of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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 SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID -27- Case PB/5-60016A To describe the sequence relationships between two or more nucleic acids or polynucleotides the following terms are used: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity".

As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full length cDNA or gene sequence, or the complete cDNA or gene sequence.

As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Preferred, non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller, 1988; the local homology algorithm of Smith et al.

1981; the homology alignment algorithm of Needleman and Wunsch 1970; the search-forsimilarity-method of Pearson and Lipman 1988; the algorithm of Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA).

Alignments using these programs can be performed using the default parameters. The -28- Case PB/5-60016A CLUSTAL program is well described by Higgins el al. 1988; Higgins et al. 1989; Corpet ct al.

1988; Huang et al. 1992; and Pearson et al. 1994. The ALIGN program is based on the algonrithm of Myers and Miller, supra. The BLAST programs of Altschul et al., 1990, are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always 0) and N (penalty score for mismatching residues; always For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, Karlin Altschul (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST can be utilized as described in Altschul et al. 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships -29- Case PB/5-60016A between molecules. See Altschul et al., supra. When utilizing BLAST, Gapped BLAST, PSI- BLAST, the default parameters of the respective programs BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength of 11, an expectation of 10, a cutoff of 100, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation of 10, and the BLOSUM62 scoring matrix (see Henikoff Henikoff, 1989). See http://www.ncbi.n m.nih.gov. Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the nucleotide sequences disclosed herein is preferably made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.

As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of I and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero Case PB/5-60016A and 1. The scoring of conservative substitutions is calculated, as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).

As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a companson window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 66%. 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, more preferably at least 80%, 90%, and most preferably at least Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions (see below). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1°C to about 20 0 C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, when a copy of a nucleic acid is created using the maximum codon degeneracy pennitted by the genetic code. One indication that two -31 Case PB/5-60016A nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

The term "substantial identity" in the context of a polypeptide indicates that a polypeptide comprises a sequence with at least 50%, 60%, 70%, 71%, 72%, 73%, 74%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%, or even more preferably, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970). An indication that two polypeptide sequences are substantially identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern -32- Case PB/5-60016A hybridization are sequence dependent, and are different under different environmental parameters. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, 1984; Tm 81.5°C 16.6 (log M) +0.41 0.61 form) 500/L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about 1°C for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 0 C. Generally, stringent conditions are selected to be about 5 0 C lower than the thermal melting point I for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 0 C lower than the thermal melting point I; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 0 C lower than the thermal melting point I; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 0 C lower than the thermal melting point I. Using the equation, hybridization and wash compositions, and desired T, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T of less than (aqueous solution) or 32 0 C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, 1993. Generally, highly stringent hybridization and wash conditions are selected to be about 5 0 C lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCI at 72 0 C for about minutes. An example of stringent wash conditions is a 0.2X SSC wash at 65 0 C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium -33- Case PB/5-60016A stringency wash for a duplex of, more than 100 nucleotides, is IX SSC at 45°C for minutes. An example low stringency wash for a duplex of, more than 100 nucleotides, is 4-6X SSC at 40°C for 15 minutes. For short probes about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C and at least about 60 0 C for long robes nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2X (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is formamide, hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37 0 C, and a wash in 0. IX SSC at 60 to 65°C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 37 0 C, and a wash in IX to 2X SSC (20X SSC 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 0 C. Exemplary moderate stringency conditions include hybridization in 40 to formamide, 1.0 M NaCI, 1% SDS at 37 0 C, and a wash in 0.5X to IX SSC at 55 to The following are examples of sets of hybridization/wash conditions that may be used to clone orthologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in 2X SSC, 0.1% SDS at 50 0 C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 1 mM EDTA at 50 0 C with washing in IX SSC, 0.1% SDS at 50 0 C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 I mM EDTA at 50 0 C with washing in 0.5X SSC, 0.1% SDS at 50 0 C, preferably in 7% sodium -34- Case PB/5-60016A dodecyl sulfate (SDS), 0.5 M NaP0 4 1 mM EDTA at 50'C with washing in 0.1X SSC, 0.1% SDS at 50 0 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0 4 1 mM EDTA at 50 0 C with washing in 0.1X SSC, 0.1% SDS at 65 0

C.

One non-limiting source of a purified polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"keto-avermectin is the cell-free extract described in the examples below. Another method for purifying a polypeptide exhibiting a P450 monooxygenase activity in accordance with the invention is to attach a tag to the protein, thereby facilitating its purification. Accordingly, the invention provides a purified polypeptide exhibiting an enzymatic activity of a P450 monooxygenase which regioselectively oxidizes avermectin to 4"-keto-avermectin, wherein the polypeptide is covalently bound to a tag. The invention further provides a nucleic acid molecule encoding such a tagged polypeptide.

As used herein, a "tag" is meant a polypeptide or other molecule covalently bound to a polypeptide of the invention, whereby a binding agent a polypeptide or molecule) specifically binds the tag. In accordance with the invention, by "specifically binds" is meant that the binding agent an antibody or Ni 2 resin) recognizes and binds to a particular polypeptide or chemical but does not substantially recognize or bind to other molecules in the sample. In some embodiments, a binding agent that specifically binds a ligand forms an association with that ligand with an affinity of at least 10 6 M or at least 10 7 M, or at least M or at least 109 M' either in water, under physiological conditions, or under conditions which approximate physiological conditions with respect to ionic strength, 140 mM NaCI, 5 mM MgCI 2 For example, a His tag is specifically bound by nickel the Ni 2 charged column commercially available as His-Bind® Resin from Novagen Inc, Madison, WI). Likewise, a Myc tag is specifically bound by an antibody that specifically binds Myc.

As described below, a His tag is attached to the purified polypeptide of the invention exhibiting an enzymatic activity of a P450 monooxygenase by generating a nucleic acid molecule encoding the His-tagged polypeptide, and expressing the polypeptide in E. coli.

These polypeptides, once expressed by E. coli, are readily purified by standard techniques using one of the His*Bind® Kits commercially available from Novagen or using the TALONTM Resin (and manufacturer's instructions) commercially available from Clontech Laboratories, Inc., Palo Alto, CA).

Case PB/5-60016A Additional tags may be attached to any or all of the polypeptides of the invention to facilitate purification. These tags include, without limitation, the HA-Tag (amino acid sequence: YPYDVPDYA (SEQ ID NO:39)), the Myc-tag (amino acid sequence: EQKLISEEDL (SEQ ID NO:40)), the HSV tag (amino acid sequence: QPELAPEDPED (SEQ ID NO:41)), and the VSV-G-Tag (amino acid sequence: YTDIEMNRLGK (SEQ ID NO:42)).

Covalent attachment via a polypeptide bond) of these tags to a polypeptide of the invention allows purification of the tagged polypeptide using, respectively, an anti-HA antibody, an anti-Myc antibody, an anti-HSV antibody, or an anti-VSV-G antibody, all of which are commercially available (for example, from MBL International Corp., Watertown, MA; Novagen Inc.; Research Diagnostics Inc., Flanders, NJ).

The tagged polypeptides of the invention exhibiting a P450 monooxygenase activity may also be tagged by a covalent bond to a chemical, such as biotin, which is specifically bound by streptavidin, and thus may be purified on a streptavidin column. Similarly, the tagged P450 monooxygenases of the invention may be covalently bound via a polypeptide bond) to the constant region of an antibody. Such a tagged P450 monooxygenase may be purified, for example, on protein A sepharose.

The tagged P450 monooxygenases of the invention may also be tagged to a GST (glutathione-S-transferase) or the constant region of an immunoglobulin. For example, a nucleic acid molecule of the invention comprising SEQ ID NO:1) can be cloned into one of the pGEX plasmids commercially available from Amersham Pharmacia Biotech, Inc.

(Piscataway NJ), and the plasmid expressed in E. coli. The resulting P450 monooxygenase encoded by the nucleic acid molecule is covalently bound to a GST (glutathione-Stransferase). These GST fusion proteins can be purified on a glutathione agarose column (commercially available from, Amersham Pharmacia Biotech), and thus purified. Many of the pGEX plasmids enable easy removal of the GST portion from the fusion protein. For example, the pGEX-2T plasmid contains a thrombin recognition site between the inserted nucleic acid molecule of interest and the GST-encoding nucleic acid sequence. Similarly, the pGES-3T plasmid contains a factor Xa site. By treating the fusion protein with the appropriate enzyme, and then separating the GST portion from the P450 monooxygenase of the invention using glutathione agarose (to which the GST specifically binds), the P450 monooxygenase of the invention can be purified.

-36- Case PB/5-60016A Yet another method to obtain a purified polypeptide of the invention exhibiting a P450 monooxygenase activity is to use a binding agent that specifically binds to such a polypeptide.

Accordingly, the invention provides a binding agent that specifically binds to a P450 monooxygenase of the invention. This binding agent of the invention may be a chemical compound a protein), a metal ion, or a small molecule.

In particular embodiments, the binding agent is an antibody. The term "antibody" encompasses, without limitation, polyclonal antibody, monoclonal antibody, antibody fragments Fab, Fv, or Fab' fragments), single chain antibody, chimeric antibody, bispecific antibody, antibody of any isotype IgG, IgA, and IgE), and antibody from any specifies rabbit, mouse, and human).

In one non-limiting example, the binding agent of the invention is a polyclonal antibody.

In another non-limiting example, the binding agent of the invention is a monoclonal antibody.

Methods for making both monoclonal and polyclonal antibodies are well known (see, e.g., Current Protocols in Immunology, ed. John E. Coligan, John Wiley Sons, Inc. 1993; Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley Sons, Inc. 2000).

The polypeptides described herein exhibiting an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin belong to a family of novel P450 monooxygenases. Accordingly, the invention also provides a family of P450 monooxygenase polypeptides, wherein each member of the family regioselectively oxidizes avermectin to 4"-keto-avermectin. In some embodiments, each member of the family comprises or consists of an amino acid sequence that is at least 50% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ D 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, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID In particular embodiments, each member of the family is encoded by a nucleic acid molecule comprising or consisting of a nucleic acid sequence that is at least 66% identical to SEQ [D NO: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ [D NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:94.

-37- Case PB/5-60016A The present invention, which provides an entire family of P450 monooxygenases, each member of which is able to regioselectively oxidize avermectin to 4"-keto-avermectin, allowed for the generation of an improved P450 monooxygenase, which may not be naturally occurring, but which regioselectively oxidizes avermectin to 4"-keto-avermectin with efficiency and with reduced undesirable side product. For instance, one of the members of the P450 monooxygenase family of the invention, P 4 50EmaI enzyme catalyzes a further oxidation that is not desirable, since the formation of 3"-O-demethyl-4"-keto-avermectin has been detected in the reaction by Streptomyces tubercidicus strain R-922 and by Streplomyces lividans containing the emal gene. The formation of 3"-O-demethyl-4"-keto-avermectin is brought about by the oxidation of the 3"-O-methyl group, whereby the hydrolytically labile 3"-O-hydroxymethyl group is formed which hydrolyzes to form formaldehyde and the 3"hydroxyl group.

By providing a family of polypeptides exhibiting an enzymatic activitiy of P450 monooxygenases that regioselectively oxidize avermectin to 4"-keto-avermectin (see, e.g., Table 3 below), individual members of the family can be subjected to family gene shuffling efforts in order to produce new hybrid genes encoding optimized P450 monooxygenases of the invention. In one non-limiting example, a portion of the emal gene encoding the 02 binding site of the P 4 50EmaI protein can be swapped with the portion of the ema2 gene encoding the 02 binding site of the P 4 50Ema2 protein. Such a chimeric emal/2 protein is within definition of a P450 monooxygenase of the invention.

Site-directed mutagenesis or directed evolution technologies may also be employed to generate derivatives of the emal gene that encode enzymes with improved properties, including higher overall activity and/or reduced side product formation. One method for deriving such a mutant is to mutate the Streptomyces strain itself, in a manner similar to the UV mutation of Streplomyces rubercidicus strain R-922 described below.

Additional derivatives may be made by making conservative or non-conservative changes to the amino acid sequence of a P450 monooxygenase. Conservative and nonconservative amino acid substitutions are well known (see, Stryer, Biochemistiy, 3 rd Ed., W.H. Freeman and Co., NY 1988). Similarly, truncations of a P450 monooxygenase of the invention may be generated by truncating the protein at its N-terminus see the emalA -38- Case PB/5-60016A gene described below), at its C-terminus, or truncating removing amino acid residues) from the middle of the protein.

Such a mutant, derivative, or truncated P450 monooxygenase is a P450 monooxygenase of the invention as long as the mutant, derivative, or truncated P450 monooxygenase is able to regioselectively oxidize avermectin to 4"-keto-avermectin.

In another aspect, the invention provides a cell genetically engineered to comprise a nucleic acid molecule encoding a polypeptide which exhibits an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin. By "genetically engineered" is meant that the nucleic acid molecule is exogenous to the cell into which it is introduced. Introduction of the exogenous nucleic acid molecule into the genetically engineered cell may be accomplished by any means, including, without limitation, transfection, transduction, and transformation.

In certain embodiments, the nucleic acid molecule is positioned for expression in the genetically engineered cell. By "positioned for expression" is meant that the exogenous nucleic acid molecule encoding the polypeptide is linked to a regulatory sequence in such a way as to permit expression of the nucleic acid molecule when introduced into a cell. By "regulatory sequence" is meant nucleic acid sequences, such as initiation signals, polyadenylation (polyA) signals, promoters, and enhancers, which control expression of protein coding sequences with which they are operably linked. By "expression" of a nucleic acid molecule encoding a protein or polypeptide fragment is meant expression of that nucleic acid molecule as protein and/or mRNA.

A genetically engineered cell of the invention may be a prokayotic cell E. coli) or a eukaryotic cell Saccharomyces cerevisiae or mammalian cell HeLa)). According to some embodiments of the invention, the genetically engineered cell is a cell wherein the wild-type not genetically engineered) cell does not naturally contain the inserted nucleic acid molecule and does not naturally express the protein encoded by the inserted nucleic acid molecule. Accordingly, the cell may be a genetically engineered Streptomyces strain, such as a Streptomyces lividans or a Streptomyces avermitilis strain. Alternatively, the cell may be a genetically engineered Pseudomonas strain, such as a Pseudomonas putida strain or a Pseudomonas fluorescens strain. In another alternative, the cell may be a genetically engineered Escherichia coli strain.

-39- Case PB/5-60016A Note that in some types of cells genetically engineered to comprise a nucleic acid molecule encoding a polypeptide which exhibits an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin, the actual genetically engineered cell, itself, may not be able to convert avermectin into 4"-ketoavermectin. Rather, the P450 monooxygenase heterogously expressed by such a genetically engineered cell may be purified from that cell, where the purified P450 monooxygenase of the invention can be used to regioselectively oxidize avermectin to 4"-keto-avermectin. Thus, the genetically engineered cell of the invention need not, itself, be able to regioselectively convert avermectin to 4"-keto-avermectin; rather, the genetically engineered cell of the invention need only comprise a nucleic acid molecule encoding a polypeptide which exhibits an enzymatic activity of a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-ketoavermectin, regardless of whether the polypeptide is active inside that cell.

In addition, a cell E. coli) geneticially engineered to comprise a nucleic acid molecule encoding a polypeptide of the invention which exhibits an enzymatic activity of a P450 monooxygenase may not be able to regioselectively oxidize avermectin to 4"-ketoavermectin, although the P450 monooxygenase purified from the genetically engineered cell is able to regioselectively oxidize avermectin to 4"-keto-avermectin. However, if the same cell were genetically engineered to comprise a polypeptide of the invention which exhibits an enzymatic activity of a P450 monooxygenase, a ferredoxin of the invention, and/or a ferredoxin reductase of the invention, then the P450 monooxygenase together with the ferredoxin and the ferredoxin reductase, all purified from that cell,and in the presence of a reducing agent NADH or NADPH), would be able to regioselectively oxidize avermectin to 4"-keto-avermectin. Furthermore the genetically engineered cell comprising a P450 monooxygenase of the invention, a ferredoxin of the invention, and a ferredoxin reductase of the invention, itself; might be able to carry out this oxidation.

Moreover, in a non-limiting example where a cell E. coli) is genetically engineered to express P450 monooxygenase, a ferredoxin, and a ferredoxin reductase proteins of the invention, all three of these proteins, when purified from the genetically engineered E. coli, are together and in the presence of a reducing agent NADH or NADPH) would be active and able to regioselectively oxidize avermectin to 4"-keto-avermectin, and so are useful in a method for making emamectin.

Case PB/5-60016A In accordance with the present invention, the following material has been deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: (1) Streptomyces lividans ZX7 (emal/fd233-TUA IA) NRRL Designation No. B-30478; and (2) Pseudomonas putida NRRL B-4067 containing plasmid pRK290-enmal/fd233, NRRL Designation No.B-30479 In identifying the novel family of polypeptides exhibiting an enzymatic activity of P450 monooxygenases that regioselectively oxidize avermectin to 4"-keto-avermectin, novel ferredoxins and novel ferredoxin reductases were also identified in the same strains of bacteria in which the P450 monooxygenases were found. Accordingly, in a further aspect, the invention provides a purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a ferredoxin, wherein the nucleic acid molecule is isolated from a Streptomyces strain comprising a polypeptide that regioselectively oxidizes avermectin to 4"-keto-avermectin. Similarly, the invention provides a purified nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a ferredoxin reductase, wherein the nucleic acid molecule is isolated from a Streptomyces strain comprising a polypeptide that regioselectively oxidizes avermectin to 4"-keto-avermectin. The invention also provides a purified protein that exhibits an enzymatic activity of a ferredoxin, as well as a purified protein that exhibits an enzymatic activity of a ferredoxin reductase, wherein the ferredoxin protein and the ferredoxin reductase protein are isolated from a Streptomyces strain comprising a polypeptide that regioselectively oxidizes avermectin to 4"-keto-avermectin.

A useful nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a ferredoxin comprises or consists essentially of a nucleic acid sequence that is at least 81% identical to SEQ ID NO:35 or SEQ ID NO:37. Alternatively, the nucleic acid molecule comprises or consists essentially of a nucleic acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 99% identical to SEQ ID NO:35 or SEQ ID NO:37. The nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that exhibits an enzymatic activity of a ferredoxin -41 Casc PB/5-60016A may comprise or consist essentially of the nucleic acid sequence of SEQ ID NO:35 or SEQ ID NO:37.

The protein of the invention exhibiting a ferredoxin activity may comprise or consist essentially of an amino acid sequence that is at least 80% identical to SEQ ID NO:36 or SEQ ID NO:38. In some embodiments, the nucleic acid molecule comprises or consists essentially an amino acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 99% identical to SEQ ID NO:36 or SEQ [D NO:38. The ferredoxin of the invention may comprise or consist essentially of the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:38.

A useful nucleic acid molecule comprising a nucleotide sequence encoding a protein of the invention exhibiting a ferredoxin reductase comprises or consists essentially of the nucleic acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 99% identical to SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, or SEQ ID NO:104. In a particular embodiment of the invention, the nucleic acid molecule encoding a ferredoxin reductase of the invention may comprise or consist essentially of the amino acid sequence of SEQ ID NO:98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.

The ferredoxin reductase of the invention may comprise or consist essentially of the amino acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 99% identical to SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105. In a particular embodiment of the invention, the ferredoxin reductase of the invention may comprise or consist essentially of the amino acid sequence of SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, or SEQ ID NO:105.

Methods for purifying ferredoxin and ferredoxin reductase proteins and nucleic acid molecules encoding such ferredoxin and ferredoxin reductase proteins are known in the art and are the same as those described above for purifying P450 monooxygenases of the invention and nucleic acid molecules encoding P450 monooxygenases of the invention.

In one non-limiting example to obtain a purified P450 monooxygenase of the invention with a purified ferredoxin, a S. lividans strain (or P. putida strain, or any other cell in which the P450 monooxygenase of the invention does not naturally occur) may be genetically engineered to contain a first nucleic acid molecule encoding a P450 monooxygenase of the invention and a second nucleic acid molecule encoding a ferredoxin protein, where both the first and second nucleic acid molecules are positioned for expression in the genetically -42- Case PB/5-60016A engineered cell. The first and the second nucleic acid molecules can be on separate plasmids, or can be on the same plasmid. Thus, the same engineered cell or strain will produce both the P450 monooxygenase of the invention and the ferredoxin protein of the invention.

In a further non-limiting example to obtain a purified P450 monooxygenase of the invention with a purified ferredoxin and with a purified ferredoxin reductase of the invention, a S. lividans strain (or P. putida strain, or any other cell in which the P450 monooxygenase of the invention does not naturally occur) may be genetically engineered to contain a first nucleic acid molecule encoding a P450 monooxygenase of the invention and a second nucleic acid molecule encoding a ferredoxin protein of the invention and a third nucleic acid molecule encoding a ferredoxin reductase protein of the invention, where all the first and second and third nucleic acid molecules are positioned for expression in the genetically engineered cell.

The first and the second and the third nucleic acid molecules may be provided on separate plasmids, or on the same plasmid. Thus, the same engineered cell or strain will produce all the P450 monooxygenase of the invention and the ferredoxin and the ferredoxin reductase proteins of the invention.

As described above for the P450 monooxygenases of the invention, the ferredoxin protein and/or the ferredoxin reductase protein may further comprise a tag. Moreover, the invention contemplates binding agents antibodies) that specifically bind to the ferredoxin protein, and binding agents that specifically bind to the ferredoxin reductase proteins of the invention. Methods for generating tagged ferredoxin protein, tagged ferredoxin reductase protein, and binding agents antibodies) that specifically bind to ferredoxin or ferredoxin reductase are the same as those as described above for generating tagged P450 monooxygenases of the invention and generating binding agents that specifically bind P450 monooxygenases of the invention.

The invention also provides a method for making emamectin. In this method, a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin is added to a reaction mixture containing avermectin. The reaction mixture is then incubated under conditions that allow the P450 monooxygenase to regioselectively oxidize avermectin to 4"keto-avermectin. The reaction mixture may further comprise a ferredoxin, such as a ferredoxin of the present invention. In particular embodiments, the reaction mixture further -43 Case PB/5-60016A comprises a ferredoxin reductase such as a ferredoxin of the present invention. The reaction mixture may further comprise a reducing agent, such as NADH or NADPH.

Additionally, the invention provides a method for making 4"-keto-avermectin. The method comprises adding a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin to a reaction mixture comprising avermectin and incubating the reaction mixture under conditions that allow the P450 monooxygenase to regioselectively oxidize avermectin to 4"-keto-avermectin. In some embodiments, the reaction mixture further comprises a ferredoxin, such as a ferredoxin of the present invention. The reaction mixture may also further comprise a ferredoxin reductase such as a ferredoxin of the present invention.

In particular embodiments, the reaction mixture further comprises a reducing agent, such as NADH or NADPH.

The invention also provides a formulation for making emamectin comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-keto-avermectin. In some embodiments, the formulation further comprises a ferredoxin, such as a ferredoxin of the present invention. In particular embodiments, the ferredoxin is isolated from the same species of cell or strain from which the P450 monooxygenase was isolated or derived. The formulation may further comprise a ferredoxin reductase such as a ferredoxin reductase of the present invention. In particular embodiments, the ferredoxin reductase is isolated from the same species of cell or strain from which the P450 monooxygenase was isolated or derived.

In some embodiments, the formulation further comprises a reducing agent, such as NADH or

NADPH.

In addition, the invention provides a formulation for making 4"-keto-avermectin comprising a P450 monooxygenase that regioselectively oxidizes avermectin to 4"-ketoavermectin. In some embodiments, the formulation further comprises a ferredoxin, such as a ferredoxin of the present invention. In particular embodiments, the ferredoxin is isolated from the same species of cell or strain from which the P450 monooxygenase was isolated or derived. In some embodiments, the formulation further comprises a ferredoxin reductase, such as a ferredoxin reductase of the present invention. In particular embodiments, the ferredoxin reductase is isolated from the same species of cell or strain from which the P450 monooxygenase was isolated or derived. The formulation may further comprise a reducing agent, such as NADH or NADPH.

-44- Case PB/5-60016A The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature.

EXAMPLE I Optimized Growth Conditions for Streptomyces tubercidicus Strain R-922 In one non-limiting example the fermentation conditions needed to provide a steady supply of cells of Streptomyces tubercidicus strain R-922 highly capable of regioselectively oxidizing avermectin to 4"-keto-avermectin were optimized.

First, the following solutions were made. For ISP-2 agar, 4 g of yeast extract (commercially available from Oxoid Ltd, Basingstoke, UK), 4 g of D(+)-glucose, 10 g of bacto malt extract (Difco No. 0186-17-7 (Difco products commercially available from, e.g., Voigt Global Distribution, Kansas City, and 20 g of agar (Difco No. 0140-01) were dissolved in one liter of demineralized water, and the pH is adjusted to 7.0. The solution was sterilized at 121 0 C for 20 min., cooled down, and kept at 55 0 C for the time needed for the immediate preparation of the agar plates.

For PHG medium, 10 g of peptone (Sigma 0521; commercially available from Sigma Chemical Co., St. Louis, MO), 10 g of yeast extract (commercially available from Difco), 10 g of D-(+)-glucose, 2 g of NaCI, 0.15 g of MgSO 4 x 7 H 2 0, 1.3 g of NaH 2

PO

4 x H 2 0, and 4.4 g of K 2

HPO

4 were dissolved in 1 liter of demineralized water, and the pH was adjusted to Streptomyces tubercidicus strain R-922 was grown in a Petri dish on ISP-2 agar at 28 0

C.

This culture was used to inoculate four 500 ml shaker flasks with a baffle, each containing 100 ml PHG medium. These pre-cultures were grown on an orbital shaker at 120 rpm at 28°C for 72 hours and then used to inoculate a 10-liter fermenter equipped with a mechanical stirrer and containing 8 liters of P'I-G medium. This main culture was grown at 28 0 C with stirring at 500 rpm and with aeration of 1.75 vvm (14 1/min.) and a pressure of 0.7 bar. At the end of the exponential growth, after about 20 hours, the cells were harvested by centrifugation. The yield of wet cells was 70-80 g/l culture.

EXAMPLE II Case 113/5-6001 6A Whole Cell Biocatalysis Assay As determined in accordance with the present invention, the following whole cell biocatalysis assay was employed to determine that the activity from Streptomyces cells capable of regioselectively oxidizing avermectin to 4"-keto-avermectin is catalyzed by a P450 monooxygenase.

Streptomyces tubercidicus strain R-922 was grown in PHG medium, and Streptomyces tubercidicus strain 1-1529 was grown in M-17 or PHG medium. PHG medium contains 10 g/1 Peptone (Sigma, 0.521), 10 g/l Yeast Extract (Difco, 0127-17-9), 10 g/l D-Glucose, 2 g/l NaCI, 0.15 g/l MgSO 4 x 7 H 2 0, 1.3 g/l NaH 2

PO

4 x 1 H20, and 4.4 g/l K 2

HPO

4 at pH 7.0. M- 17 medium contains 10 g/l glycerol, 20 g/l Dextrin white, 10 g/1 Soytone (Difco 0437-17), 3 g/1 Yeast Extract (Difco 0127-17-9), 2 g/l (NH 4 2 S0 4 and 2 g/l CaCO 3 at pH To grow the cells, an ISP2 agar plate (not older than 1-2 weeks) was inoculated and incubated for 3-7 days until good growth was achieved. Next, an overgrown agar piece was transferred (with an inoculation loop) to a 250ml Erlenmeyer flask with 1 baffle containing ml PHG medium. This pre-culture is incubated at 28'C and 120 rpm for 2-3 days. Next, 5 ml of the pre-culture were transferred to a 500 ml Erlenmeyer flask with 1 baffle containing 100 ml PHG medium. The main culture was incubated at 28 0 C and 120 rpm for 2 days. Next, the culture was centrifuged for 10 min. at 8000 rpm on a Beckman Rotor JA-14. The cells were next washed once with 50 mM potassium phosphate buffer, pH To perform the whole cell biocatalysis assay, 500 mg wet cells were placed into a ml Erlenmeyer flask, to which were added 10 ml of 50 mM potassium phosphate buffer, pH The cells were stirred with a magnetic stir bar to distribute the cells. Next, 15 pl of a solution of avermectin Bla in isopropanol (30 mg/ml) were added, and the mixture shaken on an orbital shaker at 160 rpm and 28°C. Strain R-922 was reacted for 2 hours, and strain 1- 1529 was reacted for 30 hours.

To work up the cultures in the whole cell biocatalysis assay, 10 ml methyl-t-butyl-ether was added to an Erlenmeyer flask containing the resting cells and the entire cell mixture was transferred to a 30 ml-centrifuge tube, shaken vigorously, and then centrifuged at 16000 rpm for 10 min. The ether phase was pipetted into a 50 ml pear flask, and evaporated in vacuo by means of a rotary evaporator mbar). The residue was re-dissolved in 1.2 ml acetonitrile -46- Case PB/5-60016A and transferred to an.1-PLC-sample vial. The conversion of averm-ectin B I a to 4"-hydroxyavermectin BlIa and 4"-keto-averrmecti n BlIa (also called 4"-oxo-avermecti n BlIa) and the formation of a side product fromn the biocatalysis reaction Could be observcd by HPLC analysis using 1IfPLC protocol 1.

