AU762399B2 - Recombinant narbonolide polyketide synthase - Google Patents

Description

WO 99/61599 -1 PCT/US99/11814 RECOMBINANT NARBONOLIDE POLYKETIDE SYNTHASE Reference to Government Funding This invention was supported in part by SBIR grant 1R43-CA75792-01. The U.S.

government has certain rights in this invention.

Field of the Invention The present invention provides recombinant methods and materials for producing polyketides by recombinant DNA technology. More specifically, it relates to narbonolides and derivatives thereof. The invention relates to the fields of agriculture, animal husbandry, chemistry, medicinal chemistry, medicine, molecular biology, pharmacology, and veterinary technology.

Background of the Invention Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There is a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Tetracycline, erythromycin, FK506, FK520, narbomycin, picromycin, rapamycin, spinocyn, and tylosin, are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; WO 97/02358; and WO 98/27203; United States Patent Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem.

Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.

Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs).

WO 99/61599 PCT/US99/11814 -2- Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or "modular" and Type II "iterative" PKS enzymes.

Modular PKSs are responsible for producing a large number of 12, 14, and 16membered macrolide antibiotics including methymycin, erythromycin, narbomycin, picromycin, and tylosin. These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying Bcarbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference). The modular PKS are generally encoded in multiple ORFs. Each ORF typically comprises two or more "modules" of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three or more enzymatic activities or "domains." During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Patent Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS were that it overcame the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allowed more facile construction of recombinant PKSs, and reduced the complexity of PKS analysis by providing a "clean" host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication No. WO 98/49315, incorporated herein by reference).

The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of B-carbn processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr.

Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Patent Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not WO 99/61599 PCT/US99/11814 -3produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters. It has been possible to manipulate modular PKS genes other than the narbonolide PKS using generally known recombinant techniques to obtain altered and hybrid forms. See, U.S. Patent Nos. 5,672,491 and 5,712,146 and PCT publication No. WO 98/49315. See Lau et al., 1999, "Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units" Biochemistry 38(5):1643- 1651, and Gokhale et al., 16 Apr. 1999, Dissecting and Exploiting Intermodular Communication in Polyketide Synthases", Science 284: 482-485.

The present invention provides methods and reagents relating to the modular PKS gene cluster for the polyketide antibiotics known as narbomycin and picromycin.

Narbomycin is produced in Streptomyces narbonensis, and both narbomycin and picromycin are produced in S. venezuelae. These species are unique among macrolide producing organisms in that they produce, in addition to the 14-membered macrolides narbomycin and picromycin (picromycin is shown in Figure 1, compound the 12-membered macrolides neomethymycin and methymycin (methymycin is shown in Figure 1, compound 2).

Narbomycin differs from picromycin only by lacking the hydroxyl at position 12. Based on the structural similarities between picromycin and methymycin, it was speculated that methymycin would result from premature cyclization of a hexaketide intermediate in the picromycin pathway.

Glycosylation of the C5 hydroxyl group of the polyketide precursor, narbonolide, is achieved through an endogenous desosaminyl transferase to produce narbomycin. In Streptomyces venezuelae, narbomycin is then converted to picromycin by the endogenously produced narbomycin hydroxylase. (See Figure 1) Thus, as in the case of other macrolide antibiotics, the macrolide product of the narbonolide PKS is further modified by hydroxylation and glycosylation. Figure 1 also shows the metabolic relationships of the compounds discussed above.

Picromycin (Figure 1, compound 1) is of particular interest because of its close structural relationship to ketolide compounds HMR 3004, Figure 1, compound The ketolides are a new class of semi-synthetic macrolides with activity against pathogens resistant to erythromycin (see Agouridas et al., 1998, J. Med. Chem. 41: 4080-4100, incorporated herein by reference). Thus, genetic systems that allow rapid engineering of the narbonolide PKS would be valuable for creating novel ketolide analogs for pharmaceutical WO 99/61599 PCT/US99/11814 -4applications. Furthermore, the production of picromycin as well as novel compounds with useful activity could be accomplished if the heterologous expression of the narbonolide PKS in Streptomyces lividans and other host cells were possible. The present invention meets these and other needs.

Disclosure of the Invention The present invention provides recombinant methods and materials for expressing PKSs derived in whole and in part from the narbonolide PKS and other genes involved in narbomycin and picromycin biosynthesis in recombinant host cells. The invention also provides the polyketides derived from the narbonolide PKS. The invention provides the complete PKS gene cluster that ultimately results, in Streptomyces venezuelae, in the production of picromycin. The ketolide product of this PKS is narbonolide. Narbonolide is glycosylated to obtain narbomycin and then hydroxylated at C12 to obtain picromycin. The enzymes responsible for the glycosylation and hydroxylation are also provided in recombinant form by the invention.

Thus, in one embodiment, the invention is directed to recombinant materials that contain nucleotide sequences encoding at least one domain, module, or protein encoded by a narbonolide PKS gene. The recombinant materials may be "isolated." The invention also provides recombinant materials useful for conversion of ketolides to antibiotics. These materials include recombinant DNA compounds that encode the C12hydroxylase (the picK gene), the desosamine biosynthesis and desosaminyl transferase enzymes, and the betaglucosidase enzyme involved in picromycin biosynthesis in S. venezuelae and the recombinant proteins that can be produced from these nucleic acids in the recombinant host cells of the invention.

In one embodiment, the invention provides a recombinant expression system that comprises a heterologous promoter positioned to drive expression of the narbonolide PKS, including a "hybrid" narbonolide PKS.. In a preferred embodiment, the promoter is derived from a PKS gene. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces narbonolide. In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.

In another embodiment, the invention provides a recombinant expression system that comprises the desosamine biosynthetic genes as well as the desosaminyl transferase gene. In a related embodiment, the invention provides recombinant host cells comprising a vector that WO 99/61599 PCT[US99/11814 produces the desosamine biosynthetic gene products and desosaminyl transferase gene product. In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.

In another embodiment, the invention provides a method for desosaminylating polyketide compounds in recombinant host cells, which method comprises expressing the PKS for the polyketide and the desosaminyl transferase and desosamine biosynthetic genes in a host cell. In a preferred embodiment, the host cell expresses a beta-glucosidase gene as well. This preferred method is especially advantageous when producing desosaminylated polyketides in Streptomyces host cells, because such host cells typically glucosylate desosamine residues of polyketides, which can decrease desired activity, such as antibiotic activity. By coexpression of beta-glucosidase, the glucose residue is removed from the polyketide.

In another embodiment, the invention provides the picK hydroxylase gene in recombinant form and methods for hydroxylating polyketides with the recombinant gene product. The invention also provides polyketides thus produced and the antibiotics or other useful compounds derived therefrom.

In another embodiment, the invention provides a recombinant expression system that comprises a promoter positioned to drive expression of a "hybrid" PKS comprising all or part of the narbonolide PKS and at least a part of a second PKS, or comprising a narbonolide PKS modified by deletions, insertions and/or substitutions. In a related embodiment, the invention provides recombinant host cells comprising the vector that produces the hybrid PKS and its corresponding polyketide. In a preferred embodiment, the host cell is Streptomyces lividans or S. coelicolor.

In a related embodiment, the invention provides recombinant materials for the production of libraries of polyketides wherein the polyketide members of the library are synthesized by hybrid PKS enzymes of the invention. The resulting polyketides can be further modified to convert them to other useful compounds, such as antibiotics, typically through hydroxylation and/or glycosylation. Modified macrolides provided by the invention that are useful intermediates in the preparation of antibiotics are of particular benefit.

In another related embodiment, the invention provides a method to prepare a nucleic acid that encodes a modified PKS, which method comprises using the narbonolide PKS encoding sequence as a scaffold and modifying the portions of the nucleotide sequence that encode enzymatic activities, either by mutagenesis, inactivation, insertion, or replacement.

The thus modified narbonolide PKS encoding nucleotide sequence can then be expressed in a WO 99/61599 PCTIUS99/1814 -6suitable host cell and the cell employed to produce a polyketide different from that produced by the narbonolide PKS. In addition, portions of the narbonolide PKS coding sequence can be inserted into other PKS coding sequences to modify the products thereof. The narbonolide PKS can itself be manipulated, for example, by fusing two or more of its open reading frames, particularly those for extender modules 5 and 6, to make more efficient the production of 14-membered as opposed to 12-membered macrolides.

In another related embodiment, the invention is directed to a multiplicity of cell colonies, constituting a library of colonies, wherein each colony of the library contains an expression vector for the production of a modular PKS derived in whole or in part from the narbonolide PKS. Thus, at least a portion of the modular PKS is identical to that found in the PKS that produces narbonolide and is identifiable as such. The derived portion can be prepared synthetically or directly from DNA derived from organisms that produce narbonolide. In addition, the invention provides methods to screen the resulting polyketide and antibiotic libraries.

The invention also provides novel polyketides and antibiotics or other useful compounds derived therefrom. The compounds of the invention can be used in the manufacture of another compound. In a preferred embodiment, the antibiotic compounds of the invention are formulated in a mixture or solution for administration to an animal or human.

These and other embodiments of the invention are described in more detail in the following description, the examples, and claims set forth below.

Brief Description of the Figures Figure 1 shows the structures of picromycin (compound methymycin (compound and the ketolide HMR 3004 (compound 3) and the relationship of several compounds related to picromycin.

Figure 2 shows a restriction site and function map of cosmid pKOS023-27.

Figure 3 shows a restriction site and function map of cosmid pKOS023-26.

Figure 4 has three parts. In Part A, the structures of picromycin and methymycin are shown, as well as the related structures of narbomycin, narbonolide, and methynolide. In the structures, the bolded lines indicate the two or three carbon chains produced by each module (loading and extender) of the narbonolide PKS. Part B shows the organization of the narbonolide PKS genes on the chromosome of Streptomyces venezuelae, WO 99/61599 PCT/US99/11814 -7including the location of the various module encoding sequences (the loading module domains are identified as sKS*, sAT, and sACP), as well as thepicB thioesterase gene and two desosamine biosynthesis genes (picCII and picCIII). Part C shows the engineering of the S. venezuelae host of the invention in which the picAI gene has been deleted. In the Figure, ACP is acyl carrier protein; AT is acyltransferase; DH is dehydratase; ER is enoylreductase; KR is ketoreductase; KS is ketosynthase; and TE is thioesterase.

Figure 5 shows the narbonolide PKS genes encoded by plasmid pKOS039-86, the compounds synthesized by each module of that PKS and the narbonolide (compound 4) and (compound 5) products produced in heterologous host cells transformed with the plasmid. The Figure also shows a hybrid PKS of the invention produced by plasmid pKOS038-18, which encodes a hybrid of DEBS and the narbonolide PKS. The Figure also shows the compound, 3,6-dideoxy-3-oxo-erythronolide B (compound 6), produced in heterologous host cells comprising the plasmid.

Figure 6 shows a restriction site and function map of plasmid pKOS039-104, which contains the desosamine biosynthetic, beta-glucosidase, and desosaminyl transferase genes under transcriptional control of actll-4.

Modes of Carrying out the Invention The present invention provides useful compounds and methods for producing polyketides in recombinant host cells. As used herein, the term recombinant refers to a compound or composition produced by human intervention. The invention provides recombinant DNA compounds encoding all or a portion of the narbonolide PKS. The invention also provides recombinant DNA compounds encoding the enzymes that catalyze the further modification of the ketolides produced by the narbonolide PKS. The invention provides recombinant expression vectors useful in producing the narbonolide PKS and hybrid PKSs composed of a portion of the narbonolide PKS in recombinant host cells. Thus, the invention also provides the narbonolide PKS, hybrid PKSs, and polyketide modification enzymes in recombinant form. The invention provides the polyketides produced by the recombinant PKS and polyketide modification enzymes. In particular, the invention provides methods for producing the polyketides 10-deoxymethynolide, narbonolide, YC17, narbomycin, methymycin, neomethymycin, and picromycin in recombinant host cells.

To appreciate the many and diverse benefits and applications of the invention, the description of the invention below is organized as follows. First, a general description of WO 99/61599 PCTfUS99/11814 -8polyketide biosynthesis and an overview of the synthesis of narbonolide and compounds derived therefrom in Streptomyces venezuelae are provided. This general description and overview are followed by a detailed description of the invention in six sections. In Section I, the recombinant narbonolide PKS provided by the invention is described. In Section II, the recombinant desosamine biosynthesis genes, the desosaminyl transferase gene, and the betaglucosidase gene provided by the invention are described. In Section III, the recombinant picK hydroxylase gene provided by the invention is described. In Section IV, methods for heterologous expression of the narbonolide PKS and narbonolide modification enzymes provided by the invention are described. In Section V, the hybrid PKS genes provided by the invention and the polyketides produced thereby are described. In Section VI, the polyketide compounds provided by the invention and pharmaceutical compositions of those compounds are described. The detailed description is followed by a variety of working examples illustrating the invention.

The narbonolide synthase gene, like other PKS genes, is composed of coding sequences organized in a loading module, a number of extender modules, and a thioesterase domain. As described more fully below, each of these domains and modules is a polypeptide with one or more specific functions. Generally, the loading module is responsible for binding the first building block used to synthesize the polyketide and transferring it to the first extender module. The building blocks used to form complex polyketides are typically acylthioesters, most commonly acetyl, propionyl, malonyl, methylmalonyl, and ethylmalonyl CoA. Other building blocks include amino acid like acylthioesters. PKSs catalyze the biosynthesis of polyketides through repeated, decarboxylative Claisen condensations between the acylthioester building blocks. Each module is responsible for binding a building block, performing one or more functions on that building block, and transferring the resulting compound to the next module. The next module, in turn, is responsible for attaching the next building block and transferring the growing compound to the next module until synthesis is complete. At that point, an enzymatic thioesterase activity cleaves the polyketide from the PKS. See, generally, Figure Such modular organization is characteristic of the modular class of PKS enzymes that synthesize complex polyketides and is well known in the art. The polyketide known as 6deoxyerythronolide B is a classic example of this type of complex polyketide. The genes, known as eryAI, eryAII, and eryAIII (also referred to herein as the DEBS genes, for the proteins, known as DEBS1, DEBS2, and DEBS3, that comprise the 6-dEB synthase), that WO 99/61599 PCT/US99/11814 -9code for the multi-subunit protein known as DEBS that synthesizes 6-dEB, the precursor polyketide to erythromycin, are described in U.S. Patent No. 5,824,513, incorporated herein by reference. Recombinant methods for manipulating modular PKS genes are described in U.S. Patent Nos. 5,672,491; 5,843,718; 5,830,750; and 5,712,146; and in PCT publication Nos. WO 98/49315 and WO 97/02358, each of which is incorporated herein by reference.

The loading module of DEBS consists of two domains, an acyl-transferase (AT) domain and an acyl carrier protein (ACP) domain. Each extender module of DEBS, like those of other modular PKS enzymes, contains a ketosynthase AT, and ACP domains, and zero, one, two, or three domains for enzymatic activities that modify the beta-carbon of the growing polyketide chain. A module can also contain domains for other enzymatic activities, such as, for example, a methyltransferase or dimethyltransferase activity. Finally, the releasing domain contains a thioesterase and, often, a cyclase activity.

The AT domain of the loading module recognizes a particular acyl-CoA (usually acetyl or propionyl but sometimes butyryl) and transfers it as a thiol ester to the ACP of the loading module. Concurrently, the AT on each of the extender modules recognizes a particular extender-CoA (malonyl or alpha-substituted malonyl, methylmalonyl, ethylmalonyl, and carboxylglycolyl) and transfers it to the ACP of that module to form a thioester. Once the PKS is primed with acyl- and malonyl-ACPs, the acyl group of the loading module migrates to form a thiol ester (trans-esterification) at the KS of the first extender module; at this stage, extender module 1 possesses an acyl-KS adjacent to a malonyl (or substituted malonyl) ACP. The acyl group derived from the loading module is then covalently attached to the alpha-carbon of the malonyl group to form a carbon-carbon bond, driven by concomitant decarboxylation, and generating a new acyl-ACP that has a backbone two carbons longer than the loading unit (elongation or extension). The growing polyketide chain is transferred from the ACP to the KS of the next module, and the process continues.

The polyketide chain, growing by two carbons each module, is sequentially passed as covalently bound thiol esters from module to module, in an assembly line-like process. The carbon chain produced by this process alone would possess a ketone at every other carbon atom, producing a polyketone, from which the name polyketide arises. Most commonly, however, additional enzymatic activities modify the beta keto group of each two-carbon unit just after it has been added to the growing polyketide chain, but before it is transferred to the next module. Thus, in addition to the minimal module containing KS, AT, and ACP domains necessary to form the carbon-carbon bond, modules may contain a ketodreductase (KR) that WO 99/61599 PCTIUS99/11814 reduces the keto group to an alcohol. Modules may also contain a KR plus a dehydratase (DH) that dehydrates the alcohol to a double bond. Modules may also contain a KR, a DH, and an enoylreductase (ER) that converts the double bond to a saturated single bond using the beta carbon as a methylene function. As noted above, modules may contain additional enzymatic activities as well.

Once a polyketide chain traverses the final extender module of a PKS, it encounters the releasing domain or thioesterase found at the carboxyl end of most PKSs. Here, the polyketide is cleaved from the enzyme and cyclyzed. The resulting polyketide can be modified further by tailoring enzymes; these enzymes add carbohydrate groups or methyl groups, or make other modifications, oxidation or reduction, on the polyketide core molecule.

While the above description applies generally to modular PKS enzymes, there are a number of variations that exist in nature. For example, some polyketides, such as epothilone, incorporate a building block that is derived from an amino acid. PKS enzymes for such polyketides include an activity that functions as an amino acid ligase or as a non-ribosomal peptide synthetase (NRPS). Another example of a variation, which is actually found more often than the two domain loading module construct found in DEBS, occurs when the loading module of the PKS is not composed of an AT and an ACP but instead utilizes an inactivated KS, an AT, and an ACP. This inactivated KS is in most instances called KS

Q

where the superscript letter is the abbreviation for the amino acid, glutamine, that is present instead of the active site cysteine required for activity. For example, the narbonolide PKS loading module contains a KS

Q

Yet another example of a variation has been mentioned above in the context of modules that include a methyltransferase or dimethyltransferase activity; modules can also include an epimerase activity. These variations will be described further below in specific reference to the narbonolide PKS and the various recombinant and hybrid PKSs provided by the invention.

With this general description of polyketide biosynthesis, one can better appreciate the biosynthesis of narbonolide related polyketides in Streptomyces venezuelae and S. narbonensis. The narbonolide PKS produces two polyketide products, narbonolide and deoxymethynolide. Narbonolide is the polyketide product of all six extender modules of the narbonolide PKS. 10-deoxymethynolide is the polyketide product of only the first five extender modules of the narbonolide PKS. These two polyketides are desosaminylated to yield narbomycin and YC17, respectively. These two glycosylated polyketides are the final WO 99/61599 PCTfUS99/11814 products produced in S. narbonensis. In venezuelae, these products are hydroxylated by the picK gene product to yield picromycin and either methymycin (hydroxylation at the CIO position of YC 17) or neomethymycin (hydroxylation at the C 12 position of YCl17). (See Figure 1) The present invention provides the genes required for the biosynthesis of all of these polyketides in recombinant form.

Section L: The Narbonolide PKS The narbonolide PKS is composed of a loading module, six extender modules, and two thioesterase domains one of which is on a separate protein. Figure 4, part B, shows the organization of the narbonolide PKS genes on the Strep tomyces venezuelae chromosome, as well as the location of the module encoding sequences in those genes, and the various domains within those modules. In the Figure, the loading module is not numbered, and its domains are indicated as sKS*, sAT, and ACP. Also shown in the Figure, part A, are the structures of picromycin and methymycin.

The loading and six extender modules and the thioesterase domain of the narbonolide PKS reside on four proteins, designated PICAI, PICAII, PICA!!!, and PICAIV. PICMI includes the loading module and extender modules 1 and 2 of the PKS. PICA!! includes extender modules 3 and 4. PICA!!! includes extender module 5. PICAIV includes extender module 6 and a thioesterase domain. There is a second thioesterase domain (TEII) on a separate protein, designated PICB. The amino acid sequences of these proteins are shown below.

Amino acid sequence of narbonolide synthase subunit 1, PICA! (SEQ ID NO: 1) 1 MSTVSKSESE EFVSVSNDAG SAHGTAEPVA VVGISCRVPG ARDPREFWEL 61 121 181 241 301 361 421 481 54-1 601 661 721 781 841 901 961

VPADRWNAGD

LGWEALERAG

LSYTLGLRGP

GGLSPDGRAY

MTTPDAQAQE

PLLVGSVKTN

YLPWEPEHDG

VVSAKSAAAL

VVGSGPDDLA

ECEAALS PYV

H-SQGEIAAAY

LSVAAVNGPT

AGLS PQAPRV

SAHPVLTMAL

PTYAFQTERH

VLGYATGGQT

FYDPDRSAPG

IDPSSLTGTR

SMVVDSGQSS

T FDARANGYV

AVLREAHERA

I GHLEGAAG I

QRMVVGVSSF

DAQI ERLAAF

AALAAPEGLV

DWSLEAVVRQ

VAGALSLDDA

ATVVSGDPVQ

PFFSTLEGAW

PGTVTGLATL

WLGEIEALAP

EVDRTFREAG

RSNSRWGGFI

TGVFAGAIWD

SLVAVHLACE

RGEGGGFVVL

GTAPADVRYV

AGLI KAVLAV

GMGGTNAHVV

ASRDRTDGVD

RGVAS GVGRV

APGAPTLERV

ARVVTLRSKS

IEELARACEA

ITEPVLDGGY

RRDNGGQDRL

AGE PAVQPAV

CTSLTGVDLR

EDVDRFDAAF

DYATLKHRQG

SLRRGESELA

KRLSRAVADG

ELHGTGTPVG

RGRALPASLN

LEEAPGVVEG

AGAVDAGAVD

AFVFPGQGTQ

DVVQPVTFAV

IAAHLAGKGG

DGVRARVI PV

WYRNLRHRVG

FGISPREAAE

GAAITPHTVT

LAGGVSLNLV

DPVLAVI RGS

DPIEAAALGA

YETPNPAIPF

ASVVESTVGG

AGAVARVLAG

WAGMGAELLD

MVSLARVWQH

MLSLALSEDA

DYASHSRQVE

FAPAVETLAT

LAAGGQAVTD

MDPQQRLALE

GLHRGI IANR

PDSIIGASKF

AVNNGGAAQG

ALGTGRPAGQ

EELNLRVNTE

SAVGGGVVPW

GRAQFEHRAV

S SAVFAAAMA HGVT PQAVVG

VLERLAGFDG

I IESELAEVL

DEGFTHFVEV

VASLAEAWAN GLAVDWSPLL PSATGHHSDL LRTEAAEPAE LDRDEQLRVI LDKVRAQTAQ NRINAAFGVR MAPSMIFDFP TPEALAEQLL WO 99/61599 WO 9961599PCT/US99/1 1814 -12- 1021 L 1081 E 1141 I 1201 F 1261 C 1321 C 1381 C; 1441 1 1501 V' 1561 C 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501

VVHGEAAAN

F'PQDRGWDV

JETS WEAVED

~VSYTLGLEG

~RGLAGDGRS

;LTAPNGPSQ

~PLRLGSLKS

~AVDWPEKQD

qVVSAKSAAA

;ADDLVQALA

kLSPYVDWSL iIAAAYVAGA kVNGPTATVV

?QAPRVPFFS

ILTMTL PET V

AERYWLENT

rALVDAGAKV

GIKAPLWSVT

PHLVTALSGA

A.ARWMAHHGA

PAETPLTAVV

VSSTLGIPGQ

GVPGMDPELA

RDSATSGQGG

FKDIGFDSLA

TAALPATVGAG

RGWDLDGLYD

EAFERAGIEP

FGLEGPATTV

PDGRSKAFSA

NGPSQQRVIR

GSVKSNIGHT

PAGTG PRRAA

EGSEASEAPA

RLRDVGYTLA

TGQGSQRPGA

YTQCALFALE

QELPAGGAML

SGLGRRTRAL

PEYWVRHVRG

S PAGS PADSP RVDLPTYS F1

RTHPWLADHP?

VGAPAGEPGC

PPQGAEEVPI

GGGSAAAAP

SAGTDAVSLE

DPHATS YGP1

GPADGGAEG\

AAAWGLVRTI

ASVRPETGTI

ELVHELEAL(

DVEHVLRPKM

RRRAAGLPAI

HPVLLPLRLI

DGAAETAAV'

LTAVELRNR]

TASRSTAETI

PDGAGSGAE]

PAGAEPAPVA

EGLYHPD PER

AGIDPTSLRG

PALTVDTACS

KAFAASADGT

QRVI RRALAD NIGH TQAAAG

GGLRRAAVSS

LDAQI ERLAA

DPDGLIRGTA

EAVVRQAPGA

LPLDDAARVV

SGDPVQI EEL

TLEGTWITEP

TGLGTLRREQ

PAALATGDDW

EVLTAGADDD

QGAVSVGRLD

TGEDQIAIRT

EILLLVSRSG

HTAGALDDGI

GNYAPHNAYL

LAALESALGR

SSAQGANPLA

GVELRNRLTR

AGAGAGT DAD

ADPDALGRAY

ASLRGSSTGV

DTACSSSLTA

DADGFGAAEG

QALADARLAP

QAAAGAAGI I

VSSFGISGTN

APGSREASLP

TSRTAFAHRA

GRELYDRHPV

VALFRLVESW

AVQAAEDEIR

RVSHAFHSAH

,TVRFLDGVR'V

,AGALRPRPLL

SRDRYWLDAPP

,VLGSVLLPGP

ESAGDGARPv

DGLYERLDG'

GIHPALLDAE

LTDGEGRPL\

AVLGKDELK\

RGTVARTLEI

QTENPGRFG]

APALAPEGTI

3ADVSVAACDI I DAAFLLDEL'.

SLGWGLWAE'.

SAAGLRDAAGI

r' LADRAATVD(

SNSAGGLALPA

D ALLAQLTRLI D RPWAAGDGA,

AAGAVDEPVA

PGTSYVRQGG

RQVGVFTGAN

SSLVALHLAV

SWSEGVGVLL

ARLTTSDVDV

VSGVIKMVQA

FGI SGTNAHV

FASRDRTDDA

SGVGRVAFVF

PTLERVDVVQ

TLRSKSIAAH

AQACKADGFR

VLDGTYWYRN

GGQERLVTSL

RYRI DWKRLP

REALAARLTA

TPADPDRAML

TGLHARRLAR

EQAPGATQLT

VDTLTAEQVR

DALAARRRAT

DETAITVADI

ERLAAAAPGE

ATGLQLPATL

DDP IATVANS

VREGGFLHDA

FIGLSYQDYA

LHLAVRALRS

VGLLLVERLS

GDI DAVETHG

KMVLAM~RHGT

AHVVLEQAPD

GHLPWVLSAK

AVTAADRDGF

FARALDET CA

GMRFAALLGH

VWLETEERYA

MDGMLDGFRP.

LRDLGVRTCL

VALLRRKRSE

LADTAVDTAGI

AM'VELAAHA-4

SLHSRLADAI

IGLAFGPLFQC

LHAIAVGGLN

rSVERLTLRP\

IAAALESAGVI

"LQAWLADEHI

"LDLADDASS'.

I LLTGGTGGL( I ADREALTAVI r' STPAYDLAA] r' SGMTGELGQ ,q DPAGIPALF 3 PARQRLLLE: k. TLVFDHPSP, E GALVLTGLS 3 GGSEDGAGV

IVGMACRLPG

FIENVAGFDA

THEYGPSLRD

QALRKGEVDM

VERLSDARRN

VEAHGTGTRL

MRHGLLPKTL

VLEEAPVVVE

DAGAVDAGAV

PGQGTQWAGM

PVTFAVMVS L

LAGKGGMLSL

ARI IPVDYAS

LRHRVGFAPA

AEAWVNGLPV

AAEGSERTGL

LTTGDGFTGV

WGLGRV VALE

APLHGRRPTR

AELTASGARV

RAHRAKAVGA

GRSAVSVAWG

DWDRFYLAYS

RTEILLGLVR

VFDHPT PLAL

CRYPGDIRSP

AEFDAEFFGV

ARVPNAPRGV

GECTMALAGG

DARRNGHPVL

TGTSLGDPIE

LPKTLHADEP

AAGEVLGADE

DEQSLRGQAA

LDGLATLAQG

HLDGHLELPI

SVGEIAAAHWj

GRLDVAAVNC

VLETVEFRRE

ELGPDGVLTI

TETVADALGE

GLGTADHPLI

ESAGLRDVRI

AGTAWSCHAI

LNAVWRYEGI

1DEPELVRVPI I TADQAAASRI

VGLYPDLAA]

.AGTRLLLVTI

~RTLPSVLMD

3GLVARHVVG]

SDAIPAEHPL'

VMFSSAAAV

~DLRRMSRAG

P DVVGARTVR F VVGEVAEVL P, ALASHLDAE D APGSEEVLE P DFMNASAEE GVASPEDLWR L AFFGISPREA L GGEGLDGYLL T ALAGGVAVMP

T

GHQVLAVVRG S GDPIEAQALI A HVDEPSDQID W GASVVEPSVG G A-IVLADGRAQ

F

GAELLDSSAVF

ARVWQHHGVT

P

ALNEDAVLER

I

HSRQVEIIES E IETLAVDEGF 'I AWTSLLPATA

E

SGRWLAVTPE I

VSLLDGLVPQ

HPERWAGLVD I DWQPHGTVLI I

TIAACDVADP

SVLDELTRDLI

PWDGGGMAAGI

SGRPQPLVEE

AQAAAVLRM~R

VSLLRSEFLG

EDLWRMLSEG

SPREALAMDP

EGYLLTGSTP

VAMMATPHMF

AVVRGTAVNQ

AQGLQATYGK

S PHVDWANSG VPEVSET VAN

ALHAWLSEPA

GTSAHVHLDT

LDVMFAAEGS

AGVFSLADAA

PEAAVLSGDA

SLTVVSNVTG

MAADGLADTP

AHAHGTGPDW

GAVVSLPDRD

LTLLEPLVLP

GLLATDRPEL

VFADIALPAT

HWSGVTVHAA

I GGLMHRVAWR

SQDVAAGAPA

k. GAVRDPEGSG

GLRDEPQLAL

Z WGVRRLLLVS r AVVHTAGVLS F GGAGQGAYAA I GGISDAEGIA A~ RPSAASASTT G HARGHRIDAE L PRGASDQDGA H LRSLRSMVTG L FGLLDQDPST VAGGGDAI S

AMDPQQRLL

GNTASVMSG

PGMFVE FSR

AVNQDGASN

~TYGQGRDDE

SAGAVELLT

SAVGGGVT P

'EHRAVALGA

'AAAMAECEA

~QAVVGHSQG

~SDFDGLSVA

LAQVLAGLS

~HFVEVSAHP

RPGLPTYAF

)HSAQAAAVL

IAWVQALGDA

~PAQPDAAAL

~GGTGALGSH

IAMRTLLDAI

)LDAFVLFSS

DGVAERLRNH

LPEVRRI IDA S PEDVAADRA

DEETADARRS

GEGITPFPTD

QQRMLLTTSW

SVASGRIAYT

VEFSRQRALA

DGASNGLTAP

ERPAERPLAI

LALVTEPI DW

AGTAGTSEVA

ADLSDADGPA

ARDGTTAFLF

AEAALLDETR

RLVAARGRLM

DAAREAEAYW

LAAGPDDLCD

ADSAAGSPVG

HAWFAGSGAH

GLLLTGRLSL

EHGGVELRVT

PVAPDRAANW

TNATAPATAN

GAAAARVRLA

PYALAS SGEQ

PRTVLAPLPA

ADDGGEDLSH

HDGTIRLARL

RRGTDAPGAD

DGTLPSMTTE

ANATLDALAW

LLDAALRDDR

AGTAGTPGTA

RGFLDLGFDS

GNRNGNENGT

ETGTGTASGA

D (SEQ ID NO:1) WO 99/61599 PCTIUS99/11814 13 Amino acid sequence of narbonolide, synthase subunit 2, PICAII (SEQ ID NO:2) 1 61 1211 181 C 241 3012 361 421 4811 541 601 6611 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301

TSTVNEEKYL

TAGGE DATSE kMDPQQRLLL

;NSGSVASGR

?STEFVEFSRQ

kVNQDGASSG

[YGQGRDGEQ

3AGAVELLTE

PSVGAGLVPW

ILGTUGQDDFA

ECESA-LSRYV

-SQGEIAAAY

US TAAVNGPT kGLSPRTPEV

SAHPVLTMTL

PTYAFQRRHY

EAAPVLAALS

GHPAPFTRGT

ALEHPERWGG

TASPWWQADG

LAGLVAELAD

ADALARVVTA

DALAIGQHRAD

DTAVT TADVD

AEQQRRMQEL

TLVFDYPTPR

LWRLVAGGED

REALAM'DPQQ

YVGTIGNAAS I VMST PTT VE VRG SAVNQDG

ALIATYGQGB

QI DWSAGTVE

VGGVVPWLVE

TGQDDLAAAI

AALAPYVDWS

GEIAAAYVAC

AAVNGPTAT%

SPQTPQVPFF

PVLTMALPEI]

FQTERYWPQI

AVAGTVLLP(

RTFC-LYAHPI

ANGYGYGPL1

FGAGTRLPF)

PAQLAAFSDI

TDLVEAVDR(

VTRDAVAAPJ

GDATVGGTS(

ALPLPAAPA:

ALGMYPDPA:.

WTFAQGASV

SHGKWDALR

GGRF:VEMGK

VTTWDVRRA

GVRRLLLVS

DYLRRATADL

FPQDRG WOVE

EASWEAFEHA

VAYTLGLEGP

RGLAPDGRSK

LTAPNGPSQQ

PLRLGSLKSN

AD4DWPDKGDG

LVSAKTPAAL

QALTAPEGLI

DWSLEAVVRQ

VAGALTLDDA

ATVVSGDPTQ

PFFSTLEGAW

PET VTGLGTL WLHDS PAVQG

GAGADPVQLD

GATLTLVQAL

LIDLPSDADR

TVLVTGAEEP

LGATATVVTC

KATAALHLDR

GPTVTSVAWS

WSSFAPGFTT

VREHLAVVLN

TLAEFLLAEI

AT SGFPQDRG

RLLLETSWEA

MSGRVSYTLG

FSRQRGLAED

ASNGLTAPNG

*DTEQPLRLGS

*LLTEAMDWPP

AKTPAALDAC

AAPEGLVRG'

LEA VVRQAPC ALSLDDAARv

VSGDPTQIQE

STLEGAWITE

VTGLGTLRRI

DLSAAGDIT~c 3TAFVELAFR7

DAPGEAEWTI

-QGVRGVWRR(

k WSGISLYAV(

TLDALHLLEI

3ETPAPATVLI 3GDGLRSTGQ 3 DAALGSALA' L WRLEPGTDG L MGTEGAGVV' P VVFLTAVYA h LGLDDAHIA T DVRDAERVA R. DAFRHVSQA R. RGTDAPGAG

HEARGRLREL

GLYDPN PEAT GI PAATARGT AVTVDTACS S

SFSSTADGTS

RVI RRALADA I GHTQAAAGV GLRRAAVS SF

DAQIGRLAAF

RGT PS DVGRV

APGAPTLERV

ARVVTLRSKS

IQELAQACEA

ITEPVLDGTY

RREQGGQERL

SVQDSWRYRI

VSPLGDRQRL

EDAGVAAPLW

AALDRMTTVL

AAAEAARRLA

DLTDAEAAAR

LLREAAAAGG

PWEGSRVTEG

ARPGTLLADL

HPS PEAVDTG

LGEQAGAGEQ

WDVEGLYDPD

VEDAGIDPTS

LEG PAVTVDT

GRSKAFAAS))

PSQQRVIRRP

LKSNIGHTQP

KQEGGLRRAP

IGRLAAFASC

ASGVGRVAF"%

APTLERVD\

VTLRSKSIGI

LAQACEADG\

PALDGGYWYI

NGGQHRLTT~c 3AGLGAAEHP] k GDQVGCDLVI k HATGVLAAPI 3 DEVFADVAL] 3ATALRVRLAI q TAWDGAAQA'.

I ACPAAGPGG:

AVWGLGRSA,

r ALGSGEPQL 3 LESLTAAPG T ATGPGVTHL.

L RDLADVKPG S SRTLDFESA A DHPGVGYRA R HTGKVVLTM E LVHELEALG

EAKAGEPVAI

GKSYAREAGF

SVGVFTGVM~Y

SLVALHLAVQ

WSEGVGVLLV

RLTT SDVDVV

SGVIKMVQAM

GVSGTNAHVV

ASQGRTDAAD

AFVFPGQGTQ

DVVQPVTFAV

IAAHLAGKGG

DGVRARI IPV

WYRNLRHRVG

VTSLAEAWTN

DWKRLAVADA

AATLGEALAA

CVTHGAVSVG

AGGTGEDQVA

RDGAGHLLLH

LLAGVSDAHP

RPPVLVLFSS

ATGERLRRLG

PEARRALDEQ

RAFRDLGFDS

LPVDGGVDDE

PDASGRTYCR

LQGQQVGVFA

ACSS SLVALH

DGFGPAEGVG

LADARLT TAE

AAGVSGIIK'.

LVSSFGISGTN

GRTDAADPGP

FFPGQGTQWAC

rQPVTFAVMVE

HLAGQGGMLE

7RARIIPVDYI k. NLRHRVGFAI 3LAEAWANGLI SLGAAVALADc

ELTLDAPLVI

~DRTAPVADPI

?AEVAGAEGAI

P AGPDTVSVS) U PGAVVLGGDj P EHVREALHG: D TESPGRFVL:.

P, LRDGALLVPJ.

D AETLAPEPL, A PGDRVMGLL E RLLVHSAAG F RAASGGAGM F DLGEAGPER P SGLDPEGTV A DVSVAACDV VGMACRLPGG V LYEAGEFDAD F HDYATRLTDV P ALRKGEVDMA L ERLSDARRKG H EAHGTGTRLG D RHGVLPKTLH V LEEAPAAEET P PGAVARVLAG G WAGMGAELLD V MVSLAKVWQH H MISLALSEEA TI DYASHSAHVE T FAPAVETLAT D GLTIDWAPVL P SERAGLSGRW L AGGAVDGVLS I

RADHVTSPAQ

VRASGLLARR I TTPSGSEGAE C

LSAVLHLPPT

VAAIWGGAGQ

LRPLAPATAL

QSTTAADDTV

LTAVELRNRL

PVAIVGMACR

AGGFLDEAGE

GTNGPHYEPL

LAVQALRKGE

MLLVERLSDA

VDVVEAHGTG

VQAMRHGVLP

IAHIVLEEAPV

VAR VLAGGRA

MGAELLDVSK

LAKVWQHHGV

LALSEAAVVE

SHSAHVETIE

AVETLATDEG

VDWASLLPTT

3DGCLLTGSLS

PRRGAVRVQL

AWPPPGAEPV

k FGLHPALLDA k~ ADSSGQPVFA

DGLAAALRAG

3LALMQAWLAD L DLAGEARTAG R LARAAAPAAA G PGQVRIAIRA S GAYAPVVVAD G VGMAAVQLAR D VVLNSLAREF I GEMLAEVIAL L LTGGTGALGG A DREALTAVLD

ASPEDLWRL

FGISPREAL

EGIEGYLGT

AGGVTVMST

RI LAVVRGT

PIEAQAVIA

EKPT DQVDW AS EAT PAVE

,RAEFEHRAV

SKEFAAAMA

.GVT PQAVVG

'RQRIENLHG

IESELAEVL

)EGFTHFIEV

~TATGHHPEL

~VVVPEDRSA

~LAWDESAHP

UI4VWGMGRVA

VRASLPAHG

;TSGAAEDSG

lOSE PLAATD 3AYAAGTAFL

'AILDTALGHG

.JSRELGALTG

.CNATGLALPA

UPGGVASPED

EDADFFGIS P

LRNTAEDLEG

CGLALAGGVT

RRNGHRVLAV

TRLGDPIEAQ

KTLHVDRPSD

DEDAPADEPS

QFEHRAVALG

EFAAAMAECE

TPQAVVGHSQ

RLAGFDGLSV

SELADVLAGL

FTHFVEVSAH

TTHPDLPTYA

LRTHPWLADH

SVGASDESGR

DVDGLYERFA

AVQAAGAGGA

ADSLTVLPVb GTE VLS FPDL

ERFTDGRLVL

DATAGDGLTT

DGLAAADGLA

TGLNFRDVLI

ART VARMPEG

HWGVEVHGTA

VDASLRLLGP

FEDGVLRHLP

IVARHVVGEW

SI PAEHPLTA WO 99/61599 WO 9961599PCTIUS99/1 1814 -14- 3361 3421.

