AU2002305118A1 - Biosynthetic genes for butenyl-spinosyn insecticide production - Google Patents

Description

DESCRIPTION

BIOSYNTHETIC GENES FOR BUTENYL-SPINOSYN INSECTICIDE PRODUCTION

Summary ofthe Invention [0001] The present invention provides novel butenyl-spinosyn biosynthetic genes, vectors incorporating the biosynthetic genes, Saccharopolyspora strains transformed with the biosynthetic genes, methods of using these genes to increase production of spinosyn-like insecticidal macrolides, and methods of using the genes or fragments thereof to change the metabolites made by spinosyn-producing strains of Saccharopolyspora spp.

Background ofthe invention [0002] The naturally produced spinosyn compounds consist of a 5,6,5-tricylic ring system, fused to a 12-membered macrocyclic lactone, a neutral sugar (rhamnose) and an amino sugar (forosamine) (see Kirst et al., 1991). If the amino sugar is not present, the compounds have been referred to as pseudoaglycones, and if the neutral sugar is not present then the compounds have been referred to as reverse pseudoaglycones.

[0003] A83543 spinosyns are produced by Saccharopolyspora spinosa strain NRRL18395 and derivatives thereof. The known members ofthe A83543 spinosyn family and the strains producing them have been disclosed in US Patent No. 5,362,634; 5,202,242; 5,840,861; 5,539,089 and 5,767,253. The compounds are identified by letter designation: spinosyn A, B, etc. (see Kirst et al., 1991). The structure of A83543 spinosyn A is given in Table 1 hereinafter. The A83543 spinosyn compounds are useful for the control of arachnids, nematodes and insects, in particular Lepidoptera and Diptera species, and they have favorable environmental and toxicological profiles.

[0004] DNA sequences for the genes encoding the enzymes which direct the biosynthesis of A83543 spinosyn are disclosed in U.S. Patent No. 6,143,526. The cloned genes and open reading frames are designated as spnA, spnB, spnC, spnD, spnE, spnF, spnG, spnH, spnl, spnJ, spnK, spnL, spnM, spnN, spnO, spnP, spnQ, spnR, spnS, S. spinosa gtt, S. spinosa gdh, S. spinosa epi, and S. spinosa kre.

[0005] The spinosyn biosynthetic genes, excluding those for rhamnose biosynthesis, specifically genes spnA, spnB, spnC, spnD, spnE, spnF, spnG, spnH, spnl, spnJ, spnK, spnL, spnM, spnN, spnO, spnP, spnQ, spnR, and spnS are located contiguously on an approximately 74 kb region ofthe S. spinosa chromosome. The spnA, spnB, spnC, spnD, and spnE genes were shown to be similar to genes responsible for polyketide biosynthesis and disruption of spnA, spnD, or spnE eliminated all spinosyn production. A83543 spinosyn synthesis also involves bridging ofthe lactone nucleus, an activity that is rare in macrolide producers; the spnF, spnJ, spnL, and spnM genes were believed to be involved in this biosynthetic step. The spnG, spnH, spnl, and spnK genes were reported to be involved in rhamnose addition and modification, and the spnN, spnO, spnP, spnQ, spnR and spnS genes were reported to be involved in biosynthesis and addition ofthe forosamine sugar. The genes required for rhamnose biosynthesis were not located contiguously to the rest of the A83543 spinosyn biosynthetic genes. S. spinosa gtt, and S. spinosa kre were cloned on a distinct fragment, and S. spinosa gdh and S. spinosa epi, were cloned on other distinct fragments.

[0006] Recently a second class of spinosyns, the butenyl-spinosyns, produced by a novel organism, Saccharopolyspora sp. LW107129 (NRRL 30141) or derivatives thereof, was disclosed in US Appl. No. 09/661,065, corresponding to WO 01/19840, and in U.S. Patent Appl. No. 60/277,601. Over 40 members of this chemical family have been defined in the aforementioned applications. The butenyl-spinosyn compounds produced by Saccharopolyspora sp. LW107129 (NRRL 30141) are different from the compounds in the A83543 spinosyn series. The primary difference between the two classes of spinosyns is the substitution ofthe carbon tail attached to the macrocyclic ring at C-21. The natural butenyl spinosyns are substituted with a 3-4 carbon chain at C-21, preferably butenyl, while natural A83543 spinosyns are substituted with a 1-2 carbon chain at C-21, preferably ethyl.

[0007] The butenyl-spinosyn compounds are useful as reactants in the production of synthetically modified spinosoid compounds as disclosed in the provisional U. S. Patent Application 60/277,546 on "Synthetic Derivatives of 21-Butenyl and Related Spinosyns," filed March 21, 2001. More preferably, the butenyl-spinosyn compounds and their synthetic derivatives are useful for the control of arachnids, nematodes and insects, in particular Lepidoptera and Diptera species.

[0008] In addition to the butenyl group at C-21 , butenyl-spinosyns exhibit a number of other differences from the A83543 spinosyn series. A subset ofthe butenyl-spinosyn compounds and factors which demonstrate diversity relative to A83543 spinosyns are summarized in Table 1. The butenyl spinosyns are named in Table I and referred to hereinafter by the structural acronyms "for-rham-I", "for-rham-II", "for-rham-III" and derivatives thereof. In these cases I, II, and III refer to the appropriately-substituted macrolide structure (I: R⁴=R⁵=H; LI: R⁵ = CH₃, R⁴ = H or OH; III: R⁵ = H, R⁴ = OH), 'for' represents the sugar at C-17 (for = forosamine), and 'rham' represents the sugar at C-9 (rham = tri-O-methylrhamnose). A second type of macrolide structure which is produced by strain NRRL 30141, with general Formula (2) having a 14-membered macrolide ring, is referred to hereinafter as IN and the fully glycosylated comound as "for-rharn-IN". The butenyl-spinosyn compounds of formulae (1) and (2) are useful for the control of arachnids, nematodes and insects, in particular Lepidoptera and Diptera species, and they are quite environmentally friendly and have an appealing toxicological profile.

[0009] These differences include extensive modifications at the C-21 position, hydroxylation at the C-8 position and substitution of alternate sugars, including neutral sugars, for forosamine at C-17. In addition, a compound possessing a 5,6,5-tricylic ring system, fused to a 14 membered macro-cyclic ring with forosamine and rhamnose attached at C-17 and C-9, respectively was previously disclosed in the aforementioned application.

Table 1

-A-

*R is a group having one ofthe following formulas (3 a) through (3 c)

(3a) (3b) (3c)

(3d) (^3e)

**R9 is a group having one ofthe following formulas (9a) to (9i) forosamine

H₃C H₃C 4"-N-desmethyl-1",4" diepiforosamine

N O

H₃C

OO

5"-epiforosamine [0010] Compounds 1-21 in Table I are produced by Saccharopolyspora sp. LW107129 (NRRL 30141) and are disclosed in U.S. Patent Application. 09/661,065, corresponding to WO 01/19840. Compounds 32 & 23 are disclosed in provisional U. S. Patent Application 60/277,601. on "Pesticidal Macrolides", filed March 21, 2001.

[0011] Despite the differences between the structures of butenyl-spinosyns and A83543 spinosyns, it can be deduced that some ofthe biosynthetic genes would be similar. However, as detailed above, Saccharopolyspora sp. LW107129 (NRRL 30141) produces a wide range of unique butenyl-spinosyn factors and compounds, which have not been observed in the A83543 spinosyns. Therefore, this organism must also posses novel biosynthetic enzymes, which are distinct from the A83543 spinosyn biosynthetic enzymes of S. spinosa. Specifically, relative to A83543 spinosyns, Saccharopolyspora sp. LW107129 (NRRL 30141) butenyl-spinosyn biosynthetic enzymes must be capable of extending the polyketide chain by 2 carbons (resulting in butenyl rather than ethyl at C-21). They must also be able to synthesize and attach alternate amino and neutral sugars at C-17, and hydroxylate at C-8 & C-24. In addition, rhamnose methylation must be different in Saccharopolyspora sp. LW107129 (NRRL 30141) relative to S. spinosa. Blocked mutants of S. spinosa which exhibited altered methylation ofthe rhamnose on A83543 spinosyn (as disclosed in USP 5,202,242 and 5,840,861) typically produced mono-desmethylated rhamnose derivatives of A83543 spinosyns. Di-desmethyl rhamnose derivatives of A83543 spinosyns were only detected in the presence of methylase inhibitors like sinefungin. Mutants of Saccharopolyspora sp. LW107129 (NRRL 30141) with altered methylation of rhamnose produced di- and tri-desmethyl rhamnose derivatives of butenyl-spinosyns in high amounts, in the absence of methylase inhibitors.

[0012] A challenge in producing butenyl-spinosyn compounds arises from the fact that a very large fermentation volume is required to produce a very small quantity of butenyl- spinosyns. A cloned fragment of DNA containing one or multiple genes for butenyl- spinosyn biosynthetic enzymes would enable duplication of genes to increase yield. A yield increase of this type was achieved in fermentation's of Streptomyces fradiae by duplicating the gene encoding a rate-limiting methyltransferase that converts macrocin to tylosin (Baltz et al., 1997) and in S. spinosa by duplicating the gtt & gdh genes (Baltz et al., 2000).

[0013] Cloned butenyl-spinosyn biosynthetic genes also provide a method for producing new derivatives ofthe butenyl-spinosyns, with a different spectrum of insecticidal activity. Specific intermediates (or their natural derivatives) can be synthesized by mutant strains of Saccharopolyspora sp. LW107129 (NRRL 30141) in which certain genes encoding enzymes for butenyl-spinosyn biosynthesis have been disrupted using recombinant DNA methods. Such a strategy was used effectively to generate a strain of Saccharopolyspora erythraea producing novel 6-deoxyerythromycin derivatives (Weber & McAlpine, 1992). Also butenyl-spinosyn biosynthetic genes can be expressed in other organisms, such as S. spinosa, which produce similar compounds. When expressed from the native butenyl- spinosyn promoter or a heterologous promoter these genes produce new hybrid molecules with some ofthe unique structural features of both spinosyns and butenyl-spinosyns.

[0014] Novel intermediates can also be synthesized by mutant strains of Saccharopolyspora sp. LW107129 (NRRL 30141) or S. spinosa in which parts of certain genes encoding enzymes for butenyl-spinosyn biosynthesis have been replaced with parts of the same gene which have been specifically mutated in vitro, or with conesponding parts of genes from other organisms. The hybrid gene will produce a protein with altered functions, either lacking an activity or performing a novel enzymatic transformation. A new chemical substance would accumulate upon fermentation ofthe mutant strain. Such a strategy was used to generate a strain of Saccharopolyspora erythraea producing a novel anhydroerythromycin derivative (Donadio et al, 1993).