.For B-PLC protocol 1, the following p~aramleters were used: H-Iardware Pump: Autosampler: Interface Module: Channel 1-Detector: Column Oven: Column: Adsorbent: L-6250 Merck-Hitachi AS-2000A Merck-Hitachi D-6000 Merck-Hitachi L-7450A UV-Diode Array Merck-1-lachi none 70mm x 4mm Kromnasil 100 1 &-3.5jt-C1 8 Gradient Mode: Low Pressure Limit.- Column Temperature Solvent A: Solvent B: Flow: Detection: Pump Table: 0.0 linear gradient 7.0 j .ump 9.1 12.0 Stop timne: 12 m Sampling Period: Retention time table: 5-300bar ambient (;z2 0

C)

acetonitrile water 1.5 mI/mm 243 nm min 75% min 100% min 100% min 75% Mill 75% in

A

A

A

25% B 0% B 0% B 25% B 25% B every 200 msec limie References 2.12 mnIin 4"-hydroxy- averi-ectin BlIa 47 Case PB/5-60016A 3.27 min 3.77 min 4.83 min avermectin Bla 3"-O-demethyl-4"-keto-avermectin B a 4"-keto-avermectin B I a EXAMPLE III Biotransformation With Cell-Free Extract From Streptomyces Strain R-922 To prepare an active cell-free extract from Streptomyces tubercidicus strain R-922 capable of regioselective oxidation of avermectin to 4"-keto-avermectin, the following solutions were made, stored at 4°C, and kept on ice when used.

Solution Formula PP-buffer 50 mM K 2

HPO

4

/KH

2

PO

4 (pH Disruption buffer 50 mM K 2 HPO4/KH 2

PO

4 (pH 5 mM benzamidine, 2 mM dithiothreitol, and 0.5 mM Pefabloc (from Roche Diagnostics) Substrate 10 mg avermectin were dissolved in 1 ml isopropanol Six grams of wet cells from Streptomyces strain R-922 were washed in PP-buffer and then resuspended in 35 ml disruption buffer and disrupted in a French press at 4°C. The resulting suspension was centrifuged for 1 hour at 35000 x g. The supematant of the cell free extract was collected. One p.l substrate was added to 499.tl of cleared cell free extract and incubated at 30°C for 1 hour. Then, 1 ml methyl-t-butyl ether was added to the reaction mixture and thoroughly mixed. The mixture was next centrifuged for 2 min. at 14000 rpm, and the methyl-t-butyl ether phase was transferred into a 10 ml flask and evaporated in vacuo -48- Case PB/5-60016A by means of a rotary evaporator. The residue was dissolved in 200 pl acetonitrile and transferred into an HPLC-sample vial.

For HPLC, the HPLC protocol I was used.

When 1 itl substrate was added to 499 ptl of cleared cell free extract and incubated at 0 C, no conversion of avermectin to 4"-keto-avermectin was observed by HPLC analysis using HPLC protocol 1.

However, the possibility of addition of spinach ferredoxin and spinach ferredoxin reductase and NADPH to the cell free extract to restore the biocatalytic activity was explored (see, generally, D.E. Cane and E.I. Graziani, J. Amer. Chem. Soc. 120:2682, 1998).

Accordingly, the following solutions were made: Solution Formula Substrate 10 mg avermectin were dissolved in 1 ml isopropanol Ferredoxin 5 mg ferredoxin (from spinach), solution 1-3 mg/ml in Tris/HCl-buffer (from Fluka) or 5 mg ferredoxin (from Clostridium pasteurianum), solution of 1-3 mg/ml in Tris/HCl-buffer (from Fluka) or 5 mg ferredoxin (from Porphyra umbilicalis), solution of 1-3 mg/ml in Tris/HCl-buffer (from Fluka) Ferredoxin Reductase 1 mg freeze-dried ferredoxin reductase (from spinach), solution of 3.9 U/mg in 1 ml H 2 0 (from Sigma) NADPH 100 mM NADPH in H 2 0 (from Roche Diagnostics) The substrate solution was stored at 4°C, the other solutions were stored at -20 0 C, and kept on ice when used.

Thus, to 475 il of cleared cell free extract the following solutions were added: 10 pl ferredoxin, 10 pl ferredoxin reductase and 1 ptl substrate. After the addition of substrate to the cells, the mixture was immediately and thoroughly mixed and aerated. Then, 5 pl1 of NADPH were added and the mixture incubated at 30°C for 30 min. Then,.1 ml methyl-t-butyl ether was added to the reaction mixture and thoroughly mixed. The mixture was next centrifuged for 2 min. at 14000 rpm, and the methyl-t-butyl ether phase was transferred into a 10 ml flask -49- Case PB/5-60016A and evaporated in vacuo by means of a rotary evaporator. The residue was dissolved in 200 Ptl acetonitrile and transferred into an HPLC-sample vial, and HPLC analysis performed using HPLC protocol I.

Formation of 4"-keto-avermectin was observable by HPLC analysis. Thus, addition of spinach ferredoxin and spinach ferredoxin reductase and NADPH to the cell free extract restored the biocatalytic activity.

Upon injection of a 30 il sample, a peak appeared at 4.83 min., indicating the presence of4"-keto-avermectin Bla. A mass of 870 D could be assigned to this peak by HPLC-mass spectrometry which corresponds to the molecular weight of 4"-keto-avermectin Bla.

Note that when analyzing product formation by HPLC and HPLC-mass spectrometry, in addition to the 4"-keto-avennectin, the corresponding ketohydrate 4"-hydroxy-avermectin was also found giving a peak at 2.12 min. This finding indicated that the P450 monooxygenase converts avermectin by hydroxylation to 4"-hydroxy-avermectin, from which 4"-ketoavermectin is formed by dehydration. Interestingly, when the spinach ferredoxin was replaced by ferredoxin from the bacterium Clostridium pasteurianum or from the red alga Porphyra umbilicalis, the biocatalytic conversion of avermectin to 4"-keto-avermectin still took place, indicating that the enzyme does not depend on a specific ferredoxin for receiving reduction equivalents.

EXAMPLE IV Isolation of a Mutant Streptomvces Strain R-922 With Enhanced Activity To obtain strains of Streptomyces strain R-922 that have an enhanced ability to regioselectively oxidize avermectin to 4"-keto-avermectin, UV mutants were generated. To do this, spores of Streptomyces strain R-922 were collected and stored in 15% glycerol at 0 C. This stock solution contained 2x10 9 spores.

The spore stock solution was next diluted and transferred to petri plates containing of sterile water, and the suspension was exposed to UV light in a Stratalinker UV crosslinker 2400 (commercially available from Stratagene, La Jolla, CA). The Stratalinker UV crosslinker uses a 254-nm light source and the amount of energy used to irradiate a sample can be set in the "energy mode." Case PB/5-60016A Through experimentation, it was determined that an exposure of 8000 microjoules of UV irradiation (254 nm) was required to kill 99.9% of the spores. This level of UV exposure was used in the mutagenesis.

Surviving UV-mutagenized spores were plated, cultured, and transferred to minimal media. Approximately 0.3-0.4% of the viable spores were determined to be auxotrophic, indicating a good level of mutagenesis in the population.

The mutagenized clones were screened for activity in the whole cell biocatalysis assay described in Example II. As shown in an HPLC chromatogram, one mutant ("R-922 UV mutant") showed a two to three fold increase in an ability to regioselectively oxidize avermectin to 4"-keto-avermectin as compared to wild-type strain R-922. Although the gene encoding the P450 monooxygenase responsible for the regioselectively oxidation activity, emal, is not mutated in the R-922 UV mutant, this mutant nonetheless provides an excellent source for a cell-free extract containing emal protein.

EXAMPLE V Isolation of the P450 Monooxygenase from Streptomyces Strain R-922 To enrich the P450 enzyme, 35 ml of active cell free extract were filtered through a itm filter and fractionated by anion exchange chromatography. Anion exchange chromatography conditions were as follows: FPLC instrument: Akta prime (from Pharmacia Biotech) FPLC-column: HiTrap Q (5 ml) stacked onto Resource@ Q (6 ml) (from Pharmacia Biotech) eluents buffer A: 25 mM Tris/HCI (pH buffer B: 25 mM Tris/HC (pH 7.5) containing 1 M KCI temperature eluent bottles and fractions in ice bath, flow 3 ml/min detection UV 280nm Pump table: 0.0 min 100% A 0% B linear gradient to2.0 min 90% A 10% B min 90% A 10% B -51 Case PB/5-60016A linear gradient to30.0 mmin 50% A 50% B linear gradient to40.0 min 0% A 100% B 50.0min 0% A 100% B Enzyme activity eluted with 35%-40% buffer B. The active fractions were pooled and concentrated by centrifugal filtration through Biomax T filters with an exclusion limit of (commercially available from Millipore Corp., Bedford, MA) at 5000 rpm and then rediluted in disruption buffer containing 20% glycerol to a volume of 5 ml containing 3-10 mg/ml protein. This enriched enzyme solution contained at least 25% of the original enzyme activity.

The enzyme was further purified by size exclusion chromatography. Size exclusion chromatography conditions were as follows: FPLC instrument: Akta prime (from Pharmacia Biotech) FPLC-column: HiLoad 26/60 Superdex® 200 prep grade (from Pharmacia Biotech) sample: 3-5 ml enriched enzyme solution from the anion chromatography step sample preparation: filtered through 45 .tm filter eluent buffer: PP-buffer (pH 7.0) 0.1 M KCI temperature: 4°C flow: 2 ml/min detection: UV 280nm Enzyme activity eluted between 205-235 ml eluent buffer. The active fractions were pooled, concentrated by centrifugal filtration through Biomax m filters with an exclusion limit of 5 kD (from Millipore) at 5000 rpm, and rediluted in disruption buffer containing glycerol to form a solution of 0.5-1 ml containing 2-5mg/ml protein. This enriched enzyme solution contained 10% of the original enzyme activity. This enzyme preparation, when checked for purity by SDS page, (see, generally, Laemmli, Nature 227:680-685, 1970 and Current Protocols in Molecular Biology, supra) and stained with Coomassie blue, showed one dominant protein band with a molecular weight of 45-50 kD, according to reference proteins of known molecular weight.

EXAMPLE VI -52- Case PB/5-60016A Attempted Isolation of P450 Monooxygenase Genes From Streptomvces Strains R-922 and 1-1529 Based on results described above that suggested the enzyme from strain R-922 that is responsible for the regiospecific oxidation of avennectin to 4"-keto-avermectin is a P450 monooxygenase, a direct PCR-based approach to clone P450 monooxygenase genes from this strain was initiated (see, generally, Hyun et al., J. Microbiol. Biotechnol. 8(3):295-299, 1998).

This approach is based on the fact that all P450 monooxygenase enzymes contain highly conserved oxygen-binding and heme-binding domains that are also conserved at the nucleotide level. PCR primers were designed to prime to these conserved domains and to amplify the DNA fragment from P450 genes using R-922 or 1-1529 genomic DNA as a template. The PCR primers used are shown in Table 1.

Table 1 0 2 -Binding Domain Primers to Degeneracy SEQ ID NOs I A G H E T T 43 ATC GCS GGS CAC GAG ACS AC 8 44 V A G H E T T GTS GCS GGS CAC GAG ACS AC 16 46 L A G H E T T 47 CTS GCS GGS CAC GAG ACS AC 16 48 L L L I A G H E T 49 TS CTS CTS ATC GCS GGS CAC GAG AC' 32 Heme-Binding Domain Primers to H Q C L G Q N L A 51 GTG GTC ACG GAS CCS TGC TTG GAS CG' 8 52 F G H G V H Q C 53 AAG CCS GTG CCS CAS GTG GTC ACG 8 54 F G F G V H Q C AAG GCS AAG CCS CAS GTG GTC ACG 8 56' F G H G I H Q C 57 AAG CCS GTG CCS TAG GTG GTC ACG 4 58 53- Case PB/5-60016A F G H G V H F C 59 AAG CCS GTG CCS CAS GTG AAG ACG 8 The amino acid sequence is shown on the top line and the corresponding nucleotide sequence is shown below on the second line; S=G or C.

This primer was described by Hyun et al., supra PCR amplification using any of the primers specific to nucleotide sequences encoding the 0 2 -binding domain with any of the primers specific to the nucleotide sequences encoding the heme-binding domain and genomic DNA from Streptomyces strains R-922 or 1-1529 resulted in the amplification of an approximately 350 bp DNA fragment. This is exactly the size that would be expected from this PCR amplification due to the approximately 350 bp separation in P450 genes of the gene segments encoding the 0 2 -binding and heme-binding sites.

The 350 bp PCR fragments were cloned into the pCR2.1-TOPO TA cloning plasmid (commercially available Invitrogen, Carlsbad, CA) and transformed into E. coli strain (Invitrogen, Carlsbad, CA). Approximately 150 individual clones from strains R-922 and 1- 1529 were sequenced to determine how many unique P450 gene fragments were represented.

Analysis of the sequences revealed that they included 8 unique P450 gene fragments from strain R-922 and 7 unique fragments from 1-1529.

Blast analysis (alignment of the deduced amino acid sequences of P450 gene-specific PCR fragments derived from Streptomyces tubercidicus strain R-922 and Streptomyces strain 1-1529, respectively, and the P450 monooxygenase from S. thermotolerans that is involved in the synthesis of carbomycin (Stol-ORFA) (GenBank Accession No. D30759) by the program Pretty from the University of Wisconsin Package version 10.1 (Altschul et al., Nucl. Acids Res. 25:3389-3402). demonstrated that all of the unique P450 gene fragments from both the R-922 and I-1529 strains were derived from P450 genes and encoded the region between the 0 2 -binding and heme-binding domains.

Next, in order to clone the full-length genes from which the PCR fragments were derived, the DNA fragments cloned by PCR were used as hybridization probes to gene libraries containing genomic DNA from strains R-922 and 1-1529. To do this, genomic DNA from the R-922 and 1-1529 strains was partially digested with Sau3A I, dephosphorylated with 54 Case PB/5-60016A calf intestinal alkaline phosphatase (CIP) and ligated into the cosmid pPEI-1215, a modified version of SuperCos 1 (commercially available from Stratagene, La Jolla, CA). Ligation products were packaged using the Gigapack II XL packaging extract and transfected into E.

coli XLI Blue MR host cells. Twelve cosmids that strongly hybridized to the PCR-generated P450 gene fragments were identified from the R-922 library, from which three unique P-450 genes were subcloned and sequenced. The hybridizations were performed at high stringency conditions according to the protocol of Church and Gilbert (Church and Gilbert, Proc. Natl.

Acad. Sci. USA 81:1991-1995, 1984). In brief, these high stringency conditions include Hybrid Buffer containing 500 mM Na-phosphate, 1 mM EDTA, 7% SDS, 1% BSA; Wash Buffer 1 containing 40 mM Na-phosphate, 1 mM EDTA, 5% SDS, 0.5% BSA; and Wash Buffer 2 containing 40 mM Na-phosphate, 1 mM EDTA, 1% SDS (Note that other high stringency hybridizations conditions are described, for example, in Current Protocols in Molecular Biology, supra.) Nineteen strongly hybridizing cosmids were identified from the I- 1529 library, and from these, four unique P-450 genes were subcloned and sequenced.

In yet a further approach to isolate diverse P450 monooxygenase genes from strains R- 922 and 1-1529, a known P450 gene from another bacterium was used as a hybridization probe to identify cosmid clones containing homologous P450 genes from strains R-922 and I- 1529. The epoF P450 gene from Sorangium cellulosum strain So ce90 that is involved in the synthesis of epothilones (Molnar et al., Chem Biol. 7(2):97-109, 2000) was used as a probe in this effort. Using the epoF P450 gene probe, one cosmid was identified from strain R-922 (clone LC), and threewere identified from strain 1-1529 (clones LA, LB, and EA). In each case, the homologous gene fragment was subcloned and sequenced, and found to code for P450 monooxygenase enzymes.

However, a comparison of the 17 polypeptide sequences identified in Example VII (below) failed to match any of these cloned genes. Two of the polypeptide sequences (namely, LVKDDPALLPR and AVHELMR) mapped to the region between the 02 and heme binding domains, and so these should have identified any of the partial gene fragments derived by the PCR approach. Thus, the standard approaches based on the known PCR technique of Hyun et al., supra, and using known P450 genes as hybridization probes failed to identify the gene that encodes the specific P450 monooxygenase responsible for the regioselective Case 111/5-60016A oxidation of avermectin. Accordingly, it was determined that additional experimentation was required to isolate the gene encoding the P450 monooxygenase of the invention.

EXAMPLE VII Partial Sequencing of the P450 Monooxygenase fromn Streptomivces Strain R-922 Partial amino acid sequencing of the P450 monooxygenase from Strepiomyces strain R- 922 was carried out by the Friedrich Miescher Institute, Base] Switzerland. The protein of the dominant band on the SDS page was tryptically digested and the formed peptides separated and sequenced by mass spectrometry and Edman degradation (see, generally, Zerbe-lBUrkhardt et al., J. Bi'ol. Chem. 273:6508, 1998). The sequence of the following 17 peptides were found: Sequence Sequence I.D. No.

1-IPGEPNVMDPALITDPFTGYGALR

FVNNPASPSLNYAPEDNPLTR

LLTH-YPD]SLGIAPEILER

VYLLGStLNYDAPDHTR

TWGADLISMIDPDR

EALTDDLLSELW

FMDIDSPVWLVTR

LMEMLG LPEH-LR

VEQIADALLAR

LVKDDPALLPR

DDPALLPR

TPLPGNWR

LNSLPVR

ITDLRPR

EQGPVVR

AVIELMR

(SEQ ID NO:61) (SEQ lID NO:62) (SEQ ID NO:63) (SEQ ID NO:64) (SEQ ID (SEQ TD NO:66) (SEQ ID NO:67) (SEQ lID NO:68) (SEQ MD NO:69) (SEQ MI (SEQ ID NO:7 1) (SEQ ID NO:72) (SEQ ID NO:73) (SEQ ID NO:74) (SEQ ID (SEQ ID NO:76) 56 Case PB/5-60016A

AFTAR

FEEVR

(SEQ ID NO:77) (SEQ ID NO:78) Alignment of these peptides to a selection of actinomycete P450 monooxygenase sequences indicated that all the peptides were fragments of a single P450 mono-oxygenase.

EXAMPLE VITI Cloning the P450 Monooxygenase Gene from Strain R-922 that Encodes the Enzyme Responsible for the Oxidation of Avermectin to 4"-Keto-Avermectin

J

PCR primers were designed by reverse translation from the amino acid sequences of several of the peptides derived from the P450 enzyme of strain R-922 (see Example VII and Table 2 below). Each of five forward primers (2aF, 2bF, 3F, 1F, and 7F) was paired with one reverse primer (5R) in PCR reactions with R-922 genomic DNA as a template. In each reaction, a DNA fragment of the expected size was produced.

Table 2 Primer Primer sequence and the amino acid Degen- Expected SEQ sequence to which they were designed* eracy size ID

NO:

2aF P G E D N V M 64 600 79 GGS GAR CCS AAY GTS ATG-3' 2bF A L I T D P F 32 580 81 CTS ATY ACS GAC CCS TTC-3' 82 3F F M D D S P V W 32 549 83 ATG GAC GAC WSS CCS GTS TGG-3' 84 1 F L N Y D A P D H 32 350 AAY TAY GAC GCS CCS GAC CAC-3' 86 7F V E Q I A D A L 32 300 87 GAR CAG ATY GCS GAC GCS CTS-3' 88 D L I S M D P D 64 89 3'-CTG GAS TAR WSS TAC CTG GGS CTG-5' Ambiguity codes: Y=C or T; R=A or G; S=C or G; W=A or T -57- Case PB/5-60016A Expected size of PCR product when the primer is when paired with primer The 580 and 600 bp PCR fragments generated by using primers (2bF and 5R) and (2aF and 5R), respectively, were cloned into the pCR-Blunt II-TOPO cloning plasmid (commercially available from Invitrogen, Carlsbad, CA) and transformed into E. coli strain TOPI0 (Invitrogen, Carlsbad, CA). The inserted DNA fragments were then sequenced.

Examination of the sequences revealed that the 600 and 580 bp fragments were identical in the 580 bp of sequence that they have in common. Also, there was a perfect match between the deduced amino acid sequence (SEQ ID NO:2) derived from the nucleotide sequence of the 600 bp and 580 bp fragments and the amino acid sequences of peptides isolated from the purified P 4 50Emai enzyme that aligned in this region of the isolated gene. This result strongly suggested that the gene fragments isolated in these clones are derived from the gene that encodes the P 4 50Ema, enzyme that is responsible for the oxidation of avermectin to 4"-ketoavermectin.

The 600 bp PCR fragment produced using primers 2aF (SEQ ID No:80) and 5R (SEQ ID No:90) was used as a hybridization probe to a cosmid library of genomic DNA isolated from strain R-922 (cosmid library described in Example VI). Two cosmids named pPEH249 and pPEH250 were identified that hybridized strongly with the probe. The portion of each cosmid encoding the P450 enzyme was sequenced and the sequences were found to be identical between the two cosmids. The complete coding sequence of the ernal gene was identified (SEQ ID NO:1). The amino acid sequence of all polypeptide fragments from

P

4 50Emal matched perfectly with the deduced amino acid sequence from the emal gene.

Comparison of the deduced amino acid sequence of the protein encoded by the emal gene using BLASTP (Altschul et al., supra) determined that the closest match in the databases is to a P450 monooxygenase from S. thermotolerans that has a role in the biosynthesis of carbomycin (Arisawa et al., Biosci. Biotech. Biochem. 59(4):582-588, 1995) and whose identity with emal is only 49% (Identities 202/409 Positives 271/409 Gaps 2/409 In the Blast analysis, the following settings were employed: -58- Case PB/5-60016A BLASTP 2.0.10 Lambda K H 0.322 0.140 0.428 Gapped Lambda K H 0.270 0.0470 0.230 Matrix: BLOSUM62 Gap Penalties: Existence: 11, Extension: 1 Number of Hits to DB: 375001765 Number of Sequences: 1271323 Number of extensions: 16451653 Number of successful extensions: 46738 Number of sequences better than 10.0: 2211 Number of HSP's better than 10.0 without gapping: 628 Number of HSP's successfully gapped in prelim test: 1583 Number of HSP's that attempted gapping in prelim test: 43251 Number of HSP's gapped (non-prelim): 2577 length of query: 430 length of database: 409,691,007 effective HSP length: effective length of query: 375 effective length of database: 339,768,242 effective search space: 127413090750 effective search space used: 127413090750 A similar comparison of the nucleotide sequences of these two genes demonstrated that they are 65% identical at the nucleotide level. These results demonstrate that P 4 50Ema is a new enzyme.

EXAMPLE IX Heterologous Expression of the emal Gene in Streptomyces lividans Strain ZX7 The coding sequence of the emal gene was fused to the thiostrepton-inducible promoter (tipA) (Murakami et al., J. Bacteriol. 171:1459-1466, 1989). The tipA promoter was derived from plasmid pSITII51 (Herron and Evans, FEMS Microbiology Letters 171:215-221, 1999).

The fusion of the tipA promoter and the emal coding sequence was achieved by first amplifying the emal coding sequence with the following primers to introduce a Pacl cloning site at the 5' end and a Pmel compatible end on the 3' end.

Forward Primer: The underlined sequence is a PacI recognition sequence; the sequence in bold-face type is the start of the coding sequence of emal.

GTT-3' (SEQ ID NO:91) -59- Case PB/5-60016A Reverse Primer: The underlined sequence is half of a Pmel recognition sequence; the bold-face type sequence is the reverse complement of the emal translation stop codon followed by the 3' end of the emal coding sequence.

5'-AAACTCACCCCAACCGCACCGGCAGCGAGTTC-3" (SEQ ID NO:92) The Pacl-digested PCR fragment containing the emal coding sequence was cloned into plasmid pTBBKA (see Figure 1) that was restricted digested) with PacI and Pmel, and the ligated plasmid transformed into E. coli. Four clones were sequenced. Three of the four contained the complete and correct emal coding sequence. The fourth emal gene clone contained a truncated version of the emal gene. The full-length emal gene encodes a protein that begins with the amino acid sequence MSELMNS (SEQ ID NO:93). The truncated gene encodes a protein that lacks the first 4 amino acids and begins with the second methionine residue. This gene has been named emalA. The nucleotide and amino acid sequence of emalA are provided as SEQ ID NO:33 and SEQ ID NO:34, respectively. The emal and emalA genes in these plasmids, pTBBKA-emal and pTBBKA-emalA, are in the correct juxtaposition with the tipA promoter to cause expression of the genes from this promoter.

Plasmid pTBBKA contains a gene from the Streptomyces insertion element IS117 that encodes an integrase that catalyzes site-specific integration of the plasmid into the chromosome of Streptomyces species (Henderson et al., Mol. Microbiol. 3:1307-1318, 1989 and Lydiate et al., Mol. Gen. Genet. 203:79-88, 1986). Since plasmid pTBBKA has only an E. coli replication origin and contains a mobilization site, it can be transferred from E. coli to Streptomyces strains by conjugation where it will not replicate. However, it is able to integrate into the chromosome due to the IS 117 integrase and Streptomyces clones containing chromosomal integrations can be selected by resistance to kanamycin due to the plasmidborne kanamycin resistance gene.

The emal coding sequence was also cloned into other plasmids that are either replicative in Streptomyces or, like pTBBKA, integrate into the chromosome upon introduction into a Streptomyces host. For example, emal was cloned into plasmid pEAA, which is similar to plasmid pTBBKA but the Kpnl/Pacl fragment containing the tipA promoter was replaced with the ermE gene promoter (Schmitt-John and Engels, Appl Case PB/5-60016A Microbiol Bioechnol. 36(4):493-498, 1992). In addition, pEAA does not contain the kanamrnycin resistance gene. The emal gene was cloned into pEAA as a Pacl/Pmel fragment to create plasmid pEAA-emal in which the ernal gene is expressed from the constitutive eninE promoter.

Plasmid pTUAIA is a Streptomyces-E coli shuttle plasmid (see Figure 2) that contains the tipA promoter. The emal gene was also cloned into the Pacl/PmeI site in plasmid pTUAIA to create plasmid pTUA-enmal.

The enalA gene fragment was also ligated as a Pacl/Pmel fragment into plasmids pTUA1A, and pEAA in the same way as the emnal gene fragment to create plasmids pTUAemalA, and pEAA-emalA, respectively.

The pTBBKA, pTUA 1A, and pEAA based plasmids containing the ernal or emnalA genes were introduced into S. lividans ZX7 and in each case transformants were obtained and verified lividans strains ZX7::pTBBKA-emal or emalA, ZX7 (pTUA-emal or -emalA), and ZX7::pEAA-emal or -emalA, respectively).

Wild-type Streptomyces lividans strain ZX7 was tested and found to be incapable of the oxidation of avermectin to 4"-keto-avermectin. Transformed S. lividans strains ZX7::pTBBKA-emal, ZX7::pTBBKA-enimalA, ZX7 (pTUA-emal), ZX7 (pTUA-ernmalA), ZX7::pEAA-emal, and ZX7::pEAA-enalA were each tested for the ability to oxidize avermectin to 4"-keto-avermectin using resting cells. To do this, the whole cell biocatalysis assay described above (including analysis method) was performed. Note that for the whole cell biocatalysis assay, transformed Strepromyces lividans, like strain R-922, was grown in PHIG medium and, again like strain R-922, had a reaction time of 16 hours during which time the 500 mg transformed Streptomyces lividans wet cells in 10 ml of 50 mM potassium phosphate buffer, pH 7.0, were shaken at 160 rpm at 28 0 C in the presence of 15 l11 of a solution of avermectin in isopropanol (30 mg/mI)).

In the presence of the inducer, thiostrepton (5 ug/ml), the emal- or emalA-containing strains ZX7::pTBBKA-emal, ZX7::pTBBKA-emaA, ZX7 (pTUA-enlal), ZX7 (pTUAemalA) were found to oxidize avermectin to 4"-keto-avermectin as evidenced by the appearance of the oxidized 4"-keto-avermectin compound (see Table 3).