3481 3541 3601 3661 3721 VVH TAG VLS D

GAGQGAYAAA

PMDSELTLSL

QRRAAAGGAG

TGFDSLTAVE

OTASAT DRQT ASDDDLFS FT

GTLPSMTAED

NATLDALAWR

LDAAMRRDDP

EADT DLGGRL

LRNRLNAATG

TAALAELDRL

VEHVLRPKVD

RRTAGLPALS

ALVPIALDVA

AAM'TPDDRVA

LRLPATLVFD

EGVLASLAPA

AAFLLDELTS

LGWGLWAETS

ALRAQQRDGM

HLRDLVRTHV

H PTPG ELAG H

AGGRPELAAR

T PGYDLAAFV

GMTGGLSDTD

LAPLLSGLTR

AT VLGHGT PS

LLDELATAAG

LRALAAALGD

MFSSAAAVFG

RSRLARSGAT

GSRVGGAPVN

RVDLERAFRD

GSWAEGTGSG

DGDDATDLDE

DKELGDSDF (SEQ ID NO:2) Amino acid sequence of narbonolide synthase subunit 3, PICAIII (SEQ ID NO:3) 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501

MANNEDKLRD

AGDGDAISEF

MDPQQRLSLT

SGAALGFLSG

NADLFVQFSR

SAVNQDGASN

ATYGQEKSSE

WSAGTVELLT

GVVPWPVSAK

ALRDALRMPE

RYVDWSLEAV

AAYVAGALTL

GPTATVVSGD

PH VP FF5TLE

TLPETVTGLG

RFWLQSSAPT

DVLEAGADDD

QGAVSVGRLD

TGEDQIAIRT

EHLLLVSRSG

HTAGAPGGDP

GVYAAANAHL

DELAKALSHD

VAPTGQSSAL

AVQLRNQLST

PLDRLRDAGV

YLKRVTAELQ

PQDRGWDVEG

TAWEAIESAG

RIAYVLGTDG

QRGLAADGRS

GLTAPHGPSQ

QPLRLGALKS

EAVDWPEKQD

T PAAL DAQI G GLVRGTS SDV

VRQEPGAPTL

DDAARVVTLR

PTQI EELART

GTWITEPVLD

TLRREQGGQE

SAADDWRYRV

REALAARLTA

TPADPDRAML

TGLHARRLAR

EQAPGATQLT

LDVTGPEDIA

DALAARRRAR

ET FVAVADVD

AAITALPEPE

VVGNRLPATT

LDTVLRLTGI

QNTRRLREI S

LYDPDPDASG

IDPTALKGSG

PALTVDTACS

KAFATSADGF

QRVI RRA1LAD

NIGHTQAAAG

GGLRRAAVS S

QLAAYADGRT

GRVAFVFPGQ

DRVDVVQPVT

SKS IAAHLAG

CEADGVRARI

GTYWYRNLRH

RLVTSLAEAW

EWKPLTASGQ

LTTGDGFTGV

WGLGRVVALE

APLHGRRPTR

AELTASGARV

RILGAKTSGA

GETATSVAWG

WERFAPAFTV

RRPALLTLVR

VFDHPTPAAL

EPEPGSGGSD

GRTHEPVAIV

RTYCRSGGFL

LGVFVGGWHT

SSLVALHLAV

GPAEGAGVLL

ARLAPGDVDV

VAGVI KMVQA FGI SGTNAHV

DVDPAVAARA

GTQWAGMGAE

FAVMVSLAKV

KGGMI SLALD I PVDYASHSR

RVGFAPAVET

ANGLT IDWAP

ADLSGRWIVA

VSLLDDLVPQ

HPERWAGLVD

DWQPHGTVL I

TIAACDVADP

EVLDDLLRGT

LWAGDGMGRG

SRPSLLLDGV

THAAAVLGHS

AAHLHEAYLA

GGAADPGAE P

GMACRLPGGV

HDAGEFDADF

GYTSGQTTAV

QALRKGECDM

VERLS DARRN

VEAHGTGTRL

MRHGLLPKTL

VLEEAPAVED

LVDSRTAMEH

LLDSSPEFAA

WQHHGI TPQA EAAVLKRLS D QVEI TEKELA

LAVDGFTHFI

ILPTATGHHP

VGSEPEAELL

VAWVQALGDA

LPAQPDAAAL

TGGTGALGSH

HAM'RTLLDAI

PLDAFVLYS S

ADDAYWQRRG

PEARQALAAP

SPDRVAPGRA

PAEPAPTDWE

EASIDDLDAE

ASPEDLWQLV

FGI SPREALA

QSPELEGHLV

ALAGGVTVMP

GHRILAVVRG

GDPI EAQALI HVDEPSDQI D S PAVE PPAGG

RAVAVGDSRE

SMAECETALS

VVGHSQGEIA

FDGLSVAAVN

EVLAGLAPQA

EVSAHPVLTM

ELPTYAFQTE

GALKAAGAEV

GI KAPLWSVT

AHLVTALSGA

AARWMAHHGA

PAETPLTAVV

NAGVWGSGSQ

I RPMS PDRAL

VGAPAPGDAA

FTELGFDSLT

GRVRRALAEL

ALIRMALGPR

1561 (SEQ ID NO:3) Amino acid sequence of narbonolide synthase subunit 4, PICAIV (SEQ ID NO:4) 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021

MTSSNEQLVD

AAGKDLVSEV

MDPQQRQLLE

NSSAVASGRI

GAFIEFSSQQ

INQDGASNGL

YGQGRAPGQP

AGSVELLTEA

PWPVSAKTSA

DALRMPEGLV

DWSLEAVVRQ

VAGALTLDDA

ATVVSGDPTQ

PFFSTLEGTW

PDKVTGLATL

RSYWI SPAGP CDS PEEVPVD

AERNWAVAEP

ALRASLKENE

PEERGWDIDS

ASWEVFERAG

AYSLGLEGPA

AMAADGRTKG

TAPHGPSQQR

LRLGTLKSNI

VDWPERPGRL

ALDAQI GQLA

RGTVTDPGRV

APSAPTLDRV

ARVVTLRSKS

IQELAQACEA

ITEPALDGGY

RREDGGQHRL

GEAPAHTASG

RPLREI GFDS S DHEQAEEEK

ELRKESRRRA

LYDPVPGRKG

I DPAS VRGT D VT VDTACSS S

FASAADGLAW

LIRQALADAR

GHTQAASGVA

RRAGVSAFGV

AYAEDRTDVD

AFVFPGQGTQ

DVVQPVTFAV

IAAHLAGKGG

DGIRARIIPV

WYRNLRHRVG

TTSLAEAWAN

REAVAETGLA

LTAVDFRNRV

AAAPAGARSG

DRRQEPMAIV

TTYVRNAAFL

VGVYVGCGYQ

LVALHLALKG

GEGVAVLLLE

LTS SDVDVVE GVI KMVQALR

GGTNAHVVLE

PAVAARALVD

WAGMGAELLD

MVSLAKVWQH

MI SLALSEEA

DYASHSAHVE

FAPAVETLAT

GLALDWASLL

WGPGAEDLDE

NRLTGLQLPP

ADTGAGAGMF

GMSCRFAGGI

DDAAG FDAAF

DYAPDIRVAP

LRNGDCSTAL

RLSDARRKGH

GHGTGTRLGD

HGVLPKTLHV

EAPAVEESPA

SRTAMEHRAV

SSPEFAAAMA

HG ITPEAVI G

TRQRIENLHG

TIENELADVL

DEGFTHFIEV

PATGALS PAV

EGRRSAVLAM

TVVFEHPT PV

RALFRQAVED

RSPEDLWDAV

FGISPREALA

EGTGGYVVTG

VGGVAVLATP

RVLAVVRGSA

PT EAQALLAT

DEPTDQVDWS

VEPPAGGGVV

AVGDSREALR

ECETALS PYV

HSQGETAAAY

LSIAAVNGPT

AGLS PQT PQV

SAHPVLTMTL

PDLPTYAFQH

VMRQAASVLR

ALAERI SDEL DRYGE FLDVL WO 99/61599 PCT/US99/11814 1081 AEASAFRPQF ASPEACSERL DPVLLAGGPT DRAEGRAVLV GCTGTAANGG

PH-EFLRLSTS

1141 FQEERDFLAV PLPGYGTGTG TGTALLPADL DTALDAQARA ILRAAGDAPV

VLLGHSGGAL

1201 LAHELAFRLE RAHGAPPAGI VLVDPYPPGH QEP[EVWSRQ LGEGLFAGEL EPMSDARLLA 1261 MGRYARFLAG PRPGRSSAPV LLVRASEPLG DWQEERGDWR AI-WDLPH-TVA

DVPGDHFTMM

1321 RDHAPAVAEA VLSWLDAIEG IEGAGK (SEQ ID NO:4) Amino acid sequence of typeII thioesterase, P1GB (SEQ ID 1 VTDRPLNVDS GLWIRRFHPA PNSAVRLVCL PHAGGSASYF FRFSEELHPS

VEALSVQYPG

61 RQDRRAEPCL ESVEE 'LAEHV VAATEPWWQE GRLAFFGHSL GASVAFETAR

ILEQRHGVRP

121 EGLYVSGRRA PSLAPDRLVH QLDDRAFLAE IRRLSGTDER FLQDDELLRL

VLPALRSDYK

181 AAETYLHRPS AKLTCPVMAL AGDRDPKAPL NEVAEWRRHT SGPFCLRAYS

GGHFYLNDQW

241 HEICNDISDH LLVTRGAPDA RVVQPPTSLI EGAAKRWQNP R (SEQ ID The DNA encoding the above proteins can be isolated in recombinant form from the recombinant cosmid pKOSO23-27 of the invention, which was deposited with the American Type Culture Collection under the terms of the Budapest Treaty on 20 August 1998 and is available under accession number ATCC 203141. Cosmid pKOSO23- 2 7 contains an insert of Streptomyces venezuelae DNA of -38506 nucleotides. The complete sequence of the insert from cosmid pKOS023-27 is shown below. The location of the various ORFs in the insert, as well as the boundaries of the sequences that encode the various domains of the multiple modules of the PKS, are summarized in the Table below. Figure 2 shows a restriction site and function map of pKOSO23-2 7 which contains the complete coding sequence for the four proteins that constitute narbonolide PKS and four additional ORFs. One of these additional ORFs encodes the picB gene product, the type 11 thioesterase mentioned above. P1GB shows a high degree of similarity to other type 11 thioesterases, with an identity of 51%, 49%, and 40% as compared to those of Amycolatopsis mediterranae, griseus, S. fradiae and Saccharopolyspora erythraea, respectively. The three additional ORFs in the cosmid pKOSO23-27 insert DNA sequence, from the picCIl, picCIIl, and picC V, genes, are involved in desosamine biosynthesis and transfer and described in the following section.

From Nucleotide To Nucleotide Description 13725 picA I 13725 narbonolide synthase 1 (PICAI) 148 3141 loading module 148 1434 KS loading module 1780 2802 AT loading module 2869 3141 ACP loading module 3208 7593 extender module 1 3208 4497 KS1 4828 5847

ATI

WO 99/61599 PCT/US99111814 6499 7336 7693 7693 9418 10594 12175 13063 13830 13830 13935 13935 15540 17271 18123 18447 18447 20031 21093 22620 23652 24498 25133 25133 25235 25235 26822 28474 29302 29924 29924 30026 30026 31604 32708 From Nucleotide 33068 33961 33961 34863 34863 36159 36159 37529 37529 -16- 7257 KCR1 7593 ACP1 13332 extender module 2 8974 KS2 10554 AT2 11160 DH2 12960 KR2 13332 ACP2 25049 picA 11 25049 narbonolide synthase 2 (PI 18392 extender module 3 15224 KS3 16562 AT3 18071 KR3 (inactive) 18392 ACP3 24767 extender module 4 19736 KS4 21050 AT4 21626 DH-4 23588 ER4 24423 KR4 24765 ACP4 29821 picARI1 29821 narbonolide synthase 3 (P 29567 extender module 26530 27841 29227 29569 33964 picA N 33964 narbonolide synthase 4 (P 32986 extender module 6 31312 KS6 32635 AT6 32986 ACP6 To Nucleotide Description 33961 PKS thioesterase domain 34806 picB 34806 typell thioesterase homol 36011 picCil 36011 4-keto-6-deoxyglucose is 37439 piCil 37439 desosaminyl transferase 38242 picCVI 38242 3-amino dimethyltransfe CAll)

ICAIII)

ICAIV)

og omerase rase DNA Sequence of the Insert DNA in Cosmid pKOS023-27 (SEQ ID NO: 19) 1 GATCATGCGG AGCACTCCTT CTCTCGTGCT CCTACCGGTG ATGTGCGCGC CGAATTGATT WO 99/61599 WO 9961599PCT/US99/1 1814 -17- 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2342 2401 246] 2521 2581 2641 270: 276: 282: 288: 294: 300: 3061 312 318 324 330 336 342 348 354

CGTGGAGAGA

GACGCCGGTT

GTGCCCGGCG

GTCACCGACG

GCCCCCGGCC

GCCGCCTTCT

GCCCTGGAGC

GGCACCCGCA

CGCCAGGGCG

GCGJAACCGAC

CAGTCCTCGT

GAGCTCGCCC

AGCAAGTTCG

GGCTACGTAC

GCCGACGGCG

GCCCAGGGCA

GAGCIGGGCCG

CCCGTGGGCG

GCCGGACAGC

GCCGGCATCG

AGCCTGAACT

AACACGGAGT

TCC:CGTTCG

GTCGAGGGTG

GTGCCGTGGG

GCCGCGTTCG

GCTGTCGATG

CGGGCCGTCG

GGTCTGGTCC

GGCACGCAGT

GCCATGGCCG

GTACGGCAGG

TTCG'CCGTCA

GTCGTCGGCC

GACGACGCCG

*AAGIGGCGGC1P

*TTCGACGGGC

*CCCGTACAGP

*ATTCCCGTCC

*GAGGTCCTCC

*GGCGCCTGG;

L CGTU'TGGGCI L GTCGAGGTCI

LGCGACCCTGC

LTGGGCCAACC

LTCCGACCTC(

L. CTCGCCCCG( L. CCGGCGGAG( 1 ACGGCCCAGC 1 GAGGCCGGT' 1 GGCGTACGG 1 CAGCTGCTC( 1 CCGC2TGGCGI 1 CTGCCCGGT, 1 GCGATCTCG, 1 CCCGAGCAC 1 TTCGACGCG 1 CGGCTCCTC 1 CTG'CGGGGA

TGTCGACAGT

CCGCGCACGG

CCCGGGACCC

TCCCCGCGGA

GCTCGAACAG

TCGGCATCTC

TGGGCTGGGA

CCGGCGTCTT

GCGCCGCGAT

TCTCGTACAC

CGCTCGTCGC

TCGCCGGCGG

GCGGCCTCTC

GCGGCGAGGG

ACCCGGTGCT

TGACGACCCC

GGACCGCGCC

ACCCGATCGA

CGCTCCTGGT

CCGGCCTCAT

ACGAGACCCC

ACCTGCCGTG

GCATGGGCGG

CTTCGGTCGT

TGGTGTCGGC

CCTCGCGGGA

CGGGTGCTGT

TCGTCGGCAG

GGGGCGTGGC

GGGCCGGCAT

AATGCGAGGC

CCCCCGGTGC

TGGTCTCGC'I

ACTCGCAGGC

CTCGTGTCG I

TGCTGTCCCI

TGTCCGTCGC

TCGAAGAGC']

ACTACGCGT(

CCGGGCTCA(

TCACCGAGC(

TCGCCCCGG(

GCGCCCACC(

-GTCGCGACAj

GACTCGCGG'

CCACCTACG

,CGGGCGAGCI

TCGACCGGG.

3 TGCTGGGGT.

r' GCACCTCCC k. TGGCGCCGT

TCGTCGTGC

3 CGGCCGGTG 3 GGGTCGCCT S AGTTCCCGC C CCGGCACGT G CCTTCTTCG C TCGAAACCT C GGCAGGTCC

GTCCAAGAGT

CACAGCGGAA

GAGAGAGTTC

CGGCTGGAAC

CCGGTGGGGC

GCCCCGCGAG

GGCCCTGGAG

CGCCGGCGCC

CACCCCGCAC

GCTCGGGCTC

CGTCCACCTC

CGTCTCGCTC

CCGCGACGGC

CGGCGGTTTC

CGCCGTGATC

CGACGCGCAG

GGCCGACGTG

GGCCGCTGCG

CGGCTCGGTC

CAAGGCCGTC

GAACCCGGCG

GGAGCCGGAG

CACGAACGCG

GGAGTCGACG

GAAGTCCGCT

TCGTACGGAT

CGCTCGCGTA

CGGGCCGGAC

TTCCGGTGTC

GGGTGCCGAA

CGCACTCTCC

GCCCACGCTG

GGCTCGCGTG

CGAGATCGCC

GACCCTGCGC

CGCGCTGAGC

CGCTGTGAAC

TGCTCGGGCC

CCACAGCCGC

1CCCGCAGGC'I

CGTGCTCGAC

2CGTCGAGACC 2CGTCCTCACC k. CGGCGGTCA( r CGACTGGAGC 7, GTTCCAGAC( C GGCGGTGCA( N. CGAGCAGCT( A CGCGACAGG( T GACCGGCGT( C CATGATCTTI A CGGGGAGGC' C CGTCGACGA' C GCCGGAGGA A GGACCGCGG C GTACGTCCG G GATCTCGCC 'C CTGGGAGGC ;G CGTCTTCAC

GAGTCCGAGG

CCCGTCGCCG

TGGGAACTCC

GCCGGCGACT

GGGTTCATCG

GCCGCGGAGA

CGCGCCGGGA

ATCTGGGACG

ACCGTCACCG

CGCGGCCCCA

GCGTGCGAGA

AACCTGGTGC

CGCGCCTACA

GTCGTCCTGA

CGGGGCAGCG

GCGCAGGAGG

CGGTACGTCG

CTCGGCGCCG

AAGACGAACA

CTGGCGGTCC

ATCCCGTTCG

CACGACGGGC

CATGTCGTGC

GTCGGCGGGT

GCCGCGCTGG

GGTGTCGACG

CTGGCCGGCG

GATCTGGCGG

GGGCGAGTGG

CTGCTGGACT

CCGTACGTCG

GAGCGGGTCG

TGGCAGCACC

GCGGCGTACC

AGCAAGTCCT

GAGGACGCCC

GGGCCCACCC

TGTGAGGCCC

CAGGTCGAGY

CCGCGCGTGC

GGCGGCTAC'

CTGGCCACC(

ATGGCCCTC(

3 GACCGCCTC( 2CCGCTCCTC( 2GAGCGCCACr 3 CCCGCCGTC( 3 CGCGTGATCI 2 GGGCAGATC, 3 GACCTGCGC.

Z GACTTCCCC.

S GCGGCGAAC G CCGGTGGCG C CTGTGGCGG C TGGGACGTG C CAGGGCGGT G CGCGAGGCC C GTCGAGGAC T GGGGCGATC

AATTCGTGTC

TCGTCGGCAT

TGGCGGCAGG

TCTACGACCC

AGGACGTCGA

TGGACCCGCA

TCGACCCGTC

ACTACGCCAC

GCCTCCACCG

GCATGGTCGT

GCCTGCGGCG

CGGACAGCAT

CCTTCGACGC

AG CGCCT CT C

CCGTCAACAA

CCGTGCTCCG

AGCTGCACGG

CCCTCGGCAC

TCGGCCACCT

GCGGTCGCGC

AGGAACTGAA

AGCGGATGGT

TCGAAGAGGC

CGGCGGTCGG

ACGCGCAGAT

CGGGCGCTGT

GGCGTGCTCA

CAGCGCTGGC

CGTTCGTGTI

CTTCCGCGG'I

ACTGGTCGC'I

ATGTCGTGC;

ACGGGGTGAC

TCGCCGGTGC

TCGCCGCCCI

TCCTGGAGCC

CCACCGTGG'

ATGGGGTCCC

k TCATCGAGA( 2CGTTCTTCT( C' GGTACCGCAj 3ACGAGGGCT'

CCGGGACCG'

3 TCGCCTCCC' 2 CCTCCGCGA, r GGCTGGGCG.

2: TCCGCACGG.

Z TGGACAAGG G AGGTCGACC h ACCGGATCA A CCCCCGAGG C CGGCCGGTG A TCGTCGGCA C TGGTGGCCG G AGGGGCTGI T TCATCGAGP C TCGCCATGG :G CCGGGATCC A CCCACGAGI

CGTGTCGAAC

CTCCTGCCGG

CGGCCAGGCC

GGACCGCTCC

CCGGTTCGAC

GCAGCGGCTC

CTCGCTCACC

CCTGAAGCAC

CGGCATCATC

CGACTCCGGC

CGGCGAGTCC

CATCGGGGCG

GCGCGCCAAC

CCGGGCCGTC

CGGCGGCGCC

CGAGGCCCAC

CACCGGCACC

CGGCCGCCCG

GGAGGGCGCG

GCTGCCCGCC

CCTCCGGGTG

CGTCGGCGTG

CCCGGGGGTT

CGGCGGTGTG

CGAGCGGCTT

CGATGCGGGT

GTTCGAGCAC

CGCGCCTGAG

CCCCGGGCAG

GTTCGCGGCG

GGAGGCCGTC

GCCTGTGACG

GCCCCAGGCG

CCTGAGCCTG

SCCTCGCCGGC

IACTGGCCGGG

r CTCCGGTGAC 3 TGCGCGGGTC 3 CGAGCTCGCC 2GACACTCGAA k. CCTGCGCCAT r CACCCACTTC r CACCGGTCTG r' CGCCGAAGCA C CGGCCACCAC A GATCGAGGCG A GGCGGCCGAG T CCGGGCGCAG G GACCTTCCGT A CGCCGCCTTC C TCTCGCGGAG C GGAGCCGGCT *T GGCCTGCCGC G CGGCGGGGAC 'A CCACCCGGAT A CGTCGCCGGC ;A CCCGCAGCAG ;A CCCGACCTCC 'A CGGGCCGAGC WO 99/61599 WO 9961599PCTIUS99/1 1814 18- 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5882 5942 6002 606] 6121 618, 624: 630: 636: 64 2: 648:.

654:.

660: 666: 672: 678 684 690 696 702 708

CTGCGGGAG

ATGTCGGGCC

GCCTGCTCGT

GTCGACATGG

TTCAGCCGGC

GACGGCACCA

CGCCGCAACG

GCGAGCAACG

CTGGCGGACG

ACGCGACTCG

GACGACGAAC

GCGGCCGGCG

AAGACGCTGC

CTCCTCACCG

GTCTCCTCCT

GTTGTCGAGG

GTGACGCCTT

CTTGCCGCAT

GGCGCTGTCG

CTCGGCGCCG

GGAADCGGCTT

GCTGGCATGG

TGTGAGGCCG

CCCGGTGCGC

GTCTCGCTGG

TCGCAGGGCG

CGCGTCGTCA

CTGTCCCTCG

TCCGTCGCCG

GAAGAGCTTG

TACGCGTCCC

GGTCTCAGCC

ACCGAGCCCG

GCCCCCGCCP

*GCCCACCCCC

*CGCGAACAGG

*CTTCCCGTGE

*TACGCCTTCC

*GACGACTGGC

*ACCGGCCTGI

-GCCGTGCTC

2

-GACGACGACC

L ACCGGCGTGC

LGGCGACGCGC

LCGTCTCGACZ

L GCCCTTGAGC

LGCCGCCCTC(

LATCCGCACC)

L CCCACCCGC( I. GGCAGCCAC( I CGCAGCGGC( I GCCCGCGTCI 1 GACGCCATC( 1 GACGGCATCI 1 GTCGGCGCC 1 TTCTCGTCC, 1 GCCTACCTC 1 GCCTGGGGA 1 CGCAACCAC

GCGGGGAAGG

GCGTCTCGTA

CGTCGCTGGT

CGCTCGCCGG

AGCGCGGGCT

GCTGGTCCGA

GACACCAGGT

GCCTCACGGC

CCCGGCTGAC

GCGACCCGAT

AGCCGCTGCG

TCTCGGGTGT

ACGTCGACGA

AGGCCGTCGA

TCGGGATCAG

GTGCTTCGGT

GGGTGGTGTC

TCGCCTCGCG

CTCACGTACT

GGGCGGACGA

CCGGTGTCGG

GTGCCGAACT

CGCTGTCCCC

CCACGCTGGA

CTCGCGTGTG

AGATCGCCGC

CCCTGCGCAG

CGCTGAACGA

CCGTCAACGG

CTCAGGCGTG

ACAGCCGGCP

CGCAGGCCCC

TCCTCGACGC

TCGAGACCCI

TCCTCACCAI

GAGGCCAAGT

CATGGACTTC

AGGCCGAGCC

GCTACCGCAI

CCGGCCGCTC

CCGCGCTG'

GTGAGGCCC

TCTCGCTCC'

GAATCAAGG(

k. CCCCCGCCG2

-ACCCCGAAC(

3CCCACCTCG'

CCGGACTCM

3ACTGGCAGCI 3CCGCACGCTI 3AACAAGCCC, k. CCATCGCCG,

:CCGCCGAGA

3 TGGACACGC T CGGTGCTCG G TGTCGAGCA G ACGCCCTCG C CGTGGGACG G GCGTGCCCG CCTCGACGGC T.

CACACTCGGC C CGCCCTGCAC C CGGCGTGGCC G GGCCGGGGAC G GGGCGTCGGC G CCTCGCGGTC G TCCGAACGGG C GACCTCCGAC G CGAGGCGCAG G CCTCGGGTCG T CATCAAGATG G GCCCTCGGAC C

CTGGCCGGAG

CGGCACCAAT G CGTCGAGCCG TI GGCGAAGTCC G GGATCGTACG G GGCTGACGGG C CCTCGTACAG C GCGAGTGGCC 9]

GCTGGACTCTI

GTACGTCGACI

GCGGGTCGAT

GCAGCACCAC

CGCGTACGTC

CAAGTCCATC

GGACGCCGTC

GCCCACCGCC

CAAGGCGGAC

GGTCGAGATC

GCGCGTGCCG

CACCTACTGG

GGCCGTCGAC

GACCCTCCCC

GCGTCTGGTC

GCTCCTGCCC

CTACTGGCTC

CGACTGGAAG

GCTCGCCGTC

CGACGCCGGG

r CGCCGCCCGG C' CGACGGACTC

GCCCCTGTGG

~CCCCGACCGG

3CTGGGCCGGC r CACCGCACTC k. CGCCCGCCGC

CCACGGCACC

3 GATGGCCCAC

CCGGAGCCACC

C *CTGCGACGTC C GCCCCTCACC T GACCGCCGAG A CGAGCTGACC C TCTGGGCATC C GGCTCGCCGC G TGGCGGCATG G CATGGACCCG ACCTGCTGA C TTGAGGGCC C TCGCCGTGC A TGATGCCCA C GCCGGTCGA A TCCTCCTCG T TCCGCGGCA G CCTCGCAGC A TGGACGTCG TI CCCTGATCG C

TGAAGTCCA

TCCAGGCGA TI :AGATCGACT C AGCAGGACG C ;CGCATGTGG 'I 'CGGTTGGCG C ;CTGCCGCGC9

;ATGACGCCG

]GTGCTCAGT

;CGCTGGCCG

TCGTGTTCC

CCGCGGTGT

'GGTCGCTGG

;TCGTGCAGC

3GTGTGACGC

,CCGGAGCCC

GCCGCCCACC

CTGGAGCGAC

kiCTGTCGTGT

GGATTCCGCG

FTCGAGAGCG

rTCTTCTCGA 1ACCGCAACC

GAGGGCTTCA

GAGACCGTCA

ACCTCGCTCG

GCCACGGCCT

GAGAACACTC

CGCCTCCCGG

ACGCCGGAGG

GCGAAGGTCG

CTCACCGCAC

GTACCGCAGG

TCCGTCACCC

GCCATGCTCT

CTCGTCGACC

TCCGGCGCCA

CTCGCCCGCG

GTCCTCATCA

CACGGAGCCG

CAACTCACCG

GCCGACCCCC

GCCGTCGTCC

CAGGTCCGGC

CGGGACCTCG

CCCGGTCAGG

CGGGCCACCG

GCCGCCGGTG

GAACTCGCCC

CGGCAACAC G CGCCCTGAC G *GGCCCTGCG C GCCCGGGAT G *GGCGTTCGC C 'CGAGCGCCT G CGCCGTGAA C LGCGCGTCAT C 'CGAGGCACA C CACCTACGG C LCATCGGGCA C ~GCGCCACGG A ;GTCGGCTGG C ;CGGGCTGCG C ~GCTCGAAGA G ;GTCGGCGGT C ~CGACGCGCA C kCGCCGGTGC TI rCGAGCACCG C k.TCCGGACGG C 'CGGTCAGGG C ICGCGGCGGC %GGCCGTCGT I

CTGTGACGTT

CCCAGGCGGT

rGCCCCTGGA rCGCCGGCAA

TGAGTGACTT

CGGGTGACCC

CGCGGATCAT

AGCTCGCCCA

CGCTCGAAGG

TCCGTCACCG

CGCACTTCGT

CCGGCCTCGG

CCGAGGCGTG

CCCGCCCCGG

CCGCCGCCCT

CCGCCGAGGG

ACCACTCCGC

AGGTGCTGAC

TGACGACCGG

TCGCCTGGGT

AGGGCGCGGT

GGGGCCTCGG

TCCCCGCCCA

CCGGCGAGGA

CACCCCTCCA

CCGGCGGCAC

AACACCTCCT

CCGAACTCAC

ACGCCATGCG

ACACCGCCGG

GGGCCCACCG

ACCTCGACGC

GCAACTACGC

GCCGGTCCGC

ACGGCGTGGC

TGGCCGCACT

GCCAGCGTG

GTGGACACG

AAGGGCGAG

TTCGTCGAG

GCGTCGGCG

TCGGACGCC

CAGGACGGC

CGGCGCGCG

GGCACGGGC

:CAGGGCCGT

ACCCAGGCC

~CTGCTGCCG

GCCGTGGAA

:CGGGCCGCC

;GCCCCGGTG

GGCGGCGGT

ATCGAGCGG

GTCGACGCG

;GCCGTCGCG

;CTGATACGC

ACGCAGTGG

ATGGCCGAG

~CGGCAGGCC

'GCCGTCATG

:GTCGGCCAC

:GACGCCGCC

3GGCGGCATG

CGACGGGCTG

CGTACAGATC

rCCCGTCGAC

GGTCCTCGCC

CACCTGGATC

CGTCGGCTTC

CGAGGTCAGC

CACCCTCCGT

GGTCAACGGG

TCTGCCCACC

GGCCACCGGC

GTCCGAGCGC

GCAGGCCCC

GGCCGGGGCG

TGACGGCTTC

CCAGGCGCTC

CTCCGTCGGA

CCGCGTCGTC

GCCCGATGCC

CCAGATCGCC

CGGACGTCGG

CGGAGCCCTC

CCTCGTCAGC

CGCATCGGGC

CACCCTCCTC

CGCGCTCGAC

TGCGAAGGCC

GTTCGTGCTC

CCCGCACAAC

CGTCTCGGTG

CGAGCGGCTG

GGAGTCCGCG

WO 99/61599 WO 9961599PCTIUS99/1 1814 -19- 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 8101 8161 8221 8281 8341 8401 8461 8521 8581 8641 8701 8761 8821 8881 8941 9001 9061 9121 9181 9241 9301 9361 9421 9481 9541 9601 9661 9721 9782 9842 9902 996] 1002] 1008] 1014] 1020' 1026' 1032' 1038: 1044:.

1050: 1056' 10621

CTCGGCCGGG

GCGTACTCCT

ATCGACGCAC

CCCCTGGCCG

CTCGTACGGG

GACCGCGCCT

CTGACCCGGG

CTGGCCCTCG

CGGCGGTCCG

GATGCCGACG

CGCAGCCCGG

CCCACCGACC

AGGGCGTACG

TTCGGCGTCT

ACGTCCTGGG

ACCGGTGTCT

CGTGGCGTGG

GCGTACACCT

CTGACCGCCC

GCCGGTGGCG

GCGCTCGCCC

GCGGAGGGCG

GCGGTGCTCG

ACCGCGCCCA

CTGGCACCCG

CCCATCGAGG

CTCGCCATCG

GGCATCATCA

GACGAGCCGA

ATCGACTGGC

GGGACGAACG

GCCGATGAGG

GAGGTCGCTC

TCCCTCCCCC

CAGGCCGCCC

GGACCGGCCC

CACCGCGCCC

GCCCAGGGCC

TTCCTCTTC;

CACCCCGTC]

CTGCCCCTGC

*GAGACGCGG]

*GAGAGCTGGC

*GCGCACGTC(

*CGGCTCATG(

*GAGATCCGC(

*GTCAACGGC(

*GCGTACTGG'

L TCCGCGCACJ

LCGGCGCCCC'

LCTGTGCGAC,

LGTCCGTGTC,

L CTCACCGCC.

L CCCGTCGGC L. CCGCTGCTC 1 CTCGGCAGG 1 GGGGCGCAC 1 GCCCCGGCG I CCGCTGCTC

ACGAGACCGC

CCGGTCGCCC

GGGACAGCGC

AGCGGCTGGC

CGCAGGCCGC

TCAAGGACAT

CGACCGGGCT

TGTCGCTGCT

CGGCGCTGCC

ACGATCCGAT

AGGACCTGTG

GCGGCTGGGA

TCCGCGAGGG

CGCCGCGCGA

AGGCCTTCGA

TCATCGGCCT

AGGGTTACCT

TCGGTCTCGA

TGCACCTGGC

TGGCGATGAT

CGGACGGCCG

TCGGCCTGCT

CCGTGGTCCG

ACGGACCCTC

GCGACATCGA

CCCAGGGCCT

GCTCCGTGAA

AGATGGTCCT

GCCCGCACGT

CGGCCGGCAC

CGCACGTCGT

TGCCTGAGGT

AGGGCTCTGA

GGCACCTGCC

CCCTGCACGC

*GCCTGCGGGP

CCGTGACCGC

GCACCTCGGC

SCCGGCCAGGC

TCGCCCGGGC

-TCGACGTGAI

ACACGCAGTC

,GCATGCGGCC

3 CCGGTGTGT]

AGGAGCTGCC

3 TGTGGCTGG)

CCGAGGCCGC

r CCGGGCTCG( k. TGGACGGCA' r CCCTGACCG'

ZCCGAGTACT(

C TGCGCGACC' A. TGGCGGCCG, T CTCCCGCCG G TGGCGCTGC G CGCACGCCC C GCGTGGACC G CCGACACCG G GCGCCGTGG

GATCACCGTC

GCAGCCCCTC

CACGTCCGGA

CGCGCGGCT

CGCCGTGCTC

CGGCTTCGAC

CCAGCTGCCC

CCGCAGCGAG

CGCGACTGTC

CGCGATCGTC

GCGGATGCTG

CCTCGACGGC

CGGGTTCCTG

GGCGCTGGCC

GCGGGCCGGC

CTCCTACCAG

GCTGACCGGC

AGGGCCCGCG

GGTGCGGGCG

GGCGACCCCG

CAGCAAGGCC

GCTCGTGGAG

CGGTACCGCC

GCAGCAGCGG

CGCCGTCGAG

CCAGGCCACG

GTCCAACATC

CGCGATGCC

CGACTGGGCG

CGGTCCGCGC

GCTGGAGCAG

GTCTGAGACG

GGCCTCCGACG

CTGGGTGCTC

GTGGCTGTCC

CGTCGGGTAC

CGCCGACCGC

CCACGTCCAC

CAGTCAGCGC

GCTCGACGAC

'GTTCGCGGCC

3CGCGCTGTTC

GGCCGCACTC

C' CTCGCTCGC(

CGCCGGTGG(

k GACGGAGGA(

:CGTCCTGTC(

3 CCGCAGGAC( r GCTCGACGG( r GGTCTCGAA( 3 GGTCCGGCAI r CGGCGTGCG' P, GGGCCTCGC, G CTCTCCCGC T GCGCCGCAA A CGGCACCGG T GCCCACGTA C GGTGGACAC T CAGCCTTCC

GCGGACATCG

GTCGAGGAGC

CAGGGCGGGA

CCCGGCGAC

CGGATGCGTT

TCGCTCGCCG

GCGACGCTCG

TTCCTCGGTG

GGTGCCGGTG

GCGATGAGCT

TCCGAGGGCG

CTGTACGACG

CACGACGCGG

ATGGACCCGC

ATCGAGCCGG

GACTACGCGG

AGCACGCCGA

ACGACCGTCG

CTGCGCAGCG

CACATGTTCG

TTCTCGGCGG

CGGCTCTCGG

GTCAACCAGG

GTGATCCGGC

ACGCACGGCA

TACGGCAAGG

GGACACACCC

CACGGCACCC

AACAGCGGCC

CGCGCCGCCC

GCGCCGGATC

GTAGCGATGC

GCCCCCGCGC

TCCGCCAAGC

GAGCCCGCCC

ACGCTCGCCI

GACGGGTTCC

CTGGACACCC

CCCGGCGCC(

ATCTGCGCCC

GAGGGCAGCC

GCCCTGGAG(

3CTCGGTCAC'

GACGCCGCC(

GCGATGCTCI

3 CGGTACGCGI

GGCGACGCG,

CGCGCGCTG,

3 TTCCGCGCC

:GTCACCGGC

ZGTCCGCGGC

3 ACCTGCCTG G GACACCCCC C GACTCCGCC G CGGTCGGAG A CCCGACTGG C TCCTTCCGG C GCCGGCCTC G GACCGGGAC ACTCGGACCG C TGCCCGAGGT G GCTCCGCCCA G GTACGGAGAT C CGCCGGAGGA C GTGTCGAGCT G TCTTCGACCA C ACCAGGAGAC G CCGGCGCCGG C GCCGCTACCC C CCGAGGGCAT C CCGACCCGGA C CCGAGTTCGA C AGCAGCGGAT G CATCGCTGCG C CCCGCGTCCC G GCGTCGCGTC G ACACCGCCTG C GCGAGTGCAC C TGGAGTTCAG C ACGCCGACGG C