[0015] Biosynthesis of butenyl-spinosyns proceeds via stepwise condensation and modification of 2- and 3-carbon carboxylic acid precursors, generating a linear polyketide (Figure 1 A) that is cyclized and bridged to produce the tetracyclic aglycone (Figure IB). Pseudoaglycone (containing tri-O-methylated rhamnose) is formed next, then di-N- methylated forosamine or an alternate sugar is added to complete the biosynthesis (Figure IB). Other macrolides, such as the antibiotic erythromycin, the antiparasitic avermectin and the immunosuppressant rapamycin, are synthesized in a similar fashion. In the bacteria producing these compounds, antibiotic biosynthesis is catalyzed by several very large, multifunctional proteins of a Type I polyketide synthase (PKS) (Donadio et al, 1991; Ikeda et al, 1999; Schwecke et al, 1995). Together the polypeptides form a complex consisting of an initiator module and several extender modules, each of which adds a specific acyl- CoA precursor to a growing polyketide chain, and modifies the β-keto group in a specific manner (Figure IA). The structure of a polyketide is, therefore determined by the composition and order ofthe modules in the PKS. A module comprises several domains, each of which performs a specific function. The initiator module consists of an acyl transferase (AT) domain for addition ofthe acyl group from the precursor to an acyl carrier protein (ACP) domain. The initiator module may also contain a KSQ domain, which is highly similar to β-ketosynthase (KS) domains, but an essential active site cysteine has been replaced by glutamine (Bisang, et al, 1999), so KSQ no longer has condensing activity. KSQ domains retain decarboxylase activity and determines the precursor specificity ofthe initiation module. The extender modules contain AT and ACP domains, along with an intact β-ketosynthase (KS) domain that adds the pre-existing polyketide chain to the new acyl- ACP by decarboxylative condensation. Additional domains may also be present in each extender modules to carry out specific β-keto modifications: a β-ketoreductase (KR) domain to reduce the β-keto group to a hydroxyl group, a dehydratase (DH) domain to remove the hydroxyl group and leave a double bond, and an enoyl reductase (ER) domain to reduce the double bond and leave a saturated carbon. The last extender module terminates with a thioesterase (TE) domain that liberates the polyketide from the PKS enzyme in the form of a macrocyclic lactone. Polyketide synthase enzymes are generally encoded by 3-7 large open reading frames (Donadio et al, 1991; Ikeda et al, 1999; Schwecke et al, 1995). Assembly of a functional polyketide synthase requires specific protein-protein interactions between these proteins.

[0016] Active macrolide antibiotics are derived from macrocyclic lactones by additional modifications, such as methylation and changes in reductive state, and the addition of unusual sugars. Most ofthe genes required for these modifications, and for the synthesis and attachment ofthe sugars, are clustered around the PKS genes. The genes encoding deoxysugar biosynthetic enzymes are similar in producers of macrolide antibiotics, such as erythromycin and tylosin (Donadio et al, 1993; Merson-Davies & Cundliffe, 1994), and producers of extracellular polysaccharides, such as the O-antigens of Salmonella and Yersinia (Jiang et al, 1991; Trefzer et al, 1999). All these syntheses involve activation of glucose by the addition of a nucleotide diphosphate, followed by dehydration, reduction and/or epimerization. The resultant deoxy-sugar could undergo one or more additional modifications such as deoxygenation, transamination and methylation. The sugars are incorporated into macrolides by the action of specific glycosyltransferases. Genes involved in the synthesis and attachment of a sugar may be tightly clustered - even transcribed as a single operon - or they may be dispersed (Ikeda et al, 1999; Shen et al., 2000; Aguinezabalaga et al, 1998).

[0017] The following terms are used herein as defined below:

[0018] a.a. - amino acid

[0019] AniR - the apramycin resistance-conferring gene.

[0020] ACP - acyl carrier protein domain.

[0021] AT - acyltransferase domain.

[0022] blocked mutant - mutant strain having a mutation blocking the function of a specific enzyme of a biosynthetic pathway such that a precursor or shunt product is produced. [0023] bp - base pairs.

[0024] bus - butenyl-spinosyn biosynthetic gene.

[0025] Butenyl-spinosyn - a fermentation product structurally distinct from A83543 spinosyns (Table 1), disclosed in US Appl. No. 09/661,065 and provisional U. S. Patent Appl. No. 60/277,601, or a similar macrocyclic lactone fermentation product produced by a microorganism utilizing all or most ofthe butenyl-spinosyn genes.

[0026] Butenyl-spinosyn genes- the DNA sequences that encode the products required for butenyl-spinosyn biosynthesis, more specifically the genes busA, busB, busC, busD, busE, busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, and busS, as described hereinafter, or functional equivalents thereof.

[0027] Cloning - the process of incorporating a segment of DNA into a recombinant DNA cloning vector and transforming a host cell with the recombinant DNA.

[0028] Codon bias - the propensity to use a particular codon to specify a specific amino acid. In the case of Saccharopolyspora sp. LWl 07129 (NRRL 30141) , the propensity is to use a codon having cytosine or guanine as the third base.

[0029] Complementation - the restoration of a mutant strain to its normal phenotype by a cloned gene.

[0030] Conjugation - a process in which genetic material is transferred from one bacterial cell to another.

[0031] cos - the cohesive end sequence of bacteriophage lambda.

[0032] Cosmid - a recombinant DNA cloning vector which is a plasmid that not only can replicate in a host cell in the same manner as a plasmid but also can be packaged into phage heads.

[0033] DH - dehydratase domain.

[0034] ER - enoyl reductase domain.

[0035] Gene - a DNA sequence that encodes a polypeptide.

[0036] Genomic Library - a set of recombinant DNA cloning vectors into which segments of DNA, representing substantially all DNA sequences in a particular organism have been cloned.

[0037] Homology - degree of similarity between sequences

[0038] Hybridization - the process of annealing two single stranded DNA molecules to form a double stranded DNA molecule, which may or may not be completely base paired.

[0039] In vitro packaging - the in vitro encapsulation of DNA in coat protein to produce a virus-like particle that can introduce DNA into a host cell by infection

[0040] kb - kilo base pairs.

[0041] KR - β-keto reductase domain.

[0042] KS - ketosynthase domain.

[0043] Mutagenesis - creation of changes in DNA sequence. They can be random or targeted, generated in vivo or in vitro. Mutations can be silent, or can result in changes in the amino acid sequence ofthe translation product which alter the properties ofthe protein and produce a mutant phenotype.

[0044] ORF - open reading frame.

[0045] ori - a plasmid origin of replication (oriR) or transfer (oriT).

[0046] % Identity - the % identity value give by the BLAST program when two sequences are compared.

[0047] % Similarity - the % similarity value given by the BLAST program when two sequences are compared.

[0048] PCR - polymerase chain reaction - a method to specifically amplify a region of DNA.

[0049] PKS - polyketide synthase.

[0050] Promoter - a DNA sequence that directs the initiation of transcription.

[0051] Recombinant DNA cloning vector - any autonomously replicating or integrating agent, including , but not limited to, plasmids, comprising a DNA molecule to which one or more additional DNA molecules can be or have been added.

[0052] Recombinant DNA methodology - technologies used for the creation, characterization, and modification of DNA segments cloned in recombinant DNA vectors.

[0053] Restriction fragment - any linear DNA molecule generated by the action of one or more restriction enzymes.

[0054] Spinosyn - a fermentation product also known as A83543, typically characterized by a 5,6,5-tricylic ring system, fused to a 12-membered macrocyclic lactone, with a 1-2 carbon chain at C-21, a neutral sugar (rhamnose) and an amino sugar (forosamine), or a similar macrocyclic lactone fermentation product produced by a microorganism utilizing all or most ofthe A83543 spinosyn genes.

[0055] Spinosyn genes- the DNA sequences that encode the products required for A83543 spinosyn biosynthesis, more specifically the genes spnA, spnB, spnC, spnD, spnE, spnF, spnG, spnH, spnl, spnJ, spnK, spnL, spnM, spnN, spnO, spnP, spnQ, spnR, spnS, S. spinosa gtt, S. spinosa gdh, S. spinosa epi, and S. spinosa kre, as described hereinafter, or functional equivalents thereof.

[0056] spn - A83543 spinosyn biosynthetic gene.

[0057] Subclone - a cloning vector with an insert DNA derived from another DNA of equal size or larger.

[0058] TE - thioesterase domain.

[0059] Transconjugant - recombinant strain derived from a conjugal mating.

Brief Description ofthe Figures [0060] FIG.S 1 A and IB are a diagram illustrating the butenyl-spinosyn biosynthetic pathway.

[0061] FIG. 2 is a map illustrating the anangement oϊHindlll, EcoRV and Seal fragments and open reading frames in the cloned region of Saccharopolyspora sp. LW107129 (NRRL 30141) DNA and location of butenyl-spinosyn genes on the cloned DNA.

[0062] FIG. 3 is a restriction site and functional map of Cosmid pOJ436.

[0063] FIG. 4 is a diagram illustrating the biosynthetic pathway for 17-(4"-O- methyloleandrose)-butenyl-spinosyn [Table 1; compound (11)]. Brief Description ofthe invention [0064] Butenyl-spinosyn biosynthetic genes and related ORFs were cloned and the DNA sequence of each was determined. The cloned genes and ORFs are designated hereinafter as busA, busB, busC, busD, busE, busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, busS, ORF LI, ORF LIT, ORF LUI, ORF LIN, ORF LNI, ORF LNII, ORF LNIII, ORF LLX, ORF RI, ORF RH and ORF RIII. The proposed functions ofthe cloned genes in spinosyn biosynthesis are identified FIG. 1 and in the discussion hereinafter.