Table 3 -61 Case PB/5-60016A Conversion of Averm-ectin Beispiel 1: Strain 2 houir 16 hour Streptoinyces lividans ZX7 Plasmid' None 0 0 pTBBKA-erna]A 0.5 ±0.059 1.17 ±0.112 pTBBKA-eirnal 0.21 ±0.0.356 0.65 ±0.079 pTUA-ema] 20.96 ±1.044 42.0 pEAA-ema] 3.0 ±0.232 24.1 ±0.358 pTBBKA-ema2 4.79 ±0.096 9.57 ±0.423 pTUA-erna2 0.77 ±0.138 2.05 ±0.537 pEAA-eina2 0.0 1 .73 ±3.00 pTBBKA-ernai/fd233 8.89 ±0.720 30.99 ±0.8 pTUA-emal/fd233 23.29 ±0.854 61 .2 ±3.54 8 pEAA-einal/fd233 8.26 ±0.845 10.66 ±0.858 pTUA-eina2/fd233 1.85 ±0.861 6.40 ±1 .918 Pseudomonas putida S12 Plasmid None 0 pRK-einal ND 2 18 pRK-erna]/fd233 ND 32 pTBBKA= IS 117 integrase, tipA promoter; pTUA= replicative plasm.id, tipA promoter; pEAA= JS1 117 integrase, ern-IE promoter 2 Not Determined Thesc results conclusively demonstrate that the P 4 50EmJ enzyme encoded by the emial gene is responsible for the oxidation of aven-mectin to 4"-keto-avermectin in S. tube rcidicus strain R-922. Furthermore, the data demonstrates that the einalA gene that Is 4 amino acids shorter on the N-termninuLs than the native ean gene also encodes an active P 4 5OEn,,] enzyme. As can be demonstrated by 1-IPLC analysis, oxidation of avermectin to 4"-keto-avermectin by S.

62 Case PB/5-60016A lividans strain ZX7::pTBBKA-emal following induction of emal expression with 0, 0,5, or gg/ml thiostrepton. is variable depending upon the amount of thiostrepton used to induce expression of enal. Note that S. lividans strains ZX7::pEAA-emal and ZX7::pEAA-emalA (see Table 3) demonstrated this oxidation activity in the absence of thiostrepton since in these strains the emal or emalA genes are expressed from the enmE promoter that does not require induction.

EXAMPLE X Isolation of an emal-Homologous Gene From Streptomyces tubercidicus Strain 1-1529 Streptomyces tubercidicus strain 1-1529 was also found to be active in biocatalysis of avermectin to form the 4"-keto-avermectin derivative. The cosmid library from strain 1-1529, described in Example VI, was probed at the high stringency conditions of Church and Gilbert (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984) with the 600 bp emal PCR fragment produced using primers 2aF (SEQ ID No:80) and 5R (SEQ ID described previously to identify clones containing the emal homolog from strain 1-1529.

Three strongly hybridizing cosmids were identified. The P450 gene regions in two of the cosmids, pPEH252 and pPEH253, were sequenced and found to be identical. Analysis of the DNA sequence revealed the presence of a gene with high homology to the ernal gene of strain R-922. A comparison of the deduced amino acid sequence of Ema2 P 4 50Ema2), Emal

P

4 50Emai), and a P450 monooxygenas'e from Streptomyces thennotolerans that is involved in the biosynthesis of carbomycin (Carb-450) (GenBank Accession No. D30759).

demonstrated that all of the unique P450 gene fragments from both the R-922 and 1-1529 strains were derived from P450 genes and encoded the region between the 0 2 -binding and heme-binding domains.

The gene from Streptomyces tubercidicus strain 1-1529, named ema2, encodes an enzyme with 90% identity at the amino acid level and 90.6% identity at the nucleotide level to the P 4 50Ema enzyme. The nucleotide sequence of the ema2 gene and the deduced amino acid sequence of P450Ema 2 are provided in SEQ ID NO:3 and SEQ ID NO:4, respectively.

The ema2 coding sequence was cloned in the same manner as the emal and emalA genes into plasmids pTBBKA, pTUA A, and pEAA such that the coding sequence was 63 Case PB/5-60016A functionally fused to the lipA or errnE* promoter in these plasmids. The resulting plasmids, pTBBKA-ema2, pTUA-emna2, and pEAA-ema2 were transferred from E. coli to S. lividans ZX7 by conjugation to create strains ZX7::TBBKA-ema2 and ZX7 (pTUA-ema2), and ZX7::pEAA-ema2 containing the ema2 gene integrated into the chromosome or maintained on a plasmid.

Strains ZX7::TBBKA-ema2, ZX7 (pTUA-ema2), and ZX7::pEAA-ema2 were next tested for the ability to oxidize avermectin to 4"-keto-avermectin. The ema2 gene was also shown to provide biocatalysis activity, although at a lower level compared to the emal gene (see Table 3).

These results demonstrate that the ema2 gene from S. tubercidicus strain 1-1529 also encodes a P450 enzyme (P 4 50Ema2) capable of oxidizing avermectin to 4"-keto-avermectin.

EXAMPLE XI Characterization of emal Homologs From Other Biocatalysis Strains Seventeen Streptomyces sp. strains, including strains R-922 and 1-1529, were identified that are capable of catalyzing the regiospecific oxidation of the 4"-carbinol of avermectin to a ketone. Next, the isolation and characterization of the genes encoding the biocatalysis enzyme from all of these strains was accomplished.

To do this, genomic DNA was isolated from the strains and was evaluated by restriction with several restriction endonucleases and Southern hybridization with the emal gene. A specific restriction endonuclease was identified for each DNA that would generate a single DNA fragment of a defined size to which the emal gene hybridizes. For each strain, there was only one strongly hybridizing DNA fragment, thus suggesting that other P450 genes were not detected under the high stringency hybridization conditions used in these experiments.

Each DNA was digested with the appropriate restriction endonuclease, and the DNA was subjected to agarose gel electrophoresis. DNA in a narrow size range that included the size of the emal-hybridizing fragment was excised from the gel. The size selected DNA was ligated into an appropriate cloning plasmid and this ligated plasmid was used to transform E. coli.

The E. coli clones from each experiment were screened by colony hybridization with the emal gene fragment to identify clones containing the emal-homologous DNA fragment.

-64- Case PBI5-60016A The nucleotide sequence of the cloned DNA in each emna]-homologous clone was determined and examined for the presence of a gene encoding a P450 enzyme with homnology to emia]. In this way, enia]-homologous genes were isolated from 14 of the 15 other active strains. The nucleotide and deduced amino acid sequences of these are referenced in Table 4 as SEQ ID NOS:5-32 and 94-95. The relationship of these enzymes can be shown in the form of a phylogenetic tree. Such a phylogenetic tree can be generated using the commercially available GCG Wisconsin software program version 1.0 (Madison, WI).

Table 4 Strain Number Classification SEQ ID NO (nucleotide and amino acid, respectively) R-0922 ema] Streptornyces tube rcidicus 2. 1 and 2 1-1529 ema2 Streptomyces tube rcidicus 3 and 4 1053 eina3 Streptomyces ri mosus 5 and 6 R-0401 ema4 Streptomyces lydicus 7 and 8 1-1525 emaS Streptomyces vp. 9 and DSM-40241 emia6 Streptomyces chattanoogensis* 3. 11 and 12 tI-IS-0435 eina7 Streptomyces sp. 13 and 14 C-00083 emia8 Streptomyces albofaciens I5 and 16 MAAG-7479 enia9 Streptoinyces platensis 17 and 18 A/96-1208710 ernalO Streptomyces kasugaensis 4. 19 and R-2374 einail Streptoni)'ces rimosus 21 and 22 MAAG-7027 ema]2 Streptomyces tube rcidicus 5. 23 and 24 TLIe-3077 ema]3 Strep! omnyces platensis 25 and 26 1-1548 ema]4 Streptomyces platensis 27 and 28 NRRL-2433 erna]5 Streptomyces lydicus 6. 29 and MAAG-01 14 eina]6 Streptoinyces lydicus 31 and 32 DSM-40261 einal 7 Streptoinyces tube rcidicus 94 and This strain was shown to be in the chattanoogensis species by 16s rDNA analysis; however, classical taxonornic mnethods used by the Gen-nan culture collection (DSMZ) showed it to be snarcetris.

EXAMPLE Xfl Construction of1-uis-tagged emnal and emial 1-lomologs to Facilitate Enzyme Purification 65 Case PB/5-60016A In order to purify the P450Ema enzyme and the P450 enzymes encoded by the emal homologs from other biocatalysis strains, each of the P450 genes was cloned into the E. coli expression plasmid pET-28b(+) (commercially available from Novagen, Madison, WI). The pET-28 plasmids are designed to facilitate His-tag fusions at either the or C-terminus and to provide strong expression of the genes in E. coli from the T7 phage promoter. In many cases, the coding sequence of the ema genes begins with the sequence ATGT. These genes were amplified by PCR such that the primers on the 5' end incorporated a Pcil recognition site ATATGT at the 5' terminus. The last four bases of the PciI site correspond to the ATGT at the beginning of the ema gene coding sequence.

PCR primers at the 3' end of the genes were designed to remove the translation stop codon at the end of the ema gene coding sequence and to add an Xhol recognition site to the 3' terminus. The resulting PCR fragments were restricted with Pcil and XhoI to generate PciI ends at the 5' termini and Xhol ends at the 3' termini, thereby facilitating cloning of the fragments into pET-28b(+) previously restricted with NcoI and XhoI. Since Pcil and Ncol ends are compatible, the fragments were cloned into pET-28b(+) in the proper orientation to the T7 promoter and ribosome binding site in the plasmid to provide expression of the genes.

At the 3' end of each ema gene, the coding sequence was fused in frame at the XhoI site to the His-tag sequence followed by a translation stop codon. This results in the production of an Ema enzyme with six histidine residues added to the C-terminus to facilitate purification on nickel columns.

In the case of ema genes in which the ATG translation initiation codon is not followed by a T nucleotide, the ema genes were amplified by PCR using a different strategy for the end. The primers at the 5' end were designed to incorporate a C immediately preceding the ATG translation initiation codon and the primers at the 3' end were the same as described above. The PCR fragments that were amplified were restricted with Xhol to create an Xhol end at the 3'-terminus and the 5' end was left as a blunt end. These fragments were cloned into pET-28b(+) that had been restricted with Ncol, but the Ncol ends were made blunt-ended by treatment with mung bean exonuclease, and restricted with Xhol.

In this manner, the ema genes were cloned into pET-28b(+) to create a functional fusion with the T7 promoter and the His-tag at the C-terminus as described previously. All Histagged ema genes were sequenced to ensure that no errors were introduced by PCR.

-66- Case PB/5-60016A Large amounts of the P 4 50E,ma and P 4 50Ema2 enzymes were isolated and purified by standard protocols. E. coli strain BL21 DE3 (commercially available from Invitrogen; Carlsbad, CA) containing the T7 RNA polymerase gene under the control of the inducible tac promoter and the appropriate pET-28/ema plasmid was cultured and the cells were harvested and lysed. The lysates were applied to Ni-NTA columns (commercially available from Qiagen Inc., Valencia, CA) and the protein were purified according to the procedure recommended by the manufacturer.

Purified His-tagged P 4 50Emal and P 4 50Ema2 were highly active in in vitro activity assays as evidenced by a high rate of conversion of avermectin to 4"-keto-avemectin.

EXAMPLE XImI Expression of emal in Pseudomonas The emal gene constructs were next introduced into P. putida (wildtype P. putida commercially available from the American Type Culture Collection, Manassas, Virginia; ATCC Nos. 700801 and 17453). The emal and emal/fd233 gene fragments were cloned as PacI/PmeI fragments into the plasmid pUK21 (Viera and Messing, Gene 100:189-194, 1991).

The fragments were cloned into a position located between the tac promoter (Pac) and terminator (T,ac) on pUK21 in the proper orientation for expression from the tac promoter.

The Pac-emal-TIac and Piac-emal/fd 2 3 3 -Ttac gene fragments were removed from pUK21 as BglI fragments and these were cloned into the broad host-range, transmissible plasmid, pRK290 (Ditta et al., Proc. Natl. Acad. Sci. USA 77:7347-7351, 1980) to create plasmids pRK-emal and pRK-emal/fd233 (Figure These plasmids were introduced into P. pulida strains ATCC 700801 and ATCC 17453 by conjugal transfer from E. coli hosts by standard methodology (Ditta et al., Proc. Natl. Acad. Sci. USA 77:7347-7351, 1980).

P. putida ATCC 700801 and ATCC 17453 containing plasmids pRK-emal or pRKemal/fd233 were tested for the ability to catalyze the oxidation of avermectin. The results shown in Table 3 demonstrate that these strains are able to catalyze this reaction.

EXAMPLE XIV -67- Case PB/5-60016A Identification of Genes Encoding Ferredoxins That Are Active With the

P

4 5 0Ema Monooxygenase P450 monooxygenases require two electrons for each hydroxylation reaction catalyzed (Mueller et al., "Twenty-five years of P 4 50ca,, research: Mechanistic Insights into Oxygenase Catalysis." Cytochrome P450, 2 nd Edition, P.R. Orliz de Montellano pp. 83-124; Plenum Press, NY 1995). These electrons are transferred to the P450 monooxygenase one at a time by a ferredoxin. The electrons are ultimately derived from NAD(P)H and are passed to the ferredoxin by a ferredoxin reductase. Specific P-450 monooxygenase enzymes have a higher activity when they interact with a specific ferredoxin. In many cases, the gene encoding a ferredoxin that interacts specifically with a given P450 monooxygenase is located adjacent to the gene encoding the P450 enzyme.

As described above, in addition to the emal gene, four P450 genes from strain R-922 and seven P450 genes from strain 1-1529 (see Example VI) were isolated and sequenced. In some of these, there was sufficient sequence information about the DNA flanking the P-450 genes to look for the presence of associated ferredoxin genes. By this approach, two unique ferredoxin genes were identified from each of the two strains. Ferredoxin genes fd229 and fd230 were identified from strain R-922, andfd233 andfdEA were identified from strain I- 1529. In addition, a ferredoxin reductase gene was found to reside adjacent to thefdEA gene from strain 1-1529.

In order to test the biological activity of each of these ferredoxins in combination with

P

4 50Emal, each individual ferredoxin gene was amplified by PCR to produce a gene fragment that included a blunt 5'-end, the native ribosome-binding site and ferredoxin gene coding sequence, and a Pmel restriction site on the 3'-end. Each such ferredoxin gene fragment was cloned into the Pmel site located 3' to the emal gene in plasmid pTUA-emal. In this way, artificial operons consisting of the emal gene and one of the ferredoxin genes operably linked to a functional promoter were created.

In the case of thefdEA ferredoxin gene in which a ferredoxin reductase gene,freEA, was found to be located adjacent to thefdEA gene, a DNA fragment containing both thefdEA andfreEA genes was generated by a similar PCR strategy. This gene fragment was also cloned in the Pmel site of plasmid pTUA-emal as described for the other ferredoxin genes.

-68- Case P13/5-60016A Each emal-ferredoxin gene combination was tested for biological activity by introduction of the individual emal-ferredoxin gene plasmids into S. lividans strain ZX7. The biocatalysis activity derived from each plasmid in S. lividans was determined. Of the four different constructs, only the ferredoxin genefd233 derived from strain 1-1529 provided increased activity when compared to the expression of emal alone in the same plasmid and host background (see Table The pTUA-emal/fd233 plasmid in S. lividans provided approximately 1.5 to 3- fold higher activity compared to the pTUA-emal plasmid. The other three plasmids containing the other ferredoxin genes gave results essentially the same as the plasmid with only the emal gene. Likewise, the pTUA-emal/fdEA/freEA plasmid did not yield results different from those of pTUA-eral. The nucleotide and deduced amino acid sequences of the fd233 gene are shown in SEQ ID NOs:35 and 36, respectively.

A BLAST analysis of the nucelotide and amino acid sequences offd233 revealed that the closest matches were to ferredoxins from S. coelicolor (GenBank Accession AL445945) and S. lividans (GenBank Accession AF072709). At the nucleotide level,fd233 shares 80 and 79.8 identity with the ferredoxin genes from S. coelicolor and S. lividans, respectively. At the peptide level,fd233 shares 79.4 and 77.8% identity with the ferredoxins from S. coelicolor and S. lividans, respectively.

Sincefd233 is derived from strain 1-1529 and emal is from strain R-922, the proteins encoded by the two genes cannot interact with each other in nature. In an approach designed to identify a ferredoxin gene from strain R-922 that is homologous to the fd233 gene and that might encode a ferredoxin that interacts optimally with the P 4 50EmaJ, thefd233 gene was used as a hybridization probe to a gene library of DNA from strain R-922. A strongly hybridizing cosmid, pPEH232, was identified and the hybridizing DNA was cloned and sequenced.

Comparison of the deduced amino acid sequences fromfd233 and the ferredoxin gene on cosmid pPEH232, fd232, revealed that they differed in only a single amino acid.

In a similar manner, plasmid pTUA-emal-fd232 was constructed and tested in S.

lividans ZX7. This plasmid gave similar results as those obtained with plasmid pTUA-emalfd233 (see Table The nucleotide and deduced amino acid sequences offd232 are shown in SEQ ID NOs:37 and 38, respectively.

The emal-fd233 operon was also subcloned, as a Pacl-Pmel fragment, into pTBBKA and pEAA that had been digested with the same restriction enzymes. S. lividans -69- Case PB/5-60016A ZX7::pTBBKA-emal-fd233, and S. lividans ZX7::pEAA-emal-Jd233 were tested in the avermectin conversion assay and found to have higher activities than the strains harboring the emnal gene alone in the comparable plasmids (see Table 3).

EXAMPLE XV Ileterologous Expression of P 4 50EmA and P 4 5 0E,, in OtherCells The expression constructs pRK-emnial (Example XII1) and pRK-ema2 (created in a way analogous to that described in Example XII for pRK-enzal) were mobilized by conjugation into three fluorescent soil Pseudonionas strains. Conjugation was performed according to standard methods (Ditta el al., Proc. Natl. Acad. Sci. USA 77:7347-7351, 1980). The strains were: P.fluorescensMOCGl34, P.fluorescens Pf-5, and P. fluorescens CHAO. Standard resting cell assays for the conversion of avermectin to 4"-ketoavermectin were conducted for each of the transconjugants. For strains Pf-5 and CHAO, the levels of conversion were below the detection limit. Strain MOCG 134 yielded 3% conversion for emal and 5% for ema2.

In addition, the constructs listed in the Table 5 were introduced into Streptomnyces avermitilis MOS-0001 by protoplast-mediated transformation (Kieser, Bibb, Buttner, Chater, Hopwood, D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich (England), 2000), (Stutzman-Engwall, K. et al. (1999) Strepiomyces avemnnitilis gene directing the ratio of B 2

:B

1 i avermectins, WO 99/41389).

Table Construct Conversion of avermectin, 16 hrs None 0 pTBBKA-enal 10.90 3.48 pTUA-enal 5.326 2.19 pEAA-emial 6.74 0.08 pTBBKA-enmalA/fd233 28.50 0.20 pTUA-emnalA/fd233 23.97 5.95 Case PB/5-60016A Wild-type Str. avermitilis MOS-0001 was tested and found to be incapable of the oxidation of avermectin to 4"-ketoavermectin.

Transformed S. avermitilis strains MOS-0001 ::pTBBKA-emal, MOS-0001 (pTUAemal), MOS-0001::pEAA-emal, MOS-0001::pTBBKA-emalA/fd233, and MOS-0001 (pTUA-emalA/fd233) were each tested for their ability to oxidize avermectin to 4"-ketoavermectin using resting cells. To do this, the whole cell biocatalysis assay described above (including analysis method) was performed. Note that for the whole cell biocatalysis assay, transformed Streptomyces avermitilis, like strain R-922, was grown in PHG medium and, again like strain R-922, had a reaction time of 16 hours during which time the 500 mg transformed Streptomyces avennitilis wet cells in 10 ml of 50 mM potassium phosphate buffer, pH 7.0, were shaken at 160 rpm at 28 0 C in the presence of 15 Vtl of a solution of avermectin in isopropanol (30 mg/ml)).

As shown in Table 5, in the presence of the inducer, thiostrepton (5 jig/ml), the emal- or emalA/fd233-containing strains MOS-0001::pTBBKA-emal, MOS-0001::pTBBKAemalA/fd233, MOS-0001 (pTUA-emal), MOS-0001 (pTUA-emalA/fd233) were found to oxidize avermectin to 4"-keto-avermectin as evidenced by the appearance of the oxidized 4"keto-avermectin compound. Note that the S. avermitilis strain MOS-0001::pEAA-emal demonstrated this oxidation activity in the absence of thiostrepton since in this strain the emcal gene is expressed from the ermE promoter that does not require induction.

Thus, expression of the emal P450 monooxygenase gene in various Streptomyces and Pseudomonas strains provided recombinant cells that were able to convert avermectin to 4"ketoavermectin in resting cell assays.

Next, expression and activity of P 4 50E,mna monooxygenase was tested in E. coli. To do this, the emal gene was cloned into the E. coli expression plasmid pET-28b(+) (commercially available from Novagen, Madison, WI) as described previously. E. coli strain BL21 DE3 (commercially available from Invitrogen; Carlsbad, CA) that contains the T7 RNA polymerase gene under control of the inducible tac promoter and the pET-28/emal plasmid was cultured in 50 ml LB medium containing 5 mg/I kanamycin in a 250-ml flask with one baffle, for 16 hours at 37 0 C, with shaking at 130 rpm. 0.5 ml of this culture was used to inoculate 500 ml LB medium with 5 mg/1 kanamycin in a 2-liter flask with one baffle, and the -71 Case PB/5-60016A culture was incubated for 4 hours at 37 0 C followed by 4 hours and 30 0 C, with shaking at 130 rpm throughout. The cells were harvested by centrifugation, washed in 50 mM potassium phosphate buffer, and centrifuged again.

For the resting cell assays, 90 mg wet cells were weighed into deep-well plates in triplicate and resuspended in 0.5 ml 50 mM potassium phosphate buffer. For cell-free extracts, 4 grams wet cells in 8 ml disruption buffer were disrupted in French press.

For the resting cell assays, 5 pl of substrate (2.5 mg/ml in 2-propanol) was added to the cell suspension. The plate was sealed with air permeable foil, and the reaction was incubated on an orbital shaker at 1000 rpm at 28 0 C for 22 hours. No conversion of avermectin to 4"ketoavermectin was detected.

For the cell-free assays, 100 ltl cell free extract, lpl substrate solution (20 mg/ml) in 2propanol, 5 .pl 100 mM NADPH, 10 il ferredoxin, 10 pl ferredoxin reductase, and 374 pl potassium phosphate buffer pH 7.0 were added as described in Example II, and the assay was incubated at 30°C with shaking at 600 rpm for 20 hours. 9.2% 0.3% of avermectin was converted to 4"-ketoavermectin.

Thus, expression of the emal gene in E. coli resulted in the production of the active Emal P450 monooxygenase enzyme which, when purified from the cells, was able to convert avermectin to 4"-ketoavermectin.

EXAMPLE XVI Identification and Cloning of Genes Encoding Ferredoxin Reductases that Support Increased Activity of the P 4 5 Fmni Monooxygenase The electron transport pathway that supports the activity of P450 monooxygenases also includes ferredoxin reductases. These proteins donate electrons to the ferredoxin and, as is the case with ferredoxins and P450 monooxygenases, specific ferredoxin reductases are known to be better electron donors for certain ferredoxins than others.

According, a number of ferredoxin reductase genes from Streptomyces strains were cloned and were evaluated for their impacts on the biocatalysis reaction. To do this, numerous bacterial ferredoxin reductase (Fre) protein sequences were retrieved from NCBI and aligned with the program Pretty from the GCG package. Two conserved regions, -72- Case PB/5-60016A approximately 266 amino acid residues apart, were used to make degenerate oligonucleotides for PCR. The forward primer (CGSCCSCCSCTSWSSAAS (SEQ ID NO:96; where is C or G; and is A or and the reverse primer (SASSGCSTTSBCCCARTGYTC (SEQ ID NO:97; where is C or G; is C, G, or T; is A or G; and is C or were used to amplify 800 bp products from the biocatalytically active Streptomyces strains R-922 and I- 1529. These pools of products were cloned into TOPO TA cloning vectors (commercially available from Invitrogen Inc., Carlsbad, CA), and 20 clones each from R922 and 1-1529 were sequenced according to standard methods (see, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley Sons, Inc. 2000). Sequencing revealed that 4 uniquefre gene fragments were isolated from the strains: three from R922 (fre3,frel2,fre 4) and one from 1-1529 (frel6). Thefre3,frel2,frel4, andfrel6 gene fragments were used as probes to identify full-length ferredoxin reductases from genomic clone banks of Streptomyces strains R922 and 1-1529. By this approach, the complete coding sequence of each of the 4 different fre genes was cloned and sequenced. The nucleic acid and amino acid sequences are provided as follows: fre3 (SEQ ID NOs:98 and 99);frel2 (SEQ ID NOs:100 and 101);frel4 (SEQ ID NOs:102 and 103); andfrel6 (SEQ ID NOs:104 and 105).

In order to assess the biological activity of eachfre gene in relation to the activity of Emal, each gene was inserted into the ernal/fd233 operon described above, 3' to thefd233 gene. This resulted in the formation of artificial operons consisting of the emal,fd233, and individual fre genes that were expressed from the same promoter. The emal/fd233/fre operons were cloned into the Pseudomonas plasmid pRK290 and introduced into 3 different P. putida strains. These strains were then analysed for Emal biocatalysis activity using the whole cell assay and one of the genes, thefre genefr-el6 from strain 1-1529, was found to increase the activity of P 4 50Emal monooxygenase by approximately 2-fold. This effect was strain specific, as it was seen only in one of the P. putida strains, ATCC Desposit No. 17453, and not in the other two. In P. putida strain ATCC 17453, the presence offre genefrel6 resulted in 44% conversion of avermectin to 4"-keto-avermectin, as compared to 23% without this gene. The otherfre genes had no impact on the biocatalysis activity in any of the P.

putida strains tested.

In a similar approach, each of the emal/fd233/fre operons were cloned into the Streptomyces plasmids pTUA, pTBBKA, and pEAA, and introduced into S. lividans strain -73- P:lOPER\DND\Cl.-,l12356420 d do30/11105 -74- ZX7. In each case there was no impact in S. lividans by any ofthefre genes on biocatalysis activity.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued patents, applications, and references, including GenBank database sequences, that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (19)

1. A purified nucleic acid molecule comprising a nucleic acid sequence that is at least 66% identical to SEQ ID NO:1 wherein said sequence encodes a P450 monooxygenase that regioselectively oxidizes avermectin B 1 a or avermectin B b to 4"keto-avermectin Bla or 4"keto-avermectin Bib respectively.

2. The nucleic acid molecule of claim 1, comprising a nucleic acid sequence that is selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ IDNO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 33; and SEQ ID NO: 94.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is isolated from a Streptomyces strain selected from the group consisting of: Streptomyces tubercidicus, Streptomyces lydicus, Streptomyces platensis, Streptomyces chattanoogensis, Streptomyces kasugaensis, Streptomyces rimosus and Streptomyces albofaciens.

4. The nucleic acid molecule of claim 1 further comprising a nucleic acid sequence encoding a tag which is linked to said P450 monooxygenase via a covalent bond. The nucleic acid molecule of claim 4, wherein the tag is selected from the group consisting of a His tag, a GST tag, an HA tag, a HSV tag, a Myc-tag, and VSV-G- Tag.

6. A cell genetically engineered to comprise a nucleic acid molecule according to claim 1.

7. The cell of claim 6, wherein said molecule is linked to a regulatory sequence in P:\OPER\DND\Claims\l2356420 amend doc-3011/05 -76- such a way as to permit expression of said molecule in said cell.

8. The cell of claim 6 further comprising a nucleic acid molecule encoding a ferredoxin protein.

9. The cell of claim 6, wherein the cell is a genetically engineered Streptomyces strain cell. The cell of claim 9, wherein the cell is a genetically engineered Streptomyces lividans strain.

11. The cell of claim 6, wherein the cell is a genetically engineered Pseudomonas strain.

12. The cell of claim 11, wherein the cell is a genetically engineered Pseudomonas putida strain.

13. The cell of claim 6, wherein the cell is a genetically engineered Escherichia coli strain.

14. The cell of claim 6, further comprising a nucleic acid molecule encoding a ferredoxin reductase protein. The cell of claim 6, further comprising a nucleic acid molecule encoding a ferredoxin protein and a nucleic acid molecule encoding a ferredoxin reductase protein.

16. A method for making 4"keto-avermectin Bla comprising adding a polypeptide encoded by a nucleic acid according to claim 1 or claim 2 to a reaction mixture comprising avermectin Bla and incubating the reaction mixture under conditions that allow the polypeptide to regioselectively oxidize said avermectin Bla to P'\OPER\DND\Claimr\ 2356420 Mend doc.301 I/0 -77- 4"keto-avermectin B a.

17. A method for making 4"keto-avermectin B b comprising adding a polypeptide encoded by a nucleic acid according to claim 1 or claim 2 to a reaction mixture comprising avermectin B b and incubating the reaction mixture under conditions that allow the polypeptide to regioselectively oxidize said avermectin B b to 4"keto-avermectin Bib.

18. A method according to claim 16 or 17 wherein the reaction mixture further comprises a ferredoxin protein.

19. A method according to any one of claims 16 to 18 wherein the reaction mixture further comprises a ferredoxin reductase protein.

