Polyketides and their synthesis

[0001] The present invention relates to processes and materials (including enzyme systems, nucleic acids, vectors and cultures) for preparing polyketides, particularly polyethers but including polyenes, macrolides and other polyketides by recombinant synthesis, and to the polyketides so produced, particularly novel polyketides. (N.B the term “polyketide” is being used in its conventional sense to include structures notionally derived by the reduction and/or other processing or modification of one or more Ketide units). Furthermore the invention provides the entire nucleic acid sequence of the