ACGCCGCGC

ACGGCGCCAG(

AGGCGCTCGC

CGGAACCTC

AGCGGCCCGC

IAGGCCGCGGC

TGCCGAAGAC

TGGCCCTCGT

TCTCCTCCTT

CTGCTGGTGA

CTGGGACGGC

CCCCCGGCAG

ACGAGCAGTC

CCGACCTGTC

k CGAGCCGTAC

TGGACGGGCT

]CCCGGGACGG

GCCGTGAGCT

ACCTCGACGG

3 CGGAGGCCGC 3 TCGCGCTCTT r' CGGTCGCGA

:GCCTGGTCGC

3 CCGTCCAGCC 3 CACGTCTGGA G ACI3CGGCGCG C GGGTCAGCCA G TCCTGGAGAC C TGGCCGCCGG A CCCTCCGCTT

G-AGCTGGGCCC

G CGGATTCCC G CCGGCGCGCT A CCGAGACCGT C ACGCCTGGTT C GCCACCGCTA IC GTCTCGGCAC CG GCCTGCTGCT

TTCTACCTC

CGGCGCATC

GGCGCCAAC

CTCCTCGGT

GTCGCCGCC

CGCAACAGG

CCGACGCtG

GCGGACGCC

GCCGGCACC

GGTGACATC

.ACGCCGTTC

IGCGCTCGGC

:GCGGAGTTC

;CTCCTGACG

:GGCAGCAGC

;AACGCCCCG

;GGCCCTATC

TCGTCGTCG

;ATGGCGCTC

~CGTCAGCGG

TTCGGCGCC

~AACGGTCAC

AACGGGCTG

,GACGCCCGG

3CTGGGCCAC 3GAACGGCCG

~CGTGCGGCG

CCTCCACGCC

CACCGAGCCG

CGGCATCAC

GGTGCTTGGG

TGGGACCTCC

CCGTGAGGCG

GCTGCGCGGC

GGACGCGGAC

CGCCTTCGCG

GGCCACGCTG

CACCACCGCG

GTACGACCGG

TCACCTCGAA

GCTGCTCGAC

CCGGCTCGTC

GATCGCCCC

CGCGCGCGGC

CGCGGAGGAC

CGTCGCCCC

GGAGGCGGAG

CGCCTTCCAC

GGTGGAGTTC

CCCGGACGAC

CCTCGACGGC

CGACCGCTC

TGCCGGCTCC

CCGGCCCCGG

CGCGGACGCC

CGCCGGCTCC

CTGGCTGGAC

CGCCGACCAC

CACCGCCCGC

WO 99/61599 WO 9961599PCT/US99/1 1814 10681 10741 10801 10861 10921 10981 11041 11101 11161 11221 11281 11341 11401 11461 11521 11581 11641 11701 11761 11821 11881 11941 12001 12061 12121 12181 12241 12301 12361 12421 12481 12541 12601 12661 12721 12781 12841 12901 12961 13021 13081 13141 13201 13261 13321 13381 13441 13501 13561 13621 13681 13741 13801 13861 13921 13981 14041 14101 14161 CTCTCCCTGC C CCCGGCGCCG C GTGCGGGAGC I] CGCGTGACGG I] CGGCCCGTCT C CACGCGACCG C

GCCATGTGGCC

GACGGGAACG C

GAGGGTGAGG

ACCGCGAACG

GACGCTTCGC

GTCCCCTTCC2

CGTCTCGCCT

CCGCTGGTCT

AGCCGCGTCG

GGCGAACAGG2

CTGAAGGTCG

GCCGCGCTGT

CTGCCCGCGG

CTGGAGCTGC

GTCACCCGCG

CTGTCGCACG

TTCGGCCTTC

TCCGACGGG

GCCCGCCTGG

GGCACGGTCC

GTGGGCGAGT

GGCGCCGACG

TGCGACGTCG

CCGCTCACCG

ACGACGGAGG

GAACTCACCT

GCCGTCTTCG

CTCGCCTGGC

GCCGAGACCA

GCGGGCATCG

GACGACCGCC

GCCGGGAACG

GTCCGGGCCC

GGGACGGCGG

GTGGACGGGC

GTACTCGGCC

TTCGACTCCC

CT CC CGGCGA

GCCGAGCTGC

AACGGGACGA

CGCCTGGAAG

CTGGAGCACC

TCCGGAGCCC

GGAGCCGGGG

GAGGAACTCT

CGCCTCCCGC

GGGCGCCTCC

ACTACCTGCG

AGGCGAAGGC

TCGCCTCGCC

TCCCCCAGGA

GCAAGAGTTA

TCTTCGGGAT

;CACCCACCC

~GATGGTCGA

~GACCCTCCT

"CGGGGCGCC

CCTCCACTC

;TCTGCTGGC

~GCCGCAGGG

CCTCGCCTT

rCTTCGCCGA

;CGGCGGGAG

[GCACGCCAT

k~CTGGAGCGG

'CGCGGGGAC

,CGTGGAACG

3CGGGCTGAT

%CCCGCACGC

CCGCCGCCCT

CCCAGGACGT

GTCCCGCCGA

TCCAGGCCTG

GTGCGGTGCG

CGGCCGCCTG

1CGACCTGGC

GCCTGCGCGA

CCTCCGTCCG

TGCTGACCGG

GGGGCGTACG

AGCTCGTGCA

CCGACCGCGA

CGGTCGTCCA

ACGTGGAACA

CGACGCCCGC

GTGGCGCGGG

GCCGCCGGGC

GCGGCATGAC

GCGGGATCAG

ACCCGGTCCT

ACCCGGCCGG

GGCCGTCCGC

ACGGCGCGGC

CCGCACGGCA

ACGCCCGCGG

TGACCGCCGT

CCCTGGTCTT

CGCGCGGCGC

CGGCGTCCCC

GCGCCTTGGI

TGCGGTCCCI

CGGACGGCGC

GCGGGAGTGI

TCGGCCTCCI

CCCGGACCCC

AGGAACTCAI

TCGTGCCACC

GGGCGAGCCC

CGAGGACCTC

CCGCGGCTGC

CGCCCGCGAC

CTCGCCGCG(

GTGGCTCGCG

ACTCGCCGCG

TGAACCGCTG

GGCCGGAGAG

GCGGCTCGCC

CACCGACCGG

CGCCGAGGAG

CGGTCCGCTG

CATCGCGCTC

TGCGGCGGCG

CGCGGTCGGC

TGTCACCGTG

GGACGCCGTC

GCTCACGCTG

GCACCGGGTG

CACTTCGTAC

GGAGTCCGCG

GGCGGCCGGC

CGGCGGCGCG

GCTGGCCGAC

GGACCCCGAG

GGGTCTCGTA

CGACGACGCC

CGAACCGCAG

GCCCGAGACC

CGGCACCGGC

ACGCCTGCTG

CGAGCTGGAG

AGCCCTCACC

CACGGCAGGC

CGTACTGCGG

ATACGACCTG

GCAGGGCGCC

AGCCGGACTC

CGGCGAGCTC

CGACGCCGAG

GCTGCCCCTG

AATCCCGGCG

GGCCTCCGCC

GGAAACGGCC

GCGCCTGCTC

TCACCGGATC

CGAACTCCGC

CGACCACCCI

CTCGGACCAC

GAGCACCGCC

GCTGACGGGC

GCGCTCGATC

CGGGTCCGGC

GGACGGCGCC

CGACCAGGA(

GTCCCGGGC)

GGGGACAGCC

GCGGACCTCC

3GTGGCGATC(

'TGGCGGCTG(

GACGTGGAG(

.7 GCCGGATTC(

GAGGCCCTC(

GACCACGCCG

CACGCTGCGG

GTACTGCCCG

CCCGGTGGCG

GACGCGCCCG

CCCGAGCTTC

GTGCCGCTCG

TTCCAGGGGC

CCCGCCACCA

GCCCCCTACG

GGTCTCGTCG

CACGCGGCCG

TCGCTGTCCC

CGCCCGGTCA

GCCTGGCGTC

GGGCCGACCG

GGCGTCGAAG

GCCCCGGCGC

GAGGGTGTAC

GAGCACCTCG

GGGTCCGGCG

CGGACCGCGC

TCGTCGTACC

CTCGCCCTGC

GGCACCGCCG

GGCCTGGGCG

CTGGTGAGCC

GCCCTGGGAG

GCCGTACTCG

GTCCTCTCCG

CCCAAGGTCG

GCAGCGTTCG

TACGCCGCCG

CCCGCCCTCT

GGCCAGGCGG

GGCATCGCGC

CGGCTCGACC

CTCTTCCGGC

TCGACGACAC

GCGGTCACGC

CTCGAGTTCC

GACGCCGAAC

AACCGGCTCI

AGCCCGGCGC

GACGGAGCCC

GAGACGGACC

CTCTCGGACC

GTCACGGGCC

GCCGAGGAC(

3GGAGTGCCG(

CCCAGCACGC

k CCTCGACTC( 3TGTCCACGG'

ACGAGGCCC(

3 TCGGCATGG( 3 TGGCCGGCG( 3 CCCTGTACGJ

TGTACGAGG,

3 CCATGGACC TCCTGGGGAG C AGTCCGCCGG T AGCACGGTGG C AGTCGGCCGG G CCGGTACCGC C CCGTCGCGCC C ACGGTCTCTA C TGAACGCGGT G CGAATGCGAC C GCATCCACCC C ACGAGCCCGA G GTGCCGCGGC C TGACGGACGG C CCGCCGATCA C CGTACGCCCT C CCGTCCTCGG C TCGGGCTCTA CCCGTACCGT C

GGGGCACGGT

CGGGCACCCG

CCGACGATGG

AGACCGAGAA

GGACCCTGCC

ACGACGGCAC

CACCGGCGCT

GACTGGTCGC

GGCGGGGCAC

CCGACGTCTC

ACGCCATCCC

ACGGCACCCT

ACGCCGCGTT

TCATGTTCTC

CCAACGCCAC

CCCTCGGCTG

ACCTGCGCCG

TCCTCGACGC

CCGCCGGGCT

ACGTCGTCGG

CCGGGACGGC

TCGCCGACCG

TCGTCGGCGA

GGGGCTTCCT

ACTCCGCCGG

CACTCGCCTC

GGAACCGGAA

CGCTGCTGGC

CCCCCGGGAG

3AGACCGGGAC

'GGCCCTGGGC

3 ACTTCATGAA 3 ACTGATCCCT 3 AATCACTTCA r GAACGAAGAG 3 TGGCCGCCTC

:CTGCCGCCTG

3 CGAGGACGCG P, CCCGAACCCG

CGGGCGAGTTC

C GCAGCAGCGT

:GTCCTGCTC

'CTGCGTGAC

:GTCGAGCTG

GACGGCGCA

:TGGTCCTGC

:GACCGTGCG

:GAGCGGCTC

;TGGCGGTAC

GCGCCCGCG

GCCCTGCTC

;CTCGTCCGC

;GCCCGGGTC

~GAGGGACC

;GCGGCGGCG

GCCTCGTCC

AAGGACGAG

~CCCGACCTG

CTTGCGCCG

;GCCCGGACG

'CTGCTCCTG

GGCGAGGAC

'CCCGGCCGC

3TCGGTGCTC

:ATCAGGCTG

CGCCCCGGAG

CCGGCACGTG

GGACGCCCCG

GGTGGCCGCG

CGCCGAACAC

CCCGTCCATG

CCTCCTCGAC

CTCCGCCGCC

CCTCGACGCC

GGGCCTCTGG

GATGAGCCGC

CGCCCTCCGC

GCGGGACGCG

CGCCAGGACC

CGGCACGCCG

GGCCGCCACC

GGTCGCCGAA

CGACCTCGGC

TGGCCTCGCC

CCACCTGGAC

CGGGAACGAG

ACAACTGACC

CGAAGAAGTC

CGGGACCGCG

GGCCGGGGAC

CGCCTCGGCC

GCCGCACGGT

TGCGCGCCTC

AAGTACCTCG

CGCGAGCTGG

CCCGGCGGCG

ATCTCGGAGT

GAGGCCACGG

GACGCCGACT

CTCCTCCTGG

WO 99/61599 WO 9961599PCTIUS99/I 1814 -21- 14221 14281 C 14341 C 14401 14461 14521 14581 14641 14701 14761 14821 14881 14941 15001 15061 15121 15181 15241 15301 15361 15421 15481 1554 1 15601 15661 15721 15781 15841 15901 15961 16021 16081 16141 16201 16261 16321 16381 16441 16501 16561 16621 16681 16741 16801 16861 16921 16981 17041 17101 17161 17221 17281 17341 17401 17461 17521 17581 17641 17701 GGCCTCCTG G ~GGTCGGCGT C GGAGGGCAT C ~CGCGTACAC G ,GCTGGTCGC C LCGCCGGCGG C 3CGGGCTGGC G 3GTCCGAGGG C kTCGGATCCT C TCACGGCTCC G GGCTCACGAC C kCCCGATCGA G CGCTGCGCCT C CCGGCGTGAT C rGGAGAAGCC C CCATGGACTG C GCGTCAGCGG C CTGCCTCCGA C TGGTGUTCGGC

CCTCGCAGGG

GGCG\CGCCGAC

AGGCGCTGAC

CGTTCGTGTT

TGTCGAAGGA

ACTGGTCGCT

ACGTCGTCCA

ACGGCGTGAC

TCGCCGGTGC

TCGCCGCCCA

CCCGGCAGCG

CCACCGTGGT

ACGGGGTCCG

CCATCGAGAG

CGTTCTTCTC

GGTACCGCAA

ACGAAGGCTT

CCGAGACCGT

TCACCTCACT

CCACCGCAAC

GGCTCCACGA

ACTGGAAGCG

TCGTCGTCGT

GCGCCGGCGC

CCGCGACGCT

TGCTCGCGTG

GCGCCACCCT

GCGTGACCCA

CCATGGTGTG

TGATCGACCT

CCGGCGGTAC

TCGTCCGCGC

CGGTGCTCGT

GCGACGGCGC

GCACCTCCGG

TGGGCGCGAC

TGCTCGCCGG

TCGACTCCGA

AGGCCACCGC

GTCCGCCCGT

GAGGCGTTC G TTCACCGGC G GAGGGCTAC C CTTGGCCTG G CTGCACCTC G :GTGACGGTC A CCGGACGGC C :CTCGGCGTC C :GCCGTGGTC C ~AACGGGCCG TI :TCCGACGTG G ;GCGCAGGCC G

GGGTCGTTG

AAGATGGTC C ;ACGGACCAG G ;CCGGACAAG C ACGAACGCG C ;GCGACCCG C ;AAGACTCCG C 'CGTACGGAC C 3TTCGAGCACC

'GCTCCGGAA

'CCCGGTCAG

3TTCGCGGCG

GGAGGCCGTC

GCCCGTGACC

GCCGCAGGCC

CCTCACCCTC

CCTCGCCGGC

CATCGAGAAC

I'TCGGGCGAC

CGCACGGATC

CGAACTCGCC

GACACTCGAA

CCTCCGCCAC

CACCCACTTC

CACCGGCCTC

CGCCGAAGCC

CGGCCACCAC

CTCCCCCGCC

CCTCGCGGTC

CCCCGAGGAC

CGACCCCGTA

GGGCGAGGCC

GGACGAGAGC

CACCCTGGTG

CGGCGCGGTG

GGGCATGGGC

GCCCTCGGAC

GGGTGAGGAC

CTCCCTCCCG

CACCGGTGCC

CGGACACCTC

TGCCGCCGAG

GGCCACCGTC

CGTCTCCGAC

GCCGCTCGCC

CGCGCTCCAC

CCTGGTCCTC

AGCACGCCG

TGATGTACC

TGGGCACCG

AGGGGCCGG

CCGTGCAGG

~TGTCGACGC

GGTCGAAGT

:TCCTCGTCG

GGGGCACCG

CGCAGCAGC

ACGTCGTCG

TCATCGCCA

LAGTCCAACA

AGGCGATGC

;TGGACTGGT

;GCGACGGCG

~ACGTCGTGC

;CCGTCGAGC

CCGCGCTGG

CCGCCGATC

,GGGCCGTCG

3GACTGATAC 3GCACGCAGT 3CCATGGCCG 3TCCGGCAGG rTCGCTGTCA

GTCGTCGGCC

GACGACGCCG

A.AGGGCGGCA

CTCCACGGAC

CCCACCCAGA

IkTCCCCGTCG

GAGGTCCTCG

GGCGCCTGGA

CGCGTCGGCT

A'TCGAGGTCA

GGCACCCTCC

TGGACCAACG

CCC GAGC TCC

GTCCAGGGCT

GCCGACGCG'I

CGTTCCGCCC

CAGCTGGACC

CTGGCGGCGC

GCGCACCCCC

CAGGCGCTGC

TCCGTCGGCC

CGGGTCGCCC

GCCGACCGG(

CAGGTCGCGC

GCGCACGGCI

GAGGAGCCT(

CTCCTCCACJ

GACTCCGGC(

GTGACCTGC(

GCGCACCCG(

GCGACCGAC(

CTGGACCGCI

TTCTCCTCG,

GGATCCCGGC

ACGACTACGC

GCAACTCCGG

CCGTCACGGT

CCCTGCGCAA

CCAGCACCTT

CCTTCTCGTC

AGCGCCT GT C

CCGTCAACCA.

GCGTCATCCG

AGGCCCACGG

CGTACGGGCA

TCGGACACAC

GCCACGGCGT

CCGCGGGCCC

GACTGCGCAG

TCGAAGAGGC

CGTCGGTCGG

ACGCCCAGAT

CGGCGCGGT

TCCGGCAC

GCGGCACGCC

GGGCCGGGAT

AGTGCGAGAG

CGCCGGGCGC

TGGTTTCGCT

ACTCGCAGGG

CCCGCGTCGT

TGATCTCCCT

TGTCGATCGC

TCCAAGAGCT

ACTACGCCTC

CCGGGCTCAG

TCACCGAGCC

TCGCCCCCGC

CCCCACCC

GCCGCGAACP

GCCTCACCAI

CCACCTACGC

CCGTGCAG

CCGAGCGCC

AGGCCCCCC

TGTCCCCGC9

CCGGTGGAGC

GCCACCCCGC

AGGACGCCC(

GGGCCGACC2

CCCTGGAGC)

CGGCCCTGGj 3TACGCGCCT( k. CGCCTTCGC( 3CGGCCCCCGi k CCACCCCCT(

'TCCCCGGGC'

3 ACCTCACGG,

'TCAGCGCCG'

3 CGGACGCGC

:TCCTGCGGG.

3 TCGCCGCGA GGCCACCGCC C CACCCGTCTC A CACTGTCGCC TI CGACACCCCC TI GGGCGAGGTC G CGTCGAGTTC P GACGGCCGAC G CGACCCCGT C GGACGGCGCC I ACGTCCCCTG C CACGGCTACG C GGGCCGTGAC C CCACGCCGCC C CCTGCCGAAG I

GGTCGAGCTG

GCCGCGGTC

CCCGGCGCC

CGCCGGCCTG

CGGACGCCTC

CGCTCGCGTA

CGGACAGGAC

CTCGGACGTG

GGGCGCCGAA

CGCGCTCTCC

GCCCACGCTG

GGCGAAGGTC

CGAGATCCC

CACCCTGCGC

CGCCCTCAC

CGCCGTCAAC

CGCTCAGGCG

CCACAGCCC

CCCGCGGACA

GGTGCTCGAC

CGTCGAGACC

CGTCCTCACC

GGGAGGCCAG

CGACTGGGCG

CTTCCAGCGC

CTCCTGGCGC

CGGCTGTCC

GGTGCTCCC

GGGCGACCGG

CCTCGACGC

CCCCTTCACC

3 CCTCGCCGCC k. CGTCACCTCC k. CCCCGAGCGC k. CCGCATGACC

CGGCTGCTC

GTGGTGGCAG

k. CGCCGCACC

:CGGCAGCGAA

1T CGTCGCCGAA k. CCGCAGGC T CCTCCACCTG T CGCCCGTGTC A GGCCGCGGCT T CTGGGGCGGC

:CCCGCACCT

.CCGATGTCC

CGGCCCCG

GCTCGTCCT

;ACATGGCGC

LCCCGTCAC

;GCACCAGCT

GCAAGGGCC

~GCAGCGGCC

;CGGACGCCC

~GACTCGGCG

;GCGAACAGC

CCGGTGTCT

~CGCTCCACG

TCACCGACG

rCCTCCTTCG 3AGGAGACCC 3TGCCGTGGC 3CCGCGTTCG

CTGGCCGGCG

GATTTCGCGC

GGCCGGGTGG

CTCCTCGACG

CGCTATGTCG

GAGCGGGTCG

TGGCAGCACC

GCCGCGTACG

A~GCAAGTCCA

GAGGAAGCGA

GGCCCCACCG

TGTGAGGCCG

CACGTCGAGA

CCTGAGGTC

GGCACCTACT

CTCGCCACCG

ATGACCGTCC

GAGCGTCTGG

CCCGTCCTCC

CGTCACTACT

TACCGCATCG

GGGCGCTGCC

GCGCTGTCCG

CAGCGGCTCG

GTCCTCTCC

CGGGGCACCG

CCGCTGTGGT

CCCGCCCAGG

TCGGGCCGGCC

ACGGTCCTCG

GCCCGCCGCC

GCCGACGGCA

CGGCTGGCCC

GGCGCCGAAG

CT CCGGAC C

GCCGCCCGGC

CCGCCCACCG

GTGACCGCGA

GCCGGAGGCC

GCCGGTCACG

WO 99/61599 WO 9961599PCTLJS99/1 1814 -22- 17761 G 17821 G 17881 C 17941 C 18001 G 18061 C 18121 T 18181 T 18241 G 18301 A 18361 C 18421 TI 18481 T1 18541 C 18601 C 18661 18721 C 18781 '1 18841 18901 18961 19021 19081 19141 19201 19261 19321 19381 19441 19501 19561 19621 19681 19741 19801 19861 19921 19981 20041 20101 20161 20221 20281 20341 20401 20461 20521 20581 20641 20701 20761 20821 20881 20941 21001 21061 21121 21181 21241

CGCGTACGC

CCCCACCGT

GACCGGGGA

CGCCCTGGA

GTCGAGCTT

:CGAGGCGCG

'GAGCCGCGA

CCGCGAGCA

GGCCTTCCG

~GAACGCCAC

GCTGGCGGA

'TCCGGTGGA

~GCCGGGCGG

~GATCTCCGG

~GGACGCGTC

'CGACGCCGA

;GCTCCTCCT

~TCAGGGGCA

CCCGCAACAC

CGTCGGGCCG

:CTGCTCCTC

3CGGACTGGC

TCAGCCGGCA

rCGGCTTCGG

GCCGCAACGG

CGAGCAACGG

TCGCGGACGC

CGCGACTCGC

ACACCGAACPz

CCGCCGGTGI

AGACGCTCCI

TGCTCACCGI

TCTCCTCCT]

ACGAGGACGC

CGAAGACTC(

GCCGTACGGI

AGTTCGAGCI

CCGCGCCTGJ

TCCCGGGACj

AGTTCGCGG(

TGGAGGCCG'

AGCCCGTGA(

CCCCGCAAG,

CCCTGAGCC'

ACCTCGCGG,

GACTGGCCG

TTTCGGGCG

GCGCACGGA

GCGAACTCG

CCACCCTCG

ACCTCCGCC

TCACCCACT

TCACCGGCC

TCGCCGAGG

CCACCCACC

ACCTCTCCC

TCGGCGCGC

TCCGTACGC

CGGCGTTCC

CGCCGGTACG

GACCTCGGTG

GCGGCTGCGC

CACCGCGCTC

CGCCCCCGGC

CCGCGCGCTC

GCTCGGTGCG

CCTCGCCGTG

TGACCTCGGA

CGGCCTGGCC

GTTCCTCCTC

CGGCGGGGTC

TGTCGCCTCG

CTTCCCGCAG

CGGGCGGACG

CTTCTTCGGG

GGAGACCTCC

GCAGGTCGGC

CGCCGAGGAT

TGTCTCGTAC

CTCGCTGGTC

GCTCGCGGGC

GCGCGGGCTC

CCCGGCGGAG

ACACCGTGTG

CCTGACCGCC

CCGACTGACC

CGACCCGATC

GCCGCTGCGC

CTCCGGCATC

CGTGGACCGC

GGCCATGGAC

CGGCATCAGC

CCCGGCGGAC

GGCCGCGCTC

k. CGCCGCCGA'.

k. CCGGGCCGT( k. GGGTCTGGT(

GGGCACGCA(

2GGCCATGGC( r CGTCCGACA( 2GTTCGCCGTI 2CGTCGTCGG, 1T GGACGACGC' G CCAGGGCGG G GTTCGACGG A CCCGACCCA T CATCCCCGT C CGACGTCCT A AGGCGCCTG A TCGTGTGGG T CGTCGAGGT T CGGCACCCT ;C CTGGGCCAP :C CGATCTGCC ;C CGCCGGTGP ;C CGTGGCGCI 'A CCCCTGGC I ;T GGAGCTGGC

GCCTTCCTCG

GCCTGGAGCC

CGCCTCGGCC

GGCCACGGCG

TTCACCACGG

GACGAGCAGC

CTCACCGGCG

GTCCTCAACC

TTCGACTCGC

CTCCCGGCCA

GCGGAGATCC

GACGACGAGC

CCGGAGGACC

GACCGCGGCT

TACTGCCGTG

ATCTCGCCGC

TGGGAGGCCG

GTGTTCGCGG

CTTGAGGGTT

ACCCTCGGCC

GCCCTGCACC

GGTGTGACGG

GCGGAGGACG

GGCGTCGGCA

CTGGCGGTCG

CCGAACGGGC

ACCGCCGACG

GAGGCACAGG

CTGGGGTCGT

ATCAAGATGC

CCGTCGGACC

TGGCCGAGGP

GGCACGAACC

GAGCCGTCGC

GACGCCCAG;

PCCGGGCGCGC

7 GCGCTCGGCI

CGGGGTGTGC

3 TGGGCCGGGI 2GAGTGCGAG( 7, GCCCCCGGC(

ATGGTCTCG(

Z: CACTCGCAG( C GCTCGTGTC( C ATGCTGTCC( G CTGTCCGTC, G ATCCAAGAG, C GACTACGCC G GCGGGGTTG G ATCACCGAA C TTCGCCCCG C AGCGCCCAC 'C CGCCGTGAC ,C GGCCTCACC :C ACCTACGCC LC ATCACCTCC C GCGGACTCC 'G GCGGACCAC 'G TTCCGAGCC

ACGCCCTCGC

CCTGGGAGGG

TGCGCCCCCT

ACACCGCCGT

CCCGGCCGGG

AGTCGACGAC

CCGAACAGCA

ACCCCTCCCC

TGACGGCGGT

CTCTGGTCTT

TGGGCGAGCA

CCGTCGCGAT

TGTGGCGGCT

GGGACGTGGA

CCGGTGGCTT

GCGAGGCCCT

TCGAGGACGC

GCACCAACGG

ACGTCGGGAC

TGGAGGGCCC

TCGCCGTGCA

TCATGTCGAC

GCCGGTCGAA

TGCTCCTCGT

TGCGCGGCAG

CCTCGCAGCA

TGGACGTCGI

CCCTCATCGC

TGAAGTCCAP

TCCAGGCGAI

AGATCGACTC

AGCAGGAGGC

CGCACATCG I

TCGGCGGTGI

TCGGACGCCJ

TCGCTCGCG)

k CCGGACAGGI 3CCTCCGGTG' k. TGGGTGCCGJ 3 CCGCGCTCG( 3 CGCCCACGC'

TGGCGAAGG'

3 GCGAGATCGI

TGACCCTGC'

-TCGCGCTGA

3 CCGCCGTCA C TCGCTCAGG T CCCACAGCG T CCCCCCAGA C CCGCCCTCG G CCGTCGAAA C CCGTCCTCIA A ACGGCGGAC G TCGACTGGC T TCCAGACCC :G CCGGTCTCC :G ACGGCTGCC :G CGGTGGCCC :G GGGACCAGC

CGGTCAGCAC

CAGCCGCGTC

CGCCCCCGCG

CACGATCGCC

CACCCTCCTC

GGCCGCCGAC

GCGCCGTATG

CGAGGCCGTC

CGAGCTCCGC

CGACTACCCG

GGCCGGTGCC

CGTCGGCATG

GGTGGCCGGC

GGGGCTGTAC

CCTCGACGAG

CGCCATGGAC

CGGGATCGAC

CCCCCACTAC

GGGCAACGCC

GGCCGTCACG

GGCCCTGCGC

GCCCACGACG

GGCGTTCGCC

CGAGCGCCTG

CGCGGTCAAC

GCGCGTCATC

CGAGGCCCAC

CACCTACGGC

CATCGGACAC

GCGCCACGGC

GTCGGCGGGC

CGGGCTGCGC

GCTCGAAGA-

GGTGCCGTGC

CGCCGCGTTC

rACTGGCCGGC k CGACCTGGCC P GGGTCGAGT(

ACTCCTCGA(

2TCCGTACGT( r GGAGCGGGT( r CTGGCAGCA( C CGCCGCGTA' 3 CAGCAAGTC, G CGAGGCGGC, A CGGGCCTAC C GTGTGAGGC C CCACGTCGA C ACCCCAGGT A CGGCGGCTA .C CCTGGCCAC .C CATGGCCCT 'A GCACCGCCT C CTCTCTCCT A GCGCTACTC ;G GGCGGCCGT 'T GCTCACGGC ;G CACCGTGCI ;T CGGTTGCGI

CGGGCCGACG

ACCGAGGGTG

ACGGCGCTCA

GACGTCGACT

GCCGATCTGC

GACACCGTCC

CAGGAGTTGG

GACACGGGGC

AACCGCCTCA

ACCCCCCGGA

GGCGAGCAGC

GCGTGCCGCC

GGCGAGGACG

GACCCGGACC

GCGGGCGAGT

CCGCAGCAGC

CCGACCTCCC

GAGCCGCTGC

GCCAGCATCA

GTCGACACCG

AAGGGCGAAT

TTCGTGGAGT

GCGTCGGCGG

TCGGACGCCC

CAGGACGGCG

CGGCGCGCGC

GGCACGGGCA

CAGGGGCGCG

ACCCAGGCCG

GTCCTGCCGA

ACGGTCGAGC

CGCGCGGCCG

GCCCCGGTCG

CTCGTGTCCG

3GCCTCGCAGG

'GGGCGTGCGC

3GCCGCACTGG 3 GCGTTCGTGT

GTGTCGAAGG

3 GACTGGTCGC 2GATGTCGTCC 2CACGGGGTGA Z GTCGCCGGTG C ATCGGCGCCC C GTTGTGGAGC C GCCACCGTGG C GACGGGGTCC G ACCATCGAGA C CCCTTCTTCT ,C TGGTACCGCA C GACGAAGGCT G CCCGAGACCG C ACCACCTCCC C CCCACCACGA ;G CCGCAGCCCG LG CACCCGCTGC ;G AGCCTCTCCC 'G CTGCCGGGAA T CTGGTCGAGG WO 99/61599 WO 9961599PCT/US99/1 1814 -23 21301 21361 21421 21481 21541 21601 21661 21721 21781 21841 21901 21961 22021 22081 22141 22201 22261 22321 22381 22441 22501 22561 22621 22681 22741 22801 22861 22921 22981 23041 23101 23161 23221 23281 23341 23401 23461 23521 23581 23641 23701 23761 23821 23881 23941 24001 24061 24121 24181 24241 24301 24361 24421 24481 24541 24601 24661 24721 24781

AGCTCACCCT

CCGTCGGCGC

PACGCGCCGGG

ACCGCACCGC

ACGTGGACGG

AGGGCGTCCG

CCGAGGTCGC

CCGTGCAGGC

GGAGCGGGAT

CCGGCCCGGA

CGGACTCCCT

CTCTGGACGC

CCGGCGCGGT

GCACCGAGGT

AGACCCCGGC

AGCATGTCCG

AGCGGTTCAC

GCGACGGCCT

CGGAGAGCCC

ACGCCACCGC

ACGCCGCCCT

TCCGGGACGG

ACGGCCTCGC

GGCGTCTGGA

CCGAGACCCT

CCGGTCTCAA

TGGGCACCGA

CCGGCGACCG

CGCGGACCGT

TGGTGTTCCT

GCCTCCTGGT

ACTGGGGCGT

TCGGCCTGGA

GTGCCGCTTC

TCGACGCCTC

ACGTCCGCGA

ACCTGGGCGA

TCGAGGACGG

ACGCCTTCCG

CGGGCCTCGA

TCGTGGCCCG

GGGGCACGGA

ACGTCTCGGT

CGATCCCCGC

GCACCCTCCC

CCGCGTTCCT

TGTTCTCCTC

ACGCCACCCT

TCGGCTGGGG

GCTCGCGGCT

TGGACGCGGC

CGCTCCGCGC

GATCGCGGGT

AGGCGGACAC

ACCTGCGGGA

GGGTGGACCT

TCCGCAACCG

ACCCCACCCC

GGTCCTGGGC

C.GACGCGCCG C GAGCGACGAG 9] C~GAGGCGGAG 9I CCCCGTCGCC C rCTGTACGAG rGGTGTCTGG

CGGTGCCGAG

GGCCGGTGCG

.TCCCTGTAC

.ACGGTGTCC

CACGGTGCTG

GCTGCACCTG

CGTGCTGGGC

CCTGTCCTTC

CCCGGCGACC

CGAGGCCCTG

CGATGGGCGC

GCGGTCCACG

GGGCCGGTTC

CGGGGACGGC

CGGCAGCGCC

GGCGCTCCTC

CGCGGCCGAC

GCCCGGTACG

CGCCCCGGAG

CTTCCGCGAC

GGGAGCCGGC

GGTCATGGGC

CGCGCGGATG

GACGGCCGTC

CCACTCCGCC

GGAGGTCCAC

CGACGCGCAC

CGGCGGGGCG

GCTGCGCCTG

CGCGGAGCGG

GGCCGGGCCG

GGTGCTCCGG

GCACGTCAGC

CCCGGAGGGT

GCACGTGGTG

CGCCCCGGGC

GGCCGCGTGC

CGAACACCCG

CTCGATGACA

CCTCGACGAA

CGCCGCCGCC

CGACGCCCTC

CCTCTGGGCC

GGCCCGTTCC

CATGCGCCGC

CCAGCAGCGC

CGGCGGCGCG

GGACCTCGGC

CCTCGTCCGT

GGAGCGGGCC

TCTCAACGCC

GGGGGAGCTC

GGAAGGCACC

~TCGTGCTGC C 'CCGGGCGTC G [GGACGCGGC A ;ACCCGGAGG C GCTTCGCGG C 'GGCGTGGCG T 3GCGCGCGGT TI 3GCGGGGCGT T1 3CGGTCGGCG C 3TGAGCGccG C 'CCGTCGACC C

'TGGAGTGGAC

GGCGACGCCG

CCGGACCTTAC

GTCCTGGTGGC

CACGGGTCGC I

CTGGTGCTCG

GGACAGGCCG

GTCCTGCTCG1

CTGACGACCG

CTCGCGACCG

GTACCCCGCC

GGCCTCGCCG

GACGGCAGCC

CCGCTCGGCC

GTCCTGATCG

GTGGTCACCG

CTGCTCTCCG

CCCGAGGGGT

TACGCCCTGC

GCCGGTGGCG

GGCACGGCGA

ATCGCCTCCT

GGCATGGACG

CTCGGGCCGG

GTCGCCGCCG

GAGCGGATCG

CACCTGCCCG

CAGGCCCGCC

ACGGTCCTGC

GGCGAGTGGG

GCCGGCGAGC

GACGTCGCCG

CTCACCGCGG

GCGGAGGATG

CTCACCTCGA

GTCTTCGGTG

GCCTGGCGCC

GAGACCAGCG

GGGGCGACGC

GACGACCCGG

GACGGCATGC

CCGGTCAACC

GGGCGGCTCG

ACGCACGTGG

TTCCGCGACA

GCGACCGGGC

GCCGGGCACC

GGGTCCGGAG

CCGTCGTGG C ~TACCTTCGG C LCGCCACCGG I] :GTGGCCGCC C GAACGGCTA C

LCGAGGTGTT(

'CGGCCTTCA

CGGCGCGGG

~CACCGCCCT

CGACTCCTC

~CGCGCAGCT

~CGCCTGGGA

CGGTCTCGC

~GGACCTGGT

~CTGCCCCGC

CGCGCTGAT

rGACCCGCGA

'CGTCTGGGG

kCCTCGCCGG 3GGACGCCAC

CCTCGGCTC

EGGCGCGGGC

TCTGCCGCT

TGGAGAGCCT

CGGGACAGGT

CCCTCGGCAT

CGACCGGCCC

GCGCGTACGC

GGACGTTCGC

GCGACCTGGC

TGGGCATGGC

GTCACGGGAA

CCCGCACCCT

TCGTACTGAA

GCGGCCGGTT

PCCACCCCGG

GCGAGATGCT

TCACGACCTG

ACACGGGCAA

TGACCGGCGG

GCGTACGACG

TCGTGCACGA

ACCGCGAAGC

TCGTCCACAC

TGGAACACGT

CGCCCGGCTA

GCGCGGGGCA

GCCGGACAGC

GCATGACCGG

CCATGGACAG

CGCTCGTCCC

TGGCGCCGCT

AGCGCAGGGC

CCGCGAT GAG

CGACCGTCCT

CCGGTTTCGA

TGCGGCTGCC

TGCTCGACGA

ACACGGCCTC

~GCGGTCCGT G ;CTCTACGCG C 'GTGCTGGCC G CCGGGCGCC C ~GGCTACGGC C GCCGACGTG C CCGGCGCTG C ~ACGCGGCTG C 'CGCGTGCGG C 'GGGCAGCCG C 3GCGGCCTTC IZ

MGGTGCCGCGC

'GCGGCGCTG(

3GAGGCCGTC

CGCCGGCCCC

GCAGGCCTGG

CGCGGTCGCC

CCTCGGCCGG

GGAAGCCCG

CGTCGGCGGC

GGGCGAGCCG

CGCCGCGCCC

GCCCGCCGCT

CACGGCGGCG

CCGCATCGCG

GTACCCCGAT

CGGCGTCACG

CCCGGTCGTC

CCAGGGCGCC

GGACGTCAAG

CGCCGTGCAG

GTGGGACGCC

GGACTTCGAG

CTCGCTCGCC

CGTGGAGATG

TGTCGGCTAC

CGCCGAGGTC

GGACGTGCGC

GGTCGTCCTC

CACCGGTGCG

CCTGCTGCTC

GCTGGAGGCC

CCTCACCGCC

GGCAGGCGTC

ACTGCGTCCC

CGACCTGGCA

GGGCGCCTAC

CGGACTCCCC

CGGACTCAGC

CGAGCTGACC

GATCGCCCTG

GCTCAGCGGG

AGCCGCCGGA

ACCGGACGAC

GGGACACGGC

CTCGCTCACC

GGCCACGCTG

ACTCGCCACG

GGCGACCGAT

;TGCAGCTGT

ACCCGGAGG

;CCCGTGCGG

;AGCCGGTGG

~CCCTCTTCC

;CCCTGCCGG

TCGACGCCG

~CGTTCGCCT

~TGGCCCCCG

;TGTTCGCCG

~GCGACCCGA

,AGGCCCTGC

,GCGCCGGTG

3ACCGGGGCG 3GTGGGCCGG

,TGGCCGACG

3CCCGTTCCG rCCGCGCAGA

%CGGCCGGGG

kCCTCTGGAG

CAGCTCGCCC

GCCGCGGCCG

CCGGCCCTCT

CCCGGCGACG

PTCCGGGCCA

CCGGCG CT GA

CACCTCGCCC

GTGGCGGACG

TCCGTGCCGG

CCCGGCGAGC

CTCGCCCGGC

CTGCGCGCGC

TCCGCGTTCC

CGCGAGTTCG

GGGAAGACCG

CGCGCCTTCG

ATCGCCCTCT

CGGGCCCGCG

ACGATGCCGT

CTGGGGGGCA

GTGAGCCGGC

CTGGGAGCCG

GTACTCGACT

CTCTCCGACG

AAGGTCGACG

GCGTTCGTCA

GCCGCCGCCA

GCCCTCTCCC

GACACCGACC

CTGTCCCTCC

GACGTCGCCG

CTCACCCGCG

GGCGCGGGCG

CGGGTCGCGC

ACCCCGAGCC

GCCGTCGAAC

GTCTTCGACC

GCCGCGGGCG

CGGCAGACCA

WO 99/61599 WO 9961599PCT/US99/1 1814 -24 24841 24901 24961 25021 25081 25141 25201 25261 25321 25381 25441 25501 25561 2562 1 25681 25741 25801 25861 25921 25981 26041 26101 26161 26221 26281 26341 26401 26461 26521 26581 26641 26701 26761 26821 26881 26941 27001 27061 27121 27181 27241 27301 27361 27421 27481 27541 27601 27661 27721 27781 27841 27901 27961 28021 28081 28141 28201 28261 28321

CGGCGGCCCT

CCGGCGGCCG

PCGGCGACGA

ACAAGGAGCT

PCCAGCCCCC

CAACGAAGAC

CAGGCGTCTG

CTGCCGCCTG

CGGGGACGCG

CCCCGACCCG

CGGCGAGTTC

GCAGCAGCGA

GACGGCCCTG

CTCGGGGCAG

GGCGCTGGGC

GACCGTGGAC

CCGCAAGGGC

CCTGTTCGTG

CGCCACCTCG

CCTGTCGGAC

CAACCAGGAC

CATCCGACGG

GCACGGCACG

CGGCCAGGAG

GCACACGCAG

CGGACTGCTG

GGGCACGGTG

GCGCCGCGCG

GGAGGCCCCG

GCCGTGGCCG

CGCGTACGCG

CAGCCGTACG

GGACGCCCTG

GGCGTTCGTC

CAGCTCACCG

CGACTGGTCT

CGACGTCGTC

CCACGGCATC

CGTCGCCGGT

CATCGCCGCC

CGTCCTGAAG

CGCCACCGTC

CGACGGCGTC

GATCATCGAG

GCCGTTCTTC

CTGGTACCGC

TGACGGCTTC

CGAGACCGTC

CACCTCACTC

CACCGCAACC

GCTGCAGAGC

GCCGCTGACG

CGAGCCAGAA

GGAAGCCGGG

CGGCGACGGC

GGTGCAGGCA

GGTCTCCGTC

CGGCCGCGTC

CCAGCCCGAT

'GCCGAACTC

TCCGGAGCTC

GCCACCGAC

GGGCGACTCC

CTCACACACG

DAGCTCCGCG

CGCGAGATCG

CCGGGCGGTG

ATCTCGGAGT

GACGCGTCCG

GACGCCGACT

CTGT CCCT CA

AAGGGCAGCG

PCCACCGCCG

TTCCTGTCCG

PCGGCCTGCT

GAGTGCGACA

CAGTTCAGCC

GCGGACGGCT

GCCCGCCGCA

GGCGCCAGCA

GCCCTGGCGG

GGCACGCGGC

AAGAGCAGCG

GCCGCGGCCG

CCGAAGACGC

GAACTCCTCA

GCTGTCTCCT

GCGGTCGAGG

GTGTCCGCGA

GACGGTCGTA

GCGATGGAGC

CGGATGCCGG

TTCCCCGGCC

GAGTTCGCTG

CTTGAAGCCG

CAGCCCGTGA

ACCCCCCAGG

GCACTCACCC

CACCTCGCCG

CGACTGAGCG

GTCTCCGGCG

CGTGCGCGGA

AAGGAGCTGG

TCCACCCTCG

AACCTGCGCC

ACCCACTTCA

ACCGGCCTCG

GCCGAAGCCT

GGCCACCACC

TCCGCGCCCP

GCCTCCGGCC

GCCGAGCTGC

GCGGACGACE

TTCACCGGCC

CTCGGCGACC

GGACGTCTCC

GTCGCCCTTC

GCCGCCGCCC

GACCGGCTGG

GCCGCCCGGC

CTGGACGAGG

GACTTCTGAC

GAACACGGAA

ACTACCTCAA

AGGGACGCAC

TCGCCTCGCC

TCCCGCAGGA

GCAGGACGTA

TCTTCGGGAT

CCACCGCGTG

GCCTCGGCGT

TGCAGTCGCC

GCCGTATCGC

CGTCCTCGCT

TGGCCCTCGC

GGCAGCGCGG

TCGGCCCCGC

ACGGACACCG

ACGGCCTCAC

ACGCCCGGCT

TCGGCGACCC

AACAGCCGCT

GTGTCGCAGG

TGCACGTCGA

CCGAGGCCGT

CCTTCGGCAT

ACTCCCCGGC

AGACTCCGGC

CGGACGTGGA

ACCGCGCGGT

AAGGACTGGT

AGGGCACGCA

CCTCGATGGC

TCGTCCGACA

CCTTCGCTGT

CCGTCGTCGG

TCGACGACGC

GCAAGGGCGG

ACTTCGACGG

ACCCGACCCP

TCATCCCGGI

CCGAGGTCCI

AAGGCACCTC

ATCGCGTGGC

TCGAGGTCAC

GCACCCTCCC

GGGCCAACGC

CCGAGCTCCC

LCCAGCGCCGC

AGGCGGACCI

TGGGCGCGCI

ACCGTGAGGC

TGGTCTCGC

CCGGAATCAI

ACACCCCCGC

AGCACCCCGI

TCGCCCACC'

AAGGCGTGCT

TCAGGGCGCT

CGTCCGACGA

CTGCCCGACA

CGGACAGGCG

GCGCGTCACC

GCACGAGCCG

CGAGGACCTG

CCGCGGCTGG

CTGCCGGTCC

CTCGCCGCGC

GGAGGCGATC

CTTCGTCGGC

CGAGCTGGAG

GTACGTCCTC

GGTCGCCCTG

CGGTGGTGTC

GCTGGCCGCG

GGAGGGCGCC

GATCCTCGCG

GGCTCCGCAC

CGCGCCGGGT

GATCGAGGCG

GAGGCTGGGC

TGTCATCAAG

CGAGCCCTCG

CGACTGGCCG

CAGCGGGACG

CGTCGAGCCG

CGCGCTGGAC

TCCGGCGGTG

CGCGGTCGGC

ACGCGGCACG

GTGGGCCGGC

CGAATGCGAG

GGAACCCGGC

CATGGTCTCG

CCACTCGCAG

CGCCCGCGTC

CATGATCTCC

ACTCTCCGTC

GATCGAGGAP

CGACTACGCC

CGCCGGACTC

GAT CACC GAG.