20. A purified nucleic acid molecule according to claim 1, a cell according to claim 6, or a method according to claim 16 or claim 17, substantially as herein described with reference to the Examples and accompanying drawings. DATED this 30th day of November, 2005 Syngenta Participations AG By its Patent Attorneys DAVIES COLLISON CAVE Caise SEQUENCE LISTING <110> Syngenta Participations AG <120> METHODS AND COMPOSITIONS FOR MAKING EMAMECTIN <130> PB/5-60016A <140> <141> <150> US 60/291,149 <151> 2001-05-16 <160> 105 <170> FastSEQ for Windows Version <210> 1 <211> 1293 <212> DNA <213> Streptonyces tubercidicus <400> 1 atgtcggaat aatgtgatgg cagggcccgg ttcgaggagg tccctgaact cccgagcacc acccggctgc ccccgggtcg ggcgtcgtcg ctggtcggca tcgatggatc atggtccggg catgacgacg gtcctcgccg acccaccccg cacgagc tga gacgtcgacc tcggccaac t cccgcgggcc gccacactcg gacatatcgc cggctgaact taatgaactc accccgccct tcgtacgggg tccgccaggt acgcgcccga tccgcgtcta gccgtctggt agcagatcgc acctcatcca tacccgaagc cggaccggc t aacggcgcga acggcgggcg gccacgagac accagctgcg tgcgctggtg tcgccggcac tcgacccccg acgccgagaa ccaaacagga tgggcatcgc cgctgccggt tccgttcgcc gatcaccgac ccggttcatg cctgcgcgac ggacaacccg cctgctcgga g tcgcgggcg cgacgcgc tg gcacttcgcc ggaccgcccg cggcgcctcg ggcgctcacc gc tcagcgac caccgcccac tctggtcaag cgggccggtg accgatccgc tcactacacc ccatgtgggt aggtgaagtc cccggaacac gcgg ttgggg gcgcacgtcg ccgttcaccg gacgactcgc cagcggttcg ctgacccggc tcgatcctca ttcacggccc ctggcccggc taccccctgc cagtggcgaa ttcccggcga gacgacctgc gtcgagatgg ctcatcagca gacgatccgg caca tgaccc cagggcgatg gaccccgacc t tcggcca tg gccttcggca ctggagcgga tga ggaaacaccc gctacggcgc ccgtctggct tgaacaatcc tgatggagat actacgacgc gcaagatcac tgcccgagca cgatcaccgt cgtggggcgc tga tcgagca tcagcgaact tcaccatgat acggcacggc ccctcctccc agctgcgcta ccgttcaact gcctcgatct gagcgcacta aactgctcac caccgctgcc gggcgagccg gctgcgtgag ggtgacgcgg ggcctcgccg gctgggcctc ccccgaccac cgacctgcgg cgccgaggac catctgcgaa cgacc tca tc catccatcag gatccgcacc cctcacgctc ggcgctgctc ccgtgccgtc cgccaccgcc catcctggta cacccggcac ctgcctgggc goac tacccg gggcaac tgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> 2 <211> 430 <212> PRT <213> Streptomyces tubercidicus I- Case <400> 2 Met Ser Glu Leu Met Asn Ser Pro Phe Ala Ala His Val Gly Lys His 1 5 10 Pro Gly Glu Pro Asn Val Met Asp Pro Ala Leu Ile Thr Asp Pro Phe 25 Thr Gly Tyr Gly Ala Leu Arg Glu Gin Gly Pro Val Val Arg Gly Arg 40 Phe Met Asp Asp Ser Pro Val Trp Leu Val Thr Arg Phe Glu Glu Val 55 Arg Gin Val Leu Arg Asp Gln Arg Phe Val Asn Asn Pro Ala Ser Pro 70 75 Ser Leu Asn Tyr Ala Pro Glu Asp Asn Pro Leu Thr Arg Leu Met Glu 90 Met Leu Gly Leu Pro Glu His Leu Arg Val Tyr Leu Leu Gly Ser Ile 100 105 110 Leu Asn Tyr Asp Ala Pro Asp His Thr Arg Leu Arg Arg Leu Val Ser 115 120 125 Arg Ala Phe Thr Ala Arg Lys Ile Thr Asp Leu Arg Pro Arg Val Glu 130 135 140 Gin Ile Ala Asp Ala Leu Leu Ala Arg Leu Pro Glu His Ala Glu Asp 145 150 155 160 Gly Val Val Asp Leu Ile Gin His Phe Ala Tyr Pro Leu Pro Ile Thr 165 170 175 Val Ile Cys Glu Leu Val Gly Ile Pro Glu Ala Asp Arg Pro Gin Trp 180 185 190 Arg Thr Trp Gly Ala Asp Leu Ile Ser Met Asp Pro Asp Arg Leu Gly 195 200 205 Ala Ser Phe Pro Ala Met Ile Glu His Ile His Gin Met Val Arg Glu 210 215 220 Arg Arg Glu Ala Leu Thr Asp Asp Leu Leu Ser Glu Leu Ile Arg Thr 225 230 235 240 His Asp Asp Asp Gly Gly Arg Leu Ser Asp Val Glu Met Val Thr Met 245 250 255 Ile Leu Thr Leu Val Leu Ala Gly His Glu Thr Thr Ala His Leu Ile 260 265 270 Ser Asn Gly Thr Ala Ala Leu Leu Thr His Pro Asp Gin Leu Arg Leu 275 280 285 Val Lys Asp Asp Pro Ala Leu Leu Pro Arg Ala Val His Glu Leu Met 290 295 300 Arg Trp Cys Gly Pro Val His Met Thr Gin Leu Arg Tyr Ala Thr Ala 305 310 315 320 Asp Val Asp Leu Ala Gly Thr Pro Ile Arg Gin Gly Asp Ala Val Gln 325 330 335 Leu Ile Leu Val Ser Ala Asn Phe Asp Pro Arg His Tyr Thr Asp Pro 340 345 350 Asp Arg Leu Asp Leu Thr Arg His Pro Ala Gly His Ala Glu Asn His 355 360 365 Val Gly Phe Gly His Gly Ala His Tyr Cys Leu Gly Ala Thr Leu Ala 370 375 380 Lys Gin Glu Gly Glu Val Ala Phe Gly Lys Leu Leu Thr His Tyr Pro 385 390 395 400 Asp Ile Ser Leu Gly Ile Ala Pro Glu His Leu Glu Arg Thr Pro Leu 405 410 415 Pro Gly Asn Trp Arg Leu Asn Ser Leu Pro Val Arg Leu Gly -2- Casc <210> 3 <211> 1293 <212> DNA <213> Streptornyces tubercidicus <400> 3 atgtcggcat aatgtgatgg caggccccgg ttcgaggagg cccctggccc cccgagcacc acccggc tgc ccgcgtgtcg ggcgtcgtcg ctggtcggca tcga tggacc a tgg tccggg catgacgacg gtcCtcgccg acccaccccg catgaactga gacgtcgacc gcggcgaac t cccgccggcc gccaccctcg gaga tgtccc cggctgaatt tatccagctc agccggcgct tcg tacgggg tccgccaagt ca tcggccga tccgcgtcta gccgtctggt. agcaga tcgc acctcatcca tacccgaagc cggaccggc t agcggcgcgc a tggcggccg gtcacgagac accagctgcg tgcgctggtg tcgccgg tac tcgacccccg acgccgagaa ccaagcagga tgggca tcga ccctgccgtt tccgttcgct gctcaccgac ccggttcgtg cctgcgcgac ggagaacccg catgctcggg gtcgcgcgcg cgacgagctg gcatttcgcc ggaccgcccg cggcgcaacg ggcgctcacc gctcagcgac caccgcccac cctgctcaag cgggccgg tg gcggatccac ccactacacc ccatgtgggt gggcgaagtc accggaacgt gcggctgggg gcgcatgtcg ccgttcgcgg gacgactcac cagcggttcg ctgaccaggc tcgattctca t tcacggcgc ctggcccgcc tacccgctgc cag tggcgga t tcccggcga gatgatctgc gtcgagatgg ctcatcagca gacgacccgg cagatgacgc aagggcgacg gaccccgacc t tcggccacg gcgttcggca ctggagcgat tga ggaaacaccc gctacggcgc cggtctggtt tgaacaatcc tga tggaca t actacgacgc ggaagatcac tccccgagta cgatcaccgt agtggggcgc tgatcgagca tcagcgagct tcaccatgat acggcacggc ccctgctccc agctgcgcta. ccgtacaact gtctcgatct gtgcgcatta agctgctcgc tgccgctgcc gggtgagccg gctgcgtgag cgtgacgcgc ggccgcgccg gctgggcctc ccccgaccac cga tc tgcga cgccgaggac ca tctgcgag cgacctcatc catccatgag gatccgtacc cctcacgctc ggcgctgctc ccgggccgtc cgcggccgcc cctcctggtt gacgcgtcac ctgcctggg t gcactacccg tggcaactgg 120 180 240 300 360 420 480 540 600 660 720 840 900 960 1020 1080 1140 1200 1260 1293 <210> 4 <211> 430 <212> PRT <213> Streptoiryces tubercidicus <400> 4 Met Ser Ala 1 Pro Gly Glu Leu Ser Ser Ser Pro Phe Ala Ala His Val Gly 5 Asn 10 Al a Lys His Val Met Glu Pro Gln Leu Leu Thr Ala Gly Tyr Gly Ala Leu Phe Val Asp Asp Ser Pro Arg Gln Val Leu Arg Asp 70 Pro Leu Ala Pro Ser Ala Arg Val 55 Gln Glu 40 Trp, Ala Pro Val Val1 415 Phe Asp Pro Phe Arg Gly Arg Glu Glu Val Phe Val Thr Arg Asn Arg Phe Val Asn 75 Leu Pro Ala Ala Pro Glu Glu Asn Pro Pro 90 Val1 Thr Arg Leu Met Asp Met Leu Gly Leu Asn Tyr Leu 100 Asp Glu His Leu Arg 105 Thr Tyr Met Leu Gly Ser Ile 110 Leu Val Ser Ala Pro Asp His Arg Leu Arg Arg Case 115 Arg Ala Phe 130 Gin Ile Ala 120 Ile Thr Ala Arg Lys 135 Leu Thr Asp Leu Arg 140 Glu Pro Arg Val Glu Asp Glu 145 Gly Leu 150 Ile Ala Arg Leu Pro 155 Tyr Tyr Ala Glu Asp 160 Val Val Asp Gin His Phe Ala 170 Glu Pro Leu Pro Ile Tlir 175 Val Ile Cys Arg Lys Trp 195 Ala Thr Phe Gi.u 180 Gly Val Gly Ile Ala Asp Arg Ala Asp Leu Ile 200 Met Asp Pro Asp 205 Met Pro Gin Trp 190 Arg Leu Gly Vai Arg Glu Pro Ala Met 210 Arg Arg Ala Ala Leu 225 His Thr 230 Gly Ile Glu 215 Asp Asp Arg Leu His Ile His Leu Leu Ser 235 Ser Asp Val 250 His Giu Thr Glu 220 Glu Leu Ile Arg Thr 240 Asp Asp Asp Gly 245 Val Glu Met Val Thx Met 255 Ile Leu Thr Ser Asn Gly 275 Leu Lys Asp Leu 260 Thr Leu Ala Gly Thr Ala 265 Thx Ala Ala Leu Leu 280 Leu His Pro Asp Gin 285 His His Leu Ile 270 Leu Arg Leu Glu Leu Met Asp Pro Ala 290 Arg Trp Leu 295 Gin Pro Arg Ala Cys Gly Pro 305 Asp Va1 310 Gly Met Thr Gin Leu 315 Lys Tyr Ala Ala Al a 320 Val Asp Leu Ala 325 Ala Thr Arg Ile His 330 Pro Gly Asp Ala Val Gin 335 Leu Leu Leu Asp Arg Leu 355 Val Giy The Val 340 Asp Ala Asn Phe Asp 345 Pro Arg His Tyr Leu Thr Arg His 360 His Ala Gly His Ala 365 Ala Thr Asp Pro 350 Glu Asn His Thr Leu Ala Gly His Gly 370 Lys Gln Ala 375 Ala Tyr Cys Leu Gly 380 Leu Glu Gly Glu Phe Gly Lys 385 Glu Leu 395 Leu Ala His Tyr Met Ser Leu Gly 405 Arg Ile Giu Pro Glu Leu Asn Ser Leu 425 Arg 410 Pro Glu Arg Leu Pro 415 Pro Gly Asn Trp 420 Leu Arg Leu Gly 430 <210> <211> 1413 <212> DNA <213> Streptomyces rimosus <400> atgaccacat tcgaccgctt cgcaccacgc gagctgctgg cgcgcccggt gaggtgatgc gacaaggacc cgcccaccga ccgccccgge tcccctccta agaacccgta tcatcgacga gtgaccagcg cgcgtgcccg gtcccgggcg caccacccct cgtcggcctc caccggctac ctcgcccatc gttcgtcaac gctgatcgag gccaccccgc tcggccgccg cacccgggcg ggcacgctgc tggctggtga aacccgaccc ctgttcggca ccgactccac cctctccgga agccgaacct gcgagcaggc cccgcttcga tggtgcccgg tccccgagga cgcctccccc caccaccgac gatggaaccg cccgctcgtc cgtggtgcgc catcggcgcg cctggccccg 120 180 240 300 360 420 Casc tacctcaccg gtctcccgcg accgacgagc gagcacttcg gaggatcggg atcggcgcca gcggccc tgc cggctgagcg accaccgccc cggctgatcg tgcgggccga gtccaggtcc cgccactaca gaccacgtcg gaggccgagg accccggaac cggctgccgc acaacatcct ccttcaccgc tgc tggaacg cctacccgct cgctgtggcg cca tgccgga gggacgacc C acgtcgagat acctcatcag acgaggaccc tccaggccac gccagggcga ccgggccgga gcttcggcca tggcctacgg agttggagga tgaggctgca caccagcgac acgccgtatc gc tgccggac gcccatcacg gcggtCtcggc ga tgatctcq gc tcagcggg ggtcaccctg caacggcacc g~cgctgctg ccagcttcgg ggccc tga tg gcggctcgac cggca tgcac gaagctgctc ccaggaacgc cgcgcagagc ccgccggacc caggacctgc catgccgagg gtcatctgcg gccgacctcg cacatccacg ctcatccggg gtcctgaccc ctcgccctgc ccgcgcgcgg tacgccctgg ttcagcctcg ctgacgcggc tactgcctgg acccgctacc c tgcggcagc tga acacccggct ggccgcgcg C acggcgtcgt agctgg tcgg cctcgctgaa agctgatcga cgcaggacga tggtactggc tcacccaccc tccacgagct aggacaccga tcgcggccaa agccggccgg gtgcctcact cggacctggc ccggcacctg gcgccgcc tg cgagcgga Cc cgacctcgtc ca Ccgacgag ccccaagcgc cgaacggcgc cgacggcggc cggtcacgag cgaccagcgg gatgcgctgg gg Cggccgga ccacgacccg ccgcgccgag cgcccggcag gctcgccctc gcgcc Cgcga 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1413 <210> 6 <211> 470 <212> PRT <213> Streptomyces rimosus <400> 6 Met Thr Flit Ser Pro Thr Glu Ser Arg 1 Thr Ala 5 Ala Ser Pro Ser Ala Ser Pro Asp Ala 10 Ada Thr Pro Pro Asp Ser Thx Ala Ser Thr Thr Asp 40 Glu Pro Asn Ala Pro 25 Arg Thr Leu Met Ala Thr Thr Thr Leu Pro Glu Pro Glu Pro Ser Ala Ser Tyr Val Leu Leu Glu Gly Leu His Pro Gly Asn Pro Tyr Thr Gly Tyr 70 55 Gly Gin Thr Leu Arg Arg Glu 75 Trp Ala Pro Leu Val1 Ala Arg Phe Ile Glu Asp Asp Ser Pro Ile 90 Gin Leu Val Thr Asp Val Val Thr Leu Val 115 Ile Glu Leu Arg 100 Pro Val Met Arg Asp 105 Asp Arg Phe Val Asn 110 Al a Arg Phe Asn Pro Arg Leu Gly Ile Gly Al a 120 Glu Lys Asp Pro Arg 125 Tyr Phe Gly Ile 130 Asn Ile Pro 135 Pro Asp Leu Ala Leu Thr Asp Leu Thr Ser 145 Val1 Asp 150 Thr Pro Asp His Thr 155 Gin Leu Arg Arg Ser Arg Ala Phe 165 Thr Ala Arg Arg Ile 170 Glu Asp Leu Arg Pro Arg 175 Val Glu Arg Glu Asp Gly 195 Ile Thr Val. Asp Glu Leu Leu 185 Arg Leu Pro Val Asp Leu Ile Cys Glu Leu Val Glu 200 Val Gly Asp Leu His Phe Ala Ile Asp Glu 220 Ala Ser Leu Tyr 205 Glu Asp His Ala 190 Pro Leu Pro Asp Arg Ala 210 Leu Trp 215 Ala Arg Arg Phe Gly Asn Pro Lys Arg Case 225 Ile 230 Pro 235 H is Gly Ala Thr Met 245 Al a Giu Met Ie Ser 250 Asp Ile His Giu 240 Leu Ile 255 Asp Giu Arg Arg Ala Gin 275 Thr Leu Vai Arg 260 Asp Ala Leu Arg Asp 265 Arg Leu Leu Ser Asp Asp Gly Giy 280 Leu Leu Ser Asp Vali 285 Tim Giy Leu Ile 270 Giu Met Val Tim Ala His Leu Tim Leu Aia Gly His 290 Leu Ile Giu 300 His Ser Asn Gly 305 Arg Tim 310 Asp Aia Leu Leu Tim 315 Pro Pro Asp Gin Arg 320 Leu Ile Asp Giu 325 Cys Pro Ala Leu Leu 330 Ala Arg Ala Val His Giu 335 Leu Met Arg Leu Giu Asp 355 Leu Met Phe Trp 340 Thr Giy Pro Ile Gin 345 Vali Tim Gin Leu Giu Val Ala Gly 360 Al a Gin Val Arg Gin 365 Arg Arg Tyr Ala 350 Giy Giu Ala His Tyr Thr Ser Leu Val 370 Giv Pro Al a 375 Leu Asn His Asp Pro 380 Al a Giu Arg Leu Tim Arg Gin 385 Asp Pro 395 Tyr Giy Arg Ala Giu 400 His Vai Gly Phe 405 Giu His Giy Met His 410 Tyr Cys Leu Gly Ala Ser 415 Leu Ala Arg 'Tyr Pro Asp 435 Gin 420 Leu Ala Glu Val Ala 425 Tim Gly Lys Leu Ala Leu Ala Leu 440 Tim Pro Giu Gin Leu 445 Arg Leu Tim Arg 430 Giu Asp Gin Leu Pro Leu Giu Arg 450 Arg Leu 465 <210> 7 <211> i1 <212> DI Leu Arg Gin Pro Gly 455 Trp, Arg Leu Arg 460 -is Ala Gin Ser 470 293 NIA <213> Streptomyces lydicus <400> 7 atgtcggcat aacgtgatgg cagggcccgg ttcgaggagg gcgccgggcg cccgagcacc acccggctgc ccgcgcgtca ggcgtcgtcg ctggtcggca tcgc tgcagc c tga tcgcgg catgacgacg gtcctggccg acccaccccg cacccagcaa atccggcgct tcg tgcgggg tccgcgaggt cggcccccga tgcgcgtcta gccgcctggt cacaga tagc acctgatcca tccogagga cggaccgga t cgcggcgccg acggcagccg gccacgagac accagctgcg cacgttcacc gatcggggat gcggttcatg cctgcgtgac ggacaccccg tctgctcggc o tcccgggcc cgacgagctg gcacttcgcc ggaccgcccg gagccgg tcc ggcgctcacc gc tcagcgac caccgcgcac gctgctcaag gagcacgtcg ccgttcgccg gacgactccc ccgcggt tcc o tgtcccggc tcga tootca t tcaoogogo 0 tggcoggc ta tcccc tgc cagtggcgoa ttcccggoga gacga tc tgc gtogagatgg otca toggca gacgacccgg gcaagcacc gttacggcgc ccgtgtggtt ggaacaa tcc tga tggaca t acaacgacgo gga aga tcac tgccggagca ogatcaccgt cctggggcgo tga tcgacca tcagcgagct tcaccatggt acggcacggc cgctgctgcc gggcgagocg gctgcgcgag cgtgacccgc ggtotocgog gatgggtttc ccocgacoac cga tc tgcgg cgccgaggac ca totgcgaa cgacctggtc ca tocacgag ga tooggaco ootoaocg tc ggocctgcto gcgcgcgg tg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 Case cacgagttga gacgtcgagc tcggcgaacc cctgccggcc gccacgctcg gagctgtcgc cggc tgaacg tgcgctggtg cggcccggtg cacatgaccc agctgcgcta cgccgccgag tggcgggcgt ccggatccgc acgggggacg ccgtccagct catcctggtg gcgacccgcg ccactacacc gaccccgacc ggctggacct gacccggcac acgcggagaa ccatgtgggg ttcggccacg gggcgcacta ctgtctgggc ccaagcagga gggcgaggtc gccctcggcg ccctgctcag gcacttcccc tggccgtcgc gccggaggcc ctggagcgca caccggtacc gggcagctgg cgctgccgct gcgtctgcgc tga 960 1020 1080 1140 1200 1260 1293 <210> 8 <211> 430 <212> PRT <213> Streptomyces lydicus <400> 8 Met Pro Al a Phe Arg Ala Met Leu Arg Gin 145 Gly Val1 Arg Arg Arg 225 His Val1 Gly Leu Ser Ala Gly Glu Gly Tyr Met Asp Glu Val Pro Gly Met Gly Asn Asn 115 Ala Phe 130 Ie Ala Val Val Ile Cys Thr Trp 195 Ser Phe 210 Arg Arg Asp Asp Leu Thx Asn Gly 275 Lys Asp Ser Pro Gly Asp Leu Al a Phe 100 Asp Thx Asp Asp Glu 180 Gly Pro Ala Asp Val1 260 Thr Asp Pro Asn Al a Ser Arg Al a Pro Al a Ala Giu Leu 165 Leu Al a Ala Leu Gly 245 Val1 Ala Pro Ser Val1 Leu Pro Asp 70 Pro Glu Pro Arg Leu 150 Ile Val1 Asp Met Thr 230 Ser Leu Al a Ala Asn Met Arg Val1 55 Pro Glu 1-is5 Asp Lys 135 Leu Gin Gly Leu Ile 215 Asp Arg Al a Leu Leu *Thr Asp Glu 40 Trp, Arg Asp Leu His 120 Ile Ala His Ile Val1 200 Asp Asp Leu Gly Leu 280 Leu Phe Thr Glu His Val Gly LYS His Pro 25 Gin Phe Phe Thr Arg 105 Thr Thr Arg Phe Pro 185 Ser His Leu Ser His 265 Thr Pro Ala Gly Val Arg Pro 90 Val Arg Asp Leu Ala 170 Giu Leu Ile Leu Asp 250 Glu His Arg Gin -7- Leu Pro Thr Asn