[0065] In one of its aspects, the invention provides an isolated DΝA molecule comprising a DΝA sequence that encodes a butenyl-spinosyn biosynthetic enzyme, wherein said enzyme is defined by an amino acid sequence selected from the group consisting of SEQ ID ΝOS 3-7, and 8-29, or said enzyme is defined by one of said amino acid sequences in which one or more amino acid substitutions have been made that do not affect the functional properties ofthe encoded enzyme. In a prefened embodiment, the DΝA sequence is selected from the group of genes consisting of busA, busB, busC, busD, busE, ORF RI, ORFRII, ORF RIII, busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, busS, ORF LI, ORF LII, ORF LIII, ORF LIN, ORF LNI, ORF LNII, ORF LNIII, and ORF LLX, said genes being described by, respectively, bases 1-13032, 13059-19505, 19553-29053, 29092-43890, 43945-60636, 62090-63937, 65229-66602 and 68762-69676 of SEQ ID ΝO:l and 114-938, 1389-2558, 2601-3350, 3362-4546, 4684- 6300, 6317-7507, 7555-8403, 8640-9569, 9671-10666, 10678-12135, 12867-14177, 14627- 15967, 16008-17141, 17168-17914, 18523-19932, 19982-20488, 20539-21033, 21179- 21922, 22674-23453, 23690-24886, 26180-26923, and 27646-28473 of SEQ ID NO:2.

[0066] In another of its aspects, the invention provides an isolated DNA molecule comprising a DNA sequence that encodes a butenyl-spinosyn PKS domain selected from KSi, ATi, ACPi, KSb, ATb, KRb, DHb, ACPb, KS1, ATI, KR1, and ACPI, said domains being described by, respectively, amino acids 6-423, 528-853, 895-977, 998-1413, 1495- 1836, 1846-2028, 2306-2518, 2621-2710, 2735-3160, 3241-3604, 3907-4086, and 4181- 4262 of SEQ ID NO:3. In a prefened embodiment, the DNA sequence is selected from the group consisting of bases 16-1269, 1582-2559, 2683-2931, 2992-4239, 4483-5508, 5538- 6084, 6916-7554, 7861-8130, 8203-9480, 9721-10812, 11719-12258, and 12541-12786 of SEQ ID NO:l.

[0067] In another of its aspects, the invention provides an isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS2, AT2, DH2, ER2, KR2, and ACP2, said domains being described by, respectively, amino acids 1- 421, 534-964, 990-1075, 1336-1681, 1685-1864, and 1953-2031 of SEQ ID NO:4. In a prefened embodiment the DNA sequence is selected from the group consisting of bases 13059-14321, 14658-15900, 16026-16283, 17064-18100, 18111-18650, and 18915-19151 ofSEQ DD NO:l.

[0068] In another of its aspects, the invention provides an isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS3, AT3, KR3, ACP3, KS4, AT4, KR4, and ACP4, said domains being described by, respectively, amino acids 1-421, 528-814, 1157-1335, 1422-1503, 1526-1949, 2063-2393, 2697-2877, and 2969-3049 of SEQ ID NO:5. In a prefened embodiment the DNA sequence is selected from the group consisting of bases 19553-20815, 21143-22000, 23021-23557, 23816- 24061, 24128-25399, 25739-26731, 27641-28183, and 28457-28699 of SEQ ID NO:l.

[0069] In another of its aspects the invention provides an isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS5, AT5, DH5, KR5, ACP5, KS6, AT6, KR6, ACP6, KS7, AT7, KR7, and ACP7, said domains being described by, respectively, amino acids 1-422, 537-864, 891-1076, 1382-1563, 1643- 1724, 1746-2170, 2281-2611, 2914-3093, 3186-3267, 3289-3711, 3823-4151, 4342-4636, and 4723-4804 of SEQ ID NO:6. In a prefened embodiment the DNA sequence is selected from the group consisting of bases 29092-30357, 30700-31683, 31762-32319, 33235- 33780, 34018-34263, 34327-35601, 35932-36924, 37831-38370, 38647-38892, 38956- 40224, 40560-41544, 42115-42999 and 43258-43503 of SEQ ID NO:l.

[0070] In another of its aspects, the invention provides an isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS8, AT8, DH8, KR8, ACP8, KS9, AT9, DH9, KR9, ACP9, KS10, AT10, DH10, KR10, ACP10, and TE10, said domains being described by, respectively, amino acids 1-424, 530-848, 885- 1072, 1371-1554, 1650-1728, 1751-2175, 2289-2616, 2642-2775, 3131-3315, 3396-3474, 3508-3921, 4036-4366, 4389-4569, 4876-5054, 5148-5229, and 5278-5531 of SEQ ID NO:7. In a prefened embodiment, the DNA sequence is selected from the group consisting of bases 43945-45216, 45532-46488, 46597-47160, 48055-48606, 48892-49083, 49195- 50469, 50809-51792, 51868-52269, 53335-53889, 54130-54366, 54466-55707, 56050- 57042, 57109-57651, 58570-59106, 59386-59631, and 59776-60537 of SEQ ID NO.1.

[0071] In another of its aspects the invention provides an isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS module, said module being selected from the group consisting of amino acids 6-977 of SEQ ID NO:3, 998-2710 of SEQ ID NO:3, 2735-4262 of SEQ ID NO:3, 1-2031 of SEQ ID NO:4, 1-1503 of SEQ ID NO:5, 1526-3049 of SEQ ID NO:5, 1-1724 of SEQ ID NO:6, 1746-3267 of SEQ ID NO:6, 3289- 4804 of SEQ ID NO:6, 1-1728 of SEQ ID NOJ, 1751-3474 of SEQ ID NOJ, and 3508- 5531 of SEQ ID NOJ. In a prefened embodiment the DNA sequence is selected from the group consisting of bases 16-2931, 2992-8130, 8203-12786, 13059-19151, 19553-24061, 24128-28699, 29092-34263, 34327-38892, 38956-43503, 43945-49083, 49195-54366, and 54466-60537 of SEQ ID NO:l.

[0072] In another of its aspects, the invention provides a recombinant DNA vector which comprises a DNA sequence ofthe invention as described above.

[0073] In another of its aspects the invention provides a host cell transformed with a recombinant vector ofthe invention as described above.

[0074] In another of its aspects, the invention provides a method of increasing the spinosyn-producing ability of a spinosyn-producing microorganism comprising the steps of

1 ) transforming with a recombinant DNA vector or portion thereof a microorganism that produces butenyl-spinosyn or a butenyl-spinosyn precursor by means of a biosynthetic pathway, said vector or portion thereof comprising a DNA sequence ofthe invention, as described above, that codes for the expression of an activity that is rate limiting in said pathway, and

2) culturing said microorganism transformed with said vector under conditions suitable for cell growth and division, expression of said DNA sequence, and production of spinosyn.

[0075] In another of its aspects the invention provides a spinosyn-producing microorgamsm having operative butenyl-spinosyn biosynthetic genes wherein at least one of the butenyl-spinosyn biosynthetic genes bus A, busB, busC, busD, busE, busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, and busS has been duplicated.

[0076] In another of its aspects the invention provides a butenyl-spinosyn-producing microorganism, said microorganism having butenyl-spinosyn biosynthetic genes in its genome, wherein at least one of said genes has been inactivated, the rest of said genes being operational to produce a butenyl-spinosyn other than the one that would be produced if the disrupted gene were operational. Preferably the microorganism is an Saccharopolyspora sp. LWl 07129 (NRRL 30141) or S. spinosa mutant. More preferably the microorganism is an Saccharopolyspora sp. LW107129 (NRRL 30141) mutant.

[0077] The invention also provides for expression of butenyl-spinosyn biosynthetic genes in an organism which does not normally produce butenyl-spinosyns. The genes may be expressed under natural bus gene promoters or from a heterologous promoter compatible with the recipient strain. Preferably the organism is capable of producing spinosyn-like compounds. More preferably the microorganism is S. spinosa or a derivative thereof.

[0078] The invention also provides a butenyl-spinosyn-producing microorganism having operational butenyl-spinosyn biosynthetic genes in its genome, wherein said genes a) include at least one operational PKS module more than or at least one less than is present in SEQ ID NO:l; or b) include a PKS module that differs from the conesponding module described in SEQ ID NO:l by the deletion, inactivation, or addition of a KR, DH or ER domain, or by the substitution of an AT domain. Preferably the microorganism is an Saccharopolyspora sp. LWl 07129 (NRRL 30141) mutant.

[0079] The invention also provides butenyl-spinosyns produced by cultivation ofthe novel microorganisms ofthe invention.

[0080] In another of its aspects the invention provides a process for isolating butenyl- spinosyn biosynthetic genes which comprises creating a genomic library of a butenyl- spinosyn producing microorganism, and using a labeled fragment of SEQ ID NO:l or SEQ ED NO. 2 that is at least 20 bases long as a hybridization probe.

[0081] It will be understood by those skilled in the art that substitutions can be made in the claimed amino acid sequences without substantially changing the functionality ofthe proteins. The invention encompasses such variant amino acid sequences and DNA sequences encoding the variants. Prefened amino acid sequences are those that have substantially the same functionality and are at least 98% identical to the native amino acid sequence.

Detailed Description ofthe invention

[0082] As a prerequisite for the characterization and utilization of butenyl-spinosyn genes, it is necessary to isolate and characterize the genes that encode the enzymes involved in the biosynthesis of this insect control agent. The approach described in the following

Example 1 involves the construction of a genomic cosmid library and subsequent screening via DNA-hybridization.

EXAMPLE 1 a. Isolation of total cellular DNA from Saccharopolvsyora sp. LW107129 (NRRL

3014D

[0083] Saccharopolyspora sp. LW107129 (NRRL 30141) was inoculated into 100 mL vegetative media (9.0 g/L dextrose, 30 g/L trypticase soy broth, 3.0 g/L yeast extract, 2.0 g/L magnesium sulfateJ H₂0) in a 500-mL Erlenmeyer flask and incubated shaking at 150 rpm for 72 hours at 30°C. This culture was centrifuged for 10 min. at 3,000 rpm/4°C to pellet the cells. The supernatant fluid was removed and the cell pellet was washed with 20 mL of TE buffer (10 mM Tris/HCl pH 8.0; 1 mM EDTA pH 8.0). Cells were centrifuged again at 3,000 rpm and the pellet frozen at -20°C until it was thawed for total cellular DNA isolation.