CTTCGCCCCC

CGCCCACCCC

CCGCGAACAC

CCTCACCATC

CACCTACGCC

CGACGACTGC

GTCCGGGCGC

GAAGGCCGC(

CCTCGCCGC(

CCTCGACGA(

k. GGCGCCCCTC

'CGACCCCGA(

k ACGCTGGGC( r' CGTCACCGC

CGCCTCCCTC

GGCCGCGGCC

CGACCTCTTC

CCACCGGCAC

AGAACGGGAG

GCCGAGCTGC

GTGGCGATCG

TGGCAGCTGG

GACGTGGAGG

GGCGGATTCC

GAGGCCCTCG

GAGAGCGCGG

GGCTGGCACA

GGCCACCTGG

GGTACGGACG

CACCTCGCCG

ACGGTCATGC

GACGGCCGGT

GGAGTCCTGC

GTCGTCCGCG

GGGCCCTCCC

GACGTGGACG

CAGGCCCTCA

GCGTTGAAGT

ATGGTCCAGG

GACCAGATCG

GAGAAGCAGG

AACGCGCACG

CCGGCCGGTG

GCCCAGATCG

GCCGCCCGCG

GACAGCCGGG

TCCTCGGACG

ATGGGCGCCG

ACCGCGCTCT

GCACCCACGC

CTGGCGAAGG

GGCGAGATCG

GTCACCCTGC

CTCGCCCTCG

GCCGCCGTCA

LCTCGCCCGCA

TCCCACAGCC

GCCCCGCAGG

CCGGTGCTCG

GCCGTGGAGA

GTCCTCACCA

GGAGGCCAGG

GACTGGGCGC

TTCCAGACCG

CGTTACCGCG

;TGGATCGTCG

GGAGCGGAGG

CGGCTCACCG

CTCGTGCCAC

3TGGTCCGTCA

'CGGGCCATGC

'GGCCTCGTCG

CTCTCCGGCG

3CGCCCGCCG

'TGGGGGACG

[CCTTCATCG

:ACCGGCACC

CCATGGCGAA

DGCAGAACAC

rGGGCATGGC rGGCCGGGGA

GGCTGTACGA

rGCACGACGC

CCATGGACCC

GCATCGACCC

CCGGCTACAC

I'CAGCGGCGC

GACCGGCCCT

TGCAGGCCCT

CCAACGCGGA

CGAAGGCGTT

TGGTGGAGCG

GCAGCGCGGT

A~GCAGCGCGT

TCGTCGAGGC

TCGCCACCTA

CGAACATCGG

CGATGCGCCA

ACTGGTCGGC

ACGGCGGGCT

TCGTCCTGGA

GCGGTGTGGT

GGCAGCTCGC

CCCTGGTCGA

AGGCACTGCG

TGGGCCGGGT

AACTCCTTGA

CCCGCTACGT

TCGACCGCGT

TCTGGCAGCA

CCGCCGCGTA

GCAGCAAGTC

ACGAGGCGGC.

ACGGCCCCAC

CCTGCGAGGC

GGCAGGTCGA

CTCCGCACGT

ACGGCACCTA

CCTTGGCGGT

TGACCCTCCC

AGCGTCTGGT

CCATCCTCCC

AGCGCTTCTG

TCGAGTGGAA

CCGTCGGGAG

TCGACGTACT

CACTGACGAC

AGGTCGCCTG

CCCAGGGCGC

TCTGGGGCCT

ACCTCCCCGC

CCACCGGCGA

WO 99/61599 WO 9961599PCTJUS99/1 1814 28381 C 28441 C 28501 C 28561 C 28621 C 28681 C 28741 C 28801C 28861 28921 28981 29041 29101 29161 29221 29281 29341 29401 29461 29521 29581 29641 29701 29761 29821 29881 29941 30001 30061 30121 30181 30241 30301 30361 30421 30481 30541 30601 30661 30721 30781 30841 30901 30961 31021 31081 31141 31201 31261 31321 31381 31441 31501 31561 31621 31681 31741 31801 31861

;GACCAGATC

CACGGACGT

ACCGGAGCC

CTCCTCGTC

~ACCGCATCG

;CGCACCCTC

~GGCGCACCG

'GGCGCGAAG

,GCCTTCGTC

,GCGGCGGCC

3GCGACCTCG

'GCGTACTGG

GGCCAAGGCC

GTTCGCGCCC

CCGGCAGGCG

GACCGGGCAG

GGCGCTCCTC

CCGGGTGGCC

GCTCCGCAAC

CCACCCGACG

GCCGGCCCCG

CCGGC-TGCGG

GCCGGGTTCC

GATCGACGAC

ACCCGACCGC

CACACGCCCA

ACAGTTGGTG

CCc3TCGCCGG

CGCGGGCGGA

GGTCTCCGAG

CGGGCGCAAG

CGACGCGGCC

GCAGCTCCTC

CCGCGGCACC

CCGGGTCGCC

CTCCGGGCGC

GTGCTCCTCT

CTCGACGGCP

CAGCAGCCAG

CGGCCTCGCC

GCGCAAGGGC

GAGCAACGGC

GGCCGACGCC

CCGTCTCGGC

CCCGGGGCAC

TTCGGGTGTC

GACCCTGCAC

GCTCACCGAC

CGCGTTCGGC

GGAGTCCCC'

GAAGACCTC(

CACGGACGTC

GCACCGCGC(

GGAAGGACT(

CCAGGGCAC(

CGCCGCCAT(

CGTCGTCCG,

CACCTTCGCi GGCCGT CAT,

GCCATCCGCA

CGGCCCACCC

CTCGGCAGCC

AGCCGCAGCG

GGCGCCCGCG

CTCGACGCCA

GGCGGCGATC

ACGAGCGGCG

CTCTACTCCT

AACGCCCACC

GTCGCCTGGG

CAGCGTCGCG

CTGAGCCACG

GCGTTCACGG

CTCGCCGCAC

TCGTCGGCGC

ACCCTCGTCC

CCCGGCCGTG

CAGCTCTCCA

CCCGCCGCAC

ACGGACTGGG

GACGCGGGGG

GGCGGTTCGG

CTGGACGCCG

GGTCCTGCCC

CAACCCCATC

GACGCTCTGC

GCCGACCGTC

ATCCGGTCCC

GTACCGGAGG

GGCACGACGT

TTCTTCGGGA

GAAGCCTCCT

GACGTCGGCG

CCCGAAGGCP.

ATCGCGTACI

TCGCTCGTCC

CTCGTGGGCC

CAGGCCATGC

*TGGGGCGAGC

CACCGGGTCC

CTCACGGCTC

CGGCTCACGI

-GACCCGATCC

CCGCTGCGGC

-GCCGGTGTC2

-GTGGACGAG(

3 CCGTGGAC' -GTGGGCGGGj r GCCGTCGAG( 3GCCGCACTG( 3GATCCGGCG(

IGTCGCGGTC(

3GTACGGGGC, 3CAGTGGGCC, 3GCCGAATGC, k. CAGGCTCCC.

ZGTCATGGTC

C GGCCACTCC

CCACCGGACT

GCGACTGGCA

ACGCCGCACG

GCGAACAAGC

TCACCATCGC

TCCCCGCCGA

CGCTGGACGT

CCGAGGTCCT

CGAACGCCGG

TCGACGCGCT

GCCTCTGGGC

GCATCCGTCC

ACGAGACCTT

TGTCCCGTCC

CCGTCGGTGC

TGGCCGCGAT

GTACCCACGC

CCTTCACCGA

CGGTGGTCGG

TCGCCGCGCA

AGc3GGCGGGT

TCCTCGACAC

ACGGCGGCGC

AGGCCCTGAT

CACGCGCCGC

CACGAGCc3GA

GCGCCTCTCT

GGCAGGAGCC

CCGAGGACCT

AGCGCGGCTG

ACGTCCGCAA

TCTCGCCGCG

GGGAGGTCTT

TGTACGTGGC

CCGGCGGTTP

CCCTCGGCCI

CCCTGCACCI

GCGTGGCCG1

CCGCCGACGC

GCGTCGCCGI

TGGCCGTCG]

CGCACGGGCC

CGAGCGACG]

AGGCGCAGGC

TGGGGACGC'

k TCAAGATGG'.

CGACGGACCj r GGCCGGAGC( k. CGAACGCGCA

'CGCCGGCCG

3 ACGCCCAGA' 3 TGGCCGCCC' :3 GCGACAGCC, k. CGGTCACCG.

3 GCATGGGCG G AGACCGCAC A. GCGCACCGA T CCCTCGCCA C AGGGCGAGA

CCACGCCCGC

GCCCCACGGC

CTGGATGGCC

CCCCGGAGCC

CGCCTGCGAC

GACGCCCCTC

CACCGGCCCG

CGACGACCTG

GGTCTGGGGC

CGCCGCCCGG

CGGCGACGGC

GATGAGCCCC

CGTCGCCGTG

CAGCCTTCTG

CCCGGCTCCC

CACCGCGCTC

GGCGGCCGTA

GCTCGGCTTC

CAACAGGCTC

CCTCCACGAG

GCGCCGGGCC

CGTCCTGCGC

CGCCGACCCT

CCGGATGGCT

ACCCCGCGCA

AGACCACACC

CAAGGAGAAC

CATGGCGATC

CTGGGACGCC

GGACATCGAC

CGCCGCGTTC

CGAGGCCCTC

CGAGCGGGCC

CTGTGGCTAC

CGTCGTCACC

GGAGGGACCC

CGCCCTGAAG

CCTCc3CGACC 3CCGGACCAAC

SACTCCTCCTC

'GCGCGGCAGC

CTCCCAGCAC

GGACGTCGTC

GCTGCTCGCC

C GAAGTCGAAC r GCAGGCGCTC

GGTCGACTGC

3 GCCGGGCCGC D, CGTCGTCCT( 3 TGGCGGCGT( r CGGGCAGCT( 3 CGCCCTGGT( G GGAGGCACTI A. TCCGGGCCGI C CGAACTCCT, T CTCCCCGTA, C ACTCGACCG A GGTCTGGCA T CGCCGCCGC

CGCCTCGCCC

ACCGTCCTCA

CACCACGGAG

ACCCAACTCA

c3TCGCCGACC

ACCGCCGTCG

GAGGACATCG

CTCCGCGGCA

AGCGGCAGCC

CGCCGCGCCC

ATGGGCCGGG

GACCGCGCCC

GCCGATGTCG

CTCGACGGCG

GGCGACGCCG

CCCGAGCCCG

CTCGGCCATT

GACTCGCTGA

CCCGCCACCA

GCGTACCTCG

CTGGCCGAAC

CTCACCGGCA

GGTGCGGAGC

CTCGGCCCCC

TCCCGCGCAC

CAGATGACGA

GAAGAACTCC

GTCGGCATGA

GTCGCCGCGG

TCCCTCTACG

CTCGACGACG

GCCATGGACC

GGCATCGACC

CAGGACTACG

GGCAACTCCT

GCCGTGACCC

GGCCTGCGGT

CCGGGCGCGI

GGCTTCGCCI

GAACGGCTCI

GCCATCAACC

CGCCTGATCC

GAGGGCCACC

ACGTACGGG(

ATCGGGCACI

CGCCACGGG(

3 TCGGCCGGT'.

3CTCCGCCGG( 3GAGGAGGCC( 3 GTGCCGTGG(

GCCGCATAC(

GACAGCCGT2 3 CGGGACGCC, 3; GTGGCGTTC, Z GACAGCTCA C GTCGACTGG C GTCGACGTC G CACCACGGC G TACGTCGCC

GCGCACCCCT

TCACCGGCGG

CCGAACACCT

CCGCCGAACT

CCCACGCCAT

TCCACACCGC

CCCGCATCCT

CT CCGC T GGA

AGGGCGTCTA

GGGGCGAGAC

GCGCCGACGA

TGGACGAACT

ACTGGGAGCG

TCCCGGAGGC

CCGTGGCGCC

AG CGCCGGC C

CCTCCCCCGA

CGGCCGTGCA

CGGTCTTCGA

CACCGGCCGA

TGCCCCTCGA

TCGAGCCCGA

CGGAGGCGTC

GTAACACCTG

CACCCG CCC C

GTTCCAACGA

GGAAAGAGAG

GCTGCCGGTT

GCAAGGACCT

ACCCGGTGCC

CCGCCGGATT

CGCAGCAGCG

CCGCGTCGGT

CGCCGGACAT

CCGCCGTGGC

TGGACACGGC

ACGGCGACTG

TCATCGAGTT

CGGCGGCGGA

7CCGACGCGCG

AGGACGGCGC

GCCAGGCCCT

3GCACGGGGAC

AGGGGCGCGC

k. CGCAGGCCGC 3TGCTGCCGAA r CGGTCGAGCT 3 CGGGCGTCTC

CCGGCGGTCGA

CGGTGTCCGC

3 CGGAAGACCG P. CGGCGATGGA C TGCGGATGCC G TCTTCCCCGG C CCGAATTCGC T CTCTCGAAGC G TCCAGCCCGT A TCACCCCCGA G GTGCCCTCAC WO 99/61599 WO 9961599PCTIUS99/1 1814 -26- 31921 31981 32041 32101 32161 32221 32281 32341 32401 32461 32521 32581 32641 32701 32761 32821 32881 32941 33001 33061 33121 33181 33241 33301 33361 33421 33481 33541 33601 33661 33721 33781 33841 33901 33961 34021 34081 34141 34201 34261 34321 34381 34441 34501 34561 34621 34681 34741 34801 34861 34921 34981 35041 35101 35161 35221 35281 35341 35401

CCTCGACGAC

CGGCAAGGGC

GAACCTCCAC

CGACCCCACC

GATCATCCCC

CGCCGACGTC

CGAAGGCACC

CCATCGTGTG

CTTCATCGAG

CCTGGCCACC

GGCCTGGGCC

CAGCCCCGCC

CCCCGCGGGT

GACGGGGCTC

CGTAC-TCGCG

GGTCCCCGTC

CCGCAACCGC

CCCGACGCCC

CGTCGCCGAG

GGCCCGCTCC

GGCCGTGGAG

CCGCCCGCAG

CGGCGGTCCG

GGCGAACGGC

CTTCC-TCGCC

CCCGGCCGAT

GGACGCCCCG

CTTCCGCCTG

TCCGCCGGGC

CGCGGGCGAG

GTTCCTCGCC

CGAACCGCTG

GCACACCGTC

CGT CGCC GAG

GTGACCGACA

CCGAACAGCG

TTCCGCTTCT

CGCCAGGACC

GTCGCGGCCA

GGCGCCTCCG

GAGGGCCTGT

CAGCTGGACG

TTCCTCCAGG

GCGGCGGAGA

GCCGGCGACC

AGCGGGCCGT

CACGAGATCT

CGCGTCGTGC

CGGTGACCGA

CGGTGGCCGA

ACGCCGCGAA

CCGCGTACGA

TCACCGCCGA

GCGCCGACGG

AGCG-CGAGCTA

TCGAGGGGAT

CCTTCGAGCT

TGGGTGTTCC

TGTCCGACAC

GCCGCTCGTG

GGCATGATCT

GGACTGTCGA

CAGATCCAAG

GTCGACTACG

CTGGCGGGGT

TGGATCACCG

GGCTTCGCCC

GTCAGCGCCC

CTCCGACGCG

AACGGCCTCG

GTCCCCGACC

CCCGGCGAGG

GCGTGGGGCC

ATGGTGATGC

GACCGCCCGC

GTCAACCGGC

GTCGCGCTCG

CCGTCGGATC

GGGGCCGACA

GACGACCGGT

TTCGCCTCGC

ACGGACCGGG

GGCCCGCACG

GTACCTCTCC

CTCGACACCG

GTCGTCCTGC

GAGCGGGCGC

CATCAGGAGC

CTGGAGCCGA

GGCCCGCGGC

GGCGACTGGC

GCGGACGTGC

GCCGTCCTCT

GACCTCTGAA

CGGTGCGGCT

CGGAGGAGCT

GGCGTGCCGA

CCGAACCCTG

TCGCCTTCGA

ACGTCTCCGG

ACCGGGCGTT

ACGACGAGCT

CGTACCTGCA

GTGACCCGAA

TCTGCCTCCC

GCAACGACAI

AGCCCCCGAC

CGACCTGACC

CCGTGAACTC

*CGGCGACCCC

*GCGGGTGCG I

*TCACGCCCTC

*CGTCCCGGTC

GGTGCTGCCC

CCACCGGGAC

GCTGGGCGGI

CGCGGACCG(

CCTGCTGGC(

TCGTGACCCT

CCCTCGCCCT

TCGCCGCCGT

AACTTGCTCA

CCTCCCACAG

TGTCCCCCCA

AACCCGCCCT

CGGCCGTCGA

ACCCCGTCCT

AGGACGGCGG

CCCTCGACTG

TCCCGACGTA

CGCCCGCGCA

CGGGTGCCGA

GGCAGGCGGC

TGCGGGAGAT

TGACCGGTCT

CCGAGCGCAT

ACGAGCAGGC

CCGGCGCCGG

ACGGCGAGTT

CCGAGGCCTG

CGGAAGGCCG

AGTTCCTGCG

CCGGCTACGG

CGCTCGACGC

TCGGGCACTC

ACGGCGCGCC

CCATCGAGGT

TGTCCGATGC

CGGGCCGCAG

AGGAGGAGCG

CGGGCGACCA

CCTGGCTCGA

CGTGGACAGC

GGTCTGCCTG

GCACCCCTCC

GCCGTGTCTG

GTGGCAGGAG

GACGGCCCGC

TCGGCGCGCC

CCTGGCCGAG

GCTGCGGCTG

CCGGCCGTCC

GGCGCCGCTG

GGCGTACTCC

CTCCGACCAC

CAGCCTTATC

GGGGCCCTCA

GGCACCCACC

TACGCCACCG

GCCCGCGGCG

GCGGCGAGCP

CCGCAGCAGG

GCGGCCGGTC

ACGCTGGAGG

TTCGTCCGCC

;CGCGCGGAC I

CCGCAGTCCC

CCGCAGCAAG

CAGCGAGGAA

CAACGGGCCT

GGCGTGTGAG

CGCCCACGTC

GACACCCCAG

CGACGGCGGC

GACCCTCGCC

CACCATGACC

ACAGCACCGC

GGCCTCCCTG

CGCCTTCCAG

CACCGCTTCC

GGACCTCGAC

CTCCGTGCTC

CGGCTTCGAC

CCAGCTGCCG

CAGCGACGAG

GGAGGAGGAG

CGCCGGGATG

CCTCGACGTC

CTCGGAGCGG

TGCCGTTCTC

GCTCAGCACC

CACGGGTACG

CCAGGCCCGG

CGGCGGCGCC

GCCGGCCGGG

GTGGAGCAGG

GCGGCTGCTG

CAGCGCGCCC

GGGCGACTGG

CTTCACGATG

CGCCATCGAG

GGACTGTGGA

CCGCACGCCG

GTCGAGGCCC

GAGAGCGTCG

GGCCGGCTGG

ATCCTGGAAC

CCGTCGCTGG

ATCCGGCGGC

GTGCTGCCCC

GCCAAGCTCP

AACGAGGTGC

GGCGGCCAC'I

CTGCTCGTCI

GAAGGAGCGC

CGCAGCCCCC

TCCTGGAGAC

TGCTGCGCGC

CGCTCTCCTI

TCCTCTGCTC

TCCTCTCGTI

ACGTGCCGG)

GTCTCGCGC(

CGGCGGTGA(

TCGCGGATC'

TGCGGACGG'

TCCATCGCCG

GCCACCCGGC

ACCGCCACCG

GCCGACGGCA

GAGACCATCG

GTCCCCTTCT

TACT GGTACC

ACCGACGAAG

CTCCCCGACA

CTCACCACCT

CTGCCCGCCA

CACCGCTCGT

GGGCGCGAGG

GAGGAGGGCC

CGGTGCGACT

TCGCTGACCG

CCCACCGTCG

CTGGCCGAGC

AAGGCCGCCG

TTCCGCGCCC

CTCGCCGAAG

CTCGACCCGG

GTCGGCTGCA

TCCTTCCAGG

GGCACCGGCA

GCGATCCTCC

CTGCTCGCGC

ATCGTCCTGG

CAGCTGGGCG

GCCATGGGCC

GTGCTTCTGG

CGTGCCCACT

ATGCGGGACC

GGCATCGAGG

TCCGGCGCTT

GCGGCTCCGC

TGTCGGTGCA

AGGAGCTCGC

CCTTCTTCGC-

AGCGGCACGC

CGCCGGACCC

TCAGCGGCAC

CGCTGCGCAC

CCTGCCCGG~I

CCGAGTGGCC

TCTACCTCA7

CCCGCGGCGC

CGAAGAGATC

GCTGGGCCG(

CCGCGGCATC

CCAGGCGGAC

CAGCCCGACC

GACGGACTT(

k. CGGGGAGGG( k GGGCGGGCA(

'GGACCCGTC(

'GGCCGCTGC(

r' GCTGGAGCG( r ACGGGCGGC(

CCCACCTCGC

AGCGCATCGA

TGGTTTCGGG

TCCGCGCACG

AGAACGAACT

TCTCCACCCT

GCAACCTCCG

GCTTCACCCA

AGGTCACCGG

CCCTTGCCGA

CGGGCGCCCT

ACTGGATCAG

CCGTCGCCGA

GGCGCAGCGC

CGCCCGAAGA

CCGTCGACTT

TGTTCGAGCA

GGAACTGGGC

CTCCGGCGGG

TGTTCCGGCA

CCTCCGCGTT

TGCTGCTCGC

CCGGCACCGC

AGGAGCGGGA

CGGCCCTCCT

GGGCCGCCGG

ACGAGCTGGC

TCGACCCCTA

AGGGCCTGTT

GGTACGCGCG

TCCGTGCCTC

GGGACCTTCC

ACGCGCCGGC

GGGCGGGCAA

CCACCCCGCG

CAGCTACTTC

GTATCCGGGC

CGAGCATGTG

GCACAGCCTC

GGTACGGCCC

GCTCGTCCAC

CGACGAGCGG

CGACTACAAG

GATGGCCCTG

TCGGCACACC

CGACCAGTGG

GCCCGATGCC

GCAGAACCCA

ACCGTCCGCG

CACTGGATCC

GACCCGTATC

GGCAGCTGGG

GGGGTCTCCG

TGTCCGCTGG

3CGTGCCGTGG 3 GCGTCGTACG

GCCGCCGTGC

3 CTCCGGCCGC 3GACGGCGCGC WO 99/61599 WO 9961599PCT/JS99/I 1814 -27- 35461 35521 35581 35641 35701 35761 35821 35881 35941 36001 36061 36121 36181 36241 36301 36361 36421 36481 36541 36601 36661 36721 36781 36841 36901 36961 37021 37081 37141 37201 37261 37321 37381 37441 37501 37561 37621 37681 37741 37801 37861 37921 37981 38041 38101 38161 38221 38281 38341 38401 38461 rGGCCGAGCT

CGCTCGGGGT

%TCCCGAGCA

%GACCCTCCG

kGCTGGCGGG

GCCGGGACCC

CCGCGCACCT

TCAGGCGGA

GGGACGTGCT

GCAGCTCCTG

CTCGGACCAC

CATCCCGCCC

ICGCACATCA

GGCACGAGGT

TCGCCGCGGT

GCGAGCCGCG

ACTGGGACCA

ACAACGACTC

TGCTGTGGGA

ACGCCCGGGT

TGCGGGACCG

CGCTCGACCG

ACCCGACCCC

TTCCGTACAA

GGGTCTGCCT

AGGGCGACAT

CGAGTCAGCG

CGATGCACGC

ACGCGACCGC

CGGTCAAGGC

TCACGCCGCA

CCGCCGCGCA

CCGAGCTGGA

CCGCACCCCT

CCTCCGAAAG

CCTCTTCTAC

GGTGCGCTCC

GCATCTGGAG

CATGCTCACC

GGACTTCCGG

CCTGAAGACG

CGGTGGCGTC

CAGCGCCGAC

GGAGGGGAAC

GCGGCACTTC

GTTCACGGCC

CTTCGTCGGC

GCACCAAGCP.

CCGCGGCCGC

CACCACCGTC

CCGGGCGGCC

CACGGCGCTG

CACCGCAGCC

GTGGCGGGAG

CTACGACCCG

CCGGCGGCTG

GGAGGTCTTC

CGCGCTGCAC

GGTCGCGCTG

CCGCCCCCGC

AGACACCGGG

GGGGACGGCT

CTCCACCGGC

CACGCACTAC

GCGGGTCGCC

GCCGGTCGGC

CCCGAACCAT

CGCCCTCGGC

GATGGTCGAC

GCCGACGACC

CCTGTGGGGG

GCAGCCGCCC

GTACGGCGCC

GCGGAGCCTG

CGGCACGTCG

GACCCTCGGC

CCTGGAGGCG

CGCCGAGATC

GCTCCTGCCG

CGTGATCAAC

GCGGGCCGTC

GGCCGTGCGG

CCGGCTGCGC

GCGGCTCGCC

CGCCCCAGGC

ACCGAAAGCA

CTGGGTCGCG

CGTACCCCCG

CACTTCACCA

CACGCCCGCA

CTCGGCCGGA

ACCGAGGAAC

GTCGTCGTCC

GTCGTCCGCC

GCGACGCGCP

TCCGACGTCC

GCCGGGCTGC

GTCCCCGCCI

AAGAGAGAGI

ACCACGCCCI

GCGGCCGCCC

GAGCTCGTCC

CTCGCCGATT

GTCCAGCTCA

CTGTGCGACC

CCGGTGCAGC

CCGGCCGGGG

ACGGACCCGG

CCCGCCGGTC

CGGACCCTGG

CGCGCGCCTG

GCCCCGGTCC

CAGACCGTCC

AAGGAAGGAC

TACGGCCTGG

AGCCAGCCCG

ACCGACCACC

CCGGCGATCG

ATCGAGGCGA

GACCTCGTCG

TACGCGGGCG

CCCGACGTGA

GAGCACCGCG

TCCTTCGAAG

CGCCTCGACA

GTCGTGCCGG

GTCTCCGCGG

CTCGCCGACC

CGCAACTACC

AGCTGCTCGG

GCGGTGCCGC

GCCGAGCAGG

GACGCCGTCG

GAGGAGACCT

GCGCAGCACC

CTCACCCCTG

GGAGCACCGT

GCAAGGACTA

AGGCCTCCTC

AGGAGTTCGG

AGCGGCTGCC

AGTTCTCCGC

TCGGCGCGGC

AGCCGTGGTC

GTGACGGGCC

TGGAGGTCCT

ATCTCATCAC

GCGTCGAGTI

GAGCACCGCC

AACGAACCG'I

~GGGCCTTCAC

CTCCCGGCG(

CCCAGATGA(

CGGACGACTC CCCCGGGGCC CTGCTGTCGG CCGGGAACGC GGTGCTCGCG CTCCTCGCGC GGCCCGGGCT CGCGGCGGCC GCGGTGGAGG TCGACGCCCG GGTGGTCCGC GGGGAGAGG CGCATGTCGT CGTCCTGACC GCCGCGACCG AGCGCTTCGA CCTCGCGCGC CCCGACGCCG CGTACGGCCC GGTGGCGTCC CTGGTCCGGC CCGGGCGTTT CCCCGGGCTG CGGCAGGCGG TCGGCCGCGG GCCGCTGAGC GTCCCGGTCA GCCCGGCCCC CCTTCGGACG GACCGGACGG CGTGTGTCCC CGTCCGGCTC CCGTCCGCCC ACGACGCCAT GCGCGTCCTG CTGACCTCGT TGCCCCTGGC CTGGGCGCTG CTCGCCGCCG CGCTCACGGA CACCATCACC GGGTCCGGGC TCATCCACGA GTACCGGGTG CGGATGGCGG CCTTCGACGA GGCCCGTCCC GAGCCGCTGG TCCTCGCCCC GTACTTCTAT CTGCTCGCCA ACTTCGCCCG GTCCTGGCAG CCGGACCTGG CCGTCGCCGC GCAGGTCAGC GGTGCCGCGC TGGGCAGCGC CCGCCGCAAG TTCGTCGCGG AGGACCCCAC CGCGGAGTGG CTGACGTGGA AGGAGCTGCT CACCGGCCAG TTCACGATCG CGGGCCTGCC GACCGTCGGG ATGCGTTATG ACTGGCTGAG TGAGCCGCCC GCGCGGCCCC GTGAGGTCCT CGGCGGCGAG GGCGTCTGGC TCGACATCGA GCTCGTCGCC ACGCTCGACG CGAAGCACAC CCGGTTCACG GACTTCGTGC CGATCATCCA CCACGGCGGG GCGGGCACCT AGGTCATGCT CGCCGAGCTG TGGGACGCGC GGGCGGGGTT CTTCCTGCCG CCGGCCGAGC TCCGCATCCT CGACGACCCC TCGGTCGCCA TCGGCGACCC CACCCCGGCC GGGATCGTCC GCCGCCCGCC GGCCGACGCC CGGCACTGAG TATCTGCGCC GGGGGACGCC CCCGGCCCAC GTACGAAGTC GACGACGCCG ACGTCTACGA CGCCGCCGAG GCCTCCGACA TCGCCGACCT GCTCCTGGAC GTGGCCTGCG GTACGGGCAC CGACACGGCC GGCCTGGAGC TGTCCGAGGA CGACGCCACG CTCCACCAGG GGGACATGCG CGTGGTCAGC ATGTTCAGCT CCGTCGGCTA CGTCGCCTCG TTCGCGGAGC ACCTGGAGCC GTTCCCGGAG ACCTTCGCCG ACGGCTGGGT CACCGTGGCC CGTGTCTCGC ACTCGGTGCG CTTCACCGTG GCCGACCCGG GCAAGGGCGT CCTGTTCCAC CAGGCCGAGT AGGAGGCCGC CCTGGAGGGC GGCCCGTCGG GCCGTGGCCT CAAGACCCCC CGGGGCGGGA CGTCCCGGGT GACAGGTAAG ACCCGAATAC GGCGTGTCCG CCTGGCCGTC GTCGGCACCC TGCTGGCGGG CGCCGACACG GCCAATGTTC AGTACACGAG GCTCGACGAG AAGATC (SEQ ID NO:19) Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA compounds differing in their nucleotide sequences can be used to encode a given amino acid sequence of the invention. The native DNA sequence encoding the narbonolide PKS of Streptomyces venezuelae is shown herein merely to illustrate a WO 99/61599 PCT/US99/11814 -28preferred embodiment of the invention, and the invention includes DNA compounds of any sequence that encode the amino acid sequences of the polypeptides and proteins of the invention. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The present invention includes such polypeptides with alternate amino acid sequences, and the amino acid sequences shown merely illustrate preferred embodiments of the invention.

The recombinant nucleic acids, proteins, and peptides of the invention are many and diverse. To facilitate an understanding of the invention and the diverse compounds and methods provided thereby, the following description of the various regions of the narbonolide PKS and corresponding coding sequences is provided.

The loading module of the narbonolide PKS contains an inactivated KS domain, an AT domain, and an ACP domain. The AT domain of the loading module binds propionyl CoA. Sequence analysis of the DNA encoding the KS domain indicates that this domain is enzymatically inactivated, as a critical cysteine residue in the motif TVDACSSSL, which is highly conserved among KS domains, is replaced by a glutamine and so is referred to as a

KS

Q domain. Such inactivated KS domains are also found in the PKS enzymes that synthesize the 16-membered macrolides carbomycin, spiromycin, tylosin, and niddamycin.

While the KS domain is inactive for its usual function in extender modules, it is believed to serve as a decarboxylase in the loading module.

The present invention provides recombinant DNA compounds that encode the loading module of the narbonolide PKS and useful portions thereof. These recombinant

DNA

compounds are useful in the construction of PKS coding sequences that encode all or a portion of the narbonolide PKS and in the construction of hybrid PKS encoding DNA compounds of the invention, as described in the section concerning hybrid PKSs below. To facilitate description of the invention, reference to a PKS, protein, module, or domain herein can also refer to DNA compounds comprising coding sequences therefor and vice versa.

Also, reference to a heterologous PKS refers to a PKS or DNA compounds comprising coding sequences therefor from an organism other than Streptomyces venezuelae. In addition, reference to a PKS or its coding sequence includes reference to any portion thereof.

The present invention provides recombinant DNA compounds that encode one or more of the domains of each of the six extender modules (modules 1 6, inclusive) of the narbonolide PKS. Modules 1 and 5 of the narbonolide PKS are functionally similar. Each of WO 99/61599 PCT/US99/11814 -29these extender modules contains a KS domain, an AT domain specific for methylmalonyl CoA, a KR domain, and an ACP domain. Module 2 of the narbonolide PKS contains a KS domain, an AT domain specific for malonyl CoA, a KR domain, a DH domain, and an ACP domain. Module 3 differs from extender modules 1 and 5 only in that it contains an inactive ketoreductase domain. Module 4 of the narbonolide PKS contains a KS domain, an AT domain specific for methylmalonyl CoA, a KR domain, a DH domain, an ER domain, and an ACP domain. Module 6 of the narbonolide PKS contains a KS domain, an AT domain specific for methylmalonyl CoA, and an ACP domain. The approximate boundaries of these "domains" is shown in Table 1.

In one important embodiment, the invention provides a recombinant narbonolide PKS that can be used to express only narbonolide (as opposed to the mixture of narbonolide and S0-deoxymethynolide that would otherwise be produced) in recombinant host cells. This recombinant narbonolide PKS results from a fusion of the coding sequences of the picAIII and picAIV genes so that extender modules 5 and 6 are present on a single protein. This recombinant PKS can be constructed on the Streptomyces venezuelae or S. narbonensis chromosome by homologous recombination. Alternatively, the recombinant PKS can be constructed on an expression vector and introduced into a heterologous host cell. This recombinant PKS is preferred for the expression of narbonolide and its glycosylated and/or hydroxylated derivatives, because a lesser amount or no 0-deoxymethynolide is produced from the recombinant PKS as compared to the native PKS. In a related embodiment, the invention provides a recombinant narbonolide PKS in which the picAIV gene has been rendered inactive by an insertion, deletion, or replacement. This recombinant PKS of the invention is useful in the production of 10-deoxymethynolide and its derivatives without production of narbonolide.