75. Leu Tyr Leu Leu Pro 155 Tyr Glu Gin His Ser 235 Val Thr Pro Ala Ile Val1 Arg Asn Ser Leu Arg Arg 140 Glu Pro Asp Pro Glu 220 Giu Giu Thr Asp Val1 300 Gly Val1 Phe Pro Arg Leu Arg 125 Pro His Leu Arg Asp 205 Leu Leu Met Ala Gin 285 His Asp Arg Glu Val1 Leu Gly 110 Leu Arg Al a Pro Pro 190 Arg Ile Ile Val1 His 270 Leu Glu Pro Gly Glu Ser Met Ser Val Val1 Glu Ile 175 Gin Met Ala Arg Thr 255 Leu Arg Leu Phe Arg Val1 Al a Asp Ile Ser Thr Asp 160 Thr Tr-p Ser Ala Thr 240 Met Ile Leu Met 290 Arg Trp Cys Gly Pro Val 295 His Met Thr Leu Arg Tyr Ala Ala Glu Case 305 Asp 310 Gly Val Glu Leu Ala 325 Ser Val Arg Ile Arg 330 Pro Gly Asp Ala 320 Val Gin 335 Leu Ile Leu Val Ala Asn Arg 340 Asp Asp 345 Pro Arg His Ty'r Asp Arg Leu 355 Val Cly Phe Leu Thr Arg His 360 His Ala Gly His Ala 365 Al a Thr Asp Pro 350 Glu Asn His Thr Leu Ala Gly His Gly 370 Lys Gin Ala 375 Ala Tyr Cys Leu Gly 380 Leu Glu Gly Glu 385 Glu Val1 390 Val1 Leu Gly Ala Leu 395 Leu Arg His Phe Pro 400 Val1 Leu Ser Leu Al a 405 Arg Ala Pro Glu Al a 410 Pro Glu Arg Thr Pro 415 Pro Gly Ser Trp 420 Leu Asn Ala Leu 425 Leu Arg Leu Arg 430 <210> 9 <211> 1299 <212> DNA <213> Streptomyces sp. <400> 9 atgtcagcct aacgtgatgg caggggccgg ttcgaggagg ctcctgggca cccgagcacc acccggcttc ccgcgcgtcg ggcgtcgtcg ctggtcggca tcactggagc ctgatccgcc catgacgacg gtcttcgccg acccaccccg ca tgagc tga gacatcgacc tcggcgaact cccgcaggcc gccgcgCtcg gacgtagcgc tatccagctc aaccggctct ttgtgcgtgg tccgccaagt gtcaggtcga ttcgggtcta gccgcCtcgt agcagatcgc acctcatcca tacccgaagc cggacaagct aacggcgcgg acggcagccg gtcacgagac accagctgcg tgcgctggtg tcgccggtac tcgacccccg acgccgagaa ccaggcagga tgggcg tcga tccgttcgcc gatcaacgat ccggttcatg cctgcgcgac ggagatgccg tctgctcgga. ctcgcgggcc cgacgagctg gcac ttcgcc cga tcgcccg cagcacgtcg cgcgc tcacc gctcagcgac caccgcccac cctgctcaag cgggccgg tg gccgatccgg ccac tacagc ccacg tgggc aggcqaag tg accggaagcc gcgg ttggcg gagcacatag ccgttcggcg gacgactcgc cagcggt tcg atggtcaagc tcgatcctca ttcaccgcac ctggcccggc tacccgctgc caa tggcgcg ttcccggcga gacgatc tgc gtcgagatgg ctcataggca gacgacccgg cacatgaccc aagggcgacg gaccccgatc ttcggccacg gcgttcggca c tggagcggg aagcgctaa ggaaacaccc gctacggcgc ccgtgtggtt tgaacaatcc tgctggagca acagtgacgc gtaagatcac tccccgagca cgatcacggt catggggcgc tga togacca tcagcgagct tcaccatggt acggcacggc ccctgctccc agttgcgtta ccgtccaact gcc tcgacc t gga tgcac La aactgctcgc tgccgatgcc gggcgagccg gctgcgcgag cgtgacccgc ggcgtcgccg gatgggcc tc ccccgatcac cgg tc tgcgg cgccgaggac catctgcgaa cgacctcgtg cacccatgaa gatccg tgcc gttcgctctc ggcgctgctc gcgtgccgtc cgcctccgag catcctggta gacccgtcac ctgcttgggc gcactacccg cggcagttgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1299 cggc~gaatt ccttgccgct <210> <211> 432 <212> PRT <213> Streptomyces sp. <400> Met Ser Ala Leu Ser Ser Ser Pro Phe Ala Glu His Ile Gly Lys His Case Pro Gly Glu Pro Asn Val Met Glu Pro Ala Leu Ile Asn Asp Pro Phe Gly Phe Arg Leu Gin Leu Arg Gin 145 Gly Val Arg Thr Arg 225 His Val Gly Leu Arg 305 Asp Leu Asp Val Arg 385 Asp Pro Gly Met Gin Leu Met Asn Ala 130 Ile Val Ile Ala Ser 210 Arg Asp Phe Asn Lys 290 Trp Ile Ile Arg Gly 370 Gin Val Tyr Asp Val Gly Gly Ser 115 Phe Ala Val Cys Trp 195 Phe Gly Asp Ala Gly 275 Asp Cys Asp Leu Leu 355 Phe Glu Ala Gly Asp Leu Ser Leu 100 Asp Thr Asp Asp Glu 180 Gly Pro Ala Asp Leu 260 Thr Asp Gly Leu Val 340 Asp Gly Gly Leu Ala Ser Arg Gin Pro Ala Ala Glu Leu 165 Leu Ala Ala Leu Gly 245 Val Ala Pro Pro Ala 325 Ser Leu His Glu Gly 405 Leu Pro Asp 70 Val Glu Pro Arg Leu 150 Ile Val Asp Met Thr 230 Ser Phe Ala Ala Val 310 Gly Ala Thr Gly Val 390 Val Arg Val 55 Gin Glu His Asp Lys 135 Leu Gin Gly Leu Ile 215 Asp Arg Ala Leu Leu 295 His Thr Asn Arg Met 375 Ala Glu Glu 40 Trp Arg Glu Leu His 120 Ile Ala His Ile Val 200 Asp Asp Leu Gly Leu 280 Leu Met Pro Phe His 360 His Phe Pro 25 Gin Phe Phe Met Arg 105 Thr Thr Arg Phe Pro 185 Ser His Leu Ser His 265 Thr Pro Thr Ile Asp 345 Pro Tyr Gly Glu Gly Val Val Pro 90 Val Arg Gly Leu Ala 170 Glu Leu Thr Leu Asp 250 Glu His Arg Gin Arg 330 Pro Ala Cys Lys Ala 410 Pro Thr Asn 75 Met Tyr Leu Leu Pro 155 Tyr Ala Glu His Ser 235 Val Thr Pro Ala Leu 315 Lys Arg Gly Leu Leu 395 Leu Val Arg Asn Val Leu Arg Arg 140 Glu Pro Asp Pro Glu 220 Glu Glu Thr Asp Val 300 Arg Gly His His Gly 380 Leu Glu Val Phe Pro Lys Leu Arg 125 Pro His Leu Arg Asp 205 Leu Leu Met Ala Gin 285 His Tyr Asp Tyr Ala 365 Ala Ala Arg Arg Glu Ala Leu Gly 110 Leu Arg Ala Pro Pro 190 Lys Ile Ile Val His 270 Leu Glu Ala Ala Ser 350 Glu Ala His Val Gly Glu Ser Leu Ser Val Val Glu Ile 175 Gin Leu Arg Arg Thr 255 Leu Arg Leu Ser Val 335 Asp Asn Leu Tyr Pro 415 Arg Val Pro Glu Ile Ser Glu Asp 160 Thr Trp Ser Gin Ala 240 Met Ile Leu Met Glu 320 Gin Pro His Ala Pro 400 Met Gly Ser Trp Arg Leu Asn Ser Leu Pro Leu Arg Leu Ala Lys Arg 425 430 -9- Clse <210> <211> <212> <213> 11 1293 DNA Streptornyces chattanoogenesis <400> 11 atgtcggcat aacg tga tgg cagggcccgg ttcgaggagg gcgccgggcg cccgagcacc acccggctgc ccgcgcgtca ggcg tcg tcg ctggtcggca tcgctgcagc ctga tcgcgg catgacgacg gtcctggccg acccaccccg cacgagttga gacg tcgagc tcggcgaacc cccgccgg tc gccacgctcg gagctgtcgc cggc tgaacg cacccagcaa atccggcgct tcg tgcgggg tccgcgagg t cggcccccga tgcgcgtcta gccgcctggt cacagatagc acctgatcca tccccgagga cggaccggat cgcggcgccg acggcagcag gccacgagac accagctgcg tgcgctggtg tggcgggcgt gcgacccgcg acgcggagaa ccaagcagga tggccgtcgc cgctgccgct cacgttcacc gatcggtgat gcggttcatg cctgcgtgac ggacaccccg tctgCtcggc ctcccgggcc cgacgagctg gcacttcgcc ggaccgcccg gagccgg tcc ggcgctcacc gctcagcgac caccgcgcac go tgc tcaag cggcccggtg ccgga tccgc ccactacacc ccatgtgggg gggcgagg to gccggacgcc gcgtctgggc gagcacgtcg cog ttcgcog gacgactcco ccgcggttoc ctgtocoggo tcgatcctca ttcaccgcgo c tggoooggc tatoccotgc cagtggcgca t tocoggoga gacgacc tgc gtcgagatgg ctoatcggca gacgaooogg cacatgacoo aogggggaog gaccccgac ttcggooacg gcoo tcggog ctggagcgca tga gcaagcaoc gttacggcgc ccgtgtggtt ggaacaatcc tgatggacat aoaacgacgc ggaaga toac tgccggagoa oga toacogt cc tggggcgc tga togacca tcagcgagct tcaccatggt acggcacggc cactgctgcc agctgcgcta ccgtccagct gtctggacot gggcgcacta occtgctcag caccggtacc gggcgagccg gctgcgcgag cg tgacccgc ggtCtccgcg gatgggtttc ccocgaccac cgatctgcgg cgccgaggac catctgcgaa cgacctggtc catccacgag gatccggacc cctcaccgtc ggccctgctc gcgcgcgg tg cgccgccgag catcctggtg gacccggcac ctgtotgggc gcacttcccc gggcagc tgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> 12 <211> 430 <212> PRT <213> Streptomyces chattarioogenesis <400> 12 Met Ser Ala 1 Pro Gly Glu Ala Gly Tyr Phe Met Asp Ser Pro Ser Asn Thr Phe Pro Gly 5 Asn Ala Val Met Asp Pro 25 Leu Arg Glu Gin 40 Pro Val Trp Phe Thr 10 Ala Glu His Val Gly Lys His Leu Ile Gly Asp Ser Gly Pro Val Val Thr Arg Aro Asn Asn Val Phe Asp Pro Phe Arg Gly Arg Glu Glu Val Arg Glu 55 Pro Val Leu Arg Ala Asp 70 Arg Phe Pro Val Ser 75 Leu Ala Pro Gly Ala Ala Pro Glu Asp Thr Pro 90 Val1 Ser Arg Leu Met Asp Ser Ile Met Met Gly Leu Asn Asn 115 Arg Ala Phe Pro Ala Glu His Leu Arg 105 Pro Asp His Thr 120 Arg Lys Ile Thr Tyr Leu Leu Gly Arg Leu Arg Asp Leu Arg Arg 125 Pro 110 Leu Val Ser Arg Val Thr Thr Ala Ca se 130 Ile 135 Leu Gin 145 C ly Ala Asp Giu Leu 150 Ile Ala Arg Leu Pro 155 Tyr His Ala Giu Asp 160 Val Val Asp Leu 165 Leu Gin His Phe Al a 170 Giu Pro Leu Pro Ile Thr 175 Val Ile Cys Arg Thr Trp, 195 Arg Ser Phe Val Gly Ile Pro 185 Ser Giu Asp Arg Ala Asp Leu Val1 200 Asp Leu Gin Pro Asp 205 Leu Pro Gin Tmp 190 Arg Met Ser Ile Ala Ala Pro Ala Met 210 Arg Arg Ile 215 Asp His Ile His Giu 220 Giu Arg Ala Leu Asp Leu Leu 225 His Ser 235 Val1 Leu Ile Arg Thr 240 Asp Asp Asp Gly 245 Val1 Arg Leu Ser Asp 250 Glu Giu Met Val Thr Met 255 Val Leu Thr Gly Asn Giy 275 Leu Lys Asp Val1 260 Thr Leu Ala Gly His 265 Thr Thr Thr Ala Ala Ala Leu Leu 280 Leu His Pro Asp Gin 285 His His Leu Ilie 270 Leu Arg Leu Giu Leu Met Asp Pro Ala 290 Arg Trp Leu 295 His Pro Arg Ala Val1 300 Arg Cys Oly Pro 305 Asp Val1 310 Gly Met Thr Gin Leu 315 Thr Tyr Ala Ala Glu 320 Val Giu Leu Val Arg Ile Arg 330 Pro Gly Asp Ala Val Gin 335 Leu le Leu Asp Arg Leu 355 Val Gly Phe Val1 340 Asp Ala Asn Arg Asp 345 Pro Arg His Tyr Leu Thr Arg His 360 His Ala Gly His Al a 365 Ala Thr Asp Pro 350 Glu Asn His Thr Leu Ala Gly His Gly Tyr Cys Leu 370 Lys Gin Gly 380 Leu Giu Gly Glu 385 Giu Val1 390 Val1 Leu Gly Ala Arg His Phe Pro 400 Val Leu Ser Leu Al a 405 Arg Ala Pro Asp Al a 410 Pro Giu Arg Thx Pro 415 Pro Giy Ser Tmp 420 Leu Asn Ala Leu 425 Leu Arg Leu <210> 13 <211> 1290 <212> DNA <213> Streptornyces sp. <400> 13 a tgaccgaa t aacgtgatgg cagggcccgg ttcgaggagg gcgggagg tg gagcactacc cggatccggc cggg tggagg gtcgtcgacc tagcggactc aaccggccct tggtccgcgg cccgcgaggt gaagcgg tga gggtgtacct gattggtctc aca tcgcgga tcatcaagca ccccttcagc gctcaccgat ccgg ttcgcg gctgcgcgac cacaccctcc cgccaacacc ccgggca ttc cgatctgctg ctacgcctat gagcacgtcg ccgttcaccg gacgacaccc caccggttcg aaccggc tga atcctcacca accgcccgta aggcggc tgc ccgctgccca gcaaacaccc gctacggcga ccgtgtggtt ccaa tgcccc tggaaatcat tggacgcccc agatcaccga ccgagcacgc taacggtcat cggcgagccg actgcgcgaa catcacccgc cgccttcgcg gggcc tgccc cgaccacacc tctgcgaccc cgaggacggc ctgcgaactg 120 180 240 300 360 420 480 540 CaSe gtgggaattc ctgcaaccgg atccgcgaac gacgacgacg ctcgccggtc caccccgacc gagctgatgc gtcgaggtgg gcgaaccacg gcgggccggg agcctggccc gtgtcgctgg ctggccgcac cggaggaaga a tcggc tcag ggcgcgcggc gcggccgact a tgagaccac agctgcacct gc tgg tgcgg ccgggg tgca acccccgcca ccgagaacca gccaggaggg cggtggaacc tgccgg tccq ccgactgcag caaagcg ttc gctcaccgac cagcgacgtc cgcccatctc gctgaaatcc accgg tgcag ggtcaagcag cttcgccgac cgtcggtttc cgagg tcgcc ggacgccctc gctgcgctga tggcgggat t ccggcga tga gatctgctca gaaa tgg tca a tcggcaacg ga tccggagc atgacgcagt ggcgaagcgg cccgcccggc ggccacggca ttcgggaacc cagcgggtcc gggggtccgc tcgaacacat gcgaactgat cgatggtcct gcactgccgc tgc tcccacg tgcggtacgc tgctggccat tcgacctcac tgcactactg tgctcgcgca cgctgccggg gttcgtctcc tcacgcgctg ccgggtccat gaccctcgtt gcttctcacc cgccg tgcac caccgaggac gctggtcgcg ccgccagccg cc tgggcgcc ctacccggac caactggcgg 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1290 <210> <211> <212> <213> 14 429 PRT Streptomyces sp. <400> 14 Met Thr Glu Leu Ala 1 5 Pro Gly Glu Pro Asn Asp Ser Pro Phe Ser 10 Ala Glu His Val Gly Lys His Val Met Glu Thx Gly Tryr Phe Ala Asp Gly Asp Pro 25 Gin Leu Leu Thr Glu Leu Arg Thr Pro Val 55 Arq Asp His Glu 40 Tr-p Gly Pro Val Val1 Phe Asp Pro Phe Arg Gly Arg Glu Glu Ala Phe Ile Thr Arg Glu Arg Ala Val Leu Arg Phe Ala Al a 70 Gly Asn 75 Asn Pro Ala Phe Ala Gly Gly Gly Asp Thx Pro Ser Arg Leu Met Glu Ie Met Gly Leu Thr Met Asp 115 Ala Phe Thx Pro 100 Al a His Tyr Arg Val1 105 Arg Leu Ala Asn Pro Asp His Thr 120 Thr Ile Arg Arg Leu 125 Arg Thr Ile Leu 110 Val Ser Arg Val Glu Asp Ala Arg Lys 130 Ie Ala Ile 135 Arg Asp Leu Arg Pro 140 His Asp Asp Leu 145 Val Leu 150 Lys Arg Leu Pro Glu 155 Pro Ala Glu Asp Val Asp Leu Ile 165 Val1 His Tyr Ala TrI~ 170 Glu Leu Pro Ile Thr Val 175 Ile Cys Giu Asp Trp Gly 195 Ala Phe Pro Leu 180 Ser Gly Ile Pro Giu 185 Leu Asp Arg Leu Ala Phe Val Ser 200 His Gin Pro Asp Arg 205 Ile Gln Trp Arg 190 Leu Ser Lys Arg Glu Arg Ala Met Ile 210 Giu 215 Asp Ile His Ala Leu 220 Leu Arg Ala Ala 225 Asp Asp Asp Leu Thr Leu Leu Thr Gly Gly 245 Val Leu Asp 230 Arg Leu Leu Ser Glu 235 Glu Ile Arg Val His 240 Leu Ser Asp Val1 250 Thr Met Val Thr Met Val 255 Ie Gly Ala Gly His Giu Thr Ala His Leu Case Asn Gly Lys Ser 290 Trp Cys Thr 275 Asp Ala Leu Leu Thr 280 Pro Pro Asp Gin 270 His Leu Leu Leu Met Arg Pro Glu Leu Leu 295 Met Arg Ala Val His 300 Tyr Gly Pro Val 305 Val1 Gin 310 Val1 Thr Gin Leu Arg 315 Gly Ala Thr Glu Asp 320 Glu Val Ala Gly 325 Al a Gin Val Lys Gin 330 Arg Glu Ala Val Leu Ala 335 Met Leu Val Arg Leu Asp 355 Gly Phe Gly Ala 340 Leu Asn His Asp Pro 345 Ala His Phe Ala Asp Pro Ala 350 Asn His Val Thr Arg Gln Pro 360 Gly Arg Ala Glu 365 Ser His Gly Met 370 Gin Giu His 375 Phe Cys Leu Gly Ala 380 Al a Leu Ala Arg Gly Glu Val 385 Val1 Al a 390 Glu Gly Asn Leu Leu 395 Gin His Tyr Pro Asp 400 Pro Ser Leu Ala Val1 405 Leu Pro Asp Ala Leu 410 Val1 Arg Val Pro Leu 415 Gly Asn Trp Ala Ala Leu Pro 425 Arg Leu Arg <210> <211> 1428 <212> DNA <213> Streptomyces albofaciens <400> atgaooaoa t tcgacogctg coogocacca aaootgatgg caggcgccgo t togaog tgg cccggcatcg gaggacotgg oggo tgcgcc cgcgtcgagc gtcgtcgaco 9 toggca tcg ctgaaoocca a togacgago gacgaogacg ctggccggtc ca coocg acc gagctgatgc accgaggtgg gccaaccacg gccggccgcg tcactggccc ctggcgotcg acctggcgcc cgcccaccga ccgccccggc ccgaccgcac aaccggagct tcgtccgcgc tgcgcgaggt gtgcggacca ccccgtacct gcctggtctc gga tcaccga tcg tcgagca acgaggagga agcgcatcgg ggcgtgcgga gcggccggc t acgagaccac agoggoggot gctggtgcgg ccggtgtcca acccgcgcca ccgaggacca ggcaggaggc cgctcacccc tgcgacggc t g toccgggcg caccacccot cacgctcccc go tggacaac ccggttcatc gatgcgcgac ggacccgcgc cacogacaco ccgtgccttc cgagctgctg ottogcotac ccgggogctg ogocacca tg cotgcgggac gagogaog to cgcccacctc ga togaogag googatocag ggtoogooag otacacoggo cgtcggcttc ogagg tggog ggaacagctg gccgctgagg gooaoooogo toggoogoog tcotacgtcg oogtaoaoog gaogaotogo cagcggttcg goooggotga atootoacca accgcacgcc gogcggctgc oogotgccoa tggcggoggt ooggaga tga gacctgctca gaga tggtca atoagoaaog gaoooggogo gooaoooago ggcgaggccc ccggagcggc gggoaoggga taoggoaago gaggaooagg t tgcacgcgg oogaotooao cctctoogga gcctcoacoo go taoggoao ooatotggot toaaoaaco togagctgtt gogaooogoo gtatooagga oggaooa tgo toaoggtoat toggogooga togogoaoa t gcgggotcat cgctggtgct goaccotogo tgotgoogog tgcggtaogo tgatgttoag togaootgac tgcactaotg tgo toaccog aacgcctgcg agagotga ogoctcooo cacoacotot gggogagoog gotgogogag gg tgaooogo gaoootggtg cggcatoooo ggaooaoaco cotgoggoog cgaggaoggo otgogaaotg 00 togootog ccaogagg tg oogggogoag gaccctggtg ootgotoaoo ogcggtcoao ca tggaggao ootogtogog goggoagoog cctgggtgcc o taoooggao gcagoooggo 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1428 13 Case <210> 16 <211> 475 <212> PRT <213> Streptomyces <400> 16 albofaciens Met Thr 1 Thr Ala Ala Ala Leu Pro Pro Glu Gin Ala Leu Val Phe Val Pro Arg 130 Pro Tyr 145 Arg Leu Asp Leu Leu Pro Ala Tyr 210 Glu Glu 225 Leu Asn Ile His Leu Ser Asp Val 290 Glu Thr 305 His Pro Arg Ala Gin Leu Arg Gin Thr Ser Ser Ser Leu Pro Thr Asn 115 Ala Leu Arg Arg Asp 195 Pro Asp Pro Glu Gly 275 Glu Thr Asp Val Arg 355 Gly Ser Pro Pro Tyr Leu Leu Arg 100 Asn Arg Thr Arg Pro 180 His Leu Arg Lys Val 260 Leu Met Ala Gin His 340 Tyr Glu Pro 5 Ser Asp Val Asp Val Phe Pro Leu Asp Leu 165 Arg Ala Pro Ala Arg 245 Ile Ile Val His Arg 325 Glu Ala Ala Thr Thr Thr Gly Asn 70 Arg Asp Thr Ile Thr 150 Val Val Glu Ile Leu 230 Ile Asp Arg Thr Leu 310 Arg Leu Met Leu Glu Ala Thr Leu 55 Pro Ala Val Leu Glu 135 Ile Ser Glu Asp Thr 215 Trp Gly Glu Ala Leu 295 Ile Leu Met Glu Met Ser Ala Ser 40 His Tyr Arg Val Val 120 Leu Leu Arg Arg Gly 200 Val Arg Ala Arg Gin 280 Val Ser Ile Arg Asp 360 Phe Arg Ala 25 Pro Pro Thr Phe Arg 105 Pro Phe Thr Ala Ile 185 Val Ile Arg Thr Arg 265 Asp Leu Asn Asp Trp 345 Thr Ser Ala 10 Pro Ala Gly Gly Ile 90 Glu Gly Gly Ser Phe 170 Thr Val Cys Phe Met 250 Ala Asp Thr Gly Glu 330 Cys Glu Leu Ala Ala Thr Glu Tyr 75 Asp Val Ile Ile Asp 155 Thr Asp Asp Glu Gly 235 Pro Asp Asp Leu Thr 315 Asp Gly Val Val Thr Thr Thr Pro Gly Asp Met Gly Pro 140 Pro Ala Glu Leu Leu 220 Ala Glu Leu Gly Val 300 Leu Pro Pro Ala Ala Pro Thr Asp Asn Thr Ser Arg Ala 125 Glu Pro Arg Leu Val 205 Val Asp Met Arg Gly 285 Leu Ala Ala Ile Gly 365 Ala Pro Asp Pro Ser Arg Thr Leu Met Leu Arg Pro Ile Asp Gin 110 Asp Gln Asp Leu Asp His Arg Ile 175 Leu Ala 190 Glu His Gly Ile Leu Ala Ile Ala 255 Asp Asp 270 Arg Leu Ala Gly Leu Leu Leu Leu 335 Gin Ala 350 Val Gn Asn His Ser Ala Thr Glu Glu Trp Arg Asp Ala Thr 160 Gin Arg Phe Asp Ser 240 His Leu Ser His Thr 320 Pro Thr Val Asp Case 370 Pro Arg His 385 Ala Gly Arg Cys Leu Gly Lys Leu Leu 435 Gin Leu Glu 380 Leu Tyr Thx Gly 390 Ala Glu Asp Glu Arg Leu Asp 395 Gly Thr Arg Gin Pro 400 His Val Gly His Gly met 405 Ala Ser His Tyr 415 Leu Ala Arg 420 Thr Gin 425 Leu Ala Glu Val Arg TPyr Pro Asp 440 Leu Ala Leu Ala Leu 445 iThr Ala Tyr Gly 430 Thr Pro Glu Timp Arg Leu Asp Gin Giu 450 Arg Arg 465 Arg 455 Leu Arg Gin Pro Gly 460 Leu Pro Leu Arg 470 His Ala Glu Ser 475 <210> 17 <211> 1293 <212> DNA <213> Streptornyces platensis <400> 17 atgtcggcat aatgtgatgg cagggcccgg ttcgaggagg tccctgggcc cccgagcacc acccggctgc ccgcggg tcg ggcgtcgtcg ctggtcggca tcgatggagc atggtccggg ca tgacgacg g tcc tcgccg acccaccccg cacgagc tga gacgtcgacc tcggcgaac t cccgcgggac gccacactcg gaga tggcg t cggctgaacg tacccacctc acccggcact tcgtccgcgg tccgccaagt acgcggccga tccgccccta gccgcctggt agcaga tcgc atctcatccg tacccgaagc cggaccggct agcggcgcgg acggcggccg gtcacgagac accaactgcg tgcgctggtg tggccggcac tcgacccccg a tgcggagaa ccaagcagga tgggcgtcgc cgc tgccgg t accgttcgct gatcaccgac ccgcttcgtg cctgcgcgac ggacaacccg cctcctcgga g tcgcgggcc cgacgccc tg gcacttcgcc ggaccgcccg caccgcctcg cgcgc tcacc go tcagogac caccgctcac cctgctccag cgggccggtg cacgatccac ccactacacc ccatgtgggt gggcgaag to accggagcgc gcggttgggg gcacacgtcg ccgttcaccg gacgactcac cagcggttcg ctcaccaggc tcgattctca ttcaccgccc ctggcccggc tacccgctgc cagtggcgga ttcccgccga ggcgatctgc gtcgagatgg otca toagca gacgacccgg cagatgaccc cggggcgacg gaccccgacc ttcggccatg gccttcggca 0 tggagcgga tga ggaaacacoc gctacggcgc ccgtctggct tgaacaacc tgatggacat attacgacgc gcaaga tcac tgcccgagca cgatcaccgt cgtggggcgc tgatcgagca tcagcgagct tcaccttgat acggcacggo ccctgctoc agctgcgtta ccgtccaact gcotcgatct gggcgcac ta aactgctcgc cgcccc tgcc gggcgagccg gctgcgcgag ggtgacgcga ggoggogccc gotgggcctc cocgaccac cgacctgcgg cgccgaggac catctgcgaa cgacctcgtc catccaccgg ga tccgtgoc ctcacgctc ggcgc tgc tc ccgtgccgtc cgccgccgcc catcctggtg gacccgccac otgcctgggc gcac taoccg gggcaac tgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> <211> <212> <213> 18 430 PRT Streptomyces platensis <400> 18 Met Ser Ala Leu Pro Thx Ser Pro Phe Ala Ala His Val Gly Lys His 1 5 10 Citse Pro Gly Glu Pro Asn Val Met Asp Pro Ala Leu Ie Thr Asp Pro Phe 25 Thx Phe Arg Ser Met Leu Arg Gin 145 ely Val1 Arg Al a Arg 225 H is Ile Ser Leu Arg 305 Asp Leu Asp Val1 Lys 385 Giu Gly Val1 Gin Leu Leu Asn Al a 130 Ile Val1 Ile Thr Ser 210 Arg Asp Leu Asn Gin 290 Trp Vali Ile Arg Gly 370 Gin Met Tyr Asp Val1 Giy Gly Tyr 115 Phe Ala Va i Cys Trp 195 Phe Gly Asp Thr Gly 275 Asp Cys Asp Leu Leu 355 Phe Gi u Ala Gly Asp Leu I-Iis Leu 100 Asp Thr Asp Asp Giu 180 Gly Pro Ala Asp Leu 260 Thr Asp Gly Leu Val1 340 Asp Gly Gly Leu Ala Ser Arg Ala Pro Al a Al a Ala Leu 165 Leu Al a Pro Leu Gly 245 Val1 Ala Pro Pro Ala 325 Ser Leu H is Glu Gly 405 Leu Pro Asp 70 Ala Glu Pro Arg Leu 150 Ile Val Asp Met Thr 230 Gly Leu Al a Ala Val1 310 Gly Ala Thr Gly Val1 390 Val1 Arg Val1 55 Gin Glu His Asp Lys 135 Leu Arg Gly Leu Ile 215 Gly Arg Ala Leu Leu 295 Gln Thr Asn Arg Ala 375 Ala Al a Glu 40 Trp Arg Asp Leu His 120 Ile Ala H is Ile Val 200 Glu Asp Leu Gly Leu 280 Leu Met T~hr Phe His 360 His Phe Pro Gln Leu Phe Asn Arg 105 Thr Thr Arg Phe Pro 185 Ser His Leu Ser His 265 Thr Pro Thr Ile Asp 345 Pro Tyr Gly Glu Gly Val Val1 Pro 90 Pro Arg Asp Leu Ala 170 Glu Met Ile Leu Asp 250 Glu His Arg Gin His 330 Pro Ala Cys Lys Arg 410 Pro Thr Asn 75 Leu Tyr Leu Leu Pro 155 Ala Giu His Ser 235 Val1 Thr Pro Al a Leu 315 Arg Arg Gly Leu Leu 395 Leu Val1 Arg Asn Thr Leu Arg Arg 140 Glu Pro Asp Pro Arg 220 Giu Giu Thr Asp Val1 300 Arg Gly His His Gly 380 Leu Clu Val1 Phe Pro Arg Leu Arg 125 Pro His Leu Arg Asp 205 Met Leu Met Ala Gin 285 His Tyr Asp Ala 365 Ala Ala Arg Arg Giu Al a Leu Gly 110 Leu Arg Al a Pro Pro 190 Arg Val1 Ile Val His 270 Leu Giu Al a Al a Thr 350 Glu Thr His Thr Gly Giu Al a Met Ser Val Val1 Glu Ile 175 Gin Leu Arg Arg Thr 255 Leu Arg Leu Ala Val1 335 Asp Asn Leu Tyr. Pro 415 Arg Val1 Pro Asp Ile Ser Giu Asp 160 Thr Trp Thr Giu Al a 240 Leu Ile Leu Met Al a 320 Gin Pro His Al a Pro 400O Leu Pro Gly Asn Trp Arg Leu Asn Ala Leu Pro Val Arg Leu Gly 420 425 430 16- Caise <2 10 <211> <212> <213> 19 1293 DNA Streptomyces kasugaensis <400> 19 atgtcggcat aacgtgatgg cagggcccgg ttcgaggagg gcgccgggcg cccgagcacc acccggctgc ccgcgcgtca ggcgtcgtcg ctggtcggca tcgc tgcagc c tga tcgcgg ca tgacgacg gtcctggccg acccaccoog cacgagttga gacgtcgagc tcggcgaacc cctgccggcc gccacgc tcg gagctgtcgc cggctgaacg cacccagcaa atccggcgct tcgtgcgggg tccgcgagg t cggcccccga tgcgcgtcta gccgcctggt cacaga tago acctgatcca tccccgagga cggaccgga t cgcggcgccg acggcagccg gccacgagac accagctgcg tgcgctggtg tggcgggcg t gcgacccgcg acgcggagaa ccaagcagga tggccgtcgc cgctgccgct cacgttcacc ga tcgggga t gcggttcatg cctgcgtgac ggacaccccg tctgctcggc ctcccgggcc cgacgagc tg gcacttcgcc ggaccgcccg gagccggtcc ggcgctcacc gctcagcgac caccgcgcac gctgctcaag cggCccggtg ccgga tccgc ccac tacacc ccatgtgggg gggcgagg9tc gccggaggcc gcgtctgcgc gagcacgtcg gcaagcaccc