[0084] Total cellular DNA was isolated from Saccharopolyspora sp. LW107129 (NRRL 30141) (NRRL30141) using a Genomic DNA purification kit (Qiagen Inc., Valencia, CA). Frozen bacterial cell pellets from 100 mL of culture were resuspended in 11 ml of Buffer Bl (50 mM Tris/HCl, pH 8.0; 50 mM EDTA, pH 8.0; 0.5% Tween 20, 0.5% Triton X-100) containing 11 μl of Qiagen Rnase A solution (100 mg/ml) by vortexing. To this suspension, 300 μl of a lysozyme (100 mg/ml; Sigma Chemical Co., St. Louis, MO) stock solution and 500 μl of a proteinase K (50 mg/ml; Sigma Chemical Co.) stock solution was added. The suspension was mixed by vortexing and incubated at 37°C for 30 min. Four ml of Buffer B2 (3 M guanidine HCl; 20% Tween 20) was added to the bacterial lysates and mixed into solution by gentle inversion ofthe tubes. The bacterial lysates were incubated at 50°C for 30 min. Total cellular DNA was isolated from the bacterial lysates using Qiagen Genomic-tip 500/G tips as per manufacturer's instructions. The resulting purified DNA was dissolved in 5 mL TE buffer and stored at 4°C. b. Construction of genomic cosmid library

[0085] Total cellular DNA isolated from Saccharopolyspora sp. LW107129 (NRRL 30141) was partially digested with Sau3A I based on section 3.1.3 of Ausubel, et al. (Cunent Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, NY). Small-scale (40 μg of total cellular DNA in an 80 μl reaction volume) reactions were performed to determine the proper enzyme to total cellular DNA ratio that resulted in the maximal concentration of partially-digested DNA fragments in the 25-50 Kb size range. Reactions were heated at 65°C for 15 min to inactivate the Sau3A I enzyme and aliquots of the reactions were analyzed by electrophoresis in 0.3% agarose gels to determine the relative abundance of partially-digested DNA fragments in the desired size range. Once an optimal enzyme to total cellular DNA ratio was observed, the reaction volume was increased to obtain sufficient quantities of partially-digested total cellular DNA for use as insert DNA in the construction ofthe cosmid libraries. A typical scaled reaction was 400 μg of Saccharopolyspora sp. LW107129 (NRRL 30141) total cellular DNA incubated with 9 units of Sau3A I (Gibco BRL, Gaithersburg, MD) for 15 min at 37°C in 800 μl total volume of IX React 4 Buffer (supplied as 10X by the manufacturer). The reaction was heated at 65°C for 20 min to inactivate the enzyme. The partially-digested genomic DNA was mixed with an equal volume of an equilibrated phenol-chloroform (50:50; v/v) solution and mixed by gentle inversion. After centrifugation at 14,000 x g for 15 min, the aqueous phase was removed and mixed with an equal volume of a chloroform-isoamyl alcohol (24:1; v/v) solution. After mixing the two phases by gentle inversion, the solution was centrifuged at 14,000 x g for 15 min. The aqueous phase was removed to a fresh tube and 0.1 volume of 3 M sodium acetate (pH 5.2) was added. Two volumes of ice-cold 100% ethanol were added and the solution was mixed by inversion. To aid in the precipitation ofthe DNA, the samples were placed at -70°C overnight. The precipitated DNA was pelted by centrifugation at 14,000 x g for 20 min. The DNA pellet was resuspended in 50 μl double-distilled water and stored at -20°C.

[0086] The vector used for construction ofthe cosmid library was pOJ436 (Figure 3) containing the apramycin resistance gene for selection. To minimize the re-ligation of cosmid vector DNA to itself, BamΑ I-digested pOJ436 DNA was dephosphorylated by incubating the digested DNA with 20 units of shrimp alkaline phosphatase (Roche/Boehringer Mannheim, Indianapolis, IN) for 2 hrs at 37°C in 1.2 ml total volume of IX SAP buffer (supplied as 10X by the manufacturer). Sau3A I-digested genomic DNA was ligated into the dephosphorylated BamΑ I site of pOJ436 and using a 5:1 ratio of partially-digested insert to vector DNA. For this reaction, insert and vector DNAs were incubated with 20 units of T4 DNA Ligase (New England BioLabs Inc., Beverly, MA) overnight at 16°C in IX T4 DNA Ligase Buffer (supplied as 10X by manufacturer). Ligation mixtures were packaged using Gigapack III Gold Packaging Extract (Stratagene, La Jolla, CA) and recombinant phage were titered using Escherichia coli strain DH5α- MRC⁺ cells (Gibco BRL), as described by the manufacturer's instructions. Aliquots (20-40 μl) ofthe recombinant phage and host cell culture were spread onto LB agar (10 g/1 Bacto- tryptone, 10 g/1 NaCl, 5 g/1 Bacto-yeast extract, 15g/l Bacto agar; Difco Laboratories) containing apramycin (lOOmg/1; Sigma Chemical Co.) and incubated overnight at 37°C. To construct master plates ofthe cosmid libraries for freezer storage, single colonies were picked with sterile toothpicks and inoculated into individual wells of sterile 96-well microwell plates containing 250 μl of Terrific Broth (TB media: 12 g/1 Bacto-tryptone, 24 g/1 Bacto-yeast extract, 0.4% v/v glycerol, 17 mM KH₂PO₄, 72 mM K₂HPO₄) supplemented with lOOmg/1 apramycin and incubated without shaking overnight at 37°C. To generate copy plates from the master plates, a 96-well microplate replicator (V & P Scientific, Inc., San Diego, CA) was used to inoculate a sterile 96-well microwell plate containing 250 μl of TB media containing lOOmg/1 apramycin. Copy plates are incubated without shaking at 37°C overnight.

[0087] For both master and copy plates, a 7 % (v/v) dimethylsulfoxide solution was added to the plates and the cultures and mixed using a multichannel pipette. Plates were placed at -70°C for storage.

[0088] The average insert size of selected recombinant cosmids was assessed by isolating cosmid DNA using the NucleoSpin Nucleic Acid Purification Kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and digesting the recovered DNA with 20 units ofthe restriction enzyme Eco RI (New England BioLabs) for 1 hr at 37°C. Restricted DNA was analyzed by electrophoresis in a 1.0% agarose gel. DNA fragments were visualized with UN light following 0.5% ethidium bromide (Sigma Chemical Co.) staining and relative size of fragments were estimated by comparison with 1 Kb DΝA ladder (Gibco BRL). Insert size ofthe cosmid libraries constructed ranged from 20 Kb-40 Kb. c. Screening of cosmid libraries and identification of cosmids containing butenyl-spinosyn biosynthetic gene s

[0089] Representatives of each E. coli cosmid clone were inoculated using a 96-well microplate replicator (N & P Scientific, Inc.) in duplicate onto Hybond Ν+ (Amersham Pharmacia Biotech, Piscataway, NJ) nucleic acid binding membranes. Membranes were supported on LB agar plates supplemented with 100 mg/L apramycin and incubated overnight at 37°C. Membranes were processed according the manufacturers' protocols. Inoculated membranes were placed colony-side up onto 3MM-filter paper (Whatman, Clifton, NJ) saturated with 0.5 N NaOH for 1 minute. Filters were transfened to 3MM- filter paper saturated with 1 M Tris-HCl, pH 7.6 for 1 minute to denature the DNA. Membranes were neutralized by transfer to 3 MM-filter paper saturated with 1 M Tris-HCl, pH 7.6/1.5 M NaCl for 1 minute. Final washing was performed in a solution of 1 M Tris- HCl, pH 7.6/1.5 M NaCl where remaining colony debris was removed from the membranes. DNA was cross-linked to the membrane with 1200 μjoules using an UN Stratalinker 1800 (Stratagene).

[0090] The library of recombinant bacteria thus prepared was screened for homology to any of three radiolabelled DΝA probes based upon the spn genes from S. spinosa (Baltz et al, 2000; Table 2). Pairs of oligonucleotides were used to amplify nucleotide regions specific for the spn biosynthetic gene cluster through polymerase chain reactions (PCR). Oligonucleotide primers were synthesized using a 394 DΝA/RΝA synthesizer (Applied Biosystems/PerkinΕlmer, Foster City, CA) and are listed in Table 2. PCR reactions were performed using AmpliTaq® DΝA Polymerase Kit (Perkin Elmer/Roche, Branchburg, ΝJ) according to manufacturer's protocols. DΝA fragments were amplified in a 48-sample DΝA Thermal Cycler (Perkin Elmer Cetus) under the following cycle conditions: 1) 94°C, 1 min.; 55°C, 2 min.; 72°C, 3 min.; 25 cycles 2) 72°C, 10 min.; 1 cycle. Amplified products were analyzed using 0.1% agarose gel electrophoresis and bands conesponding to the appropriate size were gel-extracted utilizing Qiagen II Gel Extraction Kit (Qiagen Inc.) as outlined according to manufacturer's directions. Table 2

[0091] Membranes were incubated at 65°C for 3 hours prior to addition of radiolabeled probe in 300 mis of pre-hybridization solution consisting of 6X SSC (52.59 g/L NaCl, 24.66 g/L sodium citrate, pH adjusted to 7.0 with 10 N NaOH), 0.1% sodium dodecyl sulfate (SDS), 10X Denhardt's Solution (50 mg/L Ficoll [Type 400, Pharmacia], 5.0 mg/L polyvinylpynolidone, 5.0 mg/L bovine serum albumin), 100 μg/ml denatured salmon sperm.

[0092] Concentration of DNA fragments was adjusted to 25 ng for all probes, denatured for 10 minutes in a boiling water bath and random-prime labeled with 50 μCi [α³²P]dCTP, 3000 Ci/mMol using 4 μl High Prime reaction mixture (Boehringer Mannheim) according to manufacturer's protocol. Separation of unincorporated nucleotides from radiolabeled DNA probes was performed using NucTrap Push Columns (Stratagene) and denatured for 10 minutes in a boiling water bath prior to addition to pre-hybridizing membranes. Approximately 2.0 x 10⁷ cpm were added to membranes for all DNA hybridizations. Hybridization conditions for all probes were for 16 hours in shaking water 65°C bath.

[0093] Hybridization solutions containing radiolabeled probes spnF, spnS, and spnE (TE) were decanted and each set of membranes washed under medium stringency conditions: 1) 15 min., room temperature in 300 ml 3X SSC/0.5 % SDS; 2) 30 min., 65°C shaking in 300 ml fresh 3X SSC/0.5 % SDS; 3) 30 min., room temperature in 300 ml IX SSC/0.5 % SDS. Membranes screened with the radiolabeled probe derived from Saccharopolyspora sp. LW107129 (NRRL 30141) cosmid 9D3 sequence were washed under stringent conditions: 1) 30 min., 65°C shaking in 300 ml fresh IX SSC/0.5 % SDS; 2) 30 min., 65°C shaking in 300 ml fresh 0.33X SSC/0.5 % SDS; 3) 30 min., 65°C shaking in 300 ml fresh 0.1X SSC/0.5 % SDS. Filters were monitored using a hand-held Geiger- Mueller counter to determine if background isotope detection was minimal. Membranes were mounted onto 3MM filter paper and covered with plastic wrap and exposed to x-ray film. Membranes were allowed to expose film for 24-72 hours at -70°C prior to development.