In similar fashion, the invention provides recombinant narbonolide PKS in which any of the domains of the native PKS have been deleted or rendered inactive to make the corresponding narbonolide or 10-deoxymethynolide derivative. Thus, the invention also provides recombinant narbonolide PKS genes that differ from the narbonolide PKS gene by one or more deletions. The deletions can encompass one or more modules and/or can be limited to a partial deletion within one or more modules. When a deletion encompasses an entire module, the resulting narbonolide derivative is at least two carbons shorter than the polyketide produced from the PKS encoded by the gene from which deleted PKS gene and corresponding polyketide were derived. When a deletion is within a module, the deletion WO 99/61599 PCT/US o/l1 1 typically encompasses a KR, DH, or ER domain, or both DH and ER domains, or both KR and DH domains, or all three KR, DH, and ER domains.

This aspect of the invention is illustrated in Figure 4, parts B and C, which shows how a vector of the invention, plasmid pKOS039-16 (not shown), was used to delete or "knock out" the picAI gene from the Streptomyces venezuelae chromosome. Plasmid pKOS039-16 comprises two segments (shown as cross-hatched boxes in Figure 4, part B) of DNA flanking the picA gene and isolated from cosmid pKOS023-27 (shown as a linear segment in the Figure) of the invention. When plasmid pKOS039-16 was used to transform S. venezuelae and a double crossover homologous recombination event occurred, the picAI gene was deleted. The resulting host cell, designated K039-03 in the Figure, does not produce picromycin unless a functional picAl gene is introduced.

This Streptomyces venezuelae K039-03 host cell and corresponding host cells of the invention are especially useful for the production of polyketides produced from hybrid PKS or narbonolide PKS derivatives. Especially preferred for production in this host cell are narbonolide derivatives produced by PKS enzymes that differ from the narbonolide PKS only in the loading module and/or extender modules 1 and/or 2. These are especially preferred, because one need only introduce into the host cell the modified picAI gene or other corresponding gene to produce the desired PKS and corresponding polyketide. These host cells are also preferred for desosaminylating polyketides in accordance with the method of the invention in which a polyketide is provided to an S. venezuelae cell and desosaminylated by the endogenous desosamine biosynthesis and desosaminyl transferase gene products.

The recombinant DNA compounds of the invention that encode each of the domains of each of the modules of the narbonolide PKS are also useful in the construction of expression vectors for the heterologous expression of the narbonolide PKS and for the construction of hybrid PKS expression vectors, as described further below.

Section II: The Genes for Desosamine Biosynthesis and Transfer and for Beta-glucosidase Narbonolide and 10-deoxymethynolide are desosaminylated in Streptomyces venezuelae and S. narbonensis to yield narbomycin and YC-17, respectively. This conversion requires the biosynthesis of desosamine and the transfer of the desosamine to the substrate polyketides by the enzyme desosaminyl transferase. Like other Streptomyces, S. venezuelae and S. narbonensis produce glucose and a glucosyl transferase enzyme that glucosylates desosamine at the 2' position. However, S. venezuelae and S. narbonensis also WO 99/61599 PCT/US99/11814 -31 produce a beta-glucosidase, which removes the glucose residue from the desosamine. The present invention provides recombinant DNA compounds and expression vectors for each of the desosamine biosynthesis enzymes, desosaminyl transferase, and beta-glucosidase.

As noted above, cosmid pKOS023-27 contains three ORFs that encode proteins involved in desosamine biosynthesis and transfer. The first ORF is from the picCII gene, also known as des VIII, a homologue of eryCII, believed to encode a 4-keto-6-deoxyglucose isomerase. The second ORF is from the picCIII gene, also known as desVII, a homologue of eryCIII, which encodes a desosaminyl transferase. The third ORF is from the picCVI gene, also known as desVI, a homologue of eryCVI, which encodes a 3-amino dimethyltransferase.

The three genes above and the remaining desosamine biosynthetic genes can be isolated from cosmid pKOS023-26, which was deposited with the American Type Culture Collection on 20 Aug 1998 under the Budapest Treaty and is available under the accession number ATCC 203141. Figure 3 shows a restriction site and function map of cosmid pKOS023-26. This cosmid contains a region of overlap with cosmid pKOS023-27 representing nucleotides 14252 to nucleotides 38506 of pKOS023-27.

The remaining desosamine biosynthesis genes on cosmid pKOS023-26 include the following genes. ORF 11, also known as desR, encodes beta-glucosidase and has no ery gene homologue. The picCI gene, also known as desV, is a homologue of eryCI. ORF14, also known as deslV, has no known ery gene homologue and encodes an NDP glucose 4,6dehydratase. ORF 13, also known as desIII, has no known ery gene homologue and encodes an NDP glucose synthase. The picCV gene, also known as desll, a homologue of eryCV is required for desosamine biosynthesis. The picCIV gene also known as desl, is a homologue of eryCIV, and its product is believed to be a 3,4-dehydratase. Other ORFs on cosmid pKOS023-26 include ORF12, believed to be a regulatory gene; ORF15, which encodes an Sadenosyl methionine synthase; and ORF16, which is a homolog of the M tuberculosis cbhK gene. Cosmid pKOS023-26 also encodes the picK gene, which encodes the cytochrome P450 hydroxylase that hydroxylates the C 12 of narbomycin and the C 10 and C 12 positions of YC- 17. This gene is described in more detail in the following section.

Below, the amino acid sequences or partial amino acid sequences of the gene products of the desosamine biosynthesis and transfer and beta-glucosidase genes are shown. These amino acid sequences are followed by the DNA sequences that encode them.

WO 99/61599 -2 C/S9111 PCT/US99/11814 Amino acid sequence of PICCI (desV) (SEQ ID NO:6) 1 61 121 181 241 301 361

VSSRAETPRV

AVG VNSGMDA

PLLVEKAITP

GSSVAAFSFY

MQAAVLRI RL

DELRSHLDAR

QALRVT DAVR

PFLDLKAAYE

LQLAILRGLGI

RTRALLPVHL

PGKNLGCFGD

XHLDSWNGRR

GIDTLTHYPV

ELRAETDAAI

GPGDEVIVPS

YGHPADMDAL

GGAVVTGDPE

SALAAEYLSG

PVHLS PAYAG

ARVLDSGRYL

HTYIASWLAV

RELADRHGLH

LAERLRMLRN

LAGLPGIGLP

EAPPEGSLPR

LGPELEGFEA

SAT GAT PVPV

IVEDAAQAHG

YGSRQKYSHE

VTAPDTDPVW

AES FARQVLS EFAAYCET DE

EPHEDHPTLD

ARYRGRRIGA

TKGTNSRLDE

HLFTVRTERR

LPIGPHLERP

EWAERVDQA (SEQ ID NO:6) Amino acid sequence of 3-keto-6-deoxyglucose isomerase, PICCII (des VIII) (SEQ ID NO:7) 1 61 121 181 241 301 361

VADRELGTHL

TAD HALAAS I

EGIHRETLEG

SDSLLAPQSL

PEQWRELCDR

RDPEVFTDPE

DVLRPRRAPV

LETRGIHWIH

LCSTDFGVSG

LAPDPSASYA

RTVRAADGAL

PGLAAAAVEE

RFDLARPDAA

GRGPLSVPVS

AANGDPYATV

ADGVPVPQQV

FELLGGFVRP

AELTALLADS

TLRYDPPVQL

AHLALH PAGP SS (SEQ ID

LRGQADDPYP

LSYGEGCPLE

AVTAAAAAVL

DDSPGALLSA

DARVVRGETE

YGPVASLVRL

NO: 7)

AYERVRARGA

REQVLPAAGD

GVPADRRADF

LGVTAAVQLT

LAGRRLPAGA

QAEVALRTLA

LSFSPTGSWV

VPEGGQRAVV

ADLLERLRPL

GNAVLALLAH

HVVVLTAATG

GRFPGLRQAG

Amino acid sequence of desosaminyl transferase, PICCILI (des VII) (SEQ ID NO: 8) 1 61 121 181 241 301 361 MRVLLTS FAH

EYRVRMAGEP

RSWQPDLVLW

TAEWLTWTLD

SEP PARPRVC TRFT DFVPMH

FFLPPAELTP

HTHYYGLVPL

RPNHPAIAFD

EPTTYAGAVA

RYGAS FEE EL

LTLGVSAREV

ALLPSCSAII

QAVRDAVVRI

AWALLAAGHE

EARPEPLDWD

AQVTGAAHAR

LTGQFTIDPT

LGGDGVSQGD

HHGGAGTYAT

LDDPSVATAA

VRVASQPALT

HALGIEAILA

VLWGPDVMGS

PPSLRLDTGL

ILEALADLDI

AVINAVPQVM

HRLREET FGD

DTITGSGLAA

PYFYLLANND

ARRKFVALRD

PTVGMRYVPY

ELVATLDASQ

LAELWDAPVK

PTPAGIVPEL

VPVGTDHLIH

SMVDDLVDFA

RQPPEHREDP

NGTSVVPDWL

RAEIRNYPKH

ARAVAEQGAG

ERLAAQHRRP

421 PADARH (SEQ ID NO:8) Partial amino acid sequence of aminotransferase-dehydrase, PICCIV (desI) (SEQ ID NO:9) 1 61 121 181 241 301 VKSALS DLAF

VAGLAGVRHA

PDTGNLDPDQ

DGRPAGSLGD

NAKM'SEAAAA

VEI DEATTGI

FGGPAAFDQP

VATCNATAGL

VAAAVT PRTS AEVFS FHATK

MGLTSLDAFP

HRDLVMEVLK

LLVGRPNRID

QLLAHAAGLT

AVVGVHLWGR

AVNAFEGGAV

EVI DRNRRNH

AEGVHTRAYF

RARLYERLDR ALDSQWLSNG GEVIMPSMTF AATPHALRWI PCAADQLRKV ADEHGLRLYF VTDDADLAAR IRALHNFGFD AXYREHLADL PGVLVADHDR S (SEQ ID NO:9)

GPLVREFEER

GLTPVFADID

DAAHALGCAV

LPGGS PAGGT

H-GLNNHQYVI

Amino acid sequence of PICCV (desII) (SEQ ID NO: 1 61 121 181 241 301 361 421 481

MTAPALSATA

SPFTPLEEAR

AALARKPVFP

SAMYFSGGLE

LYGLNDEEYE

DFIADLNDAG

DYGYALNSLR

IAGRVT PDTS RGFLR (SEQ

PAERCAHPGA

HDLGVDRDAF

YSVGLYPG PT

PLTNPGLGSL

QTTGKKAAFR

QGRTI DFVNI

TGADAELLRI

LTEVVRDFVE

ID DLGAAVI-iAVG

RRLLALFGQV

CMFRCHFCVR

AAHATDHGLR

RVRENLRRFQ

REDYSGRDDG

KPATMRPTAH

RGGEVAAVDG

QTLAAGGLVP

PELRTAVETG

VTGARYDPSA

PTVYTNSFAL

QLRAERESPI

KLPQEERAEL

PQVAVQVDLL

DEYFMDGFDQ

PDEAGTTARH

PAGAYWKNTL

LDAGNAMFRS

TERTLERQPG

NLGFAYIVLP

QEALNAFEER

GDVYLYREAG

VVTARLNQLE

LVRLAVRYGN

LPLEQRGVFD

VIDET PAGNP

LWGLHAIRTS

GRASRLLDLV

VRERTPGLHI

FPDLDGATRY

RDAADGWEEA

Amino acid sequence of 3-amino diniethyl transferase, PICCVI (des VI) (SEQ ID NO: 11) 1 VYEVDHADVY 61 GTGES 121 AVASFAEHLE 181 HFTVADPGKG ID NO:11) DLFYLGRGKD YAAEASDIAD LVRSRTPEAS SLLDVACGTG THLEHFTKEF DMLTHARKRL PDATLHQGDM RDFRLGRKFS AVVSMFSSVG YLKTTEELGA PGGVVVVEPW WFPETFADGW VSADVVRRDG RTVARVSHSV REGNATRMEV VRHFSDVHLI TLFHQAEYEA AFTAAGLRVE YLEGGPSGRG LFVGVPA (SEQ WO 99/61599 PCT/US99/11814 -33- Partial amino acid sequence of beta-glucosidase, ORFi 1 (desR) (SEQ ID NO: 12) 61 121 181 241 301 361 421 481 541 601 661 721

MTLDEKISFV

ASTFDDTMAD

AQIKGIQGAG

AYNGLNGKPS

PKGEPSPPAK

GAQAVSRKVA

APLDT IKARA

ADGEYRIAVR

SLELGWVTPA

DANPNTIVVL

AAENQHAVAG

TVVRTSTGGL

KTVTVNVDRR

HWALDPDRQN

SYGKVMGRDG

LMTTAKHFAA

CGNDELLNNV

FFGEALKTAV

ENGAVLLRNE

GAGATVTYET

ATGGYATVQL

AADAT IAKAV

NTGSSVLMPW

DPTSYPGVDN

KVTVTVRNSG

QLQFWDAAT D

VGYLPGVPRL

RALNQDMVLG

NNQENNRFSV

LRTQWGFQGW

LNGTVPEAAV

GQALPLAGDA

GEETFGTQI P

GSHTIEAGQV

ESARKARTAV

LSKTRAVLDM

QQTYREGI HV

KRAGQEVVQA

NWKTGTGNRL

GI PELRAADG

PMMNNIRVPH

NAN VDEQTLR VMS DWLATPG

TRSAERIVGQ

GKS IAVIGPT AGNLS PAFNQ

YGKVSSPLLK

VFAYDDGTEG

WY PGQAGAEA

GYRWFDKENV

YLGASPNVTA

LQTGSSSADL

PNGIRLVGQT ATALPAPVAL GGRNYETFSE DPLVSSRTAV EIEFPAFEAS SKAGAGSFMC TDAI TKGLDQ EMGVELPGDV MEKFGLLLAT PAPRPERDKA AVDPKVTGLG SAHVVPDSAA GHQLEPGKAG ALYDGTLTVP LTKGTHKLTI SGFAMSATPL VDRPNLSLPG TQDKLISAVA TAALLYGDVN PSGKLTQSFP KPLFPFGHGL SYTSFTQSAP PQAKKKLVGY TKVSLAAGEA RGSATVNVW (SEQ ID NO:12) Amino acid sequence of transcriptional activator, ORF 12 (regulatory) (SEQ ID NO: 13) 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 MNLVERDGE I

ALPGDRDIPL

TTGCS PS PAG

RAELLRQPHC

RQASHTTLGA

TTAAAVERI

EDT VARHLLV

PHLVAASWRM

P55 DNDALEL

PDNASVAQAE

WEAVFAATRA

QAERVLRQPV

IVPWRTSAAE

AEAVDMLHDS

PGRGGRRAKA

LTRVYRKLNV

AHLRAVLDAS

GVLCQLLRSA

TPFLVAVDDL

RNMWLSGLPP

AGGDEPVHGD

QELAAIGLLD

GGAPDAPWAL

NPHMTTRALA

SLTR4WLAAL

QILQGCRLSE

MIAIRCGDLP

PDAMFDS RHG

VYLRLGNRQK

GDRLEHARAL

VSTELELPGG

TRRADLPISL

AAGDGTLLLV

EQHGADTSAV

THADTASLRF

SGVRQLLAHY

AFAQAVLDCL

EDGTLGQPAI

PLLERGAQQA

LFDRLLSGEL

CPPLLESLPA

ETYEALETAL

TARERAELAL

MEYMHARGRY

ARALAEAQLA

AGMSRHQQAQ

PDVGLLSEAE

SGPAGSGKTE

RDLLDAASRR

LLYCAAHHDQ

YGPEAAERRA

HRSAEGTLET

REAALQDLPA

LFDDRLDDAF

PPSHPVMAL I

TPEPERGPVP

LVLVHADRLD

SHAAPESWGL

WLAXGRLHAA

LVRPGRSRTR

GDNYRARMTA

RRVAALAARG

LLRSLRRLAA

AGTS PPPPTR

GGIGFVMTER

PAYHATTGGN

ARWLAVLEQS

GERTELHRRA

RILEFAVRSS

RCLVWYGRLP

VRLAPRTTAL

RALFWS DALL

AVGMPLSALL

LGEFMLCGEI

GLTLRVLAAA

RLAGDMAWAC

LTNRQIARRL

ERETPVWSVR

RSASTRHTAC

ASQRAGYRVF

PLLLRALTQD

DPLLVERLTG

AEQLHRDGAD

TDNTQLARLA

EAADALSRLR

QAQAGVFQRG

AEAVERRSLG

LACTEAGEYE

LGSWNLDQPS

VDGQQAERLH

GAYPLAEEIV

CVTASTVEQH

AQDKSVTA (SEQ ID NO:13) Amino acid sequence of dNDP-glucose synthase (glucose-I1-phosphate thymidyl transferase), ORF 13 (desIII) (SEQ ID NO: 14) 1 61 121 181 241 NO: 14)

MKGIVLAGGS

FQSLLGNGRH

RDS IARLDGC VDIAKN IRPS

EERQGVWIAG

GT RLH PATS V

LGIELDYAVQ

VLFGYPVKDP

PRGELEITDV

LEEIAFRMGF

ISKQILPVYN

KEPAGIADAL

ERYGVAEVDA

NRVYLERGRA

I DAEACHGLG

KPMIYYPLSV

LVGAEHIGDD

TGRLTDLVEK

ELVNLGRGFA

EGLSRTEYGS

LMLGGIREIQ

TCALILGDNI

PVKPRSNLAV

WLDTGTHDSL

YLMEIAGREG

IISTPQHIEL

FHG PGLYTLL

TGLYLYDNDV

LRAAQYVQVL

AP (SEQ ID Amino acid sequence of dNDP-glucose 4,6-dehydratase, ORF 14 (deslV) (SEQ ID NO: 1 61 121 181 241 301

VRLLVTGGAG

BGDI RDAGLL

RVVHVSTDEV

NYGPYQHPEK

HIGGGLELTN

ADGLARTVRW

FIGSHFVRQL

ARELRGVDAI

YGSIDSGSWT

LI PLFVTNLL

RELTGILLDS

YRENRGWWEP

LAGAYPDVPA

VHFAAESHVD

ESSPLEPNSP

DGGTLPLYGD

LGADWS SVRK

LKATAPQLPA

DEVIVLDSLT YAGNRANLAP VDADPRLRFV RSIAGASVFT ETNVQGTQTL LQCAVDAGVG YAASKAGSDL VARAYHRTYG LDVRITRCCN GANVREWVHT DDHCRGIALV LAGGRAGETY VADRKGHDLR YSLDGGKIER ELGYRPQVSF TAVEVSA (SEQ ID Partial amino acid sequence of S-adenosylmethionine synthase, ORF 15 (SAM synthase) (SEQ ID NO: 16) 1 IGYDSSKKGF DGASCGVSVS IGSQSPDIAQ GVDTAYEKRV EGASQRDEGD ELDKQGAGDQ WO 99/61599 WO 9961599PCT/US99/1 1814 34- 61 121 181 241 301 GLMFGYAS DE LDT VVVS SQH

GGPMGDAGLT

RCEVQVAYAI

QTAAYGHFGR

TPELMPLPIH

ASDIDLESLL

GRKIIIDTYG

GKAEPVGLFV

ELPDFTWERT

LAHRLSRRLT

APDVRKFVVE

GMARHGGGAF

ETFGTHKIET

DRVDALKKAA

EVRKNGTT PY

HVLAQLVEDG

SGKDPSKVDR

EKIENAIGEV

GL (SEQ ID

LRPDGKTQVT

I KLDTDGYRL

SAAYAMRWVA

FDLRPAAI IR NO: 16)

IEYDGDRAVR

LVNPTGRFEI

KNVVAAGLAS

DLDLLRPIYS

Partial amino acid sequence of ORF 16 (homologous to M NO:17) 1 MPJAVTGSIA TDHLMTFPGR FAEQILPDQL AHVSLSFLVD 61 GRRPVLVGAV GKDFDGYGQL LRAAGVDTDS VRVSDRQHTA 121 MAEARDIDLG ETAGRPGGID LVLVGADDPE AMVRHTRVCR 181 SVRELVDGAE LLFTNAYERA LLLSKTGWTE QEVLARVGTW tuberculosis cbhK) (SEQ ID TLDIRHGGVA ANIAYGLGLL RFMCTTDEDG NQLAS FYAGA ELGLRRAADP SQQLARLEGD ITTLGAKGCR (SEQ ID NO:17) While not all of the insert DNA of cosmid pKOSO23-26 has been sequenced, five large contigs shown of Figure 3 have been assembled and provide sufficient sequence information to manipulate the genes therein in accordance with the methods of the invention.

The sequences of each of these five contigs are shown below.

Contig 00 1 from cosmid pKOSO23-26 contains 2401 nucleotides, the first 100 bases of which correspond to 100 bases of the insert sequence of cosmid pKOS023-27. Nucleotides 80 2389 constitute ORFI 1, which encodes 1 beta glucosidase. (SEQ ID 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861

CGTGGCGGCC

GGCGGAGCTC

GGACCCCGAC

GCTGCGTGCC

GCCCGCGCCG

GGTCATGGGC

CAACATCCGG

CTCCTCGCGC

GGCCAAGCAC

CGACGAGCAG

CGCGGGCTCC

CGAGCTCCTC

CTGGCTCGCC

CGAGCTCCCC

GGCGCTGAAG

GGAGCGGATC

GCCCGAGCGC

GGTLGCTCCTG

CG'CGGTCATC

CGTCCCGGAC

GACGGTGACG

CAGCCCGGCG

CGGCACGCTG

TTACGCCACG

GAGCAGCCCG

GA.TGAGTGCC

GACGATCGCG

CGACGACGGC

GCTGATCTCG

GTCGGTGCTG

CCAGGCGGGC

GCTCACGCAG

GCCGCTCCCG

GTCGCCCAGA

CGGCAGAACG

GCCGACGGCC

GTCGCCCTGG

CGCGACGGTC

GTGCCGCACG

ACCGCGGTCG

TTCGCGGCCA

ACGCTCCGCG

TTCATGTGTG

AACAACGTGC

ACCCCGGGCA

GGCGACGTCC

ACGGCCGTCC

GTCGGCCAGA

GACAAGGCGG

CGCAACGAGG

GGCCCGACGG

TCGGCGGCGG

TACGAGACGG

TTCAACCAGG

ACCGTGCCCG

GTGCAGCTCG

CTCCTCAAGC

ACCCCGCTCT

AAGGCCGTG

ACCGAGGGCG

GCTGTCGCGG

ATGCCGTGGC

GCCGAGGCCA

AGCTTCCCGG

GCGCCGCCGA

rGACGCTCGA

TCGGCTACCT

CGAACGGCAT

CCAGCACCTT

GCGCGCTCAA

GCGGCCGGAA

CCCAGATCAA

ACAACCAGGA

AGATCGAGTT

CCTACAACGG

TGCGCACGCA

CCGACGCCAT

CGAAGGGCGA

TGAACGGCAC

TGGAGAAGTT

GTGCCCAGGC

GCCAGGCCCT

CCGTCGACCC

CGCCACTCGA

GTGAGGAGAC

GCCACCAGCT

CCGACGGCGA

GCAGCCACAC

TGACCAAGGG

CCCTGGAGCT

AGTCGGCGCG

TCGACCGTCC

ACGCCAACCC

TGTCCAAGAC

CCGCCGCGCT

CCGCCGAGAA

CACGGCCAAT

CGAGAAGATC

TCCCGGCGTG

CCGCCTGGTG

CGACGACACC

CCAGGACATG

CTACGAGACC

GGGCATCCAG

GAACAACCGC

CCCGGCGTTC

CCTCAACGGG

GTGGGGCTTC

CACCAAGGGC

GCCCTCGCCG

GGTCCCCGAG

CGGTCTGCTC

GGTGTCCCGC

GCCGCTCGCC

CAAGGTCACC

CACCATCAAG

CTTCGGGACG

CGAGCCGGGC

GTACCGCATC

CATCGAGGCC

CACGCACAAG

GGGCTGGGTN

GAAGGCCCGT

GAACCTGTCG

GAACACGATC

CCGCGCGGTC

CCTCTACGGT

CCAGCACGCG

GTTCAGTACA

AGCTTCGTCC

CCGCGTCTGG

GGGCAGACCG

ATGGCCGACA

GTCCTGGGCC

TTCAGCGAGG

GGTGCGGGTC

T TCT CCGT GA

GAGGCGTCCT

AAGCCGTCCT

CAGGGCTGGG

CTCGACCAGG

CCGGCCAAGT

GCGGCCGTGA

CTCGCCACTC

AAGGTCGCCG

GGTGACGCCG

GGCCTGGGCA

GCCCGCGCGG

CAGATCCCGG

AAGGCGGGGG

GCGGTCCGTG

GGTCAGGTCT

CTCACGATCT

ACGCCGGCGG

ACGGCGGTCG

CTGCCGGGTA

GTGGTCCTCA

CTGGACATGT

GACGTCAACC

GTCGCCGGCG

CGAGCCGGGC

ACTGGGCGCT

GCATCCCGGA

CCACCGCGCT

GCTACGGCAA

CGATGATGAA

ACCCCCTGGT

TGATGACCAC

ACGCCAATGT

CCAAGGCCGG

GCGGCAACGA

TGATGTCCGA

AGATGGGCGT

TCTTCGGCGA

CGCGGTCGGC

CGGCGCCGCG

AGAACGGCGC

GCAAGAGCAT

GCGCCCACGT

GTGCGGGTGC

CGGGGAACCT

CGCTGTACGA

CCACCGGTGG

ACGGCAAGGT

CGGGCTTCGC

CGGCCGACGC

TCTTCGCCTA

CGCAGGACAA

ACACCGGTTC

GGTACCCGGG

CGAGCGGCAA

ACCCGACCAG

WO 99/61599 WO 9961599PCTIUS99/1 1814 1921 1981 2041 2101 2161 2221 2281 2341

CTACCCGGGC

GTTCGACAAG

GTTCACGCAG

CACGGTCCGC

CAGCCCGAAC

GCTCGCCGCG

CTGGGATGCC

TTCGTCCTCC

GTCGACAACC

GAGAACGTCA

AGCGCCCCGA

AACAGCGGGA

GTGACGGCTC

GGCGAGGCGA

GCCACGGACA

GCCGACCTGC

AGCAGACGTA

AGCCGCTGTT

CCGTCGTGCG

AGCGCGCCGG

CGCAGGCGAA

AGACGGTGAC

ACTGGAAGAC

GGGGCAGCGC

CCGCGAGGGC

CCCGTTCGGG

TACGTCCACG

CCAGGAGGTC

GAAGAAGCTC

GGTGAACGTC

GGGAACGGGC

CACGGTCAAC

ATCCACGTCG

CACGGCCTGT

GGTGGTCTGA

GTCCAGGCGT

GTGGGCTACA

GAGCCGCCGT C

AACCGCCTCC

GTCTGGTGAC

GGTACCGCTG

CGTACACCTC

AGGTCACGGT

ACCTCGGTGC

CGAAGGTCTC

AGCTGCAGTT

TGCAGACCGG

GTGACGCCGT

2401 G (SEQ ID Contig 002 from cosmid pKOSO23-26 contains 5970 nucleotides and the following ORFs: from nucleotide 995 to 1 is an ORF of picCIV that encodes a partial sequence of an amino transferase-dehydrase; from nucleotides 1356 to 2606 is an ORY of picK that encodes a cytochrome P450 hydroxylase; and from nucleotides 2739 to 5525 is ORF 12, which encodes a transcriptional activator. (SEQ ID NO:2 1) 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281

GGCGAGAAGT

CGGTGGATGC

CCGTGGCGGT

SCGGCGTGGT

CCCATGGCGG

GGCAGGTCGA

GTGACGACGG

TCGGCGTCGC

GCGTCGAAGT

CAGGGGCGGC

GCCACCTGGT

AGGCCGATCC

TCGCCGGTGA

GCCACGGCAT

GGGCCGCCGT

GCGCGGTCGA

CCGAAGAATG

CGAAATGATT

GACGAGATGG

CCGACAAGAG

TCCATCGGCG

GGAGGGCCGG

TTCCGTTCCG

CCGCTTCTCC

ATCCGACGTA

GGGACGAGGT

GGTTCAGCAA

ACCACAACAT

GTGAGTT GAG

GGCTCGTGGA

CCTGGCCGCT

CCGCCTTCCG

CCGCCATGGC

ACGGCGAGGA

CCTCCGAGGA

TCAATCTGAT

TGCGGGCCGA

CGGTGGAATC

TCCCGGCCGG

AGGCGCGGGT

CGGTGGTGGC

CGTGGTCGGC

TGCGCCGGTT

CGGCGGCCTC

AGCCGAAGTT

CGCCGCCCTC

CGAGGCTGCC

ACAGCCGCAG

CCCAGAGGTG

CCGGGTCGAG

AGCGCAGTGC

GGCCGGCGGC

GCCGGACCCC

TGGACAGCCA

TGCGGTTGGG

CGAGGTCGGA

CGCCGATCCG

TCGATTGTGG

CACGCTATGC

GCCCGAATGT

GGCGCGCTCC

CTTCCGGCCC

CCCGGTACTC

CGCGAGACTG

GTGGCTGGTC

GGACTGGCGC

GCTGGAGTCC

CAT GCGCCGG

CGCCATGCTG

GCCGATCACC

CGTCTGGACC

CGAGATGAGC

*CCTGCTCAGC

*GCTGCTCGGT

CGCCAACGGC

CATGACGCTC

CGCGACCTAC

TGACACGGTC

GTGCACGCCT

CTCGTCGATC

GACGAGGACG

CCGGTCGATG

GCTCATCTTG

GTGGAGGGCG

GAAGGCGTTG

GGCGGGCCGG

GCCGTGCTCG

GACGCCGACG

GTTGCCGGTG

GTGCGGGGTG

GTGCGCGAGG

GGCGAGCCCG

CTGGCTGTCG

CCG CCC CACG

TAAGGCGCTT

GGAATCCCGA

TGGTCGATTT

GCTCTCGATG

GATGATCCTT

GCGGAAGAGT

GGTCTGGAGT

GACCTCGGGG

CGTGCCGAGG

GTCGGCTACG

AACTCCACGA

GACCCGCCGC

GTCGAGTTGC

GCGGCGCCCG

GTGATCTCCG

GACGCCTTCG

GGCTATCTCI

GCGCTCGTGC

ATGGCCCACT

ATGTACGCGC

TTGGACGGCC

CGCTTCCCGC

CTCGTCGTCC

TCGGCCTTCA

TCGACGATCA

CCGGGGAGGT

AC CTCGGGAA

GCGTTGGTCC

CGGATCCGGG

ACGGCCTTGG

CCGTCGACCG

TCGGCGACCT

ACGGCCGAGG

TCCGGGTCGA

GCGGCGAACG

AGCTGGAGCC

GCGACGCGCT

AGGGCCCGGT

AGGAGCGGCT

TTCACGGATG

ACGAGGTCGC

CGGGGGGACT

TGCTTCGGAT

GACAGGATCC

ACGTGTGAGA

TCTCCGTGCG

CCCTGGGGCA

GTCCGGCCCA

ACCGGGCGCG

CTCCCCTGAC

GGCACACCCG

TGCGGCCCCG

ACGGCCGCGC

AACTCCTCGG

TCTTCCCGGA

CCCGGCTCAT

GGACCAGCGA

TCCTGCTCGT

TGCTCTCGCA

CGGTGGAGGPA

TCGAGCCCGI

TGGCCGACGC

GGACCTCCAT

CGTACTGGTG

CCGCGAGGTG

ACGCGTCGAG

CGCCGGCGGG

CGGCGAGGTC

TGGCGTGGAA

CGCAGCCGAG

TCCGCAGCTG

TGCGGGGTGT

TGTCGGCGAA

TCATCGACGG

CGGCCGTGGC

CCTCGAACTC

CGAGCCGCTC

GGTCGAAAGC

TTCCCTCCGG

CGCGCTCCAC

CTAATCCGCG

CACATCCGCC

GGGAATCAGC

AGTCCCGTTC

CCGTACCCAG

GGATTTCGCG

CCGGGTGCGC

GGCGGTCCTC

CGAGGCCGAG

GCTGCGCAAG

GGTCCAGGAG

CGATCTGATG

CGTGCCCGAG

*CGATCCCGCC

CGACTCCAAG

*CGAGGACGGC

CGCGGGGCAC

CCCCGACCAG

GATGTTGCGC

CGACCTGGAC

CCACCGCACC

GACGAGGTCG

GTTGTTGAGG

CTCGCGGTAG

GGAGGTGAGG

GCTGCCGCCG

GGCGTCGTCG

GCTGAAGACC

GGCGTGCGCG

GTCGGCGGCG

GACCGCGGCG

GACCGGGGTG

CATGATCACT

GTTGCAGGTG

GCGGACGAGC

GTACAGCCTG

GGCGGGGCCG

GCCACCGTCA

CGTGACGTAC

CGGAACGGGA

TCCGGGGTAT

CGAGCCGCCG

CTCTTCCCGT

CAGGGAACGA

GCCGATCCGT

ACCCCCGAGG

GCCGATCCCC

GCCGCGCTCA

CTGGTGGCCC

ATCGTCGACG

GAGTCCCTGG

CCGGACCGCG

CAGGCCCAGA

CGCGGGCAGG

TCCCGGCTGA

GAGACCACGG

CTGGCCGCCC

TACGAGGGCC

GGCACGGTCA

CCCGAGCGCT

WO 99/61599 WO 9961599PCT/US99/1 1814 -36- 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821

TCCCGGACCG

ACGGCATCCA

GCGCCCTTCT

GGTATCCGAA

GGGAGGCGGG

GAAGCCCCGG

CGAAGGGTTC

GGGAGATAGC

TACTCGTCTC

TGGCCGCCGA

TCCCCCTGGG

CCGCCGTCCG

CGACGCGCCG

CCGCCGGCAC

TGAGGTTCCT

CCGAGCGGGC

CGCACTGCCG

CCCACTACTA

GCGGGAACCC

TCGGCGCGGC

ACTGCCTGCA

AACAGTCCGA

GCCACATCCA

CCGCGATCCG

GGCGCGCCGC

TGCTGGTCGG

AGCAGGCCCT

GGTCGAGCAC

GGCGGATGAA

GTGAACTGCC

GGCTGCCCGA

TGGAGCTGTC

TGCCGGCCAC

CCGCGCTCCA

AGGCCGAACA

CGGCCCTCTT

CCCTGCTCGC

CCCGGGCGAT

TGGCGCTCTC

CGCTGCTGCT

AGCCGGTGCC

GCCGCTACTG

GGGAGATCCT

CCGCCGAGGT

AGCTCGCCCT

CGGCGGCGGT

ACGACAGCGG

AGGCCCAGGG

GGGCCTGCGG

CGAAGGCGGT

AGGCCGAACG

GCCGGCTCTG

TGAACGTGAC

CCTGAGCCAC

GGACACGCCG

AGGCGCCCGA

ACGCCAGGGA

CCCGGTGCGC

GGCGGCCGGG

GCACCGCTTC

CTTCTGCATC

CGAACGCTGC

CCCGATGATC

CCGCCGTACC

ATCGGTCCCC

GGCGCCCGGA

CCATCTCAGG

CGGACCGGCC

GCGGGAGACC

CGTCCTCTGC

CGACCTGCTG

CTCCGCGTCG

CCCGTTCCTC

CCTGTACTGC

CTCGCAGCGC

CAACATGTGG

CGGCCCCGAG

GCTGCTCCTG

CGGCGGCGAC

CCGCAGCGCC

CCCGCTCCTG

GGAGCTCGCC

CGAGGCCGCC

GGAGCAGCTG

CGGCGCCCCC

GTTCGACGAC

CGACAACACC

CCCGCACATG

GCCCAGCCAC

GGCCGCCGAC

GCTCACCCGG

GCCGGAGCCG

GGCCCAGGCC

GATCCTGCAG

GGTCCTCGTC

CGAGGCCGTG

GATCGCGATC

CCACGCGGCG

CGCCTGCACG

GGACGCGATG

GCTGGCGANC

GGGCAGCTGG

GTACCTGCGG

GGTGCGGCCC

GGACGGCCAG

CGACCGGCTC

GGACAACTAC

CGCGTACCCG

GAGCACGGAG

CCGGGTGGCG

CGTCACCGCG

CCGCCGAGCA

CCCCGGTGTC

GTGCGACACG

GGCGCCCGGT

*CCGCTGGGGA

*CCGGGGACAC

GTGTCCTTCA

3ACATCCGCC 3GCGCCCCCT

CGGACCTCG

CGCGGGCTCA

3GTTGAACCC

CCTCGCCGTA

CGAGGGGGGA

GCCGTTCTTG

GGCAGCGGGA

CCCGTCTGGT

CAGTTACTCC

GACGCCGCCT

ACGAGACACA

GTCGCCGTCG

GCCGCCCACC

GCCGGATACC

CTCTCCGGGC

GCCGCCGAGC

CGGGCG CT GA

GAGCCCGTCC

GAGGGCACAC

GTGGAGCGGC

GCCATCGGCC

CTCCAGGACC

CACCGGGACG

GACGCTCCCT

CGACTCGACG

CAGCTGGCCC

ACGACCCGGG

CCGGTCATGG

GCGCTGTCCC

ATGTGGCTCG

GAGCGGGGTC

GGCGTCTTCC

GGCTGCCGGC

CACGCCGACC

GAGCGGCGGT

CGCTGCGGCG

CCGGAGAGCT

GAGGCCGGCG

TTCGACTCGC

GGCCGGCTGC

AACCTCGACC

CTCGGCAACC

GGGCGCTCCC

CAGGCGGAGC

GAACACGCCC

CGGGCGAGGA

CTGGCCGAGG

CTGGAACTGC

GCCCTGGCAG

AGCACGGTCG

GACCTCCCGA

CCCGTGCGAC

GGGGCGCGCC

GCGGCACCCG

CACCGGGACC

CAGACCGCCG

TCGGTGGGCC

3GGACACCGC C I'GGCCCGGTT G CCCTGGACGT C kGGCCCTGCC C GCACGTCACC C k.CAAGACCTG C CTTCCGCGAT C kCGCATCCGC C 0,GACGGAGCT C CGGTCCGGGC C GCAGCGCCGA I'

CGCGGCGGGC

CCGCCTGCAC

ACGACCTGAC

ACGACCAGGG

GGGTGTTCCG

TTCCCCCCAG

GGCGGGCCCC

CCCAGGACCG

ACGGCGACGC

TGGAGACCGC

TCACGGGAAC

TCCTGGACGA

TGCCGGCCGG

GCGCCGACGA

GGGCGCTGCC

ACGCCTTCCG

GCCTCGCCCC

CCCTCGCACT

CCCTGATCCG

GGCTGCGGCC

CGGCGCTGTG

CCGTCCCCGT

AGCGGGGCCC

TGTCGGAGGA

GGCTCGACCG

CGCTCGGCTG

ACCTCCCGAC

GGGGCCTCGC

AGTACGAACA

GGCACGGCAT

ACGCGGCGCT

AGCCCTCGAT

GCCAGAAGGC

GCACCCGGGG

GGCTGCACGC

GCGCGCTCGC

TGACGGCGCG

AGATCGTGCC

CGGGCGGCCC

CCCGAGGATT

AACAGCACCT

TCAGCCTCGC

GACCCGCCGC

AGGTGCCATG

GAGACGCCAG

TCAGGGACCG

GGACCACCCG

TTCATCGGCA

GGCCATCTC G ;GAGGCCCGG P~ TCCCCCGGC C ;ATCCGCTGG C ATTACGACT C ;TTAGAGTGA I ;AATCTGGTG C ~GCAGGTGAC C ;CTGCGGTCG ;CTGcCGGGT C

.CAACACGGTC

,GGAACCTCA

;ACTGGCTGC

:CACGCCGAC I

GGCATCGGC

MGCCGAGCTG

:GGGGTACGC

'GCGTACCAC

GCAGGCCTCC

CTTCGCCCAG

CCGCTGGCTC

GACCGCCGCC

GGACGGCACC

CGAGCGCACC

GGACACCGTG

CCTGCTCGAA

GATCCTCGAG

PCACCTGGTC

CTTCGACCGG

CTGCCTCGTC

CAGCTCCGAC

CCCGCCGCTC

ACGGCTCGCG

GGACAACGCC

GACGTACGAG

GGCGCTGTTC

GGAGGCGGTC

GGCGCGGGAG

CGTGGGCATG

GGCGGAGCGG

GGAGTACATG

GGGCGAGTTC

CGTGCCCTGG

CAGGGCGCTG

TCTCACCCTG

CGAGGCGGTC

CGGGATGAGC

GCTCGCCGGC

GGGCCGCGGC

GGACGTCGGC

GACGAACCGC

GACGCGCGTC

CCAGGACAAG

ACGGGCCACC

GGGACCTCCG

GACCGCCGGG

CCGGGACCGC

AGGGTGCCCG

GGAGGAAGCG

;CCTTCGGCC

~TCGCCGTCC

;AACTCGTGT

GGCGAGGAC

~CTTGTCACG

'GGAGGACGA

;AACGCGACG

;GGACGCTCT

TCCGCCGGC

ACCGCGACA

CCGACACCT

~CTCCCCCGC

rCTCCGTCTC

~CCGCGTCCC

r'TCGTCATGA

,TCCGCCAGC

'AGTTACTCG

3CGACGACCG

'ACACCACCC

GCCGTCCTCG

GCGGTCCTCG

GCCGTCGAGC

CTGGGACAGC

GAACTGCACC

GCCCGCCACC

CGGGGCGCGC

TTCGCCGTGC

GCGGCCTCCT

CTCCTGAGCG

TGGTACGGNC

A.ACGATGCCT

CTGGAGTCCC

CCGCGGACGA

TCGGTCGCGC

GCCCTGGAGA

TGGTCGGACG

TTCGCCGCGA

CGGGCCGAGC

CCCCTCTCCG

GTCCTGCGGC

CACGCCCGGG

ATGCTCTGCG

CGGACCTCCG

GCCGAGGCCC

CGGGTCCTGG

GACATGCTGC

CGCCACCAGC

GACATGGCGT

GGCCGCCGGG

CTGCTCTCGG

CAGATAGCGC

TACCGCAAAC

TCCGTCACGG

GGGCCCGCCG

TGACCGCCCG

ACCACCGGAG

CCGAGTTGCA

GTGTGGCCCC

ACCGTGAGAC

WO 99/61599 PCT)US99/1 1814 -37- 5881 CCGTCGTGCC GTCGGCGATC AGCCGCCTGT ACGGGCGTCG GACTCCCTGG CGGTCCCGGA 5941 CCCGTCGTAC GGGCTCGCGG GACCCGGTGG (SEQ ID NO:21) Contig 003 from cosmid pKOSO23-26 contains 3292 nucleotides and the following ORFs: from nucleotide 104 to 982 is ORF 13, which encodes dNDP glucose synthase (glucose-lI-phosphate thymidyl transferase); from nucleotide 1114 to 2127 is ORF 14, which encodes dNDP-glucose 4,6-dehydratase; and from nucleotide 2124 to 3263 is the picCI ORE.