gggcgagccg ccg ttcgccg gacgactccc ccgcggttcc ctgtcccggc tcga tcc tca ttcaccgcgc ctggcccggc tatcccctgc cag tggcgca ttcccggcga gacga tctgc gtcgagatgg ctcatcggca gacgacccgg caca tgaccc acgggggacg gaccccgacc ttcggccacg gccctcggcg ctggagcgca tga gttacgqcgc ccgtgtggtt ggaacaa tcc tga tggaca t acaacgacgc ggaagatcac tgccggagca cga tcaccg t cc tggggcgc tga tcgacca tcagcgagct tcacca tgg t acggcacggc cgctgctgcc agctgcgcta ccgtccagct ggctggacct gggcgcacta ccctgctcag caccggtacc gc tgcgcgag cg tgacccgc ggtCtccgcg gatgggtttc ccccgaccac cga tc tgcgg cgccgaggac catctgcgaa cgacctggtc catccacgag gatccggacc cctcaccgtc ggccctgctc gcgcgcggtg cgccgccgag catcctggtg gacccggcac ctgtctgggc gcacttcccc gggcagctgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> <211> 430 <212> PRT <213> Streptornyces kasugaensis <400> 2 Met Ser 1 Pro Gly 0 Ala Ser Pro 5 Glu Pro Asn Ser Asn Thr Phe Thr Glu His Val Gly Lys His Val Met Asp Gly Pro 25 Gin Leu Ile Gly Ala Gly Tyr Phe Met Asp Ala Leu Arg Glu 40 Trp Gly Pro Val Val1 Phe Asp Pro Phe Arg Gly Arg Glu Glu Val Asp Ser Pro Arq Glu Val1 55 Pro Phe Val Thr Arg Asn Val Leu Arg Ala Asp 70 Pro Arg Phe Arg Asn 75 Leu Pro Val Ser Ala Pro Gly Ala Ala Pro Glu Asp Thr Pro 90 Val1 Ser Arg Leu Met Asp Met Met Gly Leu Asn Asn 115 Arg Ala Phe 130 Glu His Leu Arg 105 Thr Tyr Leu Leu Ala Pro Asp His 120 Ie k~rg Leu Arg Gly Ser Ile 110 Leu Val Ser Arg Val Thr Thr Ala Arg Lys 135 Thr Asp Leu Arg 140 17- Case Gin Ie Ala Asp Giu Leu Leu Ala Arg Leu Pro Glu His Ala 0 u Asp 145 Gly 150 Ile 155 Val Val Asp Gin His Phe Pro Leu Pro 160 Ile Thr 175 Val Ile Cys Arg Thr Trp 195 Ara Ser Phe Giu 180 Gly Val Gly Ile Pro 185 Ser Giu Asp Arg Pro Gin Trp 190 Arg Met Ser Ala Asp Leu Val1 200 Asp Leu Gin Pro Pro Ala Met 210 Arg Arg Ile 22.5 Asp 1-is Ile His Giu 220 Clu Ile Ala Ala Arg Ala Leu 225 His Thr 230 Ser Asp Leu Leu Ser 235 Val1 Leu Ile Arg Thr 240 Asp Asp Asp Gly 245 Val1 Arg Leu Ser Asp 250 Glu Giu Met Val Thr Met 255 Val Leu Thr Gly Asn Gly 275 Leu Lys Asp Leu Ala Gly His 265 Thr Thr Tlir Ala Ala Ala Leu Leu 280 Leu His Pro Asp Gin 285 His His Leu Ile 270 Leu Arg Leu Glu Leu Met Asp Pro Ala 290 Arg Tmp Leu 295 His Pro Arg Ala Cys Gly Pro 305 Asp Val1 310 Gly Met Thr Gin Leu 315 Thr Tyr Ala Ala Val Giu Leu Al a 325 Ser Val Arg Ile Arg 330 Pro Gly Asp Ala Val Gin 335 Leu Ile Leu Asp Arg Leu 355 Val Gly Phe 370 Lys Gin Giu 385 Giu Leu Ser Pro Gly Ser Val1 340 Asp Al1a Asn Arg Asp 345 Pro Arg His Tyr Leu Thx Arg His 360 His Ala Gly His Ala 365 Ala Thr Asp Pro 350 Giu Asn His Thr Leu Ala Gly His Gly Ala 375 Ala Tyr Cys Leu Gly 380 Leu Gly Glu Leu Ala 405 Tmp Arg 420 Val1 390 Val1 Leu Gly Ala Leu 395 Leu Arg His Phe Pro 400 Val1 Ala Pro Glu Ala 410 Asn Ala Leu Pro 425 Giu Arg Thr Pro 415 Leu Leu Arg Leu Arg 430 <210> 21 <211> 1428 <212> DNA <213> Streptorryces rimosus <400> 21 a tgaccaca t tcgaccgctt cccgccacca aacc tga tgg caggccccgc ttcgacgtgg cccggca tcg gaggacctga cggc tgcgcc cgcg tcgagc cgcccaccga ccgccccggc ccgaccgcac aaccggagct tcgtccgtgc tgcgcgaggt gtgcggacaa ccccgtacct gcctggtctc agatcaccga gtcccgggcg caccacccct cacgctcccc gc tggacaac ccggttcatc gatgcgcgac ggacccgcgc cgccgacacc ccgtgccttc cgcgctgctg gccaccccga tcggccgcca tcctacgtcg ccgtacaccg gacgactcgc cagcggttcg gcccggctga atcctcacca accgcgcgcc gagcgactgc ccggctccac cctcttcgga gcctccaccc gctacggcac ccatctggct tcaacaaccc tcgagctgtt gcgacccgcc gca tccagga cggaccatgc cqcc tccccc caccacc tat gggcgagccg gctgcgcgag ggtgacccgc gaccctggtg cggcatcccc ggaccacacc cc tgcggccg cgaggacggc 120 180 240 300 360 420 480 540 600 18 Case gtcgtcgacc gtcggcatcg ctgaacccca a tcgacgagc gacgacgacg ctggccggtc caccccgacc gagctga tgc accgaggtcg gccaaccacg gccggccgcg tcactcgccc ctggagctcg acctggcgcc tcg tcgagca acgaggagga agcgca tcgg ggcgcgcggc gcggccggct acgagaccac agcggcggct gctgg tgcgg ccggtgtcca acccgcgcca ccgaggacca ggcaggaggc ctctcacacc tgcggcggc t cttcgcctac ccggacgctg cgccaccatg cc tgcgggac gagcgacgtc cgcccacctc gatcgacgag gccga tocag ggtccgccag ctacaccggg cgtcggcttc cgaggtggcc ggaacagctg gccgc tgaag ccgctgccca tggcggcgg t ccggaga tga gacctgctca gagatggtca a tcagcaacg gacccggcac gccacccagc ggcgaggccc ccggagcggc gggcacggga tacgggaagc gaggaccagg o tgcacgcgc tcacggtcat tcggcgccga tcgcgcacat gcgggctcat ccctggtcct gcaccctcgc tgctgccgcg tgcggtacgc tgatgttcag tcgacctgac tgcactactg tgctcacccg aacgcctgcg ggagc tga ctgcgagctg cctcgcctca ccacgagg tg ccgggcgcag gaccctggtg cctgctcacc cgcggtccac catggaggac cctcgtcgcg gcggcagccg cctgggtgcc c taccoggac gcagcccggc 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1428 <210> 22 <211> 475 <212> PRT <213> Streptomyces rimosus <400> 22 Met Thr Thx Ser Pro 1 5 Thr Ala Ser Pro Ser Thr Giu Ser Arg Ala Ala Th-r Pro Thr Gly Ser 10 Pro Thr Ala Ser Ser Al a 25 Pro Ala Thx Thr Pro Ser Ala Arg Thr Thr Leu Met Giu Ala Thr Ser Asp Thr Thr Tyr 40 His Ala Thr Thr Asp Asn Leu Pro Pro Giu Ser Tyr Val Gly Leu 55 Pro Pro Gly Giu Pro Gly Leu Leu Asp Gin Asn 70 Arg Tyr Thr Gly Tyr 75 Asp Thr Leu Arg Giu Ala Pro Lou Val1 Phe Ala Arg Phe Ile 90 Giu Asp Sor Pro Ile Trp Lou Val Thr Phe Val Asn 115 Pro Aro Ala Asp Val Val Arg 105 Pro Val Met Arg Pro Thr Leu Val1 120 Leu Gly Ile Gly Asp Gin Arg 110 Asp Lys Asp Asp Leu Thr Arg Leu Ile Phe Gly Ile 130 Pro Tyr Pro 140 Pro Lou Ala Asp 145 Arg Thr 150 Val1 Lou Thr Sor Asp Pro Asp His 155 Thr Lou Arg Arg Lou 165 Arg Ser Arg Ala Phe 170 Thr Ala Arg Arg Ile Gin 175 Asp Lou Arg Lou Pro Asp 195 Ala Tyr Pro Pro 180 His Val Giu Gin Ile 185 Val1 Asp Ala Lou Ala Glu Asp Gly 200 Val1 Val Asp Lou Val1 205 Val1 Lou Giu Arg 190 Giu His Phe Gly Ile Asp Lou Pro Ile 210 Giu Glu Thr 215 Trp Ile Cys Glu Leu 220 Ala Asp Arg Thr 225 Lou Lou 230 Ile Arg Arg Phe Gly 235 Asp Lou Ala Sor 240 Asn Pro Lys Akrg 245 Gly Ala Thr Met Pro Giu Met Ile 250 Ala His 255 19 Case Ile His Giu Leu Ser Gly 275 Asp Val Glu Val1 260 Leu Ile Asp Glu Arg Arg 265 Asp Ala Ala Leu Arg Ie Arg Ala Gin 280 Va 1 Asp Asp Gly Gly 285 Leu Asp Asp Leu 270 Arg Leu Ser Ala Gly His Met Val Thr 290 Glu Thr Leu 295 Ile Leu Thr Leu Val1 300 Leu Thr Ala His Ser Asn Gly 305 His Thr 315 Asp Ala Leu Leu Thr 320 Pro Asp Gin Arg 325 Giu Leu Ile Asp Pro Ala Leu Leu Pro 335 Arg Ala Val Gin Leu Arg 355 Arg Gin Gly His 340 Tyr Leu Met Arg Trp 345 Thr Gly Pro Ile Ala Met Glu Asp 360 Phe Glu Val Ala Gly 365 Ala Gin Ala Thr 350 Val Gin Val Asn His Asp Giu Ala Leu 370 Arg Met 375 Pro Ser Leu Val Ala 380 Leu Pro 385 Ala His Tyr Thx Gly 390 Asp Glu Arg Lou Asp 395 Gly Thr Arg Gin Pro 400 Gly Arg Ala Glu 405 Ser His Val Gly Phe 410 Glu His Gly Met His Tyr 415 Cys Lou Gly Lys Lou Leu 435 Gin Leu Giu Lou Ala Arg Gin 425 Lou Ala Glu Val Arg Tyr Pro Asp 440 Leu Glu Lou Ala Leu Ala Tyr Gly 430 Thr Pro Glu Trp Axg Lou Asp Gin Giu 445 Thr 450 Arg Arg 465 Arg 455 Lou Arg Gin Pro Gly 460 Leu Pro Leu Lys 470 His Ala Arg <210> 23 <211> 1293 <212> DNA <213> Streptomyces tubercidicus <400> 23 atgtcggcat aatgtgatgg caaggcccgg t tcgaagagg tccctgggac cccgaccatt acccggctcc ccgcgggtcg ggtgtggtcg ctggtcggca tcgctggagc ctgatccgcg ca tgacgacg gtcctggccg acccaccccg cacgagc tga gacgtcgagg tatccaactc acccggcgct tcgtacgggg tccgccaagt gctcgatcga tccggccg ta gccgactggt agcagatcac acctcatcca tcgccgaagc cggggcggc t agcggcgcgg acggcggccg gccacgagac accagctgcg tgcgctggtg cccgc tcgcc gatcaccgac ccggttcatg cc tgcgcga t cgaaagcccc tctgctcggg ctcgcgcgcc cgacgacc tg gcacttcgcc ggaccgcccg gagcaccgcg cgcgc tcacc gctcagcgac caccgcccac cctactcaag cgggccgg tg gcacatgtcg ccgttcggcg gacgactcgc cagcggttcg gcggtcagac tcgatcctca ttcacggcac ctgacccggc taccccctgc caa tggcgga t tcccggcga gacqa tctgc a tcgaga tgg c tca taggca gacga tccgg cacatgaccc ggaaacaccc gctacggcgc ccgtctggct tgaacaaccc ttttggaaat actacgacgc gcaaga tcac t tcccgagca cgatcaccgt agtggggagc tggtcgagca tcagcgagct tcaccatgat acggcacggc cgctgctgcc agctgcggtt ccgtacaact tggcgagccg actgcgcgag gg tgacgcgc ggccgcaccg gttggggttg acccgaccac cgacctgcgg cgccgaggac gatctgcgaa cgatctcgtc ca tcca tgag ga tccgcacc cctcacgatc ggcgctgctc gcgcgccgtc cgcg tccgag catcctggta 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 tcgccgggac accgatccac aagggcgacg 20 Case tcggcgaact tcgacccccg ccactacacc gaccccgacc gtctcgacct gacccgccac cccgccggcc acgccgagaa ccatgtgggc ttcggccacg gaatgcacta ctgcctgggt gccaccctcg ccaaacagga aggcgaagtc gccttctccc gcctcttcac gcactacccg gaactgtccc tgggcgtcgc ggcggaccag ctggcgcgga cacaggtacc cggcagctgg cggctggaca ccctgccgct gcgactgggg tga 1.080 1140 1200 1.260 1293 <210> <211> <212> <213> 24 430 PRT Streptomyces tubercidicus <400> 24 Met 1 Pro Gly Phe Arg Ser Met Leu Arg Gin 145 Gly Va1 Arg Thr Arg 225 His Ile Gly Leu Arg Ser Gly Gly Met Gin Leu Leu Asn Ala 130 Ile Va1 Ile Lys Ala 210 Arg Asp Leu Asn Lys 290 Trp Ala Glu STyr Asp Va1 Gly Gly Tyr 115 The Thr Va1 Cys Trp 195 Phe Gly Asp Thr Gly 275 Asp Cys( Leu Pro Gly Asp Leu Arg Leu 100 Asp Thr Asp Asp Glu 180 Gly Pro Ala Asp Ile 260 Thr Asp Gly Ser 5 Asn Ala Ser Arg Ser Pro Ala Al a Asp Leu 165 Leu Ala Ala Leu Gly 245 Val Ala Pro Pro Asn Va1 Leu Pro Asp 70 Ile Asp Pro Arg Leu 150 Ile Va1 Asp Met Thx 230 Gly Leu Ala Ala Va1 Sei Met Arc Val 55 Gir Asp His Asp Lys 135 Leu Gin Gly Leu Va1 215 Asp Arg Ala Leu Leu 295 His Pro Leu Ala 10 Asp Pro Ala 25 Glu Gln Gly 40 Trp Leu Val i Arg Phe Val Glu Ser Pro 90 Phe Arg Pro 105 His Thr Arg 120 Ile Thr Asp Thr Arg Leu His Phe Ala 170 Ile Ala Glu 185 Val Ser Leu 200 Glu His Ile Asp Leu Leu Leu Ser Asp 250 Gly His Glu 265 Leu Thr His 280 Leu Pro Arg Met Thx Gin Pro Ile His Ala Leu Pro Thr Asn 75 Ala Tyr Leu Leu Pro 155 Tyr Ala Glu His Ser 235 lie Thr Pro a Leu 315 Lys His Ile Val Arg Asn Va1 Leu Arg Arg 140 Glu Pro .Asp Pro Glu 220 Glu Glu Thr Asp Val I 300 Arg I Gly 2 Val Thir Va1 Phe Pro Arg Leu Arg 125 Pro His Leu Arg Gly 205 Leu Leu Met a 31n 285 iis ?he ~sp Gly Asp Arg Glu Ala Leu Gly 110 Leu Arg Ala Pro Pro 190 Arg Ile Ile Val His 270 Leu Glu I Ala Ala N LyE Prc GiN Glu Ala Leu Ser Va1 Va1 Glu Ile 175 Gin Leu Arg krg Thr 255 Leu Arg Leu 3er lal His Phe Arg Val Pro I Glu Ile Ser Glu Asp 160 Thr Trp Ser Glu Thx 240 Met Ile Leu Met Glu 320 Gin 305 310 Asp Val Glu Val Ala Gly Thr -21 CaIse 325 Ser 330 Pro Leu Ilie Leu Val 340 Asp Arg Leu Asp Ala Asn Phe Asp 345 Pro Arg His Tyr 335 Thr Asp Pro 350 0 u Asn His Thx Leu Ala Leu Thr Arg 355 Val Gly Phe His 360 His Ala Gly His Ala 365 Ala Gly His Gly 370 Lys Gin Met 375 Ala Tyr Cys Leu Gly 380 Phe Glu Gly Glii 385 Giu Val1 390 Val1 Phe Ser Arg Leu 395 Leu Thr His Tyr Pro 400 Val1 Leu Ser Leu Ala Ala Asp Gin 410 Pro Ala Arg Thr Gin 415 Pro Giy Ser Trp 420 Leu Asp Thr Leu 425 Leu Arg Leu <210> <211> 1293 <212> DNA <213> Streptomyces platensis <400> atgtcggcat aatgtgatgg caagggccgg ttcgaggaag tccc tggggc cccgagcatt acccggctgc ccgcgggtcg ggcgtggtcg ctggtcggca tcgctgcagc c tga tccgcg catgacgacg gtgCtcgccg acccaccccg cacgagctgt gatgtcgaca tcggccaact cccgccggcc gccaccctcg gagg tg tcgc cggctcgatt ta tccagctc acccggcgct tcgtacgggg tccgccaagt gctcattcga tccggccgta gccgtctggt agcagatcgc acctcatcaa tagccgaagc cggaccggct agcggcgcgg acggcggccg gccacgagac accagctgcg tgcgctggtg tcgccgggac tcgacccccg acgccgagaa ccaaacagga tgggcgtcgc ccc tgccgc t accgt tcgcc gatcgccgat ccggttcatg cctgcgcgac cgacagcccc tctgCtcggt gtcgcgcgcc cgacgagctg gcacttcgcc ggatcgtccg cagcacctcg ggcgc tcacg gctcagcgac caccgcgcac gctgctcagg cgggccggtc gaagatccgt ccactacacc cca tg tgggc gggcgaag tc accggaacaa gcggttgcgg gcgcatgtcg ccgttcggtg gacgactcac cagcggttcc acggccaggc tcgattctga ttcacggcac ctgacccggc taccccctgc cagtggcgga ttcccggcga gacgatctgc gtcgagatgg ctcataggca gacgacccgg cacatgaccc aagggcgacg gaccccgaac t tcggccacg gcgttcgaga ctggaaagga taa ggaaacaccc gttatggcgc ccgtctggct tgaacgatcc tgc tggaga t acaacgacgc gcaagatcac ttcccgagta cgatcgccgt ag tgggg tgc tgatcgagca tcagcgagct tcaccatga t acggcacggc ctctgtttcc agatgcggtt ccgtacaact gtctcgacct gga tgcac ta agctcttcgc caccactgcc gggcgagccg actgcgtgag cgtgacgcgc gacggccccc gatgggactg ccccgaccac cgacc tgcgg cgccgaggac catctgcgaa cgacctcgtc catccatgag gatccgtgcc cctcacggtg ggcgctgctc ccgtgccgtc tgcg tccgag gatcctggta gacccgtcac c tgcc tgggc gcac tacccg cggcagc tgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> 26 <211> 430 <212> PRT <213> Streptomyces platensis <400> 26 Met Ser Ala Leu Ser Ser Ser Pro Phe Ala Ala His Val Cly Lys His 1 5 10 Pro Gly Giu Pro Asn Val Met Asp Pro Ala Leu Ile Ala Asp Pro Phe 22 Case Gly Gly Phe Met Arg Gin Ser Leu Met Met Leu Asn Arg Ala 130 Gin Ile 145 Gly Val Val Ile Arg Lys Thr Ser 210 Arg Arg 225 His Asp Ile Leu Gly Asn Leu Arg 290 Arg Trp 305 Asp Val Leu Ile Glu Arg Val Gly 370 Lys Gin 385 Glu Val Tyr Asp Val Gly Gly Asn 115 Phe Ala Val Cys Trp 195 Phe Gly Asp Thr Gly 275 Asp Cys Asp Leu Leu 355 Phe Glu Ser Gly Asp Leu Arg Leu 100 Asp Thr Asp Asp Glu 180 Gly Pro Ala Asp Val 260 Thr Asp Gly Ile Val 340 Asp Gly Gly Leu Ala Ser Arg Ser Pro Ala Ala Glu Leu 165 Leu Ala Ala Leu Gly 245 Val Ala Pro Pro Ala 325 Ser Leu His Glu Gly 405 Leu Pro Asp 70 Phe Glu Pro Arg Leu 150 Ile Val Asp Met Thr 230 Gly Leu Ala Ala Val 310 Gly Ala Thr Gly Val 390 Val Arg Val 55 Gin Asp His Asp Lys 135 Leu Lys Gly Leu Ile 215 Asp Arg Ala Leu Leu 295 His Thr Asn Arg Met 375 Ala Ala Glu 40 Trp Arg Asp Phe His 120 Ile Thr His Ile Val 200 Glu Asp Leu Gly Leu 280 Phe Met Lys Phe His 360 His Phe Pro 25 Gin Gly Leu Val Phe Leu Ser Pro 90 Arg Pro 105 Thr Arg Thr Asp Arg Leu Phe Ala 170 Ala Glu 185 Ser Leu His Ile Leu Leu Ser Asp 250 His Glu 265 Thr His Pro Arg Thr Gin Ile Arg 330 Asp Pro 345 Pro Ala Tyr Cys Glu Lys Glu Gin 410 Pro Thr Asn 75 Thr Tyr Leu Leu Pro 155 Tyr Ala Gin His Ser 235 Val Thr Pro Ala Met 315 Lys Arg Gly Leu Leu 395 Leu Val Arg Asp Ala Leu Arg Arg 140 Glu Pro Asp Pro Glu 220 Glu Glu Thr Asp Val 300 Arg Gly His His Gly 380 Phe Glu Val Phe Pro Arg Leu Arg 125 Pro Tyr Leu Arg Asp 205 Leu Leu Met Ala Gin 285 His Phe Asp Tyr Ala 365 Ala Ala Arg Arg Glu Thr Leu Gly 110 Leu Arg Ala Pro Pro 190 Arg Ile Ile Val His 270 Leu Glu Ala Ala Thr 350 Glu Thr His Thr Gly Glu Ala Leu Ser Val Val Glu Ile 175 Gln Leu Arg Arg Thr 255 Leu Arg Leu Ser Val 335 Asp Asn Leu Tyr Pro 415 Arg Val Pro Glu Ile Ser Glu Asp 160 Ala Trp Ser Glu Ala 240 Met Ile Leu Leu Glu 320 Gin Pro His Ala Pro 400 Leu Pro Gly Ser Trp Arg Leu Asp Ser Leu Pro Leu Arg Leu Arg 425 430 <210> 27 -23- Case <211> <212> <213> 1293 DNA Streptomyces platensis <400> 27 atgtcggcat aatgtgatgg caggccccgg ttcgaggagg cccctggccc cccgagcacc acccggctgc ccgcgtgtcg ggcgtcgtcg ctggtcggca tcgatggacc atggtccggg catgacgacg gtcCtcgccg acccaccccg catgagctga gacg tcgacc gcggcgaac t cccgccggcc gccaccctcg gagatgtccc cggctgaatt ta tccagc tc agccggcgct tcg tacgggg tccgccaagt ca tcggccga tccgcgtcta gccgtctggt agcagatcgc acctcatcca tacccgaagc cggaccggct agcggcgcgc atggcggccg gtcacgagac accagctgcg tgcgctggtg tcgccggtac tcgacccccg acgccgagaa ccaagcagga tgggcatcga ccctgccgtt tccgttcgct gctcaccgac ccggttcgtg cc tgcgcgac ggagaacccg ca tgctcggg gtcgcgcgcg cgacgagctg gcatttcgcc ggaccgcccg cggcgcaacg ggcgctcacc gc tcagcgac caccgcccac cctgctcaag cgggccggtg gcggatccac ccactacacc ccatgtgggt gggcgaagtc accggaacgt gcggc tgggg gcgcatgtcg ccg ttcgcgg gacgactcac cagcggttcg ctgaccaggc tcgattctca ttcacggcgc ctggcccgcc tacccgctgc cagtggcgga ttcccggcga gatgatctgc gtcgagatgg ctcatcagca gacgacccgg caga tgacgc aagggcgacg gaccccgacc ttcggccacg gcgttcggca ctggagcgat tga ggaaacaccc gctacggcgc cggtctggtt tgaacaatcc tgatggacat actacgacgc ggaaga tcac tccccgagta cgatcaccgt agtggggcgc tgatcgagca tcagcgagct tcaccatgat acggcacggc ccctgctccc agctgcgcta ccgtacaact gtctcgatct gtgcgcatta agctgctcgc tgccgctgcc gggtgagccg gctgcgtgag cgtgacgcgc ggccgcgccg gctgggcctc ccccgaccac cgatctgcga cgccgaggac catctgcgag cgacctcatc catccatgag ga tccgtacc cctcacgctc ggcgctgctc ccgggccgtc cgcggccgcc cctcctggtt gacgcg tcac ctgcctgggt gcactacccg tggcaac tgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> 28 <211> 430 <212> PRT <213> Streptornyces platensis <400> 2 Met Ser 1 Pro Gly Ala Gly .8 A la Leu Ser Ser Ser Pro Phe 5 Glu Pro Asn Tyr Gly Ala Ala 10 Al a Ala His Val Gly Lys His Val Met Giu Leu Arg Glu 40 Pro Val Trp Pro 25 Gln Leu Leu Thr Ala Pro Val Phe Val Asp Val1 Phe Asp Pro Phe Arg Gly Arg Glu Glu Val Asp Ser Phe Val Thr Arcj Gin 55 Gin Arg Asn Val Leu Arg Pro Asp 70 Ala Arg Phe Val Asn 75 Leu Pro Ala Ala Pro Leu Ala Pro Ser Giu Giu Asn Pro 90 Val1 Thx Arg Leu Met Asp Ser Ile Met Leu Gly Leu Asn Tyr 115 Arg Ala Phe 130 Leu Pro Giu His Leu Tyr Met Leu Gly 100 Asp Ala Pro Asp His 120 Ile Arg Leu Arg Arg 125 Thr Ala Arg Lys 135 Leu Thr Asp Leu 110 Leu Val Ser Arg Val Glu Ala Glu Asp Arg Pro 140 Glu Tyr Gin Ile Ala Asp Glu Leu Ala Arg Leu Pro 24 cilsc 145 Gly 150 Ile Val Vai Asp Leu 165 Leu Gin His Phe 155 Tyr Al a 170 Giu Pro Leu Pro 160 Ile Thr 175 Vai Ilie Cys Arg Lys rTp 195 Ala rhr Phe Giu 180 Gly Vai Giy Ie Pro 185 Ser Ala Asp Arg Ala Asp Leu Ile 200 Giu Met Asp Pro Asp 205 Met Pro Gin Trp 190 Arg Leu Giy Val Arg Glu Pro Ala Met 210 Arg Ie 215 Asp His Ile His Glu 220 Gli Arg 225 His Ala Ala Leu Thx 230 Gly Asp Leu Leu Ser 235 Val Leu Ile Arg Thr 240 Asp Asp Asp Gly 245 Val1 Arg Leu Ser Asp 250 Glu Glu Met Val Thr Met 255 Ilie Leu Thr Ser Asn Giy 275 Leu Lys Asp Leu 260 Thr Leu Ala Gly His 265 Thr Thr Thr Ala His Leu Ile 270 Leu Arg Leu Ala Ala Leu Leu 280 Leu His Pro Asp Gin 285 H is Asp Pro Ala 290 Arg Tmp Leu 295 Gin Pro Arg Ala Val1 300 Arg Giu Leu Met Cys Gly Pro 305 Asp Val1 310 Giy Met Thxr Gin Leu 315 Lys Tyr Ala Ala Val Asp Leu Thr Arg Ile His 330 Pro Gly Asp Ala Val Gin 335 Leu Leu Leu Asp Arg Leu 355 Val Gly Phe 370 Lys Gin Giu 385 Giu Met Ser Pro Gly Asn Val1 340 Asp Ala Asn Phe Asp 345 Pro Arg His Tyr Leu Thr Arg His 360 His Ala Gly His Al a 365 Al a Tlir Asp Pro 350 Glu Asn His Thr Leu Ala Gly His Gly Al a 375 Al a Tyr Cys Leu Gly 380 Leu Gly Glu Leu Gly 405 Tmp Arg 420 Val 390 Ile Phe Gly Lys Leu 395 Leu Ala His Tyr Pro 400 Leu Glu Pro Glu Arg 410 Pro Glu Arg Leu Pro 415 Leu Asn Ser Leu 425 Leu Arg Leu <210> 29 <211> 1293 <212> DNA <213> Streptornyces lydicus <400> 29 a tgtcgqca t aacgtgatgg cagggcccgg ttcgaggagg gcgccggacg cccgagcacc acccggc tgc ccgcgcgtcg ggcgtcgtcg ctggtcggca tcgctgcagc tacccagcaa atccggcgct tcgtgcgggg tccgcgaggt cggaccccga tgcgcgtcta gccgcctggt cacagatagc acctga tcca tccccgagga cggaccgga t cacgttcacc gatcggtgat gcggttcgtg cctgcgggac ggacaccccg tctgctcggc ctcccgggcc cgacgagc tg gcacttcgcc ggaccgcccg gagccgg tcc gagcacgtcg ccgttcgccg gacgactccc cagcggttcc ctgtcccggc tcgatcctca ttcaccgcgc ctggcccggc tatcccctgc cagtggcgca ttcccggcga gcaagcaccc gttacggcgc ccgtgtggtt ggaacaatcc tga tggaca t acaacgacgc ggaagatcac tgccggagca cga tcaccg t cc tggggcgc tga tcgacca gggcgaaccg gc tgcgcgag cgtgacccgc ggtctcctcg gatgggtttc ccccgaccac cga tctgcgg cgccgaggac catctgcgaa. cgacctggtc ca tccacgag 120 180 240 300 360 420 480 540 600 660 25 Oise ctgatcgcgg ca tgacgacg gtcctggccg acccaccccg cacgagttga gacgtcgagc tcggcgaacc cccgccggcc gccacgctcg gagctgtcgc cggctgaatg cgcggcgccg acggcagccg gccacgagac accagctgcg tgcgctggtg tggcgggcgt gcga tccgcg acgccgagaa ccaagcagga tggccgtcgc cgctgccgct ggcgc tcacc gctcagcgac caccgcgcac gctgctcaag cggcccggtg ccgga tccgc ccactacacc cca tg tgggg gggcgaggtc gccggacgcc gcg tc tgcgc gacgacctgc g tcgaga tgg ctcatcggca gacgacccgg cacatgaccc aagggggacg gaacccgacc ttcggccacg gccc tcggcg ctggagcgca tga tcagcgagct tcacca tgg t acggcacggc cgctgctgcc agc tgcgc ta ccgtccagct gtctggacct gggcgcac ta ccctgctcag caccggtacc gatccgaacc cctcaccgtc ggccctgctc gcgcgcggtg cgccgccgag catcctggtg gacccggcac ctgtctgggc gcac ttcccc gggcagctgg 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> <211> <212> <213> 430 PRT Streptomyces lydicus <400> Met Ser Ala 1 Pro Gly Glu Ala Gly Tyr Phe Val Asp Leu Pro 5 Pro Asn Ser Asn Thr Phe Thr 10 Ala Giu His Val Gly Lys His Val Met Asp Gly Pro 25 Gin Leu Ile Gly Ala Leu Arg Glu 40 Trp Gly Pro Val Val1 Phe Asp Pro Phe Arg Gly Arg Giu Glu Val Asp Ser Pro Arg Glu Val 55 Gin Phe Val Thr Arg Asn Val Leu Arg Ala Asp 70 Arg Phe Arg Asn 75 Leu Pro Val Ser Ser Asp Pro Asp Ala Asp Pro Pro Giu Asp Thr Pro 90 Val1 Ser Arg Leu Met Met Met Gly Leu Asn Asn 115 Arq Ala Phe Phe 100 Asp Glu His Leu Arg 105 Thr Tyr Leu Lou Ala Pro Asp His 120 Ile Arg Leu Arg Arg 125 Pro Gly Sor Ile 110 Leu Val Ser Arg Val Ala Thr Ala Arg 130 Gin Ile Lys 135 Leu Thr Asp Lou Ala Asp Giu 145 Gly Leu 150 Ile Ala Axg Lou Pro 155 His Ala Giu Asp 160 Val Val Asp Leu 165 Leu Gin His Phe Al a 170 Glu Pro Leu Pro Ile Thr 175 Val Ile Cys Arg Thr Trp 195 krg Ser Phe Giu 180 Gly Val Gly Ile Pro 185 Ser Giu Asp Arg Ala Asp Lou Val1 200 Asp Leu Gin Pro Asp 205 Lou Pro Gin Trp, 190 Arg Met Sor Ile Ala Ala Pro Ala Met 210 Arg Arg Ile 215 Asp His Ile His Giu 220 Glu Arg Ala Lou 225 H is Thr 230 Sor Asp Leu Leu Ser 235 Val1 Lou Ilie Arg Thr 240 Asp Asp Asp Gly 245 Val1 Arg