[0094] Putative positive cosmid clones were further characterized via restriction endonuclease digestion analysis and end-sequencing from the cosmid vector. Cosmid DNA was isolated using the NucleoSpin Nucleic Acid Purification Kit (CLONTECH Laboratories, Inc., Palo Alto, CA), and digested with 20 units ofthe restriction enzyme Eco RI (New England BioLabs) for 1 hr at 37 °C. Restricted DNA was electrophoresed in a 1.0% agarose gel. DNA fragments were visualized with UN light following 0.5% ethidium bromide staining and relative size of fragments were estimated by comparison with 1 Kb DΝA ladder. Additionally, Saccharopolyspora sp. LW107129 (NRRL 30141) nucleotide sequence from the cosmid/vector junctions was obtained by fluorescent cycle sequencing according to the methods of Burgett and Rosteck (1994). Sequencing reactions consisted of 3 μl (2 μg purified cosmid DΝA) template, 1 μl universal primer (4 pmole) or reverse primer (4 pmole), 8 μl Big Dye® reaction mixture, 1 μl DMSO, 7 ml H₂O under thermal cycler conditions: 96°C, 30 sec; 50°C, 15 sec; 60°C, 4 min.; 25 cycles with a 377 ABI Prism™ Sequencer (Applied Biosystems, Inc.).

[0095] Eight cosmid clones were identified as positively hybridizing to S. spinosa probes spnS, spnF and spnE (TE). Cosmid 8H3 was one of two clones that hybridized to both the spnS and spnF probes. Cosmid 9D3 was one of three clones that hybridized only to the spnF probe. Cosmid 10C1 was one of three clones that hybridized only to the spnE (TE) probe. Cosmid 9F4 was identified from the genomic library by hybidization to a radiolabeled PCR-fragment derived directly from Saccharopolyspora sp. LW107129 (ΝRRL 30141) sequence elucidated through nucleotide sequencing ofthe cosmid/vector ends from cosmid 9D3 (bases 297477-30163 in SEQ ID NO: 1). Two primers were synthesized based on the cosmid 9D3 DNA sequence (SEQ ID NO:39 & SEQ ID NO:40). A 416 bp DNA fragment was amplified from Saccharopolyspora sp. LW107129 (NRRL 30141) genomic DNA using these primes as detailed above and used for hybridization.

[0096] The complete sequences of cosmids 8H3, 9D3, 9F4 and 10C1 were determined by the method of fluorescent cycle sequencing of random DNA fragments cloned in phage Ml 3 (SeqWright, Houston, TX). The inserts in cosmids 8H3 and 9D3 overlapped, The inserts in cosmids 9D3 and 9F4 overlapped, and the insert in 9F4 and 10C1 overlapped. See Fig. 2. Together, the four cosmid inserts spanned about 111 kb of unique sequence (SEQ ID NO: 1 & 2). SEQ ID NO 1 includes the start codon of busA and all DNA to the 3' of that (see Fig. 2.). SEQ ID NO. 2 begins the base before the busA start codon and includes all DNA to the 5' side of that base. The following Table 3 identifies the portions of SEQ ID NO: 1 and SEQ ED NO. 2 included in each ofthe four inserts.

Table 3

FIG. 2 gives a graphical representation ofthe relationship ofthe four inserts to the 1 lOkb of sequence.

PKS Genes [0097] SEQ ID NO: 1 includes a central region of about 60 kb with striking homology to the DNA encoding the polyketide synthases of known macrolide producers (Donadio et al, 1991; McDaniel & Kaxz., 2001; Dehoff et al, 1997). The butenyl-spinosyn PKS DNA region consists of 5 ORFs with in-frame stop codons at the end of ACP domains, similar to the PKS ORFs in the other macrolide-producing bacteria. The five butenyl-spinosyn PKS genes are arranged head-to-tail (see FIG. 2), without any intervening non-PKS functions such as the insertion element found between the erythromycin PKS genes Al and All (Donadio et al., 1993). The PKS genes are designated busA, busB, busC, busD, and busE. The nucleotide sequence for each ofthe five spinosyn PKS genes, and the conesponding polypeptides, are identified in the following Table 4:

Table 4

[0098] busA encodes the initiator module (SEQ ED NO:l, bases 1-2931), extender module b (SEQ ED NO:l, bases 2992-8130) and extender module 1 (SEQ ED NO:l, bases 8205-13032). The nucleotide sequence and conesponding amino acid sequence for each of the functional domains within the initiator module and extender modules b and 1 are identified in the following Table 5:

Table 5

[0099] busB encodes extender module 2 (SEQ ID NO:l, bases 13059-19505). The nucleotide sequence and conesponding amino acid sequence for each ofthe functional domains within extender module 2 are identified in the following Table 6:

Table 6

[00100] busC encodes extender module 3 (SEQ ED NO: 1, bases 19553-24061) and extender module 4 (SEQ ID NO:l, bases 24128-29053). The nucleotide sequence and conesponding amino acid sequence for each ofthe functional domains within extender modules 3 and 4 are identified in the following Table 7:

Table 7

[00101] busD encodes extender module 5 (SEQ ED NO: 1, bases 29092-34263), extender module 6 (SEQ ED NO:l, bases 34327-38892), and extender module 7 (SEQ ED NO:l, bases 38956-43503). The nucleotide sequence and conesponding amino acid sequence for each ofthe functional domains within extender modules 5, 6, and 7 is identified in the following Table 8:

Table 8

[00102] spnE encodes extender module 8 (SEQ ID NO: 1 , bases 43945-49083), extender module 9 (SEQ ID NO:l, bases 49195-54366), and extender module 10 (SEQ ED NO:l, bases 54466-60707). The nucleotide sequence and conesponding amino acid sequence for each ofthe functional domains within extender modules 8, 9, and 10 is identified in the following Table 9:

Table 9

[00103] The boundaries and functions ofthe 55 domains identified in the foregoing Tables 7-11 are predicted based on similarities to the conserved amino acid sequences ofthe domains in other polyketide synthases, particularly the erythromycin polyketide synthase (Donadio et al, 1992). Like the A83543 spinosyn PKS, the bus PKS has a KSQ domain at the amino terminus ofthe initiator module. This KSQ domain cannot function as a β- ketosynthase because it contains a glutamine residue at amino acid 172, in place ofthe cysteine required for β-ketosynthase activity (Siggard- Andersen, 1993). It has been reported that KSQ domains function to decarboxylate malonyl-ACP and are chain initiation factors (Bisang, et al, 1999). The other butenyl-spinosyn PKS domains are functional. None of them has the sequence characteristics ofthe inactive domains found in the erythromycin and rapamycin PKS genes (Donadio et al, 1991; Aparicio et al, 1996).

[00104] Although busB-E are comparable in size to spnB-E, bus A is 5,244 bp larger than spnA. The first 4245 bp and the last 3,486 bp of busA have high similarity to spnA. However, bases 4246-9548 do not have counterparts in the spnA gene. This 5 kb region codes for an additional module with 5 functional domains: KSb, ATb, DHb, KRb, and ACPb. These functions together with the preceding initiation domain are responsible for the biosynthesis ofthe butenyl side chain, characteristic of butenyl-spinosyns relative to the A83543 spinosyns. The cloned bus PKS genes busB, busC, busD and busE were shown to be similar to the analogous A83543 spinosyn PKS genes spnB, spnC, spnD and spnE (table 10) (Baltz et al, 2000).

PAGE INTENTIONALLY LEFT BLANK

Table 10

butenyl- bus ORF Functional Best match in spn ORF length Functional ORF % ORF % spinosyn length Domain A83543 bp (a. a.) Domain Identity Identity

Gene bp (a.a.) spinosyn PKS (DNA) (a.a.) busA 13,032 spnA 7,788 (2,595) (4,344)

1-4,245 4,245 (1,415) KSQ-KSb 21,111-25,214 4,245 (1,415) KSQ-KS1 92 % 91.2 %

4,246-9,548 5,301 (1,767) ATb-KSl none* NA

9,549-13,032 3,486 (1,162) AT1-ACP1 26,407-28,896 3,486 (1,162) AT1-ACP1 91 % 87.6 % busB 6,450 (2,149) KS2-ACP2 spnB 6,459 (2,152) KS2-ACP2 93 % 93.1 % busC 9,546 (3,167) KS3-ACP4 spnC 9,513 (3,170) KS3-ACP4 94 % 93.5 % busD 14,805 KS5-ACP7 spnD 14,787 (4,928) KS5-ACP7 94 % 93.6 % (4,935) busE 16,692 KS8-ACP10 spnE 16,767 (5,588) KS8-ACP10 94 % 90.6 % (5,564)

Si ϊ arity to S. spinosa PKS genes was in the same range as similarity to other like domains ofthe bus & spn PKS genes.

PAGE INTENTIONALLY LEFT BLANK

[00105] The proteins, which perform similar reactions in the biosynthesis of spinosyns, share 87-93 % amino acid identity and the genes range from 93-94 % DNA sequence identity. It should be noted that the spn PKS enzymes SpnB-E and the similar bus PKS enzymes BusB-E must maintain distinct substrate specificity since, although the reactions performed by the enzymes are identical, the substrate polyketides are different. In addition, aggregation ofthe 5 PKS enzymes into a functional PKS requires specific protein-protein interactions. The residues involved in this inter-subunit molecular recognition are unknown and may not be conserved between S. spinosa and Saccharopolyspora sp. LW107129 (NRRL 30141) .

Genes Adjacent to the PKS Responsible for Additional Modifications [00106] In the DNA upstream ofthe PKS genes (cloned in cosmid 8H3) there were 22 open reading frames (ORFs), each consisting of at least 100 codons, beginning with ATG or GTG and ending with TAA, TAG or TGA, and having the codon bias expected of protein- coding regions in an organism whose DNA contains a high percentage of guanine and cytosine residues (Bibb et al, 1984). These 22 ORFs represented graphically in FIG. 2. Based on evidence that will be discussed hereinafter, 14 ofthe ORFs have been designated as butenyl-spinosyn biosynthetic genes, namely: busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, and busS (labeled F through S in FIG. 2). In the following Table 11, the DNA sequence and the amino acid sequence for the conesponding polypeptide are identified for each of these genes, as well as for ORFs found immediately downstream of spnS (ORF LI, ORF LII, ORF LIII, ORF LEV, ORF LNI, ORF LNII, ORF LNIII and ORF LIX in cosmid 8H3). Also identified in Table 11 are the nucleotide sequences for ORF RI, ORF RII and ORFRIII downstream ofthe PKS genes (in cosmid 2C10), and the amino acid sequences conesponding to them.