(SEQ ID NO:22) 61.

121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281.

2341 2401 2461 2521 2581 2641 2701

ACCCCCCAAA

ACGCCGACCG

GGCCGGCGGG

TCCGGTCTAC

TCGCGAGATT

AAACGGCAGG

CGCGGACGCA

GGGCGACAAC

CCTCGACGGC

CGAGGTGGAC

CAACCTCGCC

CATCCGGCCC

GCGGGGCCGG

CCACGACTCG

CTGGATCGCG

TCACGGCCTG

CGGCCGCGAG

GCCACCGACA

GCGACCTACA

GGCTTCATCG

GCCGATGAGG

CCGGTGGACG

CTCGCCCGGG

GACCGCTCCA

CTGCTCCAGT

GTGTACGGGT

CCCTACGCGG

GGCCTCGACG

AAGCTCATCC

GACGGCGCGA

GTCCTCGCGG

AACCGCGAAC

AAGGTCGCCG

CGCGAGCTCG

TGGTACCGGG

GCCACCGCCG

TCGACCTCAA

TCCTCGACTC

CCGCGTACTG

TCGCCCTCCG

ACATCGCCAG

ACGAGGACCA

GGGCGCTCCT

TCGCGGACCG

ACCGGGGCCC

AGAACCTCGG

GGGGTGGTGA

TTATCACCGG

AGCGGAACTC

AACAAACCGA

CAAATCATCT

CACCTGGGAA

CTTCTCGTCG

ATCTTCCACG

TGCGTGCTCT

GCGACGGGCC

GTCACCGGCC

TCGCCGCGCG

GCCGAACTCG

CTCCTGCGGG

GGCCTTGAGG

GGAGAAGGCC

GGAGCCCCGT

GTGCGACCCA

GCGCGACCGA

GCTCGCACTT

TGATCGTCCT

CGGACCCGCG

AACTGCGCGG

TCGCGGGCGC

GCGCCGTCGA

CGATCGACTC

CGTCCAAGGC

TACGGATCAC

CCCTCTTCGT

ACGTCCGCGA

GCGGCCGGGC

TCACCGGCAT

ACCGCAAGGG

GCTACCGCCC

AGAACCGCGG

TGGAGGTGTC

GGCCGCCTAC

GGGGCGCTAC

CGAGACGGAC

CGGCCTCGGC

CTGGCTCGCC

CCCCACCCTC

CCCCGTCCAC

GCACGGCCTC

GCGGATCGGC

CTGCTTCGGC

CACTCCCCCT

CGCCCTGCTG

GGCTGCATCC

TGATCTACTA

CGACCCCCCA

TAGAACTCGA

GAGCCGAGCA

GGCCCGGCCT

TCGGCTACCC

GGCTGACCGA

TCTACCTCTA

GCGAGCTGGA

TCAACCTGGG

CCGCCCAGTA

AGATCGCCTT

TCTCCCGCAC

GAGGGCACCT

CACCGCGACC

AAGGAAGACG

CGTGCGGCAG

GGACAGCCTC

ACTGCGCTTC

CGTGGACGCG

GTCCGTGTTC

CGCCGGCGTC

CGGCTCCTGG

CGGCTCCGAC

CCGCTGCTGC

GACGAACCTC

GTGGGTGCAC

CGGCGAGATC

CCTCCTGGAC

CCACGACCTG

GCAGGTCTCC

CTGGTGGGAG

CGCGTGAGCA

GAGGAGCTCC

CTCCTCGGAC

CACGCCGTCG

ATCGGACCCG

GTGTCCGCCA

GACCCGCTGC

CTCTACGGGC

CACATCGTCC

GCCGGGTCGI

GACGGCGGCC

GGGCAGCCCC

CTAGTTTCCG

GGCGACCTCG

TCCGCTGTCG

GCACATCGAA

CTATGCGGTC

CATCGGCGAC

CTACACGCTC

GGTCAAGGAC

CCTCGTCGAG

CGACAACGAC

GATCACCGAC

CCGCGGCTTC

CGTCCAGGTC

CCGCATGGGC

CGAG TAG GGC

CGCGGCCGAC

CGCACCGCCA

GCAGTGCGGC

CTCCTCGCCG

ACCTACGCGG

GTCCACGGCG

ATCGTCCACT

ACCGAGACCA

GGCCGGGTCG

ACCGAGAGCA

CTCGTTGCCC

AACAACTACG

CTCGACGGCG

ACCGACGACC

TACCACATCG

TCGCTCGGCG

CGCTACTCCC

TTCGCGGACG

CCGCTCAAGC

GCCGCGCCGP.

GCGCGGAGAC

CCGAACTCGI!

GCGTGAACAC

GGGACGAGGI

*CCGGCGCGAC

TCGTCGAGAI

ACCCCGCCGI

AGGACGCCGC

*CGGTGGCCG(

CCGTCGTCA(

TAGCGCCCCC

AGAATGAAGG

GTCATTTCGA

GTTCTCATGC

CTCTTCCAGT

CAGAAAGAGC

GACACCTGCG

CTGCGGGACA

CCCGAGCGGT

AAGCCCGTCA

GTCGTCGACA

GTCAACCGCG

GCCTGGCTGG

CTGGAGGAGC

TTCATCGACG

AGCTATCTGA

GCGTTCCCAC

CCGACAGTGC

TTCTGGTGAC

GGGCGTACCC

GCAACCGCGC

ACATCCGCGA

TCGCGGCCGA

ACGTGCAGGG

TGCACGTCTC

GCCCGCTGGA

GCGCCTACCA

GGCCGTACCA

GGACGCTCCC

ACTGCCGGGG

GCGGCGGCCT

CCGACTGGTC

TCGACGGCGG

GCCTCGCGCG-

CGACCGCCCC

LGACCCCCCGC

CGACGCCGCC

AGGATTCGAC

CGGGATGGAC

GATCGTCCCC

CCCCGTGCCC

SGGCGATCACC

SCATGGACGCC

GCAGGGCCAC

GTTCAGCTTC

CGGCGACCC(

CTAACTCGCC

GAATAGTCCT

AGCAGATTCT

TCGGCGGTAT

CGCTTCTCGG

CCGCAGGAAT

CCCTGATCCT

GCATCGCGCG

ACGGCGTCGC

AGCCGCGCTC

TCGCCAAGAA

TCTACCTGGA

ACACCGGCAC

GGCAGGGCGT

CCGAGGCCTG

TGGAGATCGC

GACCGACAGC

GACCCACACC

CGGAGGTGCG

CGACGTGCCC

CAACCTCGCC

CGCCGGCCTC

GAGCCACGTG

CACGCAGACG

CACCGACGAG

GCCCAACTCG

CCGGACGTAC

GCACCCCGAG

GCTGTACGGC

CATCGCGCTC

GGAGCTGACC

CTCGGTCCGG

CAAGATCGAG

GACCGTCCGC

GCAGCTGCCC

GTCCCCTTCC

ATCGCCCGCG

GCGGAGTTG

GCCCTCCAGC

TCGCACACGT

GTCGAGCCGC

CCCCGCACCC

CTCCGCGAGC

GGCGCCCGCT

TACCCGGGCA

GAGCTCGCCG

WO 99/61599 WO 9961599PCT/US99/1 1814 -38- 2761 2821 2881 2941 3001 3061 3121 3181 3241 NO: 22)

AACGGCTCCG

GCACCAACTC

TGGACAGCTG

GACTGCCCGG

TCACCGTGCG

ACACCCTCAC

GGCCGGAAGG

TCGGCCCGCA

CCGAGCGGGT

GATGCTCCGC

CCGCCTGGAC

GAACGGCCGC

CATCGGCCTG

CACCGAGCGC

GCACTACCCG

CTCGCTCCCG

CCTGGAGCGC

CGACCAGGCC

AACTACGGCT

GAGATGCAGG

AGGTCGGCGC

CCGGTGACCG

CGCGACGAGC

GTACCCGTGC

CGGGCCGAGA

CCGCAGGCGC

TAGTCAGGTG

CGCGGCAGAA

CCGCCGTGCT

TGGCCGCGGA

CGCCCGACAC

TGCGCAGCCA

ACCTCTCGCC

GCTTCGCGCG

TGCGGGTGAT

GTCCGGTAGA

GTACAGCCAC

GCGGATCCGG

GTACCTCTCC

CGACCCGGTC

CCTCGACGCC

GGCCTACGCG

GCAGGTCCTC

CGACGCCGTG

CCCAGCAGGC

GAGACGAAGG

CTCGNCCACC

GGGCTCGCG

TGGCACCTCT

CGCGGCATCG

GGCGAGGCAC

AGCCTGCCGA

CGCGAATGGG

CG (SEQ ID Contig 004 from cosmid pKOSO23-26 contains 1693 nucleotides and the following ORFs: from nucleotide 1692 to 694 is ORFi15, which encodes a part of S-adenosylmethionine synthetase; and from nucleotide 692 to 1 is ORF 16, which encodes a part of a protein homologous to the M tuberculosis cbhK gene. (SEQ ID NO:23) 1 ATGCGGCACC 61 TGCTCGGTCC 121 AGCTCGGCTC 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681

GAGGGGTCCG

GCCTCGGGGT

TCGCCCAGGT

TTGCCGTCCT

ACGGAGTCGG

CCGACGGCGC

GCGGCGACGC

GCGAGCTGGT

GTGGCGATCG

GTCCACGCGG

GGCGGCGGTC

GCGGAGGTCG

GCCGAAGGTC

GACCTCGCAG

GTACGCGGCG

GCGGGCCATG

CATCGGGCCG

GTCGAGCTTG

CACGTCGGGG

GACCGTGTCG

GTCGGGACGC

ACGGTGCGCG

GAACATCAGG

GGAGGCACCC

CTGCGACCCG

GTCGTACCCG

CCTTGGCGCC

AGCCCGTCTT

CGTCGACGAG

CGGCCCGGCG

CGTCCGCGCC

CGATGTCGCG

CGTCGGTGGT

TGTCGACGCC

CGACGAGGAC

CGCCGTGCCG

CCGGCAGGAT

ACCCGGTGAC

TCGGTGCGCT

TGGGAGTAGA

AAGACCTCGC

TCGACGAAGA

CGCGAGGCGA

GAGCGGTCGA

CCGCCGTAGG

CCGATCTCGA

ATGCCGTCCT

GCGAGCAGCG

AGACGGACCG

AGGTACGGGA

AGGTGGATCG

CCCTGGTCAG

TCGACCCGCT

ATGGACACCG

GAGCGTGGTG

GCTGAGCAGC

CTCCCGGACG

GAGGCCCAGC

GACGAGGACG

CGCCTCGGCC

GCACATGAAG

CGCGGCGCGG

GGGGCGGCGA

GATGTCCAGG

CTGCTCGGCG

GGCTATACGC

CCCAGGTGAA

TCGGGCGGAG

CGATGGCGTT

GGCCGACGGG

GACCGGCGGC

CCTTCGACGG

TGTCGATGAT

AGCGACCGGT

CGACGAGCTG

ACTCCAGGTC

CCCTGTCGCC

TGGTCCCGTT

GCAGCGGCAT

CGGCGCCCTG

TCTCGTACGC

ACACGCCGCA

ATCCAGGTGC

AGCGCCCGCT

CTGTCGCCCT

TCGCGGCAGA

AGGTCGATCC

ATCGCGCCCG

CGGGCGGTGT

AGCAGCTGCC

CCGAGCAGGC

GTGTCGACGA

AAGCGGCCCG

ATGTCAGAGC

GTCCGGCAGC

CAGGTCGAGG

CTCGATCTTC

CTCGGCCTTG

GACGACGTTC

GTCCTTGCCG

GATCTTGCGG

CGGGTTCACG

CGCAAGCACG

GATGTCCGAG

GTCGTACTCG

CTTGCGGACC

CAGCTCGGGC

CTTGTCGAGC

GGTGTCGACA

GGAGGCGCCG

CGACCCGGGC

CGTAGGCGTT

CCAGCCGGGC

CCCGCGTGTG

CGCCGGGCCG

CGTAGAACGA

GCTGACGGTC

CGTACCCGTC

CGAGGCCGTA

GGAACGACAG

GGAAGGTCAT

CCCGCGGCCT

TCGCGGCCGA

TCGCGGATGA

TCGGTCTCGA

CCGATCGCGT

TTCGCCACCC

GAGAAGGCGC

CCGGTGAGGC

AGCAGGCGGT

TGCTCGACGA

GCGTGCTGCG

ATGGTGACCT

TCGGTCAGGC

GTCTCGTCCG

TCGTCCCCCT

CCCTGGGCGA

TCGAAGCCCT

GAGCACCTCC

CGTGAACAGC

GAGCTGCTGC

CCGCACCATC

GCCGGCCGTC

GGCGAGCTGA

CGACACCCGC

GAAGTCCTTG

CGCGATGTTG

GGACACGTGG

CAGGTGGTCG

TCTTCAGGGC

AGTGGCCGTA

TCGCGGCCGG

TCTTGTGGGT

ACGCGACCTG

AGCGCATCGC

CGCCACCGTG

CGGCGTCGCC

AGCCGTCGGT

CGAACTTCCG.

AGGAGACGAC

GGGTCTTGCC

GGCGCGAGAG

AGGCATAGCC

CGTCCCGCTG

TGTCCGGGGA

TCTTCGAGGA

ATC (SEQ ID NO:23) Contig 005 from cosmid pKOSO23-26 contains 1565 nucleotides and contains the ORF of the picCV gene that encodes PICCY, involved in desosamine biosynthesis. (SEQ ID NO:24) 1 61 121 181 CCCCGCTCGC GGCCCCCCAG ACATCCACGC CCACGATTGG ACGCTCCCGA TGACCGCCCC CGCCCTCTCC GCCACCGCCC CGGCCGAACG CTGCGCGCAC CCCGGAGCCG ATCTGGGGGC GGCGGTCCAC GCCGTCGGCC AGACCCTCGC CGCCGGCGGC CTCGTGCCGC CCGACGAGGC CGGAACGACC GCCCGCCACC TCGTCCGGCT CGCCGTGCGC TACGGCAACA GCCCCTTCAC WO 99/61599 WO 9961599PCT/US99/1 1814 -39- 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561

CCCGCTGGAG

CGCCCTGTTC

GTACTGGAAG

CAGGAAGCCC

CTGCCACTTC

CAACGCCATG

CTTCTCCGGG

CACCGACCAC

CCTGGAGCGC

CAACGACGAG

GAACCTGCGC

CGCCTACATC

CGACCTCAAC

CAGCGGCCGT

CAACGCCTTC

CGCCCTGAAC

CATGCGGCCC

CCTGTACCGC

CGTGACCCCC

GGTGGCGGCC

CCGCCTGAAC

GCGCTGACCC

GGCCC (SEQ

GAGGCCCGCC

GGGCAGGTCC

AACACCCTGC

GTCTTCCCGT

TGCGTCCGTG

TTCCGGTCGG

GGCCTGGAGC

GGCCTGCGGC

CAGCCCGGCC

GAGTACGAGC

CGCTTCCAGC

GTGCTCCCGG

GACGCCGGGC

GACGACGGCA

GAGGAGCGGG

AGCCTGCGCA

ACCGCGCACC

GAGGCCGGCT

GACACCTCCC

GTCGACGGCG

CAGCTGGAGC

GCACCCGCCC

ID NO:24)

ACGACCTGGG

CGGAGCTCCG

TCGCGCTCGA

ACAGCGTCGG

TGACCGGCGC

TCATCGACGA

CGCTCACCAA

CCAGCGTCTA

TCTGGGGCCT

AGACCACCGG

AGCTGCGCGC

GCCGTGCCTC

AGGGCAGGAC

AGCTGCCGCA

TCCGCGAGCG

CCGGGGCCGA

CGCAGGTCGC

TCCCCGACCT

TCACCGAGGT

ACGAGTACTT

GCGACGCCGC

CGATCCCCCC

CGTCGACCGG

CACCGCGGTC

ACAGCGCGGC

CCTCTACCCC

CCGCTACGAC

GATACCCGCG

CCCCGGCCTC

CACGAACTCC

GCACGCCATC

CAAGAAGGCC

CGAGCGCGAG

CCGCCTGCTC

GATCGACTTC

GGAGGAGCGG

CACGCCCGGA

CGCCGAACTG

GGTGCAGGTC

GGACGGCGCG

CGTCAGGGAC

CATGGACGGC

GGACGGCTGG

GATCCCCCC

GACGCCTTCC

GAGACCGGCC

GTCTTCGACG

GGCCCGACCT

CCGTCCGCCC

GGCAACCCCT

GGGAGCCTGG

TTCGCGCTCA

CGCACCTCGC

GCCTTCCGCC

TCGCCGATCA

GACCTGGTCG

GTCAACATTC

GCCGAGCTCC

CTCCACATCG

CTGCGGATCA

GATCTCCTCG

ACCCGCTACA

TTCGTCGAGC

TTCGATCAGG

GAGGAGGCCC

CCACGATCCC

GGCGCCTCCT

GCGCCGGGGC

CGGCGCTCGC

GCATGTTCCG

TCGACGCCGG

CGGCGATGTA

CCGCGCACGC

CCGAGCGCAC

TCTACGGCCT

GCGTCCGCGA

ACCTCGGCTT

ACTTCATCGC

GCGAGGACTA

AGGAGGCCCT

ACTACGGCTA

AGCCCGCCAC

GCGACGTGTA

TCGCGGGCCG

GCGGCGGCGA

TCGTCACCGC

GCGGCTTCCT

CCCACCTGAG

The recombinant desosamine biosynthesis and transfer and beta-glucosidase genes and proteins provided by the invention are useful in the production of glycosylated polyketides in a variety of host cells, as described in Section IV below.

Section Ill. The Genes for Macrolide Ring Modification: the picK Hydroxylase Gene The present invention provides the picK gene in recombinant form as well as recombinant PicK protein. The availability of the hydroxylase encoded by the picK gene in recombinant form is of significant benefit in that the enzyme can convert narbomycin into picromycin and accepts in addition a variety of polyketide substrates, particularly those related to narbomycin in structure. The present invention also provides methods of hydroxylating polyketides, which method comprises contacting the polyketide with the recombinant PicK enzyme under conditions such that hydroxylation occurs. This methodology is applicable to large numbers of polyketides.

DNA encoding the picK gene can be isolated from cosmid pKOS023-26 of the invention. The DNA sequence of the picK gene is shown in the preceding section. This DNA sequence encodes one of the recombinant forms of the enzyme provided by the invention.

The amino acid sequence of this form of the picK gene is shown below. The present invention also provides a recombinant picK gene that encodes a picK gene product in which WO 99/61599 PCT/US99/11814 the PicK protein is fused to a number of consecutive histidine residues, which facilitates purification from recombinant host cells.

Amino acid sequence of picromycin/methymycin cytochrome P450 hydroxylase, PicK (SEQ ID NO:18) 1 VRRTQQGTTA SPPVLDLGAL GQDFAADPYP TYARLRAEGP AHRVRTPEGD EVWLVVGYDR 61 ARAVLADPRF SKDWRNSTTP LTEAEAALNH NMLESDPPRH TRLRKLVARE FTMRRVELLR 121 PRVQEIVDGL VDAMLAAPDG RADLMESLAW PLPITVISEL LGVPEPDRAA FRVWTDAFVF 181 PDDPAQAQTA MAEMSGYLSR LIDSKRGQDG EDLLSALVRT SDEDGSRLTS EELLGMAHIL 241 LVAGHETTVN LIANGMYALL SHPDQLAALR ADMTLLDGAV EEMLRYEGPV ESATYRFPVE 301 PVDLDGTVIP AGDTVLVVLA DAHRTPERFP DPHRFDIRRD TAGHLAFGHG IHFCIGAPLA 361 RLEARIAVRA LLERCPDLAL DVSPGELVWY PNPMIRGLKA LPIRWRRGRE AGRRTG (SEQ ID NO:18) The recombinant PicK enzyme of the invention hydroxylates narbomycin at the C 12 position and YC-17 at either the C10 or C12 position. Hydroxylation of these compounds at the respective positions increases the antibiotic activity of the compound relative to the unhydroxylated compound. Hydroxylation can be achieved by a number of methods. First, the hydroxylation may be performed in vitro using purified hydroxylase, or the relevant hydroxylase can be produced recombinantly and utilized directly in the cell that produces it.

Thus, hydroxylation may be effected by supplying the nonhydroxylated precursor to a cell that expresses the hydroxylase. These and other details of this embodiment of the invention are described in additional detail below in Section IV and the examples.

Section IV: Heterologous Expression of the Narbonolide PKS; the Desosamine Biosynthetic and Transferase Genes; the Beta-Glucosidase Gene; and the picK Hydroxylase Gene In one important embodiment, the invention provides methods for the heterologous expression of one or more of the genes involved in picromycin biosynthesis and recombinant DNA expression vectors useful in the method. Thus, included within the scope of the invention in addition to isolated nucleic acids encoding domains, modules, or proteins of the narbonolide PKS, glycosylation, and/or hydroxylation enzymes, are recombinant expression systems. These systems contain the coding sequences operably linked to promoters, enhancers, and/or termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the invention to contain these sequences either as extrachromosomal elements or integrated into the chromosome. The invention also provides Wn 00/61 A;9 PCT/US99/11814 -41methods to produce PKS and post-PKS tailoring enzymes as well as polyketides and antibiotics using these modified host cells.

As used herein, the term expression vector refers to a nucleic acid that can be introduced into a host cell or cell-free transcription and translation medium. An expression vector can be maintained stably or transiently in a cell, whether as part of the chromosomal or other DNA in the cell or in any cellular compartment, such as a replicating vector in the cytoplasm. An expression vector also comprises a gene that serves to produce RNA, which typically is translated into a polypeptide in the cell or cell extract. To drive production of the RNA, the expression vector typically comprises one or more promoter elements.

Furthermore, expression vectors typically contain additional functional elements, such as, for example, a resistance-conferring gene that acts as a selectable marker.

The various components of an expression vector can vary widely, depending on the intended use of the vector. In particular, the components depend on the host cell(s) in which the vector will be introduced or in which it is intended to function. Components for expression and maintenance of vectors in E. coli are widely known and commercially available, as are components for other commonly used organisms, such as yeast cells and Streptomyces cells.

One important component is the promoter, which can be referred to as, or can be included within, a control sequence or control element, which drives expression of the desired gene product in the heterologous host cell. Suitable promoters include those that function in eucaryotic or procaryotic host cells. In addition to a promoter, a control element can include, optionally, operator sequences, and other elements, such as ribosome binding sites, depending on the nature of the host. Regulatory sequences that allow for regulation of expression of the heterologous gene relative to the growth of the host cell may also be included. Examples of such regulatory sequences known to those of skill in the art are those that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus.

Preferred host cells for purposes of selecting vector components include fungal host cells such as yeast and procaryotic, especially E. coli and Streptomyces, host cells, but single cell cultures of, for example, mammalian cells can also be used. In hosts such as yeasts, plants, or mammalian cells that ordinarily do not produce polyketides, it may be necessary to provide, also typically by recombinant means, suitable holo-ACP synthases to convert the recombinantly produced PKS to functionality. Provision of such enzymes is described, for WO 99/61599 PCT/IIS99/11814 -42example, in PCT publication Nos. WO 97/13845 and WO 98/27203, each of which is incorporated herein by reference. Control systems for expression in yeast, including controls that effect secretion are widely available and can be routinely used. For E. coli or other bacterial host cells, promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac), and maltose, can be used. Additional examples include promoters derived from genes encoding biosynthetic enzymes, and the tryptophan (trp), the betalactamase (bla), bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter Patent No. 4,551,433), can also be used.

Particularly preferred are control sequences compatible with Streptomyces spp.

Particularly useful promoters for Streptomyces host cells include those from PKS gene clusters that result in the production of polyketides as secondary metabolites, including promoters from aromatic (Type II) PKS gene clusters. Examples of Type II PKS gene cluster promoters are act gene promoters and tcm gene promoters; an example of a Type I PKS gene cluster promoter is the spiramycin PKS gene promoter.

If a Streptomyces or other host ordinarily produces polyketides, it may be desirable to modify the host so as to prevent the production of endogenous polyketides prior to its use to express a recombinant PKS of the invention. Such hosts have been described, for example, in U.S. Patent No. 5,672,491, incorporated herein by reference. In such hosts, it may not be necessary to provide enzymatic activities for all of the desired post-translational modifications of the enzymes that make up the recombinantly produced PKS, because the host naturally expresses such enzymes. In particular, these hosts generally contain holo-ACP synthases that provide the pantotheinyl residue needed for functionality of the PKS.

Thus, in one important embodiment, the vectors of the invention are used to transform Streptomyces host cells to provide the recombinant Streptomyces host cells of the invention.

Streptomyces is a convenient host for expressing narbonolide or 10-deoxymethynolide or derivatives of those compounds, because narbonolide and 10-deoxymethynolide are naturally produced in certain Streptomyces species, and Streptomyces generally produce the precursors needed to form the desired polyketide. The present invention also provides the narbonolide PKS gene promoter in recombinant form, located upstream of the picAI gene on cosmid pKOS023-27. This promoter can be used to drive expression of the narbonolide PKS or any other coding sequence of interest in host cells in which the promoter functions, particularly S. venezuelae and generally any Streptomyces species. As described below, however, wn oo/6599 PCIT/n.99/11814 -43promoters other than the promoter of the narbonolide PKS genes will typically be used for heterologous expression.

For purposes of the invention, any host cell other than Streptomyces venezuelae is a heterologous host cell. Thus, S. narbonensis, which produces narbomycin but not picromycin is a heterologous host cell of the invention, although other host cells are generally preferred for purposes of heterologous expression. Those of skill in the art will recognize that, if a Streptomyces host that produces a picromycin or methymycin precursor is used as the host cell, the recombinant vector need drive expression of only a portion of the genes constituting the picromycin gene cluster. As used herein, the picromycin gene cluster includes the narbonolide PKS, the desosamine biosynthetic and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene. Thus, such a vector may comprise only a single ORF, with the desired remainder of the polypeptides encoded by the picromycin gene cluster provided by the genes on the host cell chromosomal DNA.

The present invention also provides compounds and recombinant DNA vectors useful for disrupting any gene in the picromycin gene cluster (as described above and illustrated in the examples below). Thus, the invention provides a variety of modified host cells (particularly, S. narbonensis and S. venezuelae) in which one or more of the genes in the picromycin gene cluster have been disrupted. These cells are especially useful when it is desired to replace the disrupted function with a gene product expressed by a recombinant DNA vector. Thus, the invention provides such Streptomyces host cells, which are preferred host cells for expressing narbonolide derivatives of the invention. Particularly preferred host cells of this type include those in which the coding sequence for the loading module has been disrupted, those in which one or more of any of the PKS gene ORFs has been disrupted, and/or those in which the picK gene has been disrupted.

In a preferred embodiment, the expression vectors of the invention are used to construct a heterologous recombinant Streptomyces host cell that expresses a recombinant PKS of the invention. As noted above, a heterologous host cell for purposes of the present invention is any host cell other than S. venezuelae, and in most cases other than S. narbonensis as well. Particularly preferred heterologous host cells are those which lack endogenous functional PKS genes. Illustrative host cells of this type include the modified Streptomyces coelicolor CH999 and similarly modified S. lividans described in PCT publication No. WO 96/40968.

WO 99/61599 PCT/US99/11814 -44- The invention provides a wide variety of expression vectors for use in Streptomyces.

For replicating vectors, the origin of replication can be, for example and without limitation, a low copy number vector, such as SCP2* (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory manual (The John Innes Foundation, Norwich, 1985); Lydiate et al., 1985, Gene 35: 223-235; and Kieser and Melton, 1988, Gene 65: 83-91, each of which is incorporated herein by reference), SLP1.2 (Thompson et al., 1982, Gene 20: 51- 62, incorporated herein by reference), and pSG5(ts) (Muth et al., 1989, Mol. Gen. Genet. 219: 341-348, and Bierman et al., 1992, Gene 116: 43-49, each of which is incorporated herein by reference), or a high copy number vector, such as pIJ101 and pJV1 (see Katz et al., 1983, J.

Gen. Microbiol. 129: 2703-2714; Vara et al., 1989, J. Bacteriol. 171: 5782-5781; and Servin- Gonzalez, 1993, Plasmid 30: 131-140, each of which is incorporated herein by reference).

High copy number vectors are generally, however, not preferred for expression of large genes or multiple genes. For non-replicating and integrating vectors and generally for any vector, it is useful to include at least an E. coli origin of replication, such as from pUC, piP, plI, and pBR. For phage based vectors, the phage phiC31 and its derivative KC515 can be employed (see Hopwood et al., supra). Also, plasmid pSET152, plasmid pSAM, plasmids pSE101 and pSE211, all of which integrate site-specifically in the chromosomal DNA of S. lividans, can be employed.

Preferred Streptomyces host cell/vector combinations of the invention include S. coelicolor CH999 and S. lividans K4-114 host cells, which do not produce actinorhodin, and expression vectors derived from the pRMI and pRM5 vectors, as described in U.S.

Patent No. 5,830,750 and U.S. patent application Serial Nos. 08/828,898, filed 31 Mar. 1997, and 09/181,833, filed 28 Oct. 1998, each of which is incorporated herein by reference.

As described above, particularly useful control sequences are those that alone or together with suitable regulatory systems activate expression during transition from growth to stationary phase in the vegetative mycelium. The system contained in the illustrative plasmid the actllactlll promoter pair and the actl-ORF4 activator gene, is particularly preferred. Other useful Streptomyces promoters include without limitation those from the ermE gene and the melC1 gene, which act constitutively, and the tipA gene and the merA gene, which can be induced at any growth stage. In addition, the T7 RNA polymerase system has been transferred to Streptomyces and can be employed in the vectors and host cells of the invention. In this system, the coding sequence for the T7 RNA polymerase is inserted into a neutral site of the chromosome or in a vector under the control of the inducible merA WO 99/61599 PCT/S99/11 814 promoter, and the gene of interest is placed under the control of the T7 promoter. As noted above, one or more activator genes can also be employed to enhance the activity of a promoter. Activator genes in addition to the actlI-ORF4 gene described above include dnrl, redD, and ptpA genes (see U.S. patent application Serial No. 09/181,833, supra).

Typically, the expression vector will comprise one or more marker genes by which host cells containing the vector can be identified and/or selected. Selectable markers are often preferred for recombinant expression vectors. A variety of markers are known that are useful in selecting for transformed cell lines and generally comprise a gene that confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.

Such markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid. Alternatively, several polyketides are naturally colored, and this characteristic can provide a built-in marker for identifying cells. Preferred selectable markers include antibiotic resistance conferring genes. Preferred for use in Streptomyces host cells are the ermE (confers resistance to erythromycin and lincomycin), tsr (confers resistance to thiostrepton), aadA (confers resistance to spectinomycin and streptomycin), aacC4 (confers resistance to apramycin, kanamycin, gentamicin, geneticin (G418), and neomycin), hyg (confers resistance to hygromycin), and vph (confers resistance to viomycin) resistance conferring genes.

To provide a preferred host cell and vector for purposes of the invention, the narbonolide PKS genes were placed on a recombinant expression vector that was transferred to the non-macrolide producing host Streptomyces lividans K4-114, as described in Example 3. Transformation ofS. lividans K4-114 with this expression vector resulted in a strain which produced two compounds in similar yield (-5-10 mg/L each). Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (Figure 5, compound 4) and 10-deoxymethynolide (Figure 5, compound the respective 14 and 12-membered polyketide precursors of narbomycin and YC17.

To provide a host cell of the invention that produces the narbonolide PKS as well as an additional narbonolide biosynthetic gene and to investigate the possible role of the PIC TEII in picromycin biosynthesis, the picB gene was integrated into the chromosome to provide the host cell of the invention Streptomyces lividans K39-18. The picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992, Gene 116: 43, incorporated herein by reference) under control of the same promoter (Pactl) as the PKS on plasmid pKOS039-86.

WO 99/619 so PCT/US99/11814 -46- A comparison of strains Streptomyces lividans K39-18/pKOS039-86 and K4- 114/pKOS039-86 grown under identical conditions indicated that the strain containing TEII produced 4-7 times more total polyketide. This increased production indicates that the enzyme is functional in this strain and is consistent with the observation that yields fall to below 5% for both picromycin and methymycin when picB is disrupted in S. venezuelae.

Because the production levels of compound 4 and 5 from K39-18/pKOS03986 increased by the same relative amounts, TEII does not appear to influence the ratio of 12 and 14membered lactone ring formation. Thus, the invention provides methods of coexpressing the picB gene product or any other type II thioesterase with the narbonolide PKS or any other PKS in heterologous host cells to increase polyketide production. However, transformation of a 6-dEB-producing Streptomyces lividans/pCK7 strain with an expression vector of the invention that produces PIC TEII resulted in little or no increase in 6-dEB levels, indicating that TEII enzymes may have some specificity for their cognate PKS complexes and that use of homologous TEII enzymes will provide optimal activity.

In accordance with the methods of the invention, picromycin biosynthetic genes in addition to the genes encoding the PKS and PIC TEII can be introduced into heterologous host cells. In particular, the picK gene, desosamine biosynthetic genes, and the desosaminyl transferase gene can be expressed in the recombinant host cells of the invention to produce any and all of the polyketides in the picromycin biosynthetic pathway (or derivatives thereof).

Those of skill will recognize that the present invention enables one to select whether only the 12-membered polyketides, or only the 14-membered polyketides, or both 12- and 14membered polyketides will be produced. To produce only the 12-membered polyketides, the invention provides expression vectors in which the last module is deleted or the KS domain of that module is deleted or rendered inactive. If module 6 is deleted, then one preferably deletes only the non-TE domain portion of that module or one inserts a heterologous TE domain, as the TE domain facilitates cleavage of the polyketide from the PKS and cyclization and thus generally increases yields of the desired polyketide. To produce only the 14-membered polyketides, the invention provides expression vectors in which the coding sequences of extender modules 5 and 6 are fused to provide only a single polypeptide.

In one important embodiment, the invention provides methods for desosaminylating polyketides or other compounds. In this method, a host cell other than Streptomyces WO 99/61599 PCT/US99/11814 -47venezuelae is transformed with one or more recombinant vectors of the invention comprising the desosamine biosynthetic and desosaminyl transferase genes and control sequences positioned to express those genes. The host cells so transformed can either produce the polyketide to be desosaminylated naturally or can be transformed with expression vectors S 5 encoding the PKS that produces the desired polyketide. Alternatively, the polyketide can be supplied to the host cell containing those genes. Upon production of the polyketide and expression of the desosamine biosynthetic and desosaminyl transferase genes, the desired desosaminylated polyketide is produced. This method is especially useful in the production of polyketides to be used as antibiotics, because the presence of the desosamine residue is known to increase, relative to their undesosaminylated counterparts, the antibiotic activity of many polyketides significantly. The present invention also provides a method for desosaminylating a polyketide by transforming an S. venezuelae or S. narbonensis host cell with a recombinant vector that encodes a PKS that produces the polyketide and culturing the transformed cell under conditions such that said polyketide is produced and desosaminylated.

In this method, use of an S. venezuelae or S. narbonensis host cell of the invention that does not produce a functional endogenous narbonolide PKS is preferred.

In a related aspect, the invention provides a method for improving the yield of a desired desosaminylated polyketide in a host cell, which method comprises transforming the host cell with a beta-glucosidase gene. This method is not limited to host cells that have been transformed with expression vectors of the invention encoding the desosamine biosynthetic and desosaminyl transferase genes of the invention but instead can be applied to any host cell that desosaminylates polyketides or other compounds. Moreover, while the beta-glucosidase gene from Streptomyces venezuelae provided by the invention is preferred for use in the method, any beta-glucosidase gene may be employed. In another embodiment, the betaglucosidase treatment is conducted in a cell free extract.

Thus, the invention provides methods not only for producing narbonolide and deoxymethynolide in heterologous host cells but also for producing narbomycin and YC-17 in heterologous host cells. In addition, the invention provides methods for expressing the picK gene product in heterologous host cells, thus providing a means to produce picromycin, methymycin, and neomethymycin in heterologous host cells. Moreover, because the recombinant expression vectors provided by the invention enable the artisan to provide for desosamine biosynthesis and transfer and/or C10 or C12 hydroxylation in any host cell, the invention provides methods and reagents for producing a very wide variety of glycosylated WO 99/61599 PCT/US99/11814 -48and/or hydroxylated polyketides. This variety of polyketides provided by the invention can be better appreciated upon consideration of the following section relating to the production of polyketides from heterologous or hybrid PKS enzymes provided by the invention.