Leu Ser Asp 250 Glu Glu Met Val Thr Met 255 Val Lou Thr Gly Asn Gly Val1 260 Thr Leu Ala Gly H is 265 Thr Thr Thr Ala His Leu Ile 270 Lou Arg Lou Ala Ala Lou Leu His Pro Asp Gin case 275 Asp 280 Leu 285 His Leu Lys 290 Arg Tr-p Asp Pro Ala Leu 295 His Pro Arg Ala Val1 300 Arg Glu Leu Met Cys Gly Pro 305 Asp Val1 310 Gly Met Thr Gin Leu 315 Lys Tyr Ala Ala Glu 320 Val Glu Leu Ala 325 Ser Val Arg Ile Arg 330 Pro Gly Asp Ala Val Gin 335 Leu Ie Leu Asp Arg Leu 355 Val Gly Phe Val1 340 Asp Ala Asn Arg Asp 345 Pro Axg His Tyr Leu 'h-r Arg His 360 His Ala Gly His Thr Glu Pro 350 Giu Asn His Thr Leu Ala Gly His Gly 370 Lys Gin Al a 375 Al a Tyr Cys Leu Gly 380 Leu Glu Gly Glu 385 Glu Val1 390 Val1 Leu Gly Ala Leu 395 Leu Arg His Phe Pro 400 Val1 Leu Ser Leu Ala 405 Arg Ala Pro Asp Ala 410 Pro Glu Arg Thr Pro 415 Pro Gly Ser Leu Asn Ala Leu 425 Leu Arg Leu <210> <211> <212> 31 1293 DNA <213> Streptornyces lydious <400> 31 atgtoggcat oatgtgatgg oagggacogg t togaggaag gogotoggog oocgagcaco acccggctgc oogcgggtgg ggcgtggtcg cttgtoggca tcgg tggac ctgatcggoo oatgaogacg gtgctggcog acocaccoog cacgagctga gacgtcgaco tcggcgaacc cccgcgggc gccagootog gaocctggogc cggctgacgt cgacoago tc atocggcgct tcgtccgcgg tccgccaggt tcgcgccgga tgoaoggo ta gccgcctggt ogoaga taao aootga tcga tcgccgcgga ccgaccggct agcggcgggo acggcagccg gccacgagac aooagctgcg tgcgctggtg tcgooggcac acgaccgcg gcgccgagaa ccaggcagga tggogg tggo cgctgccggt tccoctcago gatcagcgat aogg ttot to cc tgogogao ggaotoooog totgotoaao o tocgogoo ogoogagotg goaottogoo ggaooggooo oggooggaoo ogogotoaoo gotoagogao oaoogoaoao gotgotoaag ogggoogg tg oogga toogc ooaotaoaoo ccacgtgggo gggtgaggto gooogaggag gogootgggo goooaogtog ccgttcggog gaogaotogo oagoggttog oagctgogog togatootoa ttoaoogooo otggaoogao taooogotgo oagtggogtt ttoooggoga gaogaootgo gtogagatgg otoa toggca gaogaooogg oaogtoaooo aggggogaog gaoooogaao t togggoaog gcootgggog otggagogoa tga goaagoaooo gotacggtgo oottgtggtt tgaaoaaooo ogotggoga t ao taogaogo goaaga toao toooggagoa ogatoaoggt ootggggogo tga togaooa toagogagct toaoootggt aoggoaoogo cgotgotgoo agotgoggta oog tgoaggo goo tggaoo t gggogoaota cootgttoga oooogg tgoo gggogaaooo ootgogogag ag tgaooogo ogoogaooog gotgggoato oooogaooao ogatottogg ogoogaggao gatotgogaa ogaootggto oa tooaogog ga tooggaoo ootoacooto ggoc tgo to gogogoog to ogoogoogag ogtootggto gaoooggoag otgootgggo oogotaoooo ogg taoo tgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1293 <210> 32 <211> 430 <212> PRT <213> Streptomyoes lydious 27 Case <400> 32 Met Ser Ala Ser Thr Ser Ser Pro Leu Ser Ala His Val Gly Lys His 1 5 10 Pro Gly Glu Pro His Val Met Asp Pro Ala Leu Ile Ser Asp Pro Phe 25 Gly Gly Tyr Gly Ala Leu Arg Glu Gln Gly Pro Val Val Arg Gly Arg 40 Phe Phe Asp Asp Ser Pro Leu Trp Leu Val Thr Arg Phe Glu Glu Val 55 Arg Gln Val Leu Arg Asp Gin Arg Phe Val Asn Asn Pro Ala Asp Pro 70 75 Ala Leu Gly Val Ala Pro Glu Asp Ser Pro Gin Leu Arg Ala Leu Ala 90 Met Leu Gly Ile Pro Glu His Leu His Gly Tyr Leu Leu Asn Ser Ile 100 105 110 Leu Asn Tyr Asp Ala Pro Asp His Thr Arg Leu Arg Arg Leu Val Ser 115 120 125 Arg Ala Phe Thr Ala Arg Lys Ile Thr Asp Leu Arg Pro Arg Val Ala 130 135 140 Gin Ile Thr Ala Glu Leu Leu Asp Arg Leu Pro Glu His Ala Glu Asp 145 150 155 160 Gly Val Val Asp Leu Ile Glu His Phe Ala Tyr Pro Leu Pro Ile Thr 165 170 175 Val Ile Cys Glu Leu Val Gly Ile Ala Ala Glu Asp Arg Pro Gin Trp 180 185 190 Arg Ser Trp Gly Ala Asp Leu Val Ser Val Asp Pro Asp Arg Leu Gly 195 200 205 Arg Thr Phe Pro Ala Met Ile Asp His Ile His Ala Leu Ile Gly Gin 210 215 220 Arg Arg Ala Ala Leu Thr Asp Asp Leu Leu Ser Glu Leu Ile Arg Thr 225 230 235 240 His Asp Asp Asp Gly Ser Arg Leu Ser Asp Val Glu Met Val Thr Leu 245 250 255 Val Leu Thr Leu Val Leu Ala Gly His Glu Thr Thr Ala His Leu Ile 260 265 270 Gly Asn Gly Thr Ala Ala Leu Leu Thr His Pro Asp Gin Leu Arg Leu 275 280 285 Leu Lys Asp Asp Pro Ala Leu Leu Pro Arg Ala Val His Glu Leu Met 290 295 300 Arg Trp Cys Gly Pro Val His Val Thr Gin Leu Arg Tyr Ala Ala Glu 305 310 315 320 Asp Val Asp Leu Ala Gly Thr Arg Ile Arg Arg Gly Asp Ala Val Gin 325 330 335 Ala Val Leu Val Ser Ala Asn His Asp Pro Arg His Tyr Thr Asp Pro 340 345 350 Glu Arg Leu Asp Leu Thr Arg Gin Pro Ala Gly Arg Ala Glu Asn His 355 360 365 Val Gly Phe Gly His Gly Ala His Tyr Cys Leu Gly Ala Ser Leu Ala 370 375 380 Arg Gin Glu Gly Glu Val Ala Leu Gly Ala Leu Phe Asp Arg Tyr Pro 385 390 395 400 Asp Leu Ala Leu Ala Val Ala Pro Glu Glu Leu Glu Arg Thr Pro Val 405 410 415 Case Pro Gly Thr Trp krg Leu Thr Ser Leu Pro Val Arg Leu Gly 420 425 430 <210> <211> <212> <213> 33 1281 DNA Streptornyces tubercidicus <400> 33 atgaactctc cccgccctga gtacggggcc cgccagg tcc gcgcccgagg cgcgtctacc cgtctggtgt cagatcgccg c tca tccagc cccgaagcgg gaccggctcg cggcgcgagg ggcgggcggc cacgagacca cagctgcgtc cgctggtgcg gccggcacac gacccccgtc gccgagaacc aaacaggaag ggca tcgccc c tgccgg tgc cg ttcgccgc tcaccgaccc ggttcatgga tgcgcgacca acaacccgc t tgc tcgga tc cgcgggcgtt acgcgctgct acttcgccta accgcccgca gcgcctcgtt cgctcaccga tcagcgacgt ccgcccacc t tgg tcaagga ggccgg tgca cga tccgcca actacaccga atgtgggttt gtgaagtcgc cggaacacct ggttggggtg gcacgtcggg gttcaccggc cgactcgccc gcggttcgtg gacccggctg gatcctcaac cacggcccgc ggcccggctg ccccctgccg gtggcgaacg cccggcga tg cgacctgctc cgagatggtc catcagcaac cga tccggcc catgacccag gggcgatgcc ccccgaccgc cggcca tgga cttcggcaaa ggagcggaca aaacacccgg tacggcgcgc gtctggctgg aacaatccgg atggagatgc tacgacgccc aaga tcaccg cccgagcacg atcaccgtca tggggcgccg atcgagcaca agcgaactga accatgatcc ggcacggcgg ctcctccccc ctgcgctacg gttcaactca ctcgatctca gcgcactact ctgctcacgc ccgc tgccgg gcgagccgaa tgcgtgagca tgacgcggtt cctcgccgtc tgggcctccc ccgaccacac acctgcggcc ccgaggacgg tctgcgaact acctcatctc tccatcagat tccgcaccca tcacgc tcgt cgctgctcac gtgccgtcca ccaccgccga tcctggtatc cccggcaccc gcctgggcgc actacccgga gcaactggcg tgtgatggac gggcccggtc cgaggaggtc cctgaactac cgagcacc tc ccggctgcgc ccgggtcgag cgtcgtcgac gg tcggca ta gatggatccg ggtccgggaa tgacgacgac cc tcgccggc ccaccccgac cgagctgatg cgtcgacctc ggccaacttc cgcgggccac cacactcgcc catatcgctg gctgaactcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1281 <210> 34 <211> 426 <212> PRT <213> Streptomyces tubercidicus <400> 34 Met Asn Ser Pro Phe 1 5 Asn Val Met Asp Pro Ala Leu Arg Ciu Gin Ser Pro Val Trp Leu Arg Asp Gin Arg Phe Ala Pro Glu Asp Asn Pro Glu His Leu Arg Ala Ala His Val Gly 10 Lys His Pro Ala Leu Ile Thx Asp Pro Phe 25 Gly Pro Val Val Arg Gly Arg 40 Val Thr Arg Phe Glu Glu Val 55 Val Asn Asn Pro Ala Ser Pro 70 75 Pro Leu Thr Arg Leu Met Glu 90 Thr Phe Arg Gly Giu Pro Gly Tyr Gly Met Asp Asp Gin Val Leu Ser Leu Asn Met Leu Gly Leu Tyr Asp Val Tyr Leu Leu Gly Ser Ie Leu Asn Case 100 105 110 Ala Phe Thr Ala Pro Asp His Thr Arg Leu Arg Arg Leu Val Ser Ala Ala 145 Leu Leu Ala Ala Leu 225 Gly Val Ala Pro Pro 305 Ala Ser Leu His Glu 385 Gly Arg Arg 130 Leu Ile Val Asp Met 210 Thr Gly Leu Ala Ala 290 Val Gly Ala Thr Gly 370 Val Ile Leu 115 Lys Leu Gin Gly Leu 195 Ile Asp Arg Ala Leu 275 Leu His Thr Asn Arg 355 Ala Ala Ala Asn Ile Ala His Ile 180 Ile Glu Asp Leu Gly 260 Leu Leu Met Pro Phe 340 His His Phe Pro Ser 420 Thr Arg Phe 165 Pro Ser His Leu Ser 245 His Thr Pro Thr Ile 325 Asp Pro Tyr Gly Glu 405 Leu Asp Leu 150 Ala Glu Met Ile Leu 230 Asp Glu His Arg Gin 310 Arg Pro Ala Cys Lys 390 His Pro Leu 135 Pro Tyr Ala Asp His 215 Ser Val Thr Pro Ala 295 Leu Gin Arg Gly Leu 375 Leu Leu Val 120 Arg Glu Pro Asp Pro 200 Gin Glu Glu Thr Asp 280 Val Arg Gly His His 360 Gly Leu Glu Arg Pro His Leu Arg 185 Asp Met Leu Met Ala 265 Gin His Tyr Asp Tyr 345 Ala Ala Thr Arg Leu 425 Arg Ala Pro 170 Pro Arg Val Ile Val 250 His Leu Glu Ala Ala 330 Thr Glu Thr His Thr 410 Gly Val Glu 140 Glu Asp 155 Ile Thr Gin Trp Leu Gly Arg Glu 220 Arg Thr 235 Thr Met Leu Ile Arg Leu Leu Met 300 Thr Ala 315 Val Gln Asp-Pro Asn His Leu Ala 380 Tyr Pro 395 Pro Leu Arg 125 Gin Gly Val Arg Ala 205 Arg His Ile Ser Val 285 Arg Asp Leu Asp Val 365 Lys Ile Val Ile Thr 190 Ser Arg Asp Leu Asn 270 Lys Trp Val Ile Arg 350 Gly Gin Ala Val Cys 175 Trp Phe Glu Asp Thr 255 Gly Asp Cys Asp Leu 335 Leu Phe Glu Asp Asp 160 Glu Gly Pro Ala Asp 240 Leu Thr Asp Gly Leu 320 Val Asp Gly Gly Asp Ile Ser Leu 400 Pro Gly Asn Trp 415 <210> <211> 195 <212> DNA <213> Streptomyces tubercidicus <400> atgcggatca cgatcgacac cgacatctgt atcggcgccg gccagtgcgc cctgaccgcg ccgggagtgt tcacccagga cgacgacggc ttcagcgccc tgctgcccgg ccgcgaggac ggtgcgggcg acccgctggt gcgggaggcc gcccgcgcct gcccggtgca ggccatcacg gtcacggacg actga 120 180 195 Case <210> <211> <212> <213> 36 64 PRT Streptomyces <400> 36 Met Arg Ie Thr Ile 1 5 Ala Leu Thr Ala Pro Ala Leu Leu Pro Gly Glu Ala Ala Arg Ala tubercidicus Asp Thr Asp Ile Cys Ile Gly Ala Gly Gin Cys 10 Gly Val Phe Thr Gin Asp Asp Asp Gly Phe Ser 25 Arg Glu Asp Gly Ala Giy Asp Pro Leu Val Arg 40 Cys Pro Val Gin Ala Ile Thr Val Thr Asp Asp 55 <210> 37 <211> 195 <212> DNA <213> Streptornyces tubercidicus <400> 37 atgcggatca ccatcgacac cgacatctgc ccgggagtct tcacccagga cgacgacggt ggcgcgggcg acccgctggt gcgcgaggcc gtcacggacg actga atcggcgccg gccagtgcgc cctgaccgcg ttcagcgccc tgctgcccgg ccgcgaggac gcccgcgcct gccccgtgca ggccatttcg <210> 38 <211> 64 <212> PRT <213> Streptomryces <400> 38 Met Arg Ile Thr Ile 1 5 tubercidicus Asp Thr Asp Ile Ile Gly Ala Gly Gin Cys is Ala Leu Thr Ala Pro Gly Val Phe Ala Leu Leu Pro Gly Arg Glu Asp 40 Giu Ala Ala Arg A-la Cys Pro Val 55 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptid~e. <400> 39 Thr 25 Gly Asp Asp Asp Ala Gly Asp Pro Val1 Giy Phe Ser Leu Val Arg Thr Asp Asp Gin Ala Ile Ser -31 Case Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide. <400> Glu Gin Lys Leu Ile Ser Glu Glu Asp Leu 1 5 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide. <400> 41 Gin Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp 1 5 <210> 42 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide. <400> 42 Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 1 5 <210> 43 <211> 7 <212> PRT <213> Streptomyces <220> <221> misc feature <222> <223> Streptomyces consensus sequence <400> 43 Ile Ala Gly His Glu Thr Thr 1 -32- Case <210> <211> <212> <213> <220> <221> <222> <223> 44 DNA Artificial Sequence miscfeature Nucleotides 6, 9 and 18 are wherein g or c. <400> 44 atcgcsggsc acgagacsac <210> <211> 7 <212> PRT <213> Streptomyces <220> <221> miscfeature <222> <223> Streptomyces consensus sequence <400> Val Ala Gly His Glu Thr Thr <210> 46 <211> <212> DNA <213> Artificial Sequence <220> <221> miscfeature <222> <223> Nucleotides 3, 6, 9, and 18 are wherein g or c. <400> 46 gtsgcsggsc acgagacsac <210> <211> <212> <213> <220> <221> <222> <223> <400> 47 7 PRT Streptomyces miscfeature Streptomyces consensus sequence 47 Leu Ala Gly His Glu Thr Thr 1 -33- Case <210> <211> <212> <213> <220> <221> <222> <223> 48 DNA Artificial Sequence miscfeature Nucleotides 3, 6, 9, and 18 are wherein g or c. <400> 48 ctsgcsggsc acgagacsac <210> 49 <211> 9 <212> PRT <213> Streptomyces <220> <221> misc_feature <222> <223> Streptomyces <400> 49 Leu Leu Leu Ile Ala 1 consensus sequence Gly His Glu Thr <210> <211> <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> <223> Nucleotides 2, 5, 8, <400> tsctsctsat cgcsggscac gagac 14, and 17 are wherein g or c. <210> <211> <212> <213> <220> <221> <222> <223> 51 9 PRT Streptomyces misc_feature Streptomyces consensus sequence <400> 51 His Gin Cys Leu Gly Gin Asn Leu Ala -34- Case <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <222> <223> Nucleotides 12, 15, and 24 are wherein g or c. <400> 52 gtggtcacgg asccstgctt ggascg <210> 53 <211> 8 <212> PRT <213> Streptomyces <220> <221> misc_feature <222> <223> Streptomyces consensus sequence <400> 53 Phe Gly His Gly Val 1 His Gin Cys <210> 54 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <222> <223> Nucleotides 6, 12, and <400> 54 aagccsgtgc cscasgtggt cacg 15 are wherein g or c. <210> <211> <212> <213> <220> <221> <222> <223> 8 PRT Streptomyces misc_feature Streptomyces consensus sequence <400> Case Phe Gly Phe Gly Val His Gin Cys 1 <210> <211> <212> <213> <220> <221> <222> <223> DNA Artificial Sequence miscfeature Nucleotides 6, 12, and 15 are wherein g or c. <400> 56 aaggcsaagc cscasgtggt cacg <210> 57 <211> 8 <212> PRT <213> Streptomyces <220> <221> misc feature <222> <223> Streptomyces consensus sequence <400> 57 Phe Gly His Gly Ile His Gin Cys <210> 58 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <222> <223> Nucleotides 6 and 12 are wherein g or c. <400> 58 aagccsgtgc cstaggtggt cacg 24 <210> 59 <211> 8 <212> PRT <213> Streptomyces <220> <221> miscfeature <222> <223> Streptomyces consensus sequence -36- Case <400> 59 Phe Gly His Gly Val His Phe Cys <210> <211> <212> <213> <220> <221> <222> <223> 24 DNA Artificial Sequence misc-f eature Nucleotides 6, 12, and 15 are wherein g or c. <400> aagccsgtgc cscasgtgaa gacg 24 <210> 61 <211> 24 <212> PRT <213> Streptomyces <400> 61 His Pro Gly Glu Pro 1 5 Phe Thi- Gy Tyr Gly tu-bercidicus Asn Val Met Asp Pro Ala Leu Ie Thr Asp Pro 10 Ala Leu Arg <210> <211> <212> <213> 62 21 PRT Streptomyces tubercidicus <400> 62 Phe Val Asn Asn Pro 1 5 Asn Pro Leu Thr Arg <210> 63 <211> 19 <212> PRT <213> Streptornyces <400> 63 Leu Leu, Th-r H is Tyr Ala Ser Pro Ser Leu Asn Tyr Ala Pro Giu Asp 10 tubercidicus Pro Asp Ilie Ser Leu Gly Ile Ala Pro Glu His Leu Glu Arg 37 Case <210> 64 <211> 17 <212> PRT <213> Streptomyces <400> 64 Val TPyr Leu Leu Gly 1 5 Arg <210> <211> 13 <212> PRT <213> Streptomyces <400> Thr Trp Gly Ala Asp tubercidicus Ser Ile Leu Asn Tyr Asp Ala Pro Asp His Th-r 10 tuberc id1cu S Leu Ie Ser Met Asp Pro Asp Arg tubercidicus Asp Leu Leu Ser Glu Leu Ie Arg <210> 66 <211> 13 <212> PRT <213> Streptomyces <400> 66 Glu Ala Leu Thr Asp <210> 67 <211> 12 <212> PRT <213> Streptomyces tubercidicus <400> 67 Phe Met Asp Asp Ser 1 5 <210> 68 <211> 12 <212> PRT <213> Streptomyces <400> 68 Leu Met Glu Met Leu Pro Val Trp Leu Val Thr Arg tubercidicus Gly Leu Pro Glu His Leu Arg <210> 69 38 Case <211> 11 <212> PRT <213> Streptomyces <400> 69 Val Glu Gin Ile Ala <210> <211> 11 <212> PRT <213> Streptornyces <400> Leu Val Lys Asp Asp tubercidicus Asp Ala Leu Leu Ala Arg tubercidicus Pro Ala Leu Leu Pro Arg <210> <211> <212> <213> PRT S treptomyces tubercidicus <400> 71 Asp Asp Pro 1 Ala Leu Leu Pro Arg <210> <211> <212> <213> PRT Streptoinyces tubercidicus <400> 72 Thx Pro Leu 1 Pro Gly Asn Trp Arg <210> <211> <212> <213> 73 7 PRT Streptornyces tubercidicus <400> 73 Leu Asn Ser Leu Pro Val Arg <210> 74 <211> 7 <212> PRT <213> Streptomyces tubercidicus 39 Case <400> 74 Ile Thr Asp Leu Arg Pro Arg <210> <211> <212> 7 PRT <213> Streptomyces tubercidicus <400> Glu Gin Gly Pro Val Val Arg <210> 76 <211> 7 <212> PRT <213> Streptomyces tubercidicus <400> 76 Ala Val His Giu Leu Met Arg <210> <211> <212> <213> 77 PRT Streptomyces tubercidicus <400> 77 Ala Phe Thr Ala Arg <210> <211> <212> <213> 78 PRT Streptornyces tubercidicus <400> 78 Phe Clu Glu Val Arg <210> <211> <212> <213> 79 7 PRT Streptomyces tubercidicus <400> 79 Pro Gly Glu Asp Asn Val Met 40 Case <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> 21 DNA Artificial Sequence misc_feature Nucleotides 3, 6, 12, and 18 are wherein misc_feature Nucleotide 9 is wherein a or g. misc_feature c or g. <223> Nucleotide 15 is wherein c or t. <400> ccsggsgarc csaaygtsat g 21 <210> 81 <211> 7 <212> PRT <213> Streptomyces tubercidicus <400> 81 Ala Leu Ile Thr Asp Pro Phe 1 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <222> <223> Nucleotides 3, 6, 12, and 18 are "s"wherein c or g. <400> 82 gcsctsatya csgacccstt c <210> 83 <211> 8 <212> PRT <213> Streptomyces tubercidicus <400> 83 Phe Met Asp Asp Ser Pro Val Trp 1 -41 Case <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> 84 24 DNA Artificial Sequence misc_feature Nucleotide 13 is wherein a or t. miscfeature Nucleotides 14, 15, 18, and 21 are wherein c or g. <400> 84 ttcatggacg acwssccsgt stgg <210> <211> 8 <212> PRT <213> Streptomyces tubercidicus <400> Leu Asn Tyr Asp Ala Pro Asp His 1 <210> 86 <211> 24 <212> DNA <213> Artificial Sequence <220> <221> miscfeature <222> <223> Nucleotides 3, 15 and 18 are wherein c or g. <220> <221> miscfeature <222> <223> Nucleotides 6 and 9 are wherein c or t. <400> 86 ctsaaytayg acgcsccsga ccac <210> 87 <211> 8 <212> PRT <213> Streptomyces tubercidicus <400> 87 Val Glu Gin Ile Ala Asp Ala Leu -42- Case <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 88 24 DNA Artificial Sequence misc feature Nucleotides 3, 15, 21, and 24 are wherein c or g. misc_feature Nucleotide 12 is wherein c or t. misc_feature Nucleotide 6 is wherein a or g. <400> 88 gtsgarcaga tygcsgacgc sets <210> 89 <211> 8 <212> PRT <213> Streptomyces tubercidicus <400> 89 Asp Leu Ile Ser Met Asp Pro Asp 1 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 24 DNA Artificial Sequence misc feature Nucleotides 6, 11, 12, and 21 are wherein c or g. miscfeature Nucleotide 9 is wherein a or g. misc feature Nucleotide 10 is wherein a or t. <400> ctggastarw sstacctggg sctg <210> 91 <211> 36 -43 Case <212> DNA <213> Streptorryces tubercidicus <400> 91 agattaatta atgtcggaat taatgaactg tccgtt <210> <211> <212> <213> 92 32 DNA Streptomyces tubercidicus <400> 92 aaactcaccc caaccgcacc ggcagcgagt tc 32 <210> 93 <211> 7 <212> PRT <213> Streptoryces tubercidicus <400> 93 Met Ser Glu Leu Met Asn Ser 1 <210> 94 <211> 1293 <212> DNA <213> Streptomyces tubercidicus <400> 94 atgtcggcaa aatgtgatgg caaggccccg t tcgaagag tcaccggggc cccgaacatt acccggctgc ccgcgggtcg ggtgtggtcg ctggtcggca tcgctgcagc ctgatccgcg catgacgacg gtcctggccg acccaccccg cacgagctgc gatgtcgaga tcggcgaact cccgccggcc gccaccctcg gagctgtcgc tatccagctc acccggcgct tcctaccggg tccgccaagt attcgatcga tccggccgta gccgtctggt agcagc tcgc acctgatcaa tcccggaagc cggagcggct agcggcgcgg acggcagccg gccacgagac accagctgcg tgcgctggtg tcgccgggac tcgacccccg acgccgagaa ccaaacagga tggccgtcgc cccgttcgcc ga tcaccgac ccggttcatg cctgcgcgat cgagagcccc tctgatgggg gtcacgcgcg cgacgagctg gcacttcgcc ggaccgcccg cagcacctcg cgcgc tcacc gctcagcgac caccgcccac cctggtcaag cgggccggtc gccga tccgt ccactacacc ccatgtgggc gggcgaagtc accggacgag gcacacgtcg ccgttcggcg gacgac tcac cagcggttcc acqgccaggc tcgatcctca ttcacggcac c tggcccggc tatcccctgc caa tggcgga t tcccggcga gacgatctgc gtcgagatgg ctgataggca gacgacccgg cagatgaccc aagggcgacg gcccccgaac t tcggccacg gcgttcggca ttggagcgaa gaaagca tcc gctacggcgc ccgtctggct tgaacaaccc tgctggacat acaacgacgc gcaagatcac ttcccgagca cgatcaccgt agtggggcgc tgatcgagca tcagcgagc t tcaccatggt acggcacggc agctgcttcc agctgcggta ccgtacaact gcctcgacct gaa tgcacta agctcttcac cgccggtgcc cggcgagccg actgcgtgag cgtgacgcgc ggccgcg tcg gatggggatg ccccgaccac cgatctgcgg cgccgaggac gatctgcgaa cgacctcgtt catccatgaa gatccgtacc cctcaccgtc ggcgctgctc gcgtgccgtc cgcc tccgag catcctggta gacccgccac c tgcc tgggc gcac tacccg cggcagctgg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 44 Case cggttggatt cgctgccggt gcggttgggg tga <210> <211> 430 <212> PRT <213> Streptomyces tubercidicus 1293 <400> Met Ser 1 Pro Gly Gly Gly Phe Met Arg Gin Ser Pro Met Met Leu Asn2 Arg Ala 130 Gin Leu2 145 Gly Val Val Ie Arg Lys Thx SerI 210 Arg Arg 225 His Asp2 Val Leu Gly Asn Val Lys 1 290 Arg Tm C 305 Asp Val C Leu Ie I Al a 1lu Tyr A.sp Gly Gly %sn 115 Phe kia Ia 1 1-95 Phe .\sp rhr \sp 'ys flu ~eu Ile Pro Gly Asp Leu His Met 100 Asp Thr Asp Asp Glu 180 Gly Pro Al a Asp Val1 260 rhr Asp Gly Ile Va 1 340 Ser 5 Asn Al a Ser Arg Ser Pro Al a Aia Glu Leu 165 Leu Ala Al a Leu Gly 245 Val1 Ala Pro Pro Ala 325 Ser Ser Val1 Leu Pro Asp 70 Ile Giu Pro Arg Leu 150 Ile Val1 Asp Met Thr 230 Ser Leu Ala Giu Val1 310 Gly Al a Ser Met Arg Val1 55 Gin Asp H is Asp Lys 135 Leu Lys Gly Leu Ilie 215 Asp Arg Al a Leu Leu 295 Gin Thr Asn Pro Phe Asp Pro 25 Giu Gin 40 Tmp Leu Arg Phe Giu Ser Phe Arg 105 His Thr 120 Ile Thx Ala Arg His Phe Ile Pro 185 Val Ser 200 Glu His Asp Leu Leu Ser Gly His 265 Leu Thr 280 Leu Pro Met Thr Pro Ie Phe Asp 345 Al a Al a Gly Val1 Leu Pro 90 Pro Arg Asp Leu Ala 170 Giu Leu Ile Leu Asp 250 Glu His Arg Gin Arg 330 Pro Ala Leu Pro Thr Asn 75 Thr Tyr Leu Leu Pro 155 Ala Gin His Ser 235 Val1 Thr Pro Ala Leu 315 Lys His Ile Val1 Arg Asn Al a Leu Arg Arg 140 Glu Pro Asp Pro Giu 220 Giu Glu Thx Asp Val1 300 Arg Gly Val1 Thr Leu Phe Pro Arg Met Arg 125 Pro His Leu Arg Glu 205 Leu Leu Met Al a Gin 285 H is Tyr Asp Gly Asp Pro Giu Al a Leu Gly 110 Leu Arg Al a Pro Pro 190 Arg Ile Ile Val1 His 270 Leu Giu Ala Al a Lys Pro Gly Giu Al a Leu Ser Val1 Val1 Giu Ile 175 Gin Leu Arg Arg Thr 255 Leu Arg Leu Ser Val1 335 His Phe Arg Val1 Ser Asp Ile Ser Giu Asp 160 Thr Tmp Ser Glu Thr 240 Met Ile Leu Leu Giu 320 Gin Arg His Tyr Thr Ala Pro