Table 11

(C) indicates complementary strand is given in the sequence listing

[00107] To assign functions to the polypeptides identified in Table 11, four lines of evidence were utilized: similarity to sequences of known function, similarity to A83543 spinosyn biosynthetic genes, results of targeted gene disruption experiments, and results of bioconversion experiments.

[00108] The amino acid sequences ofthe predicted polypeptides were compared to sequences deposited in the databases at the National Center for Biotechnology Information (NCBI, Washington, DC), using the BLAST algorithm to determine how well they are related to known proteins. The BLAST searches ofthe NCBI databases were also repeated periodically to obtain new insights from additional homologies. Table 12 gives significant matches from a basic BLAST search on February 18, 2001 :

Table 12

* Greater similarity is associated with higher BLAST scores (Altschul et al, 1990). [00109] The bus open reading frames were compared directly to the sequence ofthe A83543 spinosyn biosynthetic genes (Accession number AY007564). The high degree of similarity in both the DNA and protein sequence indicated that the genes performed similar functions in biosynthesis of spinosyns. Table 13 gives the similarity comparisons between the bus and spn genes Table 13

[00110] In spite ofthe high degree of DNA and amino acid similarity between some bus & spn genes, it should be noted that some ofthe bus gene products catalyze markedly different reactions in the biosynthesis of butenyl-spinosyn relative to A83543 spinosyns. These differences are manifested in the distinct butenyl-spinosyn compounds that have been isolated from Saccharopolyspora sp. LW107129 (NRRL 30141). All natural A83543 spinosyns disclosed are substituted at C-17 with forosamine or a specific forosamine isomer (Kirst, et al, 1992). Butenyl-spinosyns, on the other hand, are also substituted at C-17 with a wider range of forosamine isomers, as well as neutral sugars like amicetose, O-methyl- glucose and O-methyloleandrose. This C-17 glycosylation diversity relative to A83543 spinosyns requires biosynthetic enzymes to make the sugars and a glycosyltransferase capable of catalyzing these glycosylations. These sugars might be synthesized by specific synthase genes located near the bus genes or elsewhere in the chromosome, or they may be synthesized by alternate substrate specificity ofthe listed butenyl-spinosyn biosynthetic genes. Amicetose could be produced by genes outside ofthe bus gene cluster or it may be an intermediate in the biosynthesis of forosamine (FIG. 4). Methyloleandrose could be produced as a byproduct of forosamine biosynthesis and the rhamnose O-methyltransferases (busH, busl& busK). This sugar could be synthesized from NDP-4-keto-2,6-deoxy-D- glucose is an intermediate in the biosynthesis of forosamine. Therefore, ketoreduction and O-methylation of this precursor by the disclosed genes and other Saccharopolyspora sp. LW 107129 (NRRL 30141) genes could lead to the biosynthesis of spinosyn derivatives containing methyloleandrose (FIG. 4).

[00111] In addition, nine genes listed in table 13 directly interact with the butenyl- spinosyn aglycone or PSA (busF, busG, busH, busl, busJ, busK, busL, busM, and busP). The aglycone and PSA substrate for these genes is distinct from the A83543 spinosyn aglycone and PSA. Therefore, these genes have distinct substrate specificity relative to their spn counterpart listed in Table 13.

[00112] Several butenyl-spinosyn analogs produced by Saccharopolyspora sp. LW107129 (NRRL 30141) are hydroxylated at C-8 or C-24 (Table 2). Macrolides can be hydroxylated post synthesis by P-450 monooxygenases as in hydroxylation at C-6 in erythromycin biosynthesis (Weber & McAlpine, 1992). ORF LNII is highly similar to P- 450 monooxygenases and it or a monooxygenase encoded elsewhere on the Saccharopolyspora sp. LW107129 (ΝRRL 30141) chromosome may be responsible for the hydroxy lations at C-8 or C-24 of butenyl-spinosyns. Alternatively, hydroxylated precursors such as glycolate or glycerol can be incorporated during polyketide synthesis as in leucomycin (Omura et al., 1983). It has been reported that the AT domain specific for addition of glycolate in the niddamycin producer (nid AT6) is similar to methyl-malonyl- CoA specific AT domains ofthe erythromycin and rapamycin PKS genes (Katz et al., 2000). PKS module 7 is responsible for addition of carbons 8 and 9 ofthe butenyl-spinosyn polyketide, however, the bus AT7 domain does not have the same methyl-malonyl-CoA specific sequences as nid AT6. There are unique sequences in bus AT7 relative to other AT domains and nid AT6 which could be responsible for glycolate specificity. The butenyl- spinosyn biosynthetic genes responsible for these modifications are unique relative to the A83543 spinosyns since no such hydroxylated spinosyns are produced by S. spinosa.

[00113] In addition, the specificity of rhamnose methylation is altered in Saccharopolyspora sp. LW107129 (NRRL 30141) relative to S. spinosa. Mutants of S. spinosa which exhibited altered methylation ofthe rhamnose on A83543 spinosyn as disclosed in US Patents 5,202,242 and 5,840,861, typically produced mono-desmethylated rhamnose derivatives of A83543 spinosyns. Di-desmethyl rhamnose derivatives of A83543 spinosyns were only detected in the presence of methyltransf erase inhibitors like sinefungin. Mutants of Saccharopolyspora sp. LWl 07129 (NRRL 30141) with altered methylation of rhamnose (Hahn et al, 2001), produced di- and τri-desmethyl rhamnose derivatives of butenyl-spinosyns in high amounts, in the absence of methyltransferase inhibitors.

[00114] For complementation studies, cosmids containing Saccharopolyspora sp. LW107129 (NRRL 30141) bus DNA were conjugated into Saccharopolyspora sp. LW107129 (NRRL 30141) mutant strains in which butenyl-spinosyn synthesis was altered. (Details are given in the following Example 4.) Transconjugants were then tested for their ability to convert the products of blocked mutants into other spinosyns. The mutant used was 30141.8 which produced 3'-O-desmethylrhamnose-butenyl-spinosyn (3- ODM) and related factors. The 30141.8/8H3 transconjugants produced butenyl-spinosyns instead of 3-ODM, so the genes responsible for methylation at the 3' position of rhamnose must be present on cosmid 8H3.

[00115] In targeted gene disruptions, internal fragments are generated by PCR amplification from the cosmid DNAs, and cloned into a plasmid. The resulting plasmids were then conjugated into Saccharopolyspora sp. LW107129 (NRRL 30141, and apramycin-resistant transconjugants were isolated and fermented. The basis of disruption experiments is that when a plasmid bearing an internal gene fragment is integrated, two incomplete copies ofthe biosynthetic gene result, thereby eliminating the enzymatic function. Fermentation products are analyzed to determine which butenyl-spinosyns accumulate. Disruption ofthe busO gene leads to the accumulation of butenyl-spinosyn PSA indicating that busO is required for synthesis or addition of forosamine (See Example 5). Compounds containing sugars at C-17 which are not synthesized using the forosamine biosynthetic genes can also be accumulated in busO mutants.

[00116] The conclusions drawn from BLAST searches, the gene disruption experiments, and the bioconversion studies will now be discussed in greater detail on a gene by gene basis.

[00117] The 14 genes upstream ofthe PKS were determined to be involved in butenyl- spinosyn biosynthesis because of their high similarity to the spnF-S genes of S. spinosa (Table 13) and because BLAST searches showed that these genes had striking similarity to enzymes known to code for functions needed for the biosynthesis of butenyl-spinosyns. busF, busJ, busL, busM [00118] The genes busF, busJ busL, and busM show high similarity to spnF, spnJ, spnL and spnM. These A83543 spinosyn genes were reported to be involved in generation ofthe aglycone from the putative monocyclic lactone product ofthe PKS genes. The busF gene product has 91 % amino acid identity to spnF, likewise the busL gene product has 94 % amino acid identity to spnL. Both spnF & spnL gene products were reported to be methyltransferases and all 4 proteins have high similarity to enzymes from Streptomyces which are known to be involved in carbon-carbon bond formation, The busJ protein had 83 % amino acid identity to spnJ, reported to be an oxidoreductase. Both bus J & spnJ are highly similar to dnrW, which is known to be involved in C-C bond formation in the biosynthesis of daunorubicin. The bws gene product was 96 % identical to spnM. The gene products of both busM & spnM are highly similar to a new class of secreted lipases from Candida albicans. The roles ofbusF, and busL as methyltransferases, bus J as an oxidase and busM as a lipase are consistent with the reported roles ofthe spnF, spnL, spnJ and spnM genes in the formation of carbon-carbon bridges. busG, busH. busL busK [00119] The busG, busH, busl, and busK had high similarity to the spnG, spnH, spnl and spnK genes of S. spinosa. These genes were reported to be involved in rhamnose addition to the A83543 spinosyn aglycone and subsequent methylation. The busG gene has 90% similarity to spnG and was highly similar to several genes involved in sugar addition to polyketide derived antibiotics (table 11). The busH, busl, and busK gene products showed high amino acid similarity to the spnH (97 %), spnl (92 %), and spnK (88 %) gene products, respectively which were reported to be involved in methylation of rhamnose in spinosyn biosynthesis. All three genes had high amino acid similarity to the tylE (busl & busK) and tylF (busH) genes from Streptomyces fradiae which have been shown experimentally to be macarocin-O-methyltransferases (Bate & Cundliffe, 1999). busN, busO, busP, busO. busR, busS [00120] The spnN, spnO, busP, busQ, busR, and busS had high similarity to the spnN, spnO, spnP, spnQ, spnR, and spnS genes of S. spinosa (Table 12). These genes were reported to be involved in the biosynthesis or addition ofthe forosamine sugar. The similarity ofbusP to other glycosyl transferases (Table 11) indicates that it encodes the butenyl-spinosyn forosamyl transferase. The high degree of similarity between busO and the urdS 2,3 dehydratase (Table 11; Hoffineister et al., 2000) indicates that it is involved in the 2'-deoxygenation step of forosamine synthesis. The similarity between the busQ gene product and the urdQ 3,4-dehydratase (S. fradiae; Hoffmeister et al, 2000), indicates that it is involved in the 3 '-dehydration step of forosamine synthesis. busR had up to 40% identity to a group of proteins proposed to function as deoxysugar transaminases (Thorson et al, 1993), indicating that busR is involved in the 4'-amination step of forosamine synthesis. Finally busS was highly similar to a group of amino methylases indicating that busS is involved in methylation ofthe 4' amino group of forosamine. Therefore, the busN, busO, busP, busQ, busR, and busS are involved in production ofthe forosamine moiety of butenyl- spinosyns.