Section V: Hybrid PKS Genes The present invention provides recombinant DNA compounds encoding each of the domains of each of the modules of the narbonolide PKS, the proteins involved in desosamine biosynthesis and transfer to narbonolide, and the PicK protein. The availability of these compounds permits their use in recombinant procedures for production of desired portions of the narbonolide PKS fused to or expressed in conjunction with all or a portion of a heterologous PKS. The resulting hybrid PKS can then be expressed in a host cell, optionally with the desosamine biosynthesis and transfer genes and/or the picK hydroxylase gene to produce a desired polyketide.

Thus, in accordance with the methods of the invention, a portion of the narbonolide PKS coding sequence that encodes a particular activity can be isolated and manipulated, for example, to replace the corresponding region in a different modular PKS. In addition, coding sequences for individual modules of the PKS can be ligated into suitable expression systems and used to produce the portion of the protein encoded. The resulting protein can be isolated and purified or can may be employed in situ to effect polyketide synthesis. Depending on the host for the recombinant production of the domain, module, protein, or combination of proteins, suitable control sequences such as promoters, termination sequences, enhancers, and the like are ligated to the nucleotide sequence encoding the desired protein in the construction of the expression vector.

In one important embodiment, the invention thus provides a hybrid PKS and the corresponding recombinant DNA compounds that encode those hybrid PKS enzymes. For purposes of the invention, a hybrid PKS is a recombinant PKS that comprises all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a first PKS and all or part of one or more extender modules, loading module, and/or thioesterase/cyclase domain of a second PKS. In one preferred embodiment, the first PKS is most but not all of the narbonolide PKS, and the second PKS is only a portion or all of a nonnarbonolide PKS. An illustrative example of such a hybrid PKS includes a narbonolide PKS in which the natural loading module has been replaced with a loading module of another WO 99/61599 PCT/US99/11814 -49- PKS. Another example of such a hybrid PKS is a narbonolide PKS in which the AT domain of extender module 3 is replaced with an AT domain that binds only malonyl CoA.

In another preferred embodiment, the first PKS is most but not all of a nonnarbonolide PKS, and the second PKS is only a portion or all of the narbonolide PKS. An illustrative example of such a hybrid PKS includes a DEBS PKS in which an AT specific for methylmalonyl CoA is replaced with the AT from the narbonolide PKS specific for malonyl CoA.

Those of skill in the art will recognize that all or part of either the first or second PKS in a hybrid PKS of the invention need not be isolated from a naturally occurring source. For example, only a small portion of an AT domain determines its specificity. See U.S.

provisional patent application Serial No. 60/091,526, and Lau et al., infra, incorporated herein by reference. The state of the art in DNA synthesis allows the artisan to construct de novo DNA compounds of size sufficient to construct a useful portion of a PKS module or domain. Thus, the desired derivative coding sequences can be synthesized using standard solid phase synthesis methods such as those described by Jaye et al., 1984, J. Biol. Chem.

259: 6331, and instruments for automated synthesis are available commercially from, for example, Applied Biosystems, Inc. For purposes of the invention, such synthetic DNA compounds are deemed to be a portion of a PKS.

With this general background regarding hybrid PKSs of the invention, one can better appreciate the benefit provided by the DNA compounds of the invention that encode the individual domains, modules, and proteins that comprise the narbonolide PKS. As described above, the narbonolide PKS is comprised of a loading module, six extender modules composed of a KS, AT, ACP, and optional KR, DH, and ER domains, and a thioesterase domain. The DNA compounds of the invention that encode these domains individually or in combination are useful in the construction of the hybrid PKS encoding DNA compounds of the invention.

The recombinant DNA compounds of the invention that encode the loading module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for the loading module of the heterologous PKS is replaced by that for the coding sequence of the narbonolide PKS loading module provides a novel PKS. Examples WO 99/i 99 PCT/STOO/1 R14 include the 6-deoxyerythronolide B, rapamycin, FK506, FK520, rifamycin, and avermectin PKS coding sequences. In another embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS loading module is inserted into a DNA compound that comprises the coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative in a different location in the modular system.

In another embodiment, a portion of the loading module coding sequence is utilized in conjunction with a heterologous coding sequence. In this embodiment, the invention provides, for example, replacing the propionyl CoA specific AT with an acetyl CoA, butyryl CoA, or other CoA specific AT. In addition, the KS o and/or ACP can be replaced by another inactivated KS and/or another ACP. Alternatively, the KS

Q

AT, and ACP of the loading module can be replaced by an AT and ACP of a loading module such as that of DEBS. The resulting heterologous loading module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

The recombinant DNA compounds of the invention that encode the first extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS first extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the first extender module of the narbonolide PKS or the latter is merely added to coding sequences for modules of the heterologous PKS, provides a novel PKS coding sequence. In another embodiment, a DNA compound comprising a sequence that encodes the first extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative or into a different location in the modular system.

In another embodiment, a portion or all of the first extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (which includes inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the WO QQ/615 Q PCT/I TS/99/11814s -51heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous first extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

In an illustrative embodiment of this aspect of the invention, the invention provides recombinant PKSs and recombinant DNA compounds and vectors that encode such PKSs in which the KS domain of the first extender module has been inactivated. Such constructs are especially useful when placed in translational reading frame with the remaining modules and domains of a narbonolide PKS or narbonolide derivative PKS. The utility of these constructs is that host cells expressing, or cell free extracts containing, the PKS encoded thereby can be fed or supplied with N-acetylcysteamine thioesters of novel precursor molecules to prepare narbonolide derivatives. See U.S. patent application Serial No. 60/117,384, filed 27 Jan.

1999, and PCT publication Nos. WO 99/03986 and WO 97/02358, each of which is incorporated herein by reference.

The recombinant DNA compounds of the invention that encode the second extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS second extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the second extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS. In another embodiment, a DNA compound comprising a sequence that encodes the second extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.

In another embodiment, a portion or all of the second extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR, the DH, or both the DH and KR; replacing the KR or the KR and DH with a KR, a KR and a DH, or a KR, DH, and ER; and/or inserting an ER. In WO 99/61599 PCT/US99/11814 -52addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous second extender module coding sequence can be utilized in conjunction with a coding sequence from a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

The recombinant DNA compounds of the invention that encode the third extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS third extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the third extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.

In another embodiment, a DNA compound comprising a sequence that encodes the third extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.

In another embodiment, a portion or all of the third extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting the inactive KR; and/or inserting a KR, or a KR and DH, or a KR, DH, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a gene for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous third extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

The recombinant DNA compounds of the invention that encode the fourth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are WO 99/6199 PCT/ITO/11R14 -53useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS fourth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fourth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.

In another embodiment, a DNA compound comprising a sequence that encodes the fourth extender module of the narbonolide PKS is inserted into a DNA compound that comprises coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.

In another embodiment, a portion of the fourth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting any one, two, or all three of the ER, DH, and KR; and/or replacing any one, two, or all three of the ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous fourth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

The recombinant DNA compounds of the invention that encode the fifth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS fifth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the fifth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.

In another embodiment, a DNA compound comprising a sequence that encodes the fifth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the WO 99/61599 PCTIUS99/11814 -54coding sequence for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.

In another embodiment, a portion or all of the fifth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; deleting (or inactivating) the KR; inserting a DH or a DH and ER; and/or replacing the KR with another KR, a DH and KR, or a DH, KR, and ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous fifth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

The recombinant DNA compounds of the invention that encode the sixth extender module of the narbonolide PKS and the corresponding polypeptides encoded thereby are useful for a variety of applications. In one embodiment, a DNA compound comprising a sequence that encodes the narbonolide PKS sixth extender module is inserted into a DNA compound that comprises the coding sequence for a heterologous PKS. The resulting construct, in which the coding sequence for a module of the heterologous PKS is either replaced by that for the sixth extender module of the narbonolide PKS or the latter is merely added to coding sequences for the modules of the heterologous PKS, provides a novel PKS.

In another embodiment, a DNA compound comprising a sequence that encodes the sixth extender module of the narbonolide PKS is inserted into a DNA compound that comprises the coding sequences for the narbonolide PKS or a recombinant narbonolide PKS that produces a narbonolide derivative.

In another embodiment, a portion or all of the sixth extender module coding sequence is utilized in conjunction with other PKS coding sequences to create a hybrid module. In this embodiment, the invention provides, for example, replacing the methylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or carboxyglycolyl CoA specific AT; and/or inserting a KR, a KR and DH, or a KR, DH, and an ER. In addition, the KS and/or ACP can be replaced with another KS and/or ACP. In each of these replacements or insertions, the WO 99/61599 PCTIUS99/11814 heterologous KS, AT, DH, KR, ER, or ACP coding sequence can originate from a coding sequence for another module of the narbonolide PKS, from a coding sequence for a PKS that produces a polyketide other than narbonolide, or from chemical synthesis. The resulting heterologous sixth extender module coding sequence can be utilized in conjunction with a coding sequence for a PKS that synthesizes narbonolide, a narbonolide derivative, or another polyketide.

The sixth extender module of the narbonolide PKS is followed by a thioesterase domain. This domain is important in the cyclization of the polyketide and its cleavage from the PKS. The present invention provides recombinant DNA compounds that encode hybrid PKS enzymes in which the narbonolide PKS is fused to a heterologous thioesterase or a heterologous PKS is fused to the narbonolide synthase thioesterase. Thus, for example, a thioesterase domain coding sequence from another PKS gene can be inserted at the end of the sixth extender module coding sequence in recombinant DNA compounds of the invention.

Recombinant DNA compounds encoding this thioesterase domain are therefore useful in constructing DNA compounds that encode the narbonolide PKS, a PKS that produces a narbonolide derivative, and a PKS that produces a polyketide other than narbonolide or a narbonolide derivative.

The following Table lists references describing illustrative PKS genes and corresponding enzymes that can be utilized in the construction of the recombinant hybrid PKSs and the corresponding DNA compounds that encode them of the invention. Also presented are various references describing tailoring enzymes and corresponding genes that can be employed in accordance with the methods of the invention.

Avermectin U.S. Pat. No. 5,252,474 to Merck.

MacNeil et al., 1993, Industrial Microorganisms: Basic and Applied Molecular Genetics, Baltz, Hegeman, Skatrud, eds. (ASM), pp. 245-256, A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin.

MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

Candicidin (FR008) Hu et al., 1994, Mol. Microbiol. 14: 163-172.

WO QQ/61 09 Pr'T/I TOQ/11 R1 -56- Epothilone U.S. patent application Serial No. 60/130,560, filed 22 Apr. 1999, and Serial No.

60/122,620, filed 3 Mar. 1999.

Erythromycin PCT Pub. No. WO 93/13663 to Abbott.

US Pat. No. 5,824,513 to Abbott.

Donadio et al., 1991, Science 252:675-9.

Cortes et al., 8 Nov. 1990, Nature 348:176-8, An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea.

Glycosylation Enzymes PCT Pat. App. Pub. No. WO 97/23630 to Abbott.

FK506 Motamedi et al., 1998, The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506, Eur. J. Biochem. 256: 528-534.

Motamedi et al., 1997, Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506, Eur. J.

Biochem. 244: 74-80.

Methyltransferase US 5,264,355, issued 23 Nov. 1993, Methylating enzyme from Streptomyces MA6858. 31-O-desmethyl-FK506 methyltransferase.

Motamedi et al., 1996, Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520, J. Bacteriol. 178: 5243-5248.

FK520 U.S. patent application Serial No. 60/123,800, filed 11 Mar. 1999.

Immunomycin Nielsen et al., 1991, Biochem. 30:5789-96.

Lovastatin U.S. Pat. No. 5,744,350 to Merck.

Nemadectin MacNeil et al., 1993, supra.

wnoo /1 oo PCT//99/11814 -57- Niddaymcin Kakavas et al., 1997, Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis, J. Bacteriol. 179: 7515-7522.

Oleandomycin Swan et al., 1994, Characterization of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence, Mol. Gen. Genet. 242: 358-362.

Olano et al., 1998, Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring, Mol. Gen. Genet. 259(3): 299-308.

U.S. patent application Serial No. 60/120,254, filed 16 Feb. 1999, and Serial No.

60/106,100, filed 29 Oct. 1998.

Platenolide EP Pat. App. Pub. No. 791,656 to Lilly.

Pradimicin PCT Pat. Pub. No. WO 98/11230 to Bristol-Myers Squibb.

Rapamycin Schwecke et al., Aug. 1995, The biosynthetic gene cluster for the polyketide rapamycin, Proc. Natl. Acad. Sci. USA 92:7839-7843.

Aparicio et al., 1996, Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase, Gene 169: 9-16.

Rifamycin August et al., 13 Feb. 1998, Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rifbiosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry Biology, 69-79.

Soraphen U.S. Pat. No. 5,716,849 to Novartis.

Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangium cellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of the Macrolide Antibiotic Soraphen A: Cloning, Characterization, and Homology to Polyketide Synthase Genes from Actinomycetes.

WO 99/61599 PCT/US99/11814 -58- Spiramycin U.S. Pat. No. 5,098,837 to Lilly.

Activator Gene U.S. Pat. No. 5,514,544 to Lilly.

Tylosin EP Pub. No. 791,655 to Lilly.

Kuhstoss et al., 1996, Gene 183:231-6., Production of a novel polyketide through the construction of a hybrid polyketide synthase.

U.S. Pat. No. 5,876,991 to Lilly.

Tailoring enzymes Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355. Analysis of five tylosin biosynthetic genes from the tylBA region of the Streptomycesfradiae genome.

As the above Table illustrates, there is a wide variety of PKS genes that serve as readily available sources of DNA and sequence information for use in constructing the hybrid PKS-encoding DNA compounds of the invention. Methods for constructing hybrid PKSencoding DNA compounds are described without reference to the narbonolide PKS in U.S.

Patent Nos. 5,672,491 and 5,712,146 and PCT publication No. WO 98/49315, each of which is incorporated herein by reference.

In constructing hybrid PKSs of the invention, certain general methods may be helpful.

For example, it is often beneficial to retain the framework of the module to be altered to make the hybrid PKS. Thus, if one desires to add DH and ER functionalities to a module, it is often preferred to replace the KR domain of the original module with a KR, DH, and ER domaincontaining segment from another module, instead of merely inserting DH and ER domains.

One can alter the stereochemical specificity of a module by replacement of the KS domain with a KS domain from a module that specifies a different stereochemistry. See Lau et al., 1999, "Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units" Biochemistry 38(5):1643-1651, incorporated herein by reference. One can alter the specificity of an AT domain by changing only a small segment of the domain. See Lau et al., supra. One can also take advantage of known linker regions in PKS proteins to link modules from two different PKSs to create a hybrid PKS. See Gokhale et al., 16 Apr. 1999, Dissecting and Exploiting Intermodular Communication in Polyketide Synthases", Science 284: 482-485, incorporated herein by reference.

WO 99/61599 PCT/US99/11814 -59- The hybrid PKS-encoding DNA compounds of the invention can be and often are hybrids of more than two PKS genes. Even where only two genes are used, there are often two or more modules in the hybrid gene in which all or part of the module is derived from a second (or third) PKS gene. Thus, as one illustrative example, the invention provides a hybrid narbonolide PKS that contains the naturally occurring loading module and thioesterase domain as well as extender modules one, two, four, and six of the narbonolide PKS and further contains hybrid or heterologous extender modules three and five. Hybrid or heterologous extender modules three and five contain AT domains specific for malonyl CoA and derived from, for example, the rapamycin PKS genes.

To construct a hybrid PKS or narbonolide derivative PKS of the invention, one can employ a technique, described in PCT Pub. No. WO 98/27203, which is incorporated herein by reference, in which the large PKS gene cluster is divided into two or more, typically three, segments, and each segment is placed on a separate expression vector. In this manner, each of the segments of the gene can be altered, and various altered segments can be combined in a single host cell to provide a recombinant PKS gene of the invention. This technique makes more efficient the construction of large libraries of recombinant PKS genes, vectors for expressing those genes, and host cells comprising those vectors.

Included in the definition of"hybrid" are PKS where alterations (including deletions, insertions and substitutions) are made directly using the narbonolide PKS as a substrate.

The invention also provides libraries of PKS genes, PKS proteins, and ultimately, of polyketides, that are constructed by generating modifications in the narbonolide PKS so that the protein complexes produced have altered activities in one or more respects and thus produce polyketides other than the natural product of the PKS. Novel polyketides may thus be prepared, or polyketides in general prepared more readily, using this method. By providing a large number of different genes or gene clusters derived from a naturally occurring PKS gene cluster, each of which has been modified in a different way from the native cluster, an effectively combinatorial library of polyketides can be produced as a result of the multiple variations in these activities. As will be further described below, the metes and bounds of this embodiment of the invention can be described on both the protein level and the encoding nucleotide sequence level.

As described above, a modular PKS "derived from" the narbonolide or other naturally occurring PKS is a subset of the "hybrid" PKS family and includes a modular PKS (or its corresponding encoding gene(s)) that retains the scaffolding of the utilized portion of the WO 99/61599 PCT/US99/11814 naturally occurring gene. Not all modules need be included in the constructs. On the constant scaffold, at least one enzymatic activity is mutated, deleted, replaced, or inserted so as to alter the activity of the resulting PKS relative to the original PKS. Alteration results when these activities are deleted or are replaced by a different version of the activity, or simply mutated in such a way that a polyketide other than the natural product results from these collective activities. This occurs because there has been a resulting alteration of the starter unit and/or extender unit, and/or stereochemistry, and/or chain length or cyclization, and/or reductive or dehydration cycle outcome at a corresponding position in the product polyketide. Where a deleted activity is replaced, the origin of the replacement activity may come from a corresponding activity in a different naturally occurring PKS or from a different region of the narbonolide PKS. Any or all of the narbonolide PKS genes may be included in the derivative or portions of any of these may be included, but the scaffolding of the PKS protein is retained in whatever derivative is constructed. The derivative preferably contains a thioesterase activity from the narbonolide or another PKS.

In summary, a PKS "derived from" the narbonolide PKS includes a PKS that contains the scaffolding of all or a portion of the narbonolide PKS. The derived PKS also contains at least two extender modules that are functional, preferably three extender modules, and more preferably four or more extender modules, and most preferably six extender modules. The derived PKS also contains mutations, deletions, insertions, or replacements of one or more of the activities of the functional modules of the narbonolide PKS so that the nature of the resulting polyketide is altered. This definition applies both at the protein and DNA sequence levels. Particular preferred embodiments include those wherein a KS, AT, KR, DH, or ER has been deleted or replaced by a version of the activity from a different PKS or from another location within the same PKS. Also preferred are derivatives where at least one noncondensation cycle enzymatic activity (KR, DH, or ER) has been deleted or added or wherein any of these activities has been mutated so as to change the structure of the polyketide synthesized by the PKS.

Conversely, also included within the definition of a PKS derived from the narbonolide PKS are functional PKS modules or their encoding genes wherein at least one portion, preferably two portions, of the narbonolide PKS activities have been inserted. Exemplary is the use of the narbonolide AT for extender module 2 which accepts a malonyl CoA extender unit rather than methylmalonyl CoA to replace a methylmalonyl specific AT in a PKS. Other examples include insertion of portions of non-condensation cycle enzymatic activities or WO 99/61599 PCTfUS99/11814 -61other regions of narbonolide synthase activity into a heterologous PKS. Again, the derived from definition applies to the PKS at both the genetic and protein levels.

Thus, there are at least five degrees of freedom for constructing a hybrid PKS in terms of the polyketide that will be produced. First, the polyketide chain length is determined by the number of modules in the PKS. Second, the nature of the carbon skeleton of the PKS is determined by the specificities of the acyl transferases that determine the nature of the extender units at each position, malonyl, methylmalonyl, ethylmalonyl, or other substituted malonyl. Third, the loading module specificity also has an effect on the resulting carbon skeleton of the polyketide. The loading module may use a different starter unit, such as acetyl, butyryl, and the like. As noted above and in the examples below, another method for varying loading module specificity involves inactivating the KS activity in extender module 1 (KS 1) and providing alternative substrates, called diketides that are chemically synthesized analogs of extender module 1 diketide products, for extender module 2. This approach was illustrated in PCT publication Nos. WO 97/02358 and WO 99/03986, incorporated herein by reference, wherein the KS 1 activity was inactivated through mutation.

Fourth, the oxidation state at various positions of the polyketide will be determined by the dehydratase and reductase portions of the modules. This will determine the presence and location of ketone and alcohol moieties and C-C double bonds or C-C single bonds in the polyketide. Finally, the stereochemistry of the resulting polyketide is a function of three aspects of the synthase. The first aspect is related to the AT/KS specificity associated with substituted malonyls as extender units, which affects stereochemistry only when the reductive cycle is missing or when it contains only a ketoreductase, as the dehydratase would abolish chirality. Second, the specificity of the ketoreductase may determine the chirality of any beta- OH. Finally, the enoylreductase specificity for substituted malonyls as extender units may influence the result when there is a complete KR/DH/ER available.

Thus, the modular PKS systems, and in particular, the narbonolide PKS system, permit a wide range of polyketides to be synthesized. As compared to the aromatic PKS systems, a wider range of starter units including aliphatic monomers (acetyl, propionyl, butyryl, isovaleryl, etc.), aromatics (aminohydroxybenzoyl), alicyclics (cyclohexanoyl), and heterocyclics (thiazolyl) are found in various macrocyclic polyketides. Recent studies have shown that modular PKSs have relaxed specificity for their starter units (Kao et al., 1994, Science, supra). Modular PKSs also exhibit considerable variety with regard to the choice of extender units in each condensation cycle. The degree of beta-ketoreduction following a W) 99/6159 Q PCT/US99/11814 -62condensation reaction has also been shown to be altered by genetic manipulation (Donadio et al., 1991, Science, supra; Donadio et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7119-7123).

Likewise, the size of the polyketide product can be varied by designing mutants with the appropriate number of modules (Kao et al., 1994, J. Am. Chem. Soc. 116:11612-11613).

Lastly, these enzymes are particularly well known for generating an impressive range of asymmetric centers in their products in a highly controlled manner. The polyketides and antibiotics produced by the methods of the invention are typically single stereoisomeric forms. Although the compounds of the invention can occur as mixtures of stereoisomers, it may be beneficial in some instances to generate individual stereoisomers. Thus, the combinatorial potential within modular PKS pathways based on any naturally occurring modular, such as the narbonolide, PKS scaffold is virtually unlimited.

The combinatorial potential is increased even further when one considers that mutations in DNA encoding a polypeptide can be used to introduce, alter, or delete an activity in the encoded polypeptide. Mutations can be made to the native sequences using conventional techniques. The substrates for mutation can be an entire cluster of genes or only one or two of them; the substrate for mutation may also be portions of one or more of these genes. Techniques for mutation include preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene encoding a PKS subunit using restriction endonuclease digestion. See, Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82: 448; Geisselsoder et al., 1987, BioTechniques 5:786. Alternatively, the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) that hybridizes to the native nucleotide sequence, at a temperature below the melting temperature of the mismatched duplex. The primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located.

See Zoller and Smith, 1983, Methods Enzymol. 100:468. Primer extension is effected using DNA polymerase, the product cloned, and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Identification can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, Dalbie-McFarland et al., 1982, Proc. Natl. Acad. Sci.

USA 79: 6409. PCR mutagenesis can also be used to effect the desired mutations.

Random mutagenesis of selected portions of the nucleotide sequences encoding enzymatic activities can also be accomplished by several different techniques known in the art, by inserting an oligonucleotide linker randomly into a plasmid, by irradiation with WO 99/61599 PCT/US99/11814 -63- X-rays or ultraviolet light, by incorporating incorrect nucleotides during in vitro DNA synthesis, by error-prone PCR mutagenesis, by preparing synthetic mutants, or by damaging plasmid DNA in vitro with chemicals. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, nitrosoguanidine, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as 5-bromouracil, 2-aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like. Generally, plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.

In constructing a hybrid PKS of the invention, regions encoding enzymatic activity, regions encoding corresponding activities from different PKS synthases or from different locations in the same PKS, can be recovered, for example, using PCR techniques with appropriate primers. By "corresponding" activity encoding regions is meant those regions encoding the same general type of activity. For example, a KR activity encoded at one location of a gene cluster "corresponds" to a KR encoding activity in another location in the gene cluster or in a different gene cluster. Similarly, a complete reductase cycle could be considered corresponding. For example, KR/DH/ER corresponds to KR alone.

If replacement of a particular target region in a host PKS is to be made, this replacement can be conducted in vitro using suitable restriction enzymes. The replacement can also be effected in vivo using recombinant techniques involving homologous sequences framing the replacement gene in a donor plasmid and a receptor region in a recipient plasmid.

Such systems, advantageously involving plasmids of differing temperature sensitivities are described, for example, in PCT publication No. WO 96/40968, incorporated herein by reference. The vectors used to perform the various operations to replace the enzymatic activity in the host PKS genes or to support mutations in these regions of the host PKS genes can be chosen to contain control sequences operably linked to the resulting coding sequences in a manner such that expression of the coding sequences can be effected in an appropriate host.

However, simple cloning vectors may be used as well. If the cloning vectors employed to obtain PKS genes encoding derived PKS lack control sequences for expression operably linked to the encoding nucleotide sequences, the nucleotide sequences are inserted into appropriate expression vectors. This need not be done individually, but a pool of isolated encoding nucleotide sequences can be inserted into expression vectors, the resulting vectors WO 99/61599 PCT[US99/11814 -64transformed or transfected into host cells, and the resulting cells plated out into individual colonies.

The various PKS nucleotide sequences can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, a single promoter. The PKS subunit encoding regions can include flanking restriction sites to allow for the easy deletion and insertion of other PKS subunit encoding sequences so that hybrid PKSs can be generated. The design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as sitedirected mutagenesis and PCR.

The expression vectors containing nucleotide sequences encoding a variety of PKS enzymes for the production of different polyketides are then transformed into the appropriate host cells to construct the library. In one straightforward approach, a mixture of such vectors is transformed into the selected host cells and the resulting cells plated into individual colonies and selected to identify successful transformants. Each individual colony has the ability to produce a particular PKS synthase and ultimately a particular polyketide. Typically, there will be duplications in some, most, or all of the colonies; the subset of the transformed colonies that contains a different PKS in each member colony can be considered the library.

Alternatively, the expression vectors can be used individually to transform hosts, which transformed hosts are then assembled into a library. A variety of strategies are available to obtain a multiplicity of colonies each containing a PKS gene cluster derived from the naturally occurring host gene cluster so that each colony in the library produces a different PKS and ultimately a different polyketide. The number of different polyketides that are produced by the library is typically at least four, more typically at least ten, and preferably at least 20, and more preferably at least 50, reflecting similar numbers of different altered PKS gene clusters and PKS gene products. The number of members in the library is arbitrarily chosen; however, the degrees of freedom outlined above with respect to the variation of starter, extender units, stereochemistry, oxidation state, and chain length is quite large.

Methods for introducing the recombinant vectors of the invention into suitable hosts are known to those of skill in the art and typically include the use of CaC12 or agents such as other divalent cations, lipofection, DMSO, protoplast transformation, infection, transfection, and electroporation. The polyketide producing colonies can be identified and isolated using known techniques and the produced polyketides further characterized. The polyketides WO 99/61599 PCT/US99/11814 produced by these colonies can be used collectively in a panel to represent a library or may be assessed individually for activity.

The libraries of the invention can thus be considered at four levels: a multiplicity of colonies each with a different PKS encoding sequence; colonies that contain the proteins that are members of the PKS library produced by the coding sequences; the polyketides produced; and antibiotics or compounds with other desired activities derived from the polyketides. Of course, combination libraries can also be constructed wherein members of a library derived, for example, from the narbonolide PKS can be considered as a part of the same library as those derived from, for example, the rapamycin PKS or DEBS.

Colonies in the library are induced to produce the relevant synthases and thus to produce the relevant polyketides to obtain a library of polyketides. The polyketides secreted into the media can be screened for binding to desired targets, such as receptors, signaling proteins, and the like. The supematants per se can be used for screening, or partial or complete purification of the polyketides can first be effected. Typically, such screening methods involve detecting the binding of each member of the library to receptor or other target ligand. Binding can be detected either directly or through a competition assay. Means to screen such libraries for binding are well known in the art. Alternatively, individual polyketide members of the library can be tested against a desired target. In this event, screens wherein the biological response of the target is measured can more readily be included.

Antibiotic activity can be verified using typical screening assays such as those set forth in Lehrer et al., 1991, J. Immunol. Meth. 137:167-173, incorporated herein by reference, and in the examples below.

The invention provides methods for the preparation of a large number of polyketides.

These polyketides are useful intermediates in formation of compounds with antibiotic or other activity through hydroxylation and glycosylation reactions as described above. In general, the polyketide products of the PKS must be further modified, typically by hydroxylation and glycosylation, to exhibit antibiotic activity. Hydroxylation results in the novel polyketides of the invention that contain hydroxyl groups at C6, which can be accomplished using the hydroxylase encoded by the eryF gene, and/or C12, which can be accomplished using the hydroxylase encoded by the picK or eryK gene. The presence of hydroxyl groups at these positions can enhance the antibiotic activity of the resulting compound relative to its unhydroxylated counterpart.

WO 99/61599 PCT/US99/11814 -66 Gycosylation is important in conferring antibiotic activity to a polyketide as well.

Methods for glycosylating the polyketides are generally known in the art; the glycosylation may be effected intracellularly by providing the appropriate glycosylation enzymes or may be effected in vitro using chemical synthetic means as described herein and in PCT publication No. WO 98/49315, incorporated herein by reference. Preferably, glycosylation with desosamine is effected in accordance with the methods of the invention in recombinant host cells provided by the invention. In general, the approaches to effecting glycosylation mirror those described above with respect to hydroxylation. The purified enzymes, isolated from native sources or recombinantly produced may be used in vitro. Alternatively and as noted, glycosylation may be effected intracellularly using endogenous or recombinantly produced intracellular glycosylases. In addition, synthetic chemical methods may be employed.

The antibiotic modular polyketides may contain any of a number of different sugars, although D-desosamine, or a close analog thereof, is most common. Erythromycin, picromycin, narbomycin and methymycin contain desosamine. Erythromycin also contains Lcladinose (3-O-methyl mycarose). Tylosin contains mycaminose (4-hydroxy desosamine), mycarose and 6-deoxy-D-allose. 2-acetyl-1-bromodesosamine has been used as a donor to glycosylate polyketides by Masamune et al., 1975, J. Am. Chem. Soc. 97: 3512-3513. Other, apparently more stable donors include glycosyl fluorides, thioglycosides, and trichloroacetimidates; see Woodward et al., 1981, J. Am. Chem. Soc. 103: 3215; Martin et al., 1997, J. Am. Chem. Soc. 119: 3193; Toshima et al., 1995, J. Am. Chem. Soc. 117: 3717; Matsumoto et al., 1988, Tetrahedron Lett. 29: 3575. Glycosylation can also be effected using the polyketide aglycones as starting materials and using Saccharopolyspora erythraea or Streptomyces venezuelae to make the conversion, preferably using mutants unable to synthesize macrolides.

To provide an illustrative hybrid PKS of the invention as well as an expression vector for that hybrid PKS and host cells comprising the vector and producing the hybrid polyketide, a portion of the narbonolide PKS gene was fused to the DEBS genes. This construct also allowed the examination of whether the TE domain of the narbonolide PKS (pikTE) could promote formation of 12-membered lactones in the context of a different PKS. A construct was generated, plasmid pKOS039-18, in which the pikTE ORF was fused with the DEBS genes in place of the DEBS TE ORF (see Figure To allow the TE to distinguish between substrates most closely resembling those generated by the narbonolide PKS, the fusion junction was chosen between the AT and ACP to eliminate ketoreductase activity in DEBS WO 99/61599 PCTIUS99/11814 -67extender module 6 (KR 6 This results in a hybrid PKS that presents the TE with a B-ketone heptaketide intermediate and a B-(S)-hydroxy hexaketide intermediate to cyclize, as in narbonolide and 10-deoxymethynolide biosynthesis.

Analysis of this construct indicated the production of the 14-membered ketolide 3,6dideoxy-3-oxo-erythronolide B (Figure 5, compound Extracts were analyzed by LC/MS.

The identity of compound 6 was verified by comparison to a previously authenticated sample (see PCT publication No. WO 98/49315, incorporated herein by reference). The predicted 12membered macrolactone, (8R,9S)-8,9-dihydro-8-methyl-9-hydroxy-10-deoxymethynolide (see Kao et al. J. Am. Chem. Soc. (1995) 117:9105-9106 incorporated herein by reference) was not detected. Because the 12-membered intermediate can be formed by other recombinant PKS enzymes, see Kao et al., 1995, supra, the PIC TE domain appears incapable of forcing premature cyclization of the hexaketide intermediate generated by DEBS. This result, along with others reported herein, suggests that protein interactions between the narbonolide PKS modules play a role in formation of the 12 and 14-membered macrolides.

The above example illustrates also how engineered PKSs can be improved for production of novel compounds. Compound 6 was originally produced by deletion of the KR 6 domain in DEBS to create a 3-ketolide producing PKS (see U.S. patent application Serial No.

09/073,538, filed 6 May 1998, and PCT publication No. WO 98/49315, each of which is incorporated herein by reference). Although the desired molecule was made, purification of compound 6 from this strain was hampered by the presence of 2-desmethyl ketolides that could not be easily separated. Extracts from Streptomyces lividans K4-114/pKOS039-18, however, do not contain the 2-desmethyl compounds, greatly simplifying purification. Thus, the invention provides a useful method of producing such compounds. The ability to combine the narbonolide PKS with DEBS and other modular PKSs provides a significant advantage in the production of macrolide antibiotics.

Two other hybrid PKSs of the invention were constructed that yield this same compound. These constructs also illustrate the method of the invention in which hybrid PKSs are constructed at the protein, as opposed to the module, level. Thus, the invention provides a method for constructing a hybrid PKS which comprises the coexpression of at least one gene from a first modular PKS gene cluster in a host cell that also expresses at least one gene from a second PKS gene cluster. The invention also provides novel hybrid PKS enzymes prepared in accordance with the method. This method is not limited to hybrid PKS enzymes composed WO 99/61599 PCT/US99/11814 -68of at least one narbonolide PKS gene, although such constructs are illustrative and preferred.

Moreover, the hybrid PKS enzymes are not limited to hybrids composed of unmodified proteins; as illustrated below, at least one of the genes can optionally be a hybrid PKS gene.

In the first construct, the eryAI and eryAII genes were coexpressed with picAIV and a gene encoding a hybrid extender module 5 composed of the KS and AT domains of extender module 5 of DEBS3 and the KR and ACP domains of extender module 5 of the narbonolide PKS. In the second construct, the picAJV coding sequence was fused to the hybrid extender module 5 coding sequence used in the first construct to yield a single protein. Each of these constructs produced 3-deoxy-3-oxo-6-deoxyerythronolide B. In a third construct, the coding sequence for extender module 5 of DEBS3 was fused to the picAN coding sequence, but the levels of product produced were below the detection limits of the assay.

A variant of the first construct hybrid PKS was constructed that contained an inactivated DEBS1 extender module 1 KS domain. When host cells containing the resultant hybrid PKS were supplied the appropriate diketide precursor, the desired 13-desethyl-13propyl compounds were obtained, as described in the examples below.

Other illustrative hybrid PKSs of the invention were made by coexpressing the picAI and picAH genes with genes encoding DEBS3 or DEBS3 variants. These constructs illustrate the method of the invention in which a hybrid PKS is produced from coexpression of PKS genes unmodified at the modular or domain level. In the first construct, the eryAIII gene was coexpressed with the picAI and picAII genes, and the hybrid PKS produced 10,11 -anhydro-6-deoxyerythronolide B in Streptomyces lividans. Such a hybrid PKS could also be constructed in accordance with the method of the invention by transformation of S.

venezuelae with an expression vector that produces the eryAIII gene product, DEBS3. In a preferred embodiment, the S. venezuelae host cell has been modified to inactivate the picAIII gene.

In the second construct, the DEBS3 gene was a variant that had an inactive KR in extender module 5. The hybrid PKS produced 5,6-dideoxy-5-oxo-10-desmethyl-10,l 1anhydroerythronolide B in Streptomyces lividans.

In the third construct, the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS. This construct produced 5,6-dideoxy-5-oxo- 10-desmethyl-10,11 anhydroerythronolide B and 5,6-dideoxy-4,5-anhydro- 10-desmethyl-10,11- WO 99/61599 PCT/US99/11814 -69anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH and KR domains functioned only inefficiently in this construct.

In the fourth construct, the DEBS3 gene was a variant in which the KR domain of extender module 5 was replaced by the DH, KR, and ER domains of extender module 1 of the rapamycin PKS. This construct produced 5,6-dideoxy-5-oxo-10-desmethyl-10,11anhydroerythronolide B as well as 5,6-dideoxy- 10-desmethyl-10,11-anhydroerythronolide B in Streptomyces lividans, indicating that the rapamycin DH, KR, and ER domains functioned only inefficiently in this construct.

In the fifth construct, the DEBS3 gene was a variant in which the KR domain of extender module 6 was replaced by the DH and KR domains of extender module 4 of the rapamycin PKS. This construct produced 3,6-dideoxy-2,3-anhydro- 10-desmethyl-10,11anhydroerythronolide B in Streptomyces lividans.

In the sixth construct, the DEBS3 gene was a variant in which the AT domain of extender module 6 was replaced by the AT domain of extender module 2 of the rapamycin PKS. This construct produced 2,10-didesmethyl-10,11 -anhydro-6-deoxyerythronolide B in Streptomyces lividans.

These hybrid PKSs illustrate the wide variety ofpolyketides that can be produced by the methods and compounds of the invention. These polyketides are useful as antibiotics and as intermediates in the synthesis of other useful compounds, as described in the following section.

Section VI: Compounds The methods and recombinant DNA compounds of the invention are useful in the production of polyketides. In one important aspect, the invention provides methods for making ketolides, polyketide compounds with significant antibiotic activity. See Griesgraber et al., 1996, J. Antibiot. 49: 465-477, incorporated herein by reference. Most if not all of the ketolides prepared to date are synthesized using erythromycin A, a derivative of 6-dEB, as an intermediate. While the invention provides hybrid PKSs that produce a polyketide different in structure from 6-dEB, the invention also provides methods for making intermediates useful in preparing traditional, 6-dEB-derived ketolide compounds.

Because 6-dEB in part differs from narbonolide in that it comprises a group, the novel hybrid PKS genes of the invention based on the narbonolide PKS provide many novel ketolides that differ from the known ketolides only in that they lack a WO 99/61599 PCT/US99/l1814 group. Thus, the invention provides the 10-desmethyl analogues of the ketolides and intermediates and precursor compounds described in, for example, Griesgraber et al., supra; Agouridas et al., 1998, J. Med. Chem. 41: 4080-4100, U.S. Patent Nos. 5,770,579; 5,760,233; 5,750,510; 5,747,467; 5,747,466; 5,656,607; 5,635,485; 5,614,614; 5,556,118; 5,543,400; 5,527,780; 5,444,051; 5,439,890; 5,439,889; and PCT publication Nos. WO 98/09978 and WO 98/28316, each of which is incorporated herein by reference. Because the invention also provides hybrid PKS genes that include a methylmalonyl-specific AT domain in extender module 2 of the narbonolide PKS, the invention also provides hybrid PKS that can be used to produce the 10-methyl-containing ketolides known in the art.