350. 45 Case Clu Arg Leu 355 Asp Leu Thx Arg H is 360 His Pro Ala Gly His Ala 365 Ala Glu Asn His Thr Leu, Ala Val Gly Phe Gly His Gly 370 Lys Gin Glu Gly Glu Val 385 390 Glu Leu Ser Leu Ala Val Met 375 Ala Tyr Cys Leu Gly 380 Phe Phe Gly Lys Thr His Tyr Pro 400 Val1 Ala Pro Asp 405 Arg Gl u 410 Pro Glu Arg Thr Pro 415 Pro Gly Ser Trp 420 Leu Asp Ser Leu 425 Val Arg Leu <210> 96 <211> 18 <212> DNA <213> Artificial Sequence <400> 96 cgsccsccsc tswssaas <210> 97 <211> 21 <212> DNA <213> Artificial Sequence <400> 97 sassgcstts bcccartgyt c <210> 98 <211> 1266 <212> DNA <213> Streptornyces tubercidicus <400> 98 gtggtcgacg gcggagac tc gaccacccg t gacagcgtgt ggccagcccg ctgatcgcct cccggcaccg cgcggcgtcc tggatcggcc qaggccgccc gatctgcacc ggagagggcg gatgtgctcg ga tctcgccg gacccgtaca cacaccagac tccgcgcgga acgagcgccc tcgtcca tga ccgtccgcct acgacaagct gcc tggccgg tggcctccct tggaggtcgc cgacgccgc t gcgagcacgg gcatggtgct ccgcga tcgg acccggagac tc tacgccgc gttcgtcatc ggggt tcacc cccgc tc tcc gcccgcctgg cgaccccgag gc tgc tggcc cgtgcaccac cggccgcgac cgccgcggcc gcacggcatc cgtccgcttc cgccg tgcgc cgccgccccg cggcggcggg cggtgacgtc gtcgggggtg ggccggg tga aaggggt tcc tacgcccagg ggcaggaccg accggcgccg ctgcgccgcc aacggcca tc cgctcctacg ctggggcccg cacttcggcg accgacgacg cgcaccgcgc gtggccgtcg gccgccgccg gcctggccgg tcctcatctg tgctcggcaa cacagatcga tccgcctcgg aaccgcggcg tcgcccacgc tggtgatcgc gcgccgaggt aactcggcgg cccgcttcac gcgaggaaca tcgccgaaca acgcggcgct accacccgc t cgcaaaggcc tgacgagcgc ggaagagcgc actgcacctg cgacggcacc cctggacatc cgaacggctg cqgagccggc gaccg tcg tc tctgttcacc cgaga tcg tc ccccgcccac ggccgggctg gcgcacctcc gc tggacacc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 46 Case cggctgcggg tcgaacactg ggccaacgcc ctcaacggcg gcccggccgc cgcccgcgcc 960 atgctcggcc aggacatcag ctacgaccqc atcccgtact tcttctccga ccagtacgac 1020 gtcggcatgg agtactccgg ctacgccccg cccggctcgt acgcccaggt cgtctgccgc 1080 ggcgacgtcg ccaagcggga gttcatcgcc ttctggctgg cggcggacgg ccggctgctc 1140 gcgggcatga acgtcaacgt ctgggacgtc gccgagtcca tccagcaact catccgctcc 1200 ggggcgccgt tggagcccgg cgcactggcc gatccgcagg ttccgctggc ggcactgctc 1260 ccgtag 1266 <210> 99 <211> 421 <212> PRT <213> Streptomyces tubercidicus <400> 99 Val Val Asp 1 Gly Ala Lys Val Ile Leu Leu Ser Lys Val His Glu Gly Gin Pro Gly Asp Gly Ala Glu Pro 115 His His Leu 130 Ala Ser Leu 145 Trp Ile Gly Val Thx Val Pro Glu Leu 195 Arg Phe His 210 Met Val Leu 225 Asp Val Leu Gin Ala Gly Val Asp Ala 275 Asp Val Ala 290 Ala Ala Ile Gly Pro Al a Thr 100 Arg Arg Gly Leu Val 180 Gly Phe Ala Ala Leu 260 Ala Al a His 5 Ala Cys Phe Ala Va1 Leu Arg Arg Arg Glu 165 Glu Gly Gly Va1 Ala 245 Asp Leu Ala Gin Glu Asp Leu Trp 70 Arg Ile Leu Leu Asp 150 Va1 Ala Leu Ala Arg 230 Ile Leu Arg Asp Thx Thr Glu Leu 55 Tyr Leu Ala Asp Ala 135 Asn Ala Ala Phe Arg 215 Thr Gly Ala Thr His 295 Phe Leu Arg 40 Gly Ala Asp Tyr Ile 120 His Gly Ala Pro Thr 200 Phe Asp Ala Asp Ser 280 Pro Val Ile Val Gly Gly Gly Leu Ala 10 Arg 25 Asp Lys Gin Pro Asp 105 Pro A a His Ala Thr 185 Asp Thr Asp Ala Pro 265 Asp Leu Ala His Glu Ala Glu 90 Lys Gly Glu Leu Ala 170 Pro Leu Glu Gly Pro 250 Glu Pro Leu Glu Pro Glu Gin 75 Gly Leu Thr Arg Va1 155 Arg Leu His Ile Glu 235 Arg Thr Tyr Asp Gly Tyr Arg Ile Arg Leu Gly Leu 140 Ile Ser His Arg Va1 220 Glu Thr Gly Ile Thr 300 Phe Thr Glu Arg Asp Ser Glu Leu Thr Val Leu Ala 110 Leu Ala 125 Axg Gly Ala Gly Tyr Gly Gly Ile 190 Glu His 205 Gly Glu His Pro Ala Leu Gly Gly 270 Tyr Ala 285 Arg Leu Gly Arg Pro Pro Val Phe His Leu Arg Leu Thr Gly Gly Val Val Leu Ala Gly 160 Ala Glu 175 Leu Gly Gly Val Gly Gly Ala His 240 Ala Glu 255 Val Ala Ala Gly Arg Val 47- case Glu 305 Met His Trp Ala Asn Al a 310 Ile Leu Asn Gly Gly Pro 315 Ile Ala Ala Ala Arg Ala 320 Leu Gly Gln Asp 325 Val1 Ser Tyr Asp Arg 330 Ser Pro Tyr Phe Phe Ser 335 Asp Gln Tyr Ser Tlyr Ala 355 Ile Ala Phe Asp 340 Gln Gly Met Glu Tyr Gly Tyr Ala 345 Gly Val Val Cys Arg 360 Asp Asp Val Ala Lys 365 Ala Pro Pro Gly 350 Arg Glu Phe Gly Met Asn Trp Leu Ala 370 Val Asn Ala 375 Ala Gly Arg Leu Leu 380 Gln Val Trp Asp 385 Gly Val1 390 Pro Glu Ser Ile Gln 395 Asp Leu Ile Arg Ser 400 Leu Ala Pro Leu Glu 405 Pro Gly Ala Leu Al a 410 Pro Gln Val Pro 415 Ala Ala Leu Leu 420 <210> 100 <211> 1314 <212> DNA <213> Streptomyces tubercidicus <400> 100 atgcccgctg ccgccgggcc cgccgcggag ggcctgtacg ggggacgag t gccaccgacc gaatggctgc gatggcggcc ctgctccccg gcgCtccgtg a tcggcgccg gccgcgccgc ctgcatgcgg gagggcgacg gacgtggtcg ctgccgCtcg gtggccgtcg accgaacagg cggagcctgc cggctgccca gcctgttacg ttca tgcggc cacgccgccg gtgcgcaccc gagggcaccg ccgcgcgg tc gccacggccc cgggccgac t tgggcacccg ggtccctgac gaccggtgcc cggatctggc aggtcgcctc tccccctcgt accacggcg t gccggcgcgt tcg tcggca t acgacggtgt gcgacg tcgc ccgccg tggc cgtacttctg ccgacacacc aacgggacgg tccgccgcga ccttcgacct accccg tgac ccgcatgaag cctgcgttcc ctacgaccgg cgccctggcc ggccaccggg caccgacggc cgccggggtc gccggcgccg gtcctgcgcc cccccaactc cacgctgctc caccggcgtc cggggtacgc gctctgcgac caggg tggac ggcgcggaac gtccgaccag gcgcgtcctc acgcaccacc actcgcccgc ccgcaccgga cgtccaggac tcggtcgctg caggggt tcg cccccgctgt gacgccgagg ctcgacaccg gtggtcCtcg cacaccctgc gtccgggtcg gccctaggcc ggccacgcca accggaaccg gagc tgaccg ccccgcaccg gcgggctgtg ggcgcccgtg c tgc tggccg tacggcg tcc gaaggctccc gcggtgCtcg accgccctgt gcggcgacct ggcatgatct tcatcggggc acggccgcct ccaaggactt agatcgccga gcggacgcac ccaccggcgc gcaccctcga tgg tga tcgg atgacgtcac tggccgagat gtgtcgcccg acggccgcct cctggctcac tcaccccgct ccgagcactg gcagcaccg t gca tccagt t ccgacgaccg ccc tcaaccg cggccaccac gcctgcccgc acgtccatca ctcgctggcg ggtgatcgtc cctcaccggc ac tcgacgcc ggtgctgCtc cgccccgcgc cgacgcccag cggcggcttc cgtggtcgag ctgcgccgcc gc tgcgcagc gctccccgcc ggactccgga gcccgccgc gaccagcgcc cgcgacccac cgcgggccac cagcttcctc gccccgcccc ctga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1314 <210> 101 <211> 437 <212> PRT <213> Streptomyces tubercidicus 48 Case <400> 101 Met Pro Ala Ala Arg Arg Arg Leu Arg Pro Pro His Arg Ser Gly Asp Leu Gly Met Ala Gly Phe Glu Thr Ser 145 Leu Asp Val Cys Pro 225 Leu Arg Thr Val Asp 305 Val Trp Ala Asp Asp 385 Pro Arg Lys Arg Asp Leu Glu Gly 130 Leu Leu Asp Val Ala 210 Leu His Leu Asp Arg 290 Gly Ala Thr Gly Gln 370 Thr Ala His Ser Ser Glu Thr Ile 115 Leu Thr Pro Ala Val 195 Ala Val Ala Arg Gly 275 Pro Val Val Ser Ser 355 Tyr Pro Arg Asp Val. Leu Cys Gly 100 Ala Asp Thr Gly Gin 180 Ile Leu Pro Asp Ser 260 Arg Arg Leu Gly Ala 340 Thr Gly Arg Pro Leu Ala Arg His Ala Glu Thr Asp Pro 165 Ala Gly Gly Gin His 245 Glu Leu Thr Cys Asp 325 Thr Val Val Val Pro Arg Val Ser 70 Gly Thr Leu Gly Gly 150 Val Leu Gly His Leu 230 Gly Gly Leu Ala Asp 310 Val Glu Ala Arg Leu 390 Gly Pro Ile 55 Gin Pro Asp Asp Gly 135 Val Pro Arg Gly Asp 215 Gly Val Asp Pro Trp 295 Ala Ala Gin Thr Ile 375 Glu SArg SSer 40 Gly Gly Tyr Pro SAla 120 Arg Val Ala Ala Phe 200 Val His Thr Gly Ala 280 Leu Gly Arg Ala His 360 Gin Gly Ale 25 Arc Ale Phe Asp Gly 105 Glu Thr Leu Gly Asp 185 Ile Thr Ala Leu Arg 265 Asp Thr Cys Val Ala 345 Arg Phe Ser SHis Pro Arg Gly Ser Leu SAsp Gly 75 Arg Pro 90 Arg Leu Trp Leu SVal Leu SAla Thr 155 Val His 170 SLeu Ala Gly Ala Val Val Met Ala 235 Leu Thr 250 Arg Val Val Val Asp Ser Val Thr 315 Asp Gly 330 Val Ala Ser Leu Ala Gly Pro Asp 395 Thr Ala 410 Pro Gly Ala Arg Pro Ala Leu Leu 140 Gly Thr Pro Glu Glu 220 Glu Gly Thr Val Gly 300 Pro Ala Ala Pro His 380 Asp Arg SGly Gly Leu Leu Leu Gly 125 Asp Ala Leu Ala Val 205 Ala Ile Thr Gly Val 285 Leu Leu Arg Arg Tyr 365 Arg I Arg Asr His Leu Val Ser Ala 110 Thr Gly Ala Arg Pro 190 Ala Ala Cys Gly Val 270 Gly Pro Pro Ala Asn 350 Phe Leu Ser Arg Arg STyr Ile Lys Asp Arg SGly Pro Thr 175 Val Ser Pro Ala Val 255 Glu Ile Leu Ala Glu 335 Leu Trp Pro Phe Pro SArg SAla SVal Asp SAla Ala Arg Arg 160 Leu Arg Ser Leu Ala 240 Ala Leu Gly Asp Val 320 His Leu Ser Thr Leu 400 Ala Cys Tyr Glu Arg Asp Gly Arg Thr 405 Val Leu Ala Leu Asn 415 49- Case Arg Pro Arg Pro Phe Met Arg Leu Arg Arg Clu Leu Ala Arg Thir Ala 420 425 430 Leu Ser Ala Thr Thr 435 <210> 102 <211> 1233 <212> DNA <213> Streptornyces tubercidicus <400> 102 a tggcccaga gagacactgc cgtccctacg aaggcgtacg aacgccgtca ctgggctacg ggcgccgacc gacctcttcc accacggccg cccctgctgg cacggtgtcg gtcgacgggg gtcgggatca gtcgtcgtgg gccaacgcct ctccaccagc ctgccgtact ccgggcggg t ttctggCtct gacccga tcc gcggacgtac acacggcatt gcgcggaggg agcggccgcc tcca tccgcc ccgccctcga ccaagctgct tcgacgggg t ggtccgcgtc ccgcgcgtgc gggtgctggg cgctgcgctg tacggctggc cccccaactc acgagcggct accaccccct cgaagaccgc tcttcaccga acgaccgcgt ccggcggccg gggCcctggt cgc tggcgga ca tca tcgcg cttcggcggc gctgtccaag ccagtggtac cccggccggc gctggccacc ccacacgc tg ccggatcgtg ggcgggggtc ccgcgaggtc cgacacccag cgacggcacc cgagacggcc gtgctcctcc cctgggcaag ggcccgggcc ccagtacgac ggtgttccgc ggtgctggcc ggcgagcggg tctggtcccc ggagcggggc cccgtcctgc ggctacctct gccgagcacg cacgagg tga ggctccactc cgg tacc tgg gtgatcggcg gaggtgaccg gcccaggtct gtcacggaga cggatcgcgg gccgcgggcg cacccggaca cacc tccgcg atgctgggcg ctgggcatgg ggcgacaccg gggatgaatg cgggccgtgg tga tggccggggc tgctgggcga tgggcacctc acgtcgatct ccctcgccga cgcgccggc t cggacagcga gcggctggat tgctggagtc tcgccga tc t tcaccggcac ccgacgcgg t ggctcaaggt tctacgccgc tcgagcactg gggaggccgg agtacacggg gtgcccgcga tgaacg ta tg accccgagcg gaaggccgcg cgagcgcgag cgagcgggag gcggctgggc cggcagccgg gccggtgccc ccgcctcaag cggcc tggag ggcgccgc tg gcacaccgag gaacggcgcg gatcgtcggc cgacaacggc cggcgacg tc ggccaacgcc ctacgaccgg gca tg tggag gttcatcgcc ggacgtcacc gc tcgccgac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1233 <210> 103 <211> 410 <212> PRT <213> Streptomyces tu-bercidicus <400> 103 Met Ala Gin Asn Thm Ala Phe Ie 1 5 Ala Lys Ala Ala Glu Tim Leu Arg Leu Leu Leu Gly Asp Glu Arg Glu 40 Ser Lys Gly Tyr Leu Leu Gly Tim His Pro Pro Gin Trp Tyr Ala Glu 70 Asn Ala Val Tim Ala Leu Asp Pro Ile Ala 10 Ala Glu Gly Ala Gly Leu Ala Gly Gly Phe Gly Arg Pro Tyr Glu Ser Glu Arg Glu Arg Lys Gly Pro Val Pro Pro Leu Ala Tyr Val His Asp Val Asp Leu Arg Leu 75 Ala Gly His Glu Val Tim Leu Gly Ala Case 90 Asp Gly Ser Arg Leu Gly Tyr Ala Lys Leu Leu Leu Ala Thr Gly Ser 100 105 110 Thr Pro Arg Arg Leu Pro Val Pro Gly Ala Asp Leu Asp Gly Val His 115 120 125 Thr Leu Arg Tyr Leu Ala Asp Ser Asp Arg Leu Lys Asp Leu Phe Arg 130 135 140 Ser Ala Ser Arg Ile Val Val Ile Gly Gly Gly Trp Ile Gly Leu Glu 145 150 155 160 Thr Thr Ala Ala Ala Arg Ala Ala Gly Val Glu Val Thr Val Leu Glu 165 170 175 Ser Ala Pro Leu Pro Leu Leu Gly Val Leu Gly Arg Glu Val Ala Gin 180 185 190 Val Phe Ala Asp Leu His Thr Glu His Gly Val Ala Leu Arg Cys Asp 195 200 205 Thr Gin Val Thr Glu Ile Thr Gly Thr Asn Gly Ala Val Asp Gly Val 210 215 220 Arg Leu Ala Asp Gly Thr Arg Ile Ala Ala Asp Ala Val Ile Val Gly 225 230 235 240 Val Gly Ile Thr Pro Asn Ser Glu Thr Ala Ala Ala Ala Gly Leu Lys 245 250 255 Val Asp Asn Gly Val Val Val Asp Glu Arg Leu Cys Ser Ser His Pro 260 265 270 Asp Ile Tyr Ala Ala Gly Asp Val Ala Asn Ala Tyr His Pro Leu Leu 275 280 285 Gly Lys His Leu Arg Val Glu His Trp Ala Asn Ala Leu His Gin Pro 290 295 300 Lys Thr Ala Ala Arg Ala Met Leu Gly Gly Glu Ala Gly Tyr Asp Arg 305 310 315 320 Leu Pro Tyr Phe Phe Thr Asp Gln Tyr Asp Leu Gly Met Glu Tyr Thr 325 330 335 Gly His Val Glu Pro Gly Gly Tyr Asp Arg Val Val Phe Arg Gly Asp 340 345 350 Thr Gly Ala Arg Glu Phe Ile Ala Phe Trp Leu Ser Gly Gly Arg Val 355 360 365 Leu Ala Gly Met Asn Val Asn Val Trp Asp Val Thr Asp Pro Ile Arg 370 375 380 Ala Leu Val Ala Ser Gly Arg Ala Val Asp Pro Glu Arg Leu Ala Asp 385 390 395 400 Ala Asp Val Pro Leu Ala Asp Leu Val Pro 405 410 <210> 104 <211> 1266 <212> DNA <213> Streptomyces tubercidicus <400> 104 gtggtcgacg cacaccagac gttcgtcatc gtcgggggtg gcctggccgg cgcaaaggcc gcggagactc tccgcgcgga agggttcacc ggccgggtga tcctcatctg tgacgagcgc 120 gaccacccgt acgagcgccc cccgctctcc aaggggttcc tgctcggcaa ggaagagcgc 180 gacagcgttt tcgtccacga acccgcctgg tacgcccagg cacagatcga actgcacctg 240 -51 Casc ggccagcccg ctgatcgcct cccggcaccg cgcggcg tcc tggatcggcc gaggccgccc gaactgcacc ggacaggacg gacgtgctcg gacctcgccg gacccgtaca cggc tgcgcg a tgc tcggcc gtcggcatgg ggcgacgtcg gcggggatga ggggtgcggt ccgtag ccg tccgcc t acgacaagct gcctggccgg tggcctccct tggaggtcgc cgacaccgct gcgcacacgg gcatggtgct ccgcga tcgg acccggaggc tctacgccgc tcgaacactg aggaca tcag agtactccgg ccaaacggga acgtcaacgt tggagcccgg cgaccccgag gc tgc tggcc cgtgcaccac cgggcgggac cgccgcggcc gcacggca tc cgtgcgcttc cgccg tgcgc cgccgccccg cggcggcggc cggcgacgtg ggccaacgcc ctacgaccgc c tacgccccg gttcatcgcg ctgggacgtc cgagctggct gcgaagaccg accggcgccg ctgcgccgcc aacgggcatc cgctcctacg ctggggcccg cacttcggcg accgacgacg cgcaccgcac g tggccgtcg gccgccgccg c tcaacggcg gtcccgtact cccggctcct ttctggctcg gccgaaacca gatccggagg tccgcctcgg agccgcgccg tcgcccacgc tggtgatcgc gcgccgaggt aactcggcgg cccgtttcac gcgaggagca tcgccgaaca acgcgacgc t accaccccct gcccggccgc tcttctccga acgcacaggt gcgaggacgg tccagcaac t t tccgc tgac cgacggcacc cctggacatc cgaacggc tg cggcgccggc caccgtcgtc cctgttcacc cgagatcgtc ccccgcccac ggccggactc gcgcacctcc cc tggacacc cgcgcgcgcc ccagtacgac cgtctgccgc acggctgctc catccgcggc ctcactgctc 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1266 <210> 105 <211> 421 <212> PRT <213> Streptoryces tubercidicus <400> 105 Val Val Asp Ala 1 Gly Ala Lys Ala Gin Thr Phe Val Ile Val Gly Gly Gly Leu Ala Giu T Lr Leu Ile Arg 25 Asp Giu Gly Phe Val Ilie Leu Leu Ser Lys Cys Asp Giu Arg 40 Gly His Pro Tyr Giu Asp Thr Gly Arg Arg Pro Pro Ser Val Phe Gly Phe Leu Val His Leu 55 Tyr Lys Giu Giu Arg Ile Glu Pro Ala Gly Trp 70 Arg Ala Gin Ala Gin 75 Ala Glu Leu His Leu Gin Pro Ala Val1 Leu Leu Asp Pro Glu 90 Lys Lys Thx Val Arg Leu Gly Asp Gly Ala Giu Pro 115 His His Leu Ile Ala Tyr Asp 105 Pro Leu Leu Leu Ala Thr Gly Ala Giy Val Arg Leu Asp Ilie 120 His Gly Thr Gly Leu 125 Arg Arg A xg Leu 130 Ala Ser Al a 135 Asn Ala Glu Arg Leu 140 Ile Gly Val Leu Leu Gly Arg 145 Trp, Asp 150 Gly His Leu Ala Gly Ala Gly 160 Ile Gly Leu Giu Vai Ala 165 Ala Ala Al a 170 Pro Ser Tyr Gly Ala Giu 175 Val Thr Val Pro Giu Leu 195 Arg Phe His Val1 180 Gly Glu Gly Ala Ala Pro Thr 185 Leu Phe Thr Giu 200 Ala Arg Phe Thr Leu His Gly Ile Leu Gly 190 His Gly Val Leu His Arg Glu Ile Val Al a 205 Gly Phe Gly Gin Asp Gly Case 210 215 Net Val Leu Ala Val Arg Th-r Asp Asp Gly Giu Giu His Pro Ala His 225 Asp Gin Val1 Asp Giu 305 Met Asp Ser Ile Val1 385 Gly Thr Val1 Al a Asp Val1 290 His Leu Gin Tyr Ala 370 Asn Val Ser Leu Gly Ala 275 Al a Trp Gly Al a 355 Phe Val1 Arg Leu Ala Al1a 245 Leu Asp 260 Thr Leu Ala Ala Ala Asn Gin Asp 325 Asp Val 340 Gin Val Trp Leu Trp Asp Leu Glu 405 Leu Pro 420 230 Ie Leu Arg Asp Al a 310 Ile Gly Val1 Gly Val1 390 Pro Gly Al a Th-r His 295 Leu Ser Met Cys Glu 375 Al a Al a Asp Ser 280 Pro Asn Tyr Glu Arg 360 Asp Glu Al a Pro 265 Asp Leu Gly Asp Tyr 345 Gly Gly Thr Pro 250 Giu Pro Leu Gly Arg 330 Ser Asp Arg Ie Al a 410 235 Arg Al a Asp Pro 315 Val1 Gly Val1 Leu Gin 395 Asp Thr Gly Ile Thxr 300 Al a Pro Tyr Al a Leu 380 Gin Pro Ai a Gly Tyr 285 Arg Ala Tyr Ala Lys 365 Ala Leu Glu Leu Gly 270 Ala Leu Al a Phe Pro 350 Arg Gly Ile Val Al a 255 Val Ala Arg Arg Phe 335 Pro Glu Met Arg Pro 240 Giu Al a Gly Vali Ala 320 Ser Gly Phe Asn Gly 400 Leu Gly Glu Leu 53