[00121] Thus 19 genes from Saccharopolyspora sp. LW107129 (NRRL 30141) can be assigned roles in butenyl-spinosyn biosynthesis: 5 PKS genes to produce a macrocyclic lactone, 4 genes to modify this to the aglycone, 4 genes to add and methylate the rhamnose, and 6 genes to synthesize and add forosamine. The hypothetical biosynthetic pathway is summarized in FIGS. IA and IB.

Utility [00122] There are many uses for the cloned Saccharopolyspora sp. butenyl-spinosyn DNA. The cloned genes can be used to improve yields of butenyl-spinosyns and to produce new butenyl-spinosyns. Improved yields can be obtained by integrating into the genome of a particular butenyl-spinosyn producing strain, a duplicate copy of one or more butenyl- spinosyn biosynthetic genes. In the extreme case where the biosynthetic pathway is blocked in a particular mutant strain due to lack of a required enzyme, production ofthe desired spinosyns can be restored by integrating a copy ofthe required gene.

[00123] Novel compounds can be produced using fragments ofthe cloned DNA to disrupt steps in the biosynthesis of butenyl-spinosyns. Such disruption may lead to the accumulation of precursors or "shunt" products (the naturally-processed derivatives of precursors). Modified spinosyns produced by disrupting genes may be insect control agents themselves, or serve as substrates for further chemical modification, creating new semi- synthetic spinosyns with unique properties and spectra of activity. A disruption ofthe busO gene results in the accumulation of butenyl-spinosyn PSA. Butenyl-spinosyn PSA is useful as a starting material for the synthesis of spinosyn analogs containing novel groups at C-17.

[00124] Novel butenyl-spinosyns can also be produced by transfer of one or more ofthe cloned bus genes, or a part thereof, into a heterologous host. These genes may provide enzymatic functions not present in the recipient host. Such genes might provide alternate sugars, modify existing sugars or aglycone carbons, allow alternate sugars to be attached to an aglycone or alter the base structure ofthe aglycone itself. Compounds produced by transfer ofthe cloned bus genes into a heterologous host may be insect control agents themselves, or serve as substrates for further chemical modification, creating new semi- synthetic spinosyns with unique properties and spectra of activity. Saccharopolyspora sp. LW107129 (NRRL 30141) DNA from cosmids 8H3 and 9D3 can be transfened into S. spinosa, producer of A83543 spinosyns, and the transconjugants produce novel spinosyns.

[00125] Novel butenyl-spinosyns can also be produced by mutagenesis ofthe cloned genes, and substitution ofthe mutated genes for their unmutated counterparts in a butenyl- spinosyn-producing organism. Mutagenesis may involve, for example: 1) deletion or inactivation of a KR, DH or ER domain so that one or more of these functions is blocked and the strain produces a spinosyn having a lactone nucleus with a double bond, a hydroxyl group, or a keto group that is not present in the nucleus of spinosyn A (see Donadio et al, 1993); 2) replacement of an AT domain so that a different carboxylic acid is incorporated in the lactone nucleus (see Ruan et al, 1997); 3) addition of a KR, DH, or ER domain to an existing PKS module so that the strain produces a spinosyn having a lactone nucleus with a saturated bond, hydroxyl group, or double bond that is not present in the nucleus of spinosyn A (MacDaniel & Katz, 2001); or 4) addition or subtraction of a complete PKS module so that the cyclic lactone nucleus has a greater or lesser number of carbon atoms.

[00126] The DNA from the butenyl-spinosyn gene cluster region can be used as a hybridization probe to identify homologous sequences. Thus, the DNA cloned here could be used to locate additional plasmids from the Saccharopolyspora sp. LWl 07129 (NRRL 30141) gene libraries which overlap the region described here but also contain previously uncloned DNA from adjacent regions in the genome of Saccharopolyspora sp. LW107129 (NRRL 30141). Also, comparisons ofthe Saccharopolyspora sp. LW107129 (NRRL 30141 bus genes with the S. spinosa spn genes leads to the identification of regions of conserved sequence which are distinct from non-spinosyn producing biosynthetic genes such as the biosynthetic genes for erythromycin, rapamycin, tylosin and others. These spinosyn-specific gene probes as well as all DNA from the region cloned here may be used to identify non-identical but similar sequences in other organisms. Hybridization probes are normally at least about 20 bases long and are labeled to permit detection.

[00127] The modified strains provided by the invention may be cultivated to provide spinosyns using conventional protocols such as those disclosed in U. S. Patent No.

5,362,634 or provisional U. S. Patent Application 60/277,601 on "Pesticidal Macrolides", filed March 21, 2001. The above examples are non-limiting and should not be construed as limitations ofthe invention.

EXAMPLE 2 LC MS Method for Analysis of Fermentation Broth for Butenyl-Spinosvn Metabolites

[00128] The following method utilizes HPLCseparation with electrospray (ESI) mass spectrometry to monitor fermentation for the production of Formula (1) and other components. Such a system was also used for determining molecular weights ofthe purified factors, by deduction from the electrospray adduct ions. These data are summarized in Table 15.

[00129] Add a volume of denatured ethanol equal to that of fermentation broth. Shake the mixture for 1 hour, then centrifuge and filtere (0.22 μm pore size) to remove the bulk cell debris. Microfuge a 1-mL aliquot, then analyze the clarified extract by the following LC-MS system:

[00130] HPLC system: Column stationary phase: 250 x 4.6-mm column, base- deactiviated silica gel, 5 μm C8 (Hypersil-C8-BDS). Mobile phase: 10 mM ammonium acetate-methanol-acetonitrile linear gradient summarized below:

TABLE 14

Time (mins) Percent solvent A Percent solvent B

0 100 0

20 0 100

25 0 100

30 100 0

35 100 0 where solvent A is 10 mM ammonium acetate and solvent B is methanol- acetonitrile (l:l);

Flow rate: 1 mL/min; split after UN detector such that MS:waste ratio is approx. 5:95;

Detection: positive ESI acquired in low and high cone voltage modes

Characteristic LC retention times and mass spectrometry ions as summarized in Table 15.

TABLE 15

Compound No. LC retention time m/z for [M+H]^{+ a} m/z for secondary

(from Table 1) ion^b

1 23.8 758.4 142.0 (forosamine)

4 22.9 744.4 142.0

5 24.3 772.4 142.0

6 22.1 774.4 142.0

9 21.4 810.4 [M+NH₄] 189.0 (tri-O- methyl-rhamnose)

13 22.0 617.3 189.0 ^a m/z for parent ion observed under +ESI mode, low cone voltage ^b m/z for most abundant fragment or adduct ion observed under +ESI, high cone voltage mode

EXAMPLE 3 Preparation Of Butenyl-Spinosyn Metabolites Through Fermentation

[00131] Metabolites of Formula (1) are produced by cultivation ofthe desired strain of

Saccharopolyspora chosen from one ofthe following strains NRRL 30141, NRRL 30421, or derivatives thereof in fermentation medium as described below. A 1.8-mL frozen vegetative culture was thawed, inoculated into 25 mL vegetative media in a 125-mL

Erlenmeyer baffled flask and grown at 30° C shaking at 150 rpm for 72-96 hours. TABLE 16

Vegetative Medium

Ingredient Amount (g)

Dextrose 9.0 Trypticase Soy Broth 30.0

Yeast Extract 3.0

Magnesium Sulfate. 7 H₂O 2.0

De-ionized water 1000.0

Shake Flask Fermentation

[00132] Twelve milliliters of mature first stage seed was used to inoculate 50-mL fermentation medium in a 500-mL baffled Erlenmeyer fermentation flask.

TABLE 17

Fermentation Medium (per liter of water)

Ingredient Amount (g)

Dextrose 80.0

Cottonseed Flour 32.0

Soybean Flour 8.0

Corn Steep Powder 8.0

Calcium Carbonate 5.0

Yeast Extract 2.0

[00133] Fermentation was maintained at 30° C, 200 φm (50 mm stroke) for 7-12 days.

Mature fermentation beer can be extracted with a suitable solvent and the metabolites recovered by chromatographic separation, as disclosed in Example 1.

EXAMPLE 4 Complementation of Rhamnose Methylation Defect in Strain NRRL 30421 by Cosmid 8H3

[00134] Strain NRRL 30421 is a mutant of Saccharopolyspora sp. NRRL 30141 which is unable to fully methylate the rhamnose on butenyl-spinosyns. Strain NRRL 30421 accumulates compound 4 and other butenyl-spinosyns which lack O-methylation at the 3' position ofthe rhamnose (3'-ODM). This methylation defect is presumed to be the result of a mutation in one ofthe O-methyltransferases encoded by the busH, busl or busK genes. All of these genes are present in cosmid 8H3 (Figure 2)

[00135] Cosmid 8H3 (Figure 3) was transfened from Escherichia coli ATCC 47055 into strain NRRL 30421 by conjugal transfer (Matsushima et al, (1994)). Two independent isolates transformed with cosmid 8H3 were fermented as in Example 2 and analyzed for production of compound 1 and compound 4 as exemplified in Example 1. TABLE 18

Strain (Genotype) compound 4 compound 1 ratio of μg/ml μg/ml compounds 4

NRRL 30421 (3'-ΟDM*) 1.0 0.7 0.7

NRRL 30421 (3'- 0.5 8.9 17.8 ODM*)/8H3-42

NRRL 30421 (3'- 0.1 3.0 30.0

ODM*)/8H3-45

NRRL 30141 0.4 9.7 24.3

* = mutation preventing methylation of rhamnose at 3' position

[00136] While NRRL 30421 produced predominantly compound 4, strains of NRRL

30421 containing cosmid 8H3 produced mostly compound 1 [Table 18]. The production of compounds 1 and 4 in NRRL 30421 containing cosmid 8H3 is similar to the non-mutant culture NRRL 30141 (Table 18). It has therefore been demonstrated that transformation with cosmid 8H3 is able to overcome a methylation defect in strain NRRL 30421 to restore enhanced production of compound 1.

EXAMPLE 5 Accumulationation of Butenyl-Spinosyn Precursor and Shunt Product Caused by Disruption ofbusO

[00137] The busO gene was inactivated by integration of a cloned internal fragment of the busO gene. A pair of oligonucleotides (the first conesponding to bases 11882- 11861 in SEQ ID NO: 2 and the second conesponding to bases 10970-10993 in SEQ ED NO: 2) were used to amplify a 912 bp region internal to the 1,457 bp busO gene conesponding to bases 10970-11882 in SEQ ID NO: 2. Transformation of Saccharopolyspora sp. LW107129

(NRRL 30141) with a plasmid containing the fragment would result in partial duplication ofthe busO gene, to yield two truncated copies ofthe gene flanking the plasmid and antibiotic resistance gene.

[00138] The 912 bp internal busO PCR fragment was generated with primers SEQ ID NO: 33 & 34 using FailSafe™PCR (Epicenter) and cloned into pCREI according to the manufacturer's instructions (Invitrogen). The resulting plasmid was digested with EeoRI and the busO fragment was cloned into the EcøRI site of pOJ260 (Figure 3). The resultant plasmid was conjugated from Escherichia coli ATCC 47055 into a derivative of Saccharopolyspora sp. NRRL 30121 by conjugal transfer (Matsushima et al, (1994)). Six independent apramycin resistant exconjugants were fermented as in Example 2 and analyzed for production of compound 1 and other spinosyn derivatives as in Example 1.

[00139] The parental strain, NRRL 30141 produced high levels of compound 1 and low levels ofthe pseudoaglycone (PSA; compound 13). It also produced a small amount of compound 9 [Table 19]. Compound 1 could not be detected in any ofthe six busO mutants, indicating that busO is required for complete butenyl spinosyn biosynthesis. In addition, levels of PSA were increased in all six busO mutants, as would be predicted from a deficiency in forosamine supply. The levels of compound 9 which has a sugar other than forosamine at C17, also increased in the busO mutants.

TABLE 19

Strain (Genotype) compound 1* compound 13 compound 9

NRRL 30141 366.3 1.0 0.4

NRRL 30141 nd 13.8 1.7 bus065

NRRL 30141 nd 12.3 3.7 bus067

NRRL 30141 nd 6.7 3.8 bus068

NRRL 30141 nd 9.3 1.3 busO70

NRRL 30141 nd 12.3 2.4 bus071

NRRL 30141 nd 5.4 1.6 bus072

* amounts reported are relative to compound 13 in NRRL 30141; nd — not detected

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Claims (22)

CLAEMS

1. An isolated DNA molecule comprising a DNA sequence that encodes a butenyl- spinosyn biosynthetic enzyme, wherein said enzyme is comprised of an amino acid sequence at least 98% identical to one selected from the group consisting of SEQ ID NOS 3-7, and 8-29, provided that if the sequence is less than 100% identical to the selected sequence, then the differences do not substantially affect the functional properties ofthe encoded enzyme.

2. An isolated DNA molecule ofclaim 1 wherein the DNA sequence is selected from the group of genes consisting of busA, busB, busC, busD, busE, ORF RI, ORFRII, ORF RIII, busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, busS, ORF LI, ORF LEI, ORF LIII, ORF LEV, ORF LNI, ORF LNII, ORF LNIII, and ORF LEX, said genes being described by, respectively, bases 1-13032, 13059-19505, 19553- 29053, 29092-43890, 43945-60636, 62090-63937, 65229-66602 and 68762-69676 of SEQ ID ΝO:l and 114-938, 1389-2558, 2601-3350, 3362-4546, 4684-6300, 6317-7507, 7555-8403, 8640-9569, 9671-10666, 10678-12135, 12867-14177, 14627-15967, 16008- 17141, 17168-17914, 18523-19932, 19982-20488, 20539-21033, 21179-21922, 22674- 23453, 23690-24886, 26180-26923, and 27646-28473 of SEQ ED NO:2.

3. An isolated DNA molecule comprising a DNA sequence that encodes a butenyl- spinosyn PKS domain selected from KSi, ATi, ACPi, KSb, ATb, KRb, DHb, ACPb, KSl, ATI, KR1, and ACPI, said domains being described by, respectively, amino acids 7-423, 528-853, 895-977, 998-1413, 1495-1836, 1846-2028, 2306-2518, 2621-2710, 2735-3160, 3241-3604, 3907-4086, and 4181-4262 of SEQ ED NO:3.

4. An isolated DNA molecule ofclaim 3 wherein the DNA sequence is selected from the group consisting of bases 16-1269, 1582-2559, 2683-2931, 2992-4239, 4483-5508, 5538- 6084, 6916-7554, 7861-8130, 8203-9480, 9721-10812, 11719-12258, and 12541-12786 of SEQ ID NO:l.

5. An isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS2, AT2, DH2, ER2, KR2, and ACP2, said domains being described by, respectively, amino acids 1-421, 534-964, 990-1075, 1336-1681, 1685- 1864, and 1953-2031 of SEQ ED NO:4.

6. An isolated DNA molecule ofclaim 5 wherein the DNA sequence is selected from the group consisting of bases 13059-14321, 14658-15900, 16026-16283, 17064-18100, 18111-18650, and 18915-19151 of SEQ ED NO:l.

7. An isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS3, AT3, KR3, ACP3, KS4, AT4, KR4, and ACP4, said domains being described by, respectively, amino acids 1-421, 528-814, 1157-1335, 1422-1503, 1526-1949, 2063-2393, 2697-2875, and 2969-3049 of SEQ ED NO:5.

8. An isolated DNA molecule ofclaim 7 wherein the DNA sequence is selected from the group consisting of bases 19553-20815, 21143-22000, 23021-23557, 23816-24061, 24128-25399, 25739-26731, 27641-28183, and 28457-28699 of SEQ ED NO:l.

9. An isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS5, AT5, DH5, KR5, ACP5, KS6, AT6, KR6, ACP6, KS7, AT7, KR7, and ACP7, said domains being described by, respectively, amino acids 1-422, 537- 864, 891-1076, 1382-1563, 1643-1724, 1746-2170, 2281-2611, 2914-3093, 3186-3267, 3289-3711, 3823-4151, 4342-4636, and 4723-4804 of SEQ ID NO:6.

10. An isolated DNA molecule ofclaim 9 wherein the DNA sequence is selected from the group consisting of bases 29092-30357, 30700-31683, 31762-32319, 33235-33780, 34018-34263, 34327-35601, 35932-36924, 37831-38370, 38647-38892, 38956-40224, 40560-41544, 42115-42999 and 43258-43503 of SEQ ED NO: 1.

11. An isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS domain selected from KS8, AT8, DH8, KR8, ACP8, KS9, AT9, DH9, KR9, ACP9, KS10, AT10, DH10, KR10, ACP10, and TE10, said domains being described by, respectively, amino acids 1-424, 530-848, 885-1072, 1371-1554, 1650-1728, 1751-2175, 2289-2616, 2642-2775, 3131-3315, 3396-3474, 3508-3921, 4036-4366, 4389-4569, 4876-5054, 5148-5229, and 5278-5531 of SEQ ED NOJ.

12. An isolated DNA molecule ofclaim 11 wherein the DNA sequence is selected from the group consisting of bases 43945-45216, 45532-46488, 46597-47160, 48055-48606, 48892-49083, 49195-50469, 50809-51792, 51868-52269, 53335-53889, 54130-54366, 54466-55707, 56050-57042, 57109-57651, 58570-59106, 59386-59631, and 59776-

60537 ofSEQ ED NO:l.

13. An isolated DNA molecule comprising a DNA sequence that encodes a spinosyn PKS module, said module being selected from the group consisting of amino acids 6-977 of SEQ ID NO:3, 998-2710 of SEQ ED NO:3, 2735-4262 of SEQ ED NO:3, 1-2031 of SEQ ED NO:4, 1-1503 of SEQ ED NO:5, 1526-3049 of SEQ ED NO:5, 1-1724 of SEQ ED NO:6, 1746-3267 of SEQ ED NO:6, 3289-4804 of SEQ ED NO:6, 1-1728 of SEQ ED NOJ, 1751-3474 of SEQ ED NOJ, and 3508-5531 ofSEQ ID NO:7.

14. An isolated DNA molecule ofclaim 13 wherein the DNA sequence is selected from the group consisting of bases 16-2931, 2992-8130, 8203-12786, 13059-19151, 19553- 24061, 24128-28699, 29092-34263, 34327-38892, 38956-43503, 43945-49083, 49195- 54366, and 54466-60537 of SEQ ID NO:l.

15. A recombinant DNA vector which comprises a DNA sequence of any one of claims 1-14.

16. A host cell transformed with a recombinant vector of claim 15.

17. A method of increasing the spinosyn-producing ability of a spinosyn-producing microorganism comprising the steps of

1) transforming with a recombinant DNA vector or portion thereof a microorgamsm that produces butenyl-spinosyn or a butenyl-spinosyn precursor by means of a biosynthetic pathway, said vector or portion thereof comprising a DNA sequence ofthe invention, as described above, that codes for the expression of an activity that is rate limiting in said pathway, and

2) culturing said microorganism transfonned with said vector under conditions suitable for cell growth and division, expression of said DNA sequence, and production of spinosyn.

18. A process for preparing a butenyl-spinosyn which comprises cultivating a microorganism having operative butenyl-spinosyn biosynthetic genes in its genome, provided that the genome ofthe organism has been modified so that duplicate copies of at least one ofthe butenyl-spinosyn biosynthetic genes bus A, busB, busC, busD, busE, busF, busG, busH, busl, busJ, busK, busL, busM, busN, busO, busP, busQ, busR, and busS has are present.

19. A process for preparing a butenyl-spinosyn which comprises cultivating a microorganism having butenyl-spinosyn biosynthetic genes in its genome, provided that at least one of said genes has been inactivated, the rest of said genes being operational to produce a butenyl-spinosyn other than the one that would be produced if the disrupted gene were operational.

20. A process for preparing a butenyl spinosyn which comprises cultivating a heterologous microorganism that has been transformed so that its genome contains operative butenyl spinosyn biosynthetic genes.

21. A process for preparing a butenyl spinosyn which comprises cultivating a microorganism having operational butenyl-spinosyn biosynthetic genes in its genome, wherein said genes a) include at least one operational PKS module more than or at least one less than is present in SEQ ID NO:l; or b) include a PKS module that differs from the conesponding module described in SEQ ED NO:l by the deletion, inactivation, or addition of a KR, DH or ER domain, or by the substitution of an AT domain.

22. A process for isolating butenyl-spinosyn biosynthetic genes which comprises creating a genomic library of a butenyl-spinosyn producing microorganism, and using a labeled fragment of SEQ ED NO:l or SEQ ED NO. 2 that is at least 20 bases long as a hybridization probe.