Thus, a hybrid PKS of the invention that produces 10-methyl narbonolide is constructed by substituting the malonyl-specific AT domain of the narbonolide PKS extender module 2 with a methylmalonyl specific AT domain from a heterologous PKS. A hybrid narbonolide PKS in which the AT of extender module 2 was replaced with the AT from DEBS extender module 2 was constructed using boundaries described in PCT publication No. WO 98/49315, incorporated herein by reference. However, when the hybrid PKS expression vector was introduced into Streptomyces venezuelae, detectable quantities of methyl picromycin were not produced. Thus, to construct such a hybrid PKS of the invention, an AT domain from a module other than DEBS extender module 2 is preferred. One could also employ DEBS extender module 2 or another methylmalonyl specific AT but utilize instead different boundaries than those used for the substitution described above. In addition, one can construct such a hybrid PKS by substituting, in addition to the AT domain, additional extender module 2 domains, including the KS, the KR, and the DH, and/or additional extender module 3 domains.

Although modification of extender module 2 of the narbonolide PKS is required, the extent of hybrid modules engineered need not be limited to module 2 to make narbonolide. For example, substitution of the KS domain of extender module 3 of the narbonolide PKS with a heterologous domain or module can result in more efficient processing of the intermediate generated by the hybrid extender module 2. Likewise, a heterologous TE domain may be more efficient in cyclizing 10-methyl narbonolide.

Substitution of the entire extender module 2 of the narbonolide PKS with a module encoding the correct enzymatic activities, a KS, a methylmalonyl specific AT, a KR, a DH, and an ACP, can also be used to create a hybrid PKS of the invention that produces a methyl ketolide. Modules useful for such whole module replacements include extender WO 99/61599 PCT/US99/11814 -71modules 4 and 10 from the rapamycin PKS, extender modules 1 and 5 from the FK506 PKS, extender module 2 of the tylosin PKS, and extender module 4 of the rifamycin PKS. Thus, the invention provides many different hybrid PKSs that can be constructed starting from the narbonolide PKS that can be used to produce 10-methyl narbonolide. While narbonolide is referred to in describing these hybrid PKSs, those of skill recognize that the invention also therefore provides the corresponding derivatives produces by glycosylation and hydroxylation. For example, if the hybrid PKS is expressed in Streptomyces narbonensis or S. venezuelae, the compounds produced are 10-methyl narbomycin and picromycin, respectively. Alternatively, the PKS can be expressed in a host cell transformed with the vectors of the invention that encode the desosamine biosynthesis and desosaminyl transferase and picK hydroxylase genes.

Other important compounds provided by the invention are the 6-hydroxy ketolides.

These compounds include 3-deoxy-3-oxo erythronolide B, 6-hydroxy narbonolide, and 6narbonolide. In the examples below, the invention provides a method for utilizing EryF to hydroxylate 3-ketolides that is applicable for the production of any 6hydroxy-3-ketolide.

Thus, the hybrid PKS genes of the invention can be expressed in a host cell that contains the desosamine biosynthetic genes and desosaminyl transferase gene as well as the required hydroxylase gene(s), which may be either picK (for the C12 position) or eryK (for the C12 position) and/or eryF (for the C6 position). The resulting compounds have antibiotic activity but can be further modified, as described in the patent publications referenced above, to yield a desired compound with improved or otherwise desired properties. Alternatively, the aglycone compounds can be produced in the recombinant host cell, and the desired glycosylation and hydroxylation steps carried out in vitro or in vivo, in the latter case by supplying the converting cell with the aglycone.

The compounds of the invention are thus optionally glycosylated forms of the polyketide set forth in formula below which are hydroxylated at either the C6 or the C12 or both. The compounds of formula can be prepared using the loading and the six extender modules of a modular PKS, modified or prepared in hybrid form as herein described. These polyketides have the formula: WO 99/61599 PCT/US99/11814 -72- R2 b s t

R

13 or unsubstituted hydrocarbyl of 1-15C;

X

1 R 6 each of R'-R 6 is independently H or alkyl (1-4C) wherein any alkyl at R' may optionally be substituted; each ofX'-X 5 is independently two H, H and OH, or or each of X'-X 5 is independently H and the compound of formula contains a doublebond in the ring adjacent to the position of said X at 2-3, 4-5, 6-7, 8-9 and/or 10-11; with the proviso that: at least two ofR'-R 6 are alkyl (1-4C).

Preferred compounds comprising formula 2 are those wherein at least three ofR'-R are alkyl preferably methyl or ethyl; more preferably wherein at least four of R -R are alkyl preferably methyl or ethyl. Also preferred are those wherein X 2 is two H, or H and OH, and/or X 3 is H, and/or X' is OH and/or X 4 is OH and/or X 5 is OH. Also preferred are compounds with variable R* when R'-R 5 is methyl, X 2 is and X 4 and

X

5 are OH. The glycosylated forms of the foregoing are also preferred.

The invention also provides the 12-membered macrolides corresponding to the compounds above but produced from a narbonolide-derived PKS lacking extender modules and 6 of the narbonolide PKS.

The compounds of the invention can be produced by growing and fermenting the host cells of the invention under conditions known in the art for the production of other polyketides. The compounds of the invention can be isolated from the fermentation broths of these cultured cells and purified by standard procedures. The compounds can be readily WO 99/61599 PCT/US99/11814 -73formulated to provide the pharmaceutical compositions of the invention. The pharmaceutical compositions of the invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid, or liquid form. This preparation will contain one or more of the compounds of the invention as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.

The carriers which can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquefied form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used. For example, the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Patent No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference.

Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6:17-22, incorporated herein by reference. Dosage forms for external application may be prepared essentially as described in EPO patent publication No. 423,714, incorporated herein by reference. The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the disease process or condition.

For the treatment of conditions and diseases caused by infection, a compound of the invention may be administered orally, topically, parenterally, by inhalation spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvant, and vehicles. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intramuscular, and intrasternal injection or infusion techniques.

Dosage levels of the compounds of the invention are of the order from about 0.01 mg to about 50 mg per kilogram of body weight per day, preferably from about 0.1 mg to about 10 mg per kilogram of body weight per day. The dosage levels are useful in the treatment of the above-indicated conditions (from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg patient). In addition, the compounds of the invention may be administered on an intermittent basis, at semi-weekly, weekly, semi-monthly, or monthly intervals.

74 The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain from 0.5 mg to 5 mg of active agent compounded with an appropriate and convenient amount of carrier material, which may vary from about 5 percent to about 95 percent of the total composition. Dosage unit forms will generally contain from about 0.5 mg to about 500 mg of active ingredient.

For external administration, the compounds of the invention may be formulated within the range of, for example, 0.00001% to 60% by weight, preferably from 0.001% to 10% by weight, and most preferably from 0.005% to 0.8% by weight.

It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and the severity of the particular disease or condition for which therapy is sought.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

~Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

A detailed description of the invention having been provided above, the following examples are given for the purpose of illustrating the invention and shall not be construed as being a limitation on the scope of the invention or claims.

Example 1 General Methodology Bacterial strains, plasmids, and culture conditions. Streptomyces coelicolor CH999 described in WO 95/08548, published 30 March 1995, or S. lividans K4-114, described in Ziermann and Betlach, Jan. 99, Recombinant Polyketide Synthesis in Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, Streptomyces: Engineering of Improved Host Strains, BioTechniques 26:106-110, m:\specifications\090000\93000\93075resmro.doc 74a incorporated herein by reference, was used as an expression host. DNA manipulations were performed in Escherichia coli XL1-Blue, available from Stratagene. E. coli MC1061 is also suitable for use as a host for plasmid manipulation. Plasmids were passaged through E. coli ET12567 (dam dcm hsdS Cmr) (MacNeil, 1988, J Bacteriol.

170: 5607, incorporated herein by reference) to generate unmethylated DNA prior to transformation of S. coelicolor. E. coli strains were grown under standard conditions.

S. coelicolor strains were grown on R2YE agar m:\specifications\090000\93000\93O75resmro. doc WO 99/61599 PCT/US99/11814 plates (Hopwood et al., Genetic manipulation ofStreptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985, incorporated herein by reference).

Many of the expression vectors of the invention illustrated in the examples are derived from plasmid pRM5, described in WO 95/08548, incorporated herein by reference.

This plasmid includes a colEI replicon, an appropriately truncated SCP2* Streptomyces replicon, two act-promoters to allow for bidirectional cloning, the gene encoding the actll- ORF4 activator which induces transcription from act promoters during the transition from growth phase to stationary phase, and appropriate marker genes. Engineered restriction sites in the plasmid facilitate the combinatorial construction of PKS gene clusters starting from cassettes encoding individual domains of naturally occurring PKSs. When plasmid pRM5 is used for expression of a PKS, all relevant biosynthetic genes can be plasmid-borne and therefore amenable to facile manipulation and mutagenesis in E. coli. This plasmid is also suitable for use in Streptomyces host cells. Streptomyces is genetically and physiologically well-characterized and expresses the ancillary activities required for in vivo production of most polyketides. Plasmid pRM5 utilizes the act promoter for PKS gene expression, so polyketides are produced in a secondary metabolite-like manner, thereby alleviating the toxic effects of synthesizing potentially bioactive compounds in vivo.

Manipulation of DNA and organisms. Polymerase chain reaction (PCR) was performed using Pfu polymerase (Stratagene; Taq polymerase from Perkin Elmer Cetus can also be used) under conditions recommended by the enzyme manufacturer. Standard in vitro techniques were used for DNA manipulations (Sambrook et al. Molecular Cloning: A Laboratory Manual (Current Edition)). E. coli was transformed using standard calcium chloride-based methods; a Bio-Rad E. coli pulsing apparatus and protocols provided by Bio- Rad could also be used. S. coelicolor was transformed by standard procedures (Hopwood et al. Genetic manipulation ofStreptomyces. A laboratory manual. The John Innes Foundation: Norwich, 1985), and depending on what selectable marker was employed, transformants were selected using 1 mL of a 1.5 mg/mL thiostrepton overlay, 1 mL of a 2 mg/mL apramycin overlay, or both.

Example 2 Cloning of the Picromycin Biosynthetic Gene Cluster from Streptomyces venezuelae Genomic DNA (100 Ag) isolated from Streptomyces venezuelae ATCC15439 using standard procedures was partially digested with Sau3AI endonuclease to generate fragments WO 99/61599 PCT/US99/11814 -76kbp in length. SuperCosI (Stratagene) DNA cosmid arms were prepared as directed by the manufacturer. A cosmid library was prepared by ligating 2.5 pg of the digested genomic DNA with 1.5 gg of cosmid arms in a 20 pL reaction. One microliter of the ligation mixture was propagated in E. coli XL1-Blue MR (Stratagene) using a GigapackIII XL packaging extract kit (Stratagene). The resulting library of -3000 colonies was plated on a 10x150 mm agar plate and replicated to a nylon membrane.

The library was initially screened by direct colony hybridization with a DNA probe specific for ketosynthase domain coding sequences of PKS genes. Colonies were alkaline lysed, and the DNA was crosslinked to the membrane using UV irradiation. After overnight incubation with the probe at 42 0 C, the membrane was washed twice at 25 0 C in 2xSSC buffer 0.1% SDS for 15 minutes, followed by two 15 minute washes with 2xSSC buffer at 55 0

C.

Approximately 30 colonies gave positive hybridization signals with the degenerate probe.

Several cosmids were selected and divided into two classes based on restriction digestion patterns. A representative cosmid was selected from each class for further analysis. The representative cosmids were designated pKOS023-26 and pKOS023-27. These cosmids were determined by DNA sequencing to comprise the narbonolide PKS genes, the desosamine biosynthesis and transferase genes, the beta-glucosidase gene, and the picK hydroxylase gene.

These cosmids were deposited with the American Type Culture Collection in accordance with the terms of the Budapest Treaty. Cosmid pKOS023-26 was assigned accession number ATCC 203141, and cosmid pKOS023-27 was assigned accession number ATCC 203142.

To demonstrate that the narbonolide PKS genes had been cloned and to illustrate how the invention provides methods and reagents for constructing deletion variants of narbonolide PKS genes, a narbonolide PKS gene was deleted from the chromosome of Streptomyces venezuelae. This deletion is shown schematically in Figure 4, parts B and C. A -2.4 kb EcoRI KpnI fragment and a -2.1 kb KpnI Xhol fragment, which together comprise both ends of the picAI gene (but lack a large portion of the coding sequence), were isolated from cosmid pKOS023-27 and ligated together into the commercially available vector pLitmus 28 (digested with restriction enzymes EcoRI and Xhol) to give plasmid pKOS039-07. The -4.5 kb HindIII-Spel fragment from plasmid pKOS039-07 was ligated with the 2.5 kb HindIII-Nhel fragment of integrating vector pSET152, available from the NRRL, which contains an E. coli origin of replication and an apramycin resistance-conferring gene to create WO 99/61599 PCT/US99/11814 -77plasmid pKOS039-16. This vector was used to transform S. venezuelae, and apramycinresistant transformants were selected.

Then, to select for double-crossover mutants, the selected transformants were grown in TSB liquid medium without antibiotics for three transfers and then plated onto nonselective media to provide single colony isolates. The isolated colonies were tested for sensitivity to apramycin, and the apramycin-sensitive colonies were then tested to determine if they produced picromycin. The tests performed included a bioassay and LC/MS analysis of the fermentation media. Colonies determined not to produce picromycin (or methymycin or neomethymycin) were then analyzed using PCR to detect an amplification product diagnostic of the deletion. A colony designated K39-03 was identified, providing confirmation that the narbonolide PKS genes had been cloned. Transformation of strain K39-03 with plasmid pKOS039-27 comprising an intactpicA gene under the control of the ermE* promoter from plasmid pWHM3 (see Vara et al., J. Bact. (1989) 171: 5872-5881, incorporated herein by reference) was able to restore picromycin production.

To determine that the cosmids also contained the picK hydroxylase gene, each cosmid was probed by Southern hybridization using a labeled DNA fragment amplified by PCR from the Saccharopolyspora erythraea C12-hydroxylase gene, eryK. The cosmids were digested with BamHI endonuclease and electrophoresed on a 1% agarose gel, and the resulting fragments were transferred to a nylon membrane. The membrane was incubated with the eryK probe overnight at 42 0 C, washed twice at 25 0 C in 2XSSC buffer with 0.1% SDS for minutes, followed by two 15 minute washes with 2XSSC buffer at 50 0 C. Cosmid pKOS023- 26 produced an -3 kb fragment that hybridized with the probe under these conditions. This fragment was subcloned into the PCRscriptTM (Stratagene) cloning vector to yield plasmid pKOS023-28 and sequenced. The -1.2 kb gene designated picK above was thus identified.

The picK gene product is homologous to eryK and other known macrolide cytochrome P450 hydroxylases.

By such methodology, the complete set of picromycin biosynthetic genes were isolated and identified. DNA sequencing of the cloned DNA provided further confirmation that the correct genes had been cloned. In addition, and as described in the following example, the identity of the genes was confirmed by expression of narbomycin in heterologous host cells.

WO 99/61599 PCT/US99/11814 -78- Example 3 Heterologous Expression of the Narbonolide PKS and the Picromycin Biosynthetic Gene Cluster To provide a preferred host cell and vector for purposes of the invention, the narbonolide PKS was transferred to the non-macrolide producing host Streptomyces lividans K4-114 (see Ziermann and Betlach, 1999, Biotechniques 26, 106-110, and U.S. patent application Serial No. 09/181,833, filed 28 Oct. 1998, each of which is incorporated herein by reference). This was accomplished by replacing the three DEBS ORFs on a modified version of pCK7 (see Kao et al., 1994, Science 265, 509-512, and U.S. Patent No. 5,672,491, each of which is incorporated herein by reference) with all four narbonolide PKS ORFs to generate plasmid pKOS039-86 (see Figure The pCK7 derivative employed, designated pCK7'Kan', differs from pCK7 only in that it contains a kanamycin resistance conferring gene inserted at its HindII restriction enzyme recognition site. Because the plasmid contains two selectable markers, one can select for both markers and so minimize contamination with cells containing rearranged, undesired vectors.

Protoplasts were transformed using standard procedures and transformants selected using overlays containing antibiotics. The strains were grown in liquid R5 medium for growth/seed and production cultures at 30 0 C. A 2 L shake flask culture ofS. lividans K4- 114/pKOS039-86 was grown for 7 days at 30 0 C. The mycelia was filtered, and the aqueous layer was extracted with 2 x 2 L ethyl acetate. The organic layers were combined, dried over MgSO4, filtered, and evaporated to dryness. Polyketides were separated from the crude extract by silica gel chromatography (1:4 to 1:2 ethyl acetate:hexane gradient) to give an mg mixture of narbonolide and 10-deoxymethynolide, as indicated by LC/MS and 1H NMR.

Purification of these two compounds was achieved by HPLC on a C-18 reverse phase column (20-80% acetonitrile in water over 45 minutes). This procedure yielded -5 mg each of narbonolide and 10-deoxymethynolide. Polyketides produced in the host cells were analyzed by bioassay against Bacillus subtilis and by LC/MS analysis. Analysis of extracts by LC/MS followed by 1H-NMR spectroscopy of the purified compounds established their identity as narbonolide (Figure 5, compound 4; see Kaiho et al., 1982, J. Org. Chem. 47: 1612-1614, incorporated herein by reference) and 10-deoxymethynolide (Figure 5, compound 5; see Lambalot et al., 1992, J. Antibiotics 45, 1981-1982, incorporated herein by reference), the respective 14 and 12-membered polyketide aglycones of YC17, narbomycin, picromycin, and methymycin.

WO 99/61599 PCT/sI9Q/1 d14 -79 The production of narbonolide in Streptomyces lividans represents the expression of an entire modular polyketide pathway in a heterologous host. The combined yields of compounds 4 and 5 are similar to those obtained with expression of DEBS from pCK7 (see Kao et al., 1994, Science 265: 509-512, incorporated herein by reference). Furthermore, based on the relative ratios of compounds 4 and 5 produced, it is apparent that the narbonolide PKS itself possesses an inherent ability to produce both 12 and 14-membered macrolactones without the requirement of additional activities unique to S. venezuelae.

Although the existence of a complementary enzyme present in S. lividans that provides this function is possible, it would be unusual to find such a specific enzyme in an organism that does not produce any known macrolide.

To provide a heterologous host cell of the invention that produces the narbonolide PKS and the picB gene, the picB gene was integrated into the chromosome of Streptomyces lividans harboring plasmid pKOS039-86 to yield S. lividans K39-18/pKOS039-86. To provide the integrating vector utilized, the picB gene was cloned into the Streptomyces genome integrating vector pSET152 (see Bierman et al., 1992, Gene 116, 43, incorporated herein by reference) under control of the same promoter (Pactl) as the PKS on plasmid pKOS039-86.

A comparison of strains K39-18/pKOS039-86 and K4-114/pKOS039-86 grown under identical conditions indicated that the strain containing TEII produced 4-7 times more total polyketide. Each strain was grown in 30 mL of R5 (see Hopwood et al., Genetic Manipulation of Streptomyces: A Laboratory Manual; John Innes Foundation: Norwich, UK, 1985, incorporated herein by reference) liquid (with 20 pg/mL thiostrepton) at 30 0 C for 9 days. The fermentation broth was analyzed directly by reverse phase HPLC. Absorbance at 235 nm was used to monitor compounds and measure relative abundance. This increased production indicates that the enzyme is functional in this strain. As noted above, because the production levels of compound 4 and 5 from K39-18/pKOS03986 increased by the same relative amounts, TEII does not appear to influence the ratio of 12 and 14-membered lactone ring formation.

To express the glycosylated counterparts of narbonolide (narbomycin) and deoxymethynolide (YC 17) in heterologous host cells, the desosamine biosynthetic genes and desosaminyl transferase gene were transformed into the host cells harboring plasmid pKOS039-86 (and, optionally, the picB gene, which can be integrated into the chromosome as described above).

WO 99/61599 PCT/US99/11814 Plasmid pKOS039-104, see Figure 6, comprises the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene. This plasmid was constructed by first inserting a polylinker oligonucleotide, containing a restriction enzyme recognition site for Pad, a Shine-Dalgarno sequence, and restriction enzyme recognition sites for Ndel, BglII, and HindIII, into a pUC19 derivative, called pKOS24-47, to yield plasmid pKOS039-98.

An -0.3 kb PCR fragment comprising the coding sequence for the N-terminus of the desI gene product and an ~0.12 kb PCR fragment comprising the coding sequence for the Cterminus of the desR gene product were amplified from cosmid pKOS23-26 (ATCC 203141) and inserted together into pLitmus28 treated with restriction enzymes Nsil and EcoRI to produce plasmid pKOS039-101. The ~6 kb SphI-PstI restriction fragment of pKOS23-26 containing the desI, deslI, desIII, deslV, and des V genes was inserted into plasmid pUC19 (Stratagene) to yield plasmid pKOS039-102. The -6 kb SphI-EcoRI restriction fragment from plasmid pKOS039-102 was inserted into pKOS039-101 to produce plasmid pKOS039-103.

The -6 kb BglII-PstI fragment from pKOS23-26 that contains the desR, des VI, des VII, and desVIII genes was inserted into pKOS39-98 to yield pKOS39-100. The -6 kb Pacl-Pstl restriction fragment of pKOS39-100 and the -6.4 kb Nsil-EcoRI fragment of pKOS39-103 were cloned into pKOS39-44 to yield pKOS39-104.

When introduced into Streptomyces lividans host cells comprising the recombinant narbonolide PKS of the invention, plasmid pKOS39-104 drives expression of the desosamine biosynthetic genes, the beta-glucosidase gene, and the desosaminyl transferase gene. The glycosylated antibiotic narbomycin was produced in these host cells, and it is believed that YC17 was produced as well. When these host cells are transformed with vectors that drive expression of the picK gene, the antibiotics methymycin, neomethymycin, and picromycin are produced.

In similar fashion, when plasmid pKOS039-18, which encodes a hybrid PKS of the invention that produces 3-deoxy-3-oxo-6-deoxyerythronolide B was expressed in Streptomyces lividans host cells transformed with plasmid pKOS39-104, the desosaminylated analog was produced. Likewise, when plasmid pCK7, which encodes DEBS, which produces 6-deoxyerythronolide B, was expressed in Streptomyces lividans host cells transformed with plasmid pKOS39-104, the 5-desosaminylated analog was produced.

These compounds have antibiotic activity and are useful as intermediates in the synthesis of other antibiotics.

WO 99/61599 PCT/US99/11814 -81- Example 4 Expression Vector for Desosaminyl Transferase While the invention provides expression vectors comprising all of the genes required for desosamine biosynthesis and transfer to a polyketide, the invention also provides expression vectors that encode any subset of those genes or any single gene. As one illustrative example, the invention provides an expression vector for desosaminyl transferase.

This vector is useful to desosaminylate polyketides in host cells that produce NDPdesosamine but lack a desosaminyl transferase gene or express a desosaminyl transferase that does not function as efficiently on the polyketide of interest as does the desosaminyl transferase of Streptomyces venezuelae. This expression vector was constructed by first amplifying the desosaminyl transferase coding sequence from pKOS023-27 using the primers: N3917: 5'-CCCTGCAGCGGCAAGGAAGGACACGACGCCA-3' (SEQ ID NO:25); and N3918: 5'-AGGTCTAGAGCTCAGTGCCGGGCGTCGGCCGG-3' (SEQ ID NO:26), to give a 1.5 kb product. This product was then treated with restriction enzymes PstI and Xbal and ligated with HindIII and Xbal digested plasmid pKOS039-06 together with the 7.6 kb PstI-Hindlll restriction fragment of plasmid pWHM 104 to provide plasmid pKOS039- 14. Plasmid pWHM1104, described in Tang et al., 1996, Molec. Microbiol. 22(5): 801-813, incorporated herein by reference, encodes the ermE* promoter. Plasmid pKOS039-14 is constructed so that the desosaminyl transferase gene is placed under the control of the ermE* promoter and is suitable for expression of the desosaminyl transferase in Streptomyces, Saccharopolyspora erythraea, and other host cells in which the ermE* promoter functions.

Example Heterologous Expression of the picK Gene Product in E. coli The picK gene was PCR amplified from plasmid pKOS023-28 using the oligonucleotide primers: N024-36B (forward): 5'-TTGCATGCATATGCGCCGTACCCAGCAGGGAACGACC (SEQ ID NO:27); and N024-37B (reverse): (SEQ ID NO:28). These primers alter the Streptomyces GTG start codon to ATG and introduce a Spel site at the C- WO 99/61599 PCT/US99/11814 -82terminal end of the gene, resulting in the substitution of a serine for the terminal glycine amino acid residue. The blunt-ended PCR product was subcloned into the commercially available vector pCRscript at the Srfl site to yield plasmid pKOS023-60. An -1.3 kb Ndel- Xhol fragment was then inserted into the NdeIIXhoI sites of the T7 expression vector pET22b (Novagen, Madison, WI) to generate pKOS023-61. Plasmid pKOS023-61 was digested with restriction enzymes Spel and EcoRI, and a short linker fragment encoding 6 histidine residues and a stop codon (composed ofoligonucleotides 30-85a: CTAGTATGCATCATCATCATCATCATTAA-3' (SEQ ID NO:29); and 30-85b: 5'-AATTTTAATGATGATGATGATGATGCATA-3' (SEQ ID NO:30) was inserted to obtain plasmid pKOS023-68. Both plasmid pKOS023-61 and pKOS023-68 produced active PicK enzyme in recombinant E. coli host cells.

Plasmid pKOS023-61 was transformed into E. coli BL21-DE3. Successful transformants were grown in LB-containing carbenicillin (100 Rg/ml) at 37 0 C to an OD600 of 0.6. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown for an additional 3 hours before harvesting. The cells were collected by centrifugation and frozen at -80 0 C. A control culture of BL21-DE3 containing the vector plasmid pET21c (Invitrogen) was prepared in parallel.

The frozen BL21-DE3/pKOS023-61 cells were thawed, suspended in 2 tL of cold cell disruption buffer (5 mM imidazole, 500 mM NaC1, 20 mM Tris/HC1, pH 8.0) and sonicated to facilitate lysis. Cellular debris and supernatant were separated by centrifugation and subjected to SDS-PAGE on 10-15% gradient gels, with Coomassie Blue staining, using a Pharmacia Phast Gel Electrophoresis system. The soluble crude extract from BL21- DE3/pKOS023-61 contained a Coomassie stained band of Mr-46 kDa, which was absent in the control strain BL21-DE3/pET21c.

The hydroxylase activity of the picK protein was assayed as follows. The crude supernatant (20 pL) was added to a reaction mixture (100 LL total volume) containing mM Tris/HCl (pH 20 pM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6phosphate, and 20 nmol of narbomycin. The narbomycin was purified from a culture ofStreptomyces narbonensis, and upon LC/MS analysis gave a single peak of The reaction was allowed to proceed for 105 minutes at 30 0 C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by evaporative light scattering (ELSD) and mass spectrometry. The control extract (BL21-DE3/pET21c) was WO 99/61599 PCT/UJS99/11814 -83processed identically. The BL21-DE3/pKOS023-61 reaction contained a compound not present in the control having the same retention time, molecular weight and mass fragmentation pattern as picromycin The conversion of narbomycin to picromycin under these conditions was estimated to be greater than 90% by ELSD peak area.

The poly-histidine-linked PicK hydroxylase was prepared from pKOS023-68 transformed into E. coli BL21 (DE3) and cultured as described above. The cells were harvested and the PicK protein purified as follows. All purification steps were performed at 4°C. E. coli cell pellets were suspended in 32 pL of cold binding buffer (20 mM Tris/HCl, pH 8.0, 5 mM imidazole, 500 mM NaC1) per mL of culture and lysed by sonication. For analysis ofE. coli cell-free extracts, the cellular debris was removed by low-speed centrifugation, and the supernatant was used directly in assays. For purification of PicK/6- His, the supernatant was loaded (0.5 mL/min.) onto a 5 mL HiTrap Chelating column (Pharmacia, Piscataway, New Jersey), equilibrated with binding buffer. The column was washed with 25 pL of binding buffer and the protein was eluted with a 35 jpL linear gradient (5-500 mM imidazole in binding buffer). Column effluent was monitored at 280 nm and 416 nm. Fractions corresponding to the 416 nm absorbance peak were pooled and dialyzed against storage buffer (45 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 0.2 mM DTT, glycerol). The purified 46 kDa protein was analyzed by SDS-PAGE using Coomassie blue staining, and enzyme concentration and yield were determined.

Narbomycin was purified as described above from a culture of Streptomyces narbonensis ATCC 19790. Reactions for kinetic assays (100 pLL) consisted of 50 mM Tris/HCl (pH 100 LM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 U glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, 20-500 pM narbomycin substrate, and 50-500 nM of PicK enzyme. The reaction proceeded at 30 0 C, and samples were withdrawn for analysis at 5, 10, 15, and minutes. Reactions were stopped by heating to 100°C for 1 minute, and denatured protein was removed by centrifugation. Depletion of narbomycin and formation of picromycin were determined by high performance liquid chromatography (HPLC, Beckman C-18 0.46x15 cm column) coupled to atmospheric pressure chemical ionization (APCI) mass spectroscopic detection (Perkin Elmer/Sciex API 100) and evaporative light scattering detection (Alltech 500 ELSD).

WO 99/61599 PCT/US99/11814 -84- Example 6 Expression of the picK Gene Encoding the Hydroxylase in Streptomyces narbonensis To produce picromycin in Streptomyces narbonensis, a host that produces narbomycin but not picromycin, the methods and vectors of the invention were used to express the picK gene in this host.

The picK gene was amplified from cosmid pKOS023-26 using the primers: N3903: 5'-TCCTCTAGACGTTTCCGT-3' (SEQ ID NO:31); and N3904: 5'-TGAAGCTTGAATTCAACCGGT-3' (SEQ ID NO:32) to obtain an -1.3 kb product. The product was treated with restriction enzymes Xbal and HindIland ligated with the 7.6 kb XbaI-HindII restriction fragment of plasmid pWHM 1104 to provide plasmid pKOS039-01, placing the picK gene under the control of the ermE* promoter. The resulting plasmid was transformed into purified stocks of S. narbonensis by protoplast fusion and electroporation. The transformants were grown in suitable media and shown to convert narbomycin to picromycin at a yield of over Example 7 Construction of a Hybrid DEBS/Narbonolide PKS This example describes the construction of illustrative hybrid PKS expression vectors of the invention. The hybrid PKS contains portions of the narbonolide PKS and portions of rapamycin and/or DEBS PKS. In the first constructs, pKOS039-18 and pKOS039-19, the hybrid PKS comprises the narbonolide PKS extender module 6 ACP and thioesterase domains and the DEBS loading module and extender modules 1-5 as well as the KS and AT domains of DEBS extender module 6 (but not the KR domain of extender module In pKOS039-19, the hybrid PKS is identical except that the KS1 domain is inactivated, the ketosynthase in extender module 1 is disabled. The inactive DEBS KS1 domain and its construction are described in detail in PCT publication Nos. WO 97/02358 and WO 99/03986, each of which is incorporated herein by reference. To construct pKOS039-18, the 2.33 kb BamHI-EcoRI fragment of pKOS023-27, which contains the desired sequence, was amplified by PCR and subcloned into plasmid pUC19. The primers used in the PCR were: N3905: 5'-TTTATGCATCCCGCGGGTCCCGGCGAG-3' (SEQ ID NO:33); and N3906: 5'-TCAGAATTCTGTCGGTCACTTGCCCGC-3' (SEQ ID NO:34).

WO 99/61599 PCT/US99/11814 The 1.6 kb PCR product was digested with PstI and EcoRI and cloned into the corresponding sites ofplasmid pKOS015-52 (this plasmid contains the relevant portions of the coding sequence for the DEBS extender module 6) and commercially available plasmid pLitmus 28 to provide plasmids pKOS039-12 and pKOS039-13, respectively. The BglII EcoRI fragment of plasmid pKOS039-12 was cloned into plasmid pKOS01 1-77, which contains the functional DEBS gene cluster and into plasmid pJRJ2, which contains the mutated DEBS gene that produces a DEBS PKS in which the KS domain of extender module I has been rendered inactive. Plasmid pJRJ2 is described in PCT publication Nos. WO 99/03986 and WO 97/02358, incorporated herein by reference.

Plasmids pKOS039-18 and pKOS039-19, respectively, were obtained. These two plasmids were transformed into Streptomyces coelicolor CH999 by protoplast fusion.

The resulting cells were cultured under conditions such that expression of the PKS occurred.

Cells transformed with plasmid pKOS039-18 produced the expected product 3-deoxy-3-oxo- 6-deoxyerythronolide B. When cells transformed with plasmid pKOS039-19 were provided (2S,3R)-2-methyl-3-hydroxyhexanoate NACS, 13-desethyl-13-propyl-3-deoxy-3-oxo- 6 deoxyerythronolide B was produced.

Example 8 6-Hydroxylation of 3,6-dideoxy-3-oxoerythronolide B using the eryF hydroxylase Certain compounds of the invention can be hydroxylated at the C6 position in a host cell that expresses the eryF gene. These compounds can also be hydroxylated in vitro, as illustrated by this example.

The 6-hydroxylase encoded by eryF was expressed in E. coli, and partially purified.

The hydroxylase (100 pmol in 10 gL) was added to a reaction mixture (100 gl total volume) containing 50 mM Tris/HCl (pH 20 pM spinach ferredoxin, 0.025 Unit of spinach ferredoxin:NADP+ oxidoreductase, 0.8 Unit of glucose-6-phosphate dehydrogenase, 1.4 mM NADP+, 7.6 mM glucose-6-phosphate, and 10 nmol 6-deoxyerythronolide B. The reaction was allowed to proceed for 90 minutes at 30 0 C. Half of the reaction mixture was loaded onto an HPLC, and the effluent was analyzed by mass spectrometry. The production of erythronolide B as evidenced by a new peak eluting earlier in the gradient and showing Conversion was estimated at 50% based on relative total ion counts.

Those of skill in the art will recognize the potential for hemiketal formation in the above compound and compounds of similar structure. To reduce the amount of hemiketal WO 99/61599 PCT/US99/11814 -86formed, one can use more basic (as opposed to acidic) conditions or employ sterically hindered derivative compounds, such as 5-desosaminylated compounds.

Example 9 Measurement of Antibacterial Activity Antibacterial activity was determined using either disk diffusion assays with Bacillus cereus as the test organism or by measurement of minimum inhibitory concentrations (MIC) in liquid culture against sensitive and resistant strains of Staphylococcuspneumoniae.

The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.

Claims (36)

1. A recombinant DNA compound that comprises a sequence encoding a domain of a narbonolide polyketide synthase (PKS) of Streptomyces or an isolated DNA compound comprising a degenerate sequence to said Streptomyces sequence.

2. A recombinant DNA compound according to claim 1 wherein Streptomyces is Streptomyces narbonensis.

3. A recombinant DNA compound according to claim 1 wherein the Streptomyces is Streptomyces venezuelae.

4. The recombinant DNA compound according to any one of claims 1 to 3, wherein said domain is selected from the group consisting of a thioesterase domain, a KSQ domain, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain, and an ER domain. The recombinant DNA compound of claim 4 that comprises the coding sequence of a loading module, thioesterase domain, and all six extender modules of the narbonolide PKS.

6. The recombinant DNA compound of claim 4 that comprises a hybrid PKS.

7. The recombinant DNA compound of claim 6 wherein said hybrid PKS 25 comprises at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.

8. The recombinant DNA compound of claim 7 wherein said second PKS gene is a DEBS gene.

9. The recombinant DNA compound of claim 8 wherein said hybrid PKS is composed of a loading module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS. A recombinant DNA compound that comprises a coding sequence of a desosamine biosynthetic gene or a desosaminyl transferase gene or a beta-glucosidase gene of Streptomyces venezuelae or a degenerate sequence to said S. venezuelae sequence.

11. A recombinant DNA compound that comprises a coding sequence of a picK hydroxylase gene of Streptomyces venezuelae or a degenerate sequence to said S. venezuelae sequence.

12. The DNA compound of any one of claims 1 to 11 that further comprises a promoter operably linked to said coding sequence.

13. The recombinant DNA compound of claim 12, wherein said promoter is a promoter derived from a cell other than a Streptomyces venezuelae cell.

14. The recombinant DNA compound of claim 13 that is a recombinant DNA expression vector. The expression vector of claim 14 that expresses a PKS in a Streptomyces host. cell.

16. A recombinant host cell, which in its untransformed state does not produce i: deoxymethynolide or narbonolide, wherein said cell comprises the recombinant DNA expression vector of claim 14 encoding a narbonolide PKS and wherein said cell 25 produces 10-deoxymethynolide or narbonolide.

17. The recombinant host cell of claim 16 that further comprises apicB gene.

18. The recombinant host cell of claim 16 or 17 that further comprises desosamine biosynthetic genes and a gene for desosaminyl transferase and produces YC17 or narbomycin.

19. The recombinant host cell of claim 18 that further comprises a picK gene and produces methymycin, neomethymycin, or picromycin. 89 The recombinant host cell of claim 19 that is Streptomyces coelicolor or Streptomyces lividans.

21. A recombinant host cell other than a Streptomyces venezuelae cell that expresses a picK hydroxylase gene of S. venezuelae or a degenerate sequence of S. venezuelae.

22. A recombinant host cell other than a Streptomyces venezuelae host cell that expresses a desosamine biosynthetic gene or desosaminyl transferase gene of S. venezuelae or a degenerate sequence ofS. venezuelae.

23. A method of increasing the yield of a desosaminylated polyketide in a cell, which method comprises transforming the cell with a recombinant expression vector that encodes a functional beta-glucosidase gene.

24. A hybrid PKS which comprises at least one domain of a Streptomyces narbonolide PKS. The hybrid PKS of claim 24 wherein said hybrid PKS comprises at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.

26. The hybrid PKS of claim 25 wherein said second PKS gene is a DEBS gene.

27. The hybrid PKS of claim 26 wherein said hybrid PKS is comprised of a loading S• 25 module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS.

28. A method of producing a polyketide which comprises providing starter, extender and/or intermediate ketide units to the hybrid PKS of claim 24.

29. A polyketide produced by the method of claim 28.

30. A method of desosaminylating a polyketide compound comprising expressing a recombinant DNA compound according to claim 1 and/or claim 10 and/or claim 11 in a host cell.

31. A method according to claim 30 Streptomyces venezuelae.

32. A method according to claim 30 narbonensis.

33. A method according to any one of Streptomyces lividans. 0

34. A method according to any one of Streptomyces coelicolor. wherein the recombinant DNA is from wherein the recombinant DNA is from claims 30 to 32 wherein the host cell is claims 30 to 32 wherein the host cell is A method according to any one of claims 30 to 34 wherein a beta-glucosidase gene is also expressed in the host cell.

36. A host cell that has been transformed with the recombinant DNA according to any one of claims 1 to 14 such that it expresses a Streptomyces narbonolide PKS and/or a Streptomyces desoaminyl transferase and/or a Streptomyces desosamine biosynthetic enzyme.

37. A host cell according to claim 36 wherein the Streptomyces is Streptomyces venezuelae. 25 38. A host cell according to claim 36 wherein the Streptomyces is Streptomyces nabonensis.

39. A host cell according to any one of claims 36 to 38 wherein the cell also expresses a beta-glucosidase enzyme. A recombinant DNA compound according to any one of claims 1 to 14 or the expression vector of claim 15 substantially as hereinbefore described with reference to the examples and/or the drawings. 9 9* 9 9 9* 9

41. A recombinant host cell according to any one of claims 16 to 22 or a host cell according to any one of claims 36 to 39 substantially as hereinbefore described with reference to the examples and/or the drawings.

42. A method for increasing the yield of a desosaminylated polyketide in a cell according to claim 23 substantially as hereinbefore described with reference to the examples and/or the drawings.

43. A hybrid PKS according to any one of claims 24 to 27 substantially as hereinbefore described with reference to the examples and/or the drawings.

44. A method of producing a polyketide according to claim 28 or a polyketide according to claim 29 substantially as hereinbefore described with reference to the examples and/or the drawings. A method of desosaminylating a polyketide compound according to any one of claims 30 to 35 substantially as hereinbefore described with reference to the examples and/or the drawings. Dated this TWENTY EIGHTH day of APRIL 2003 KOSAN Biosciences, Inc. i Patent Attorneys for the Applicant: F B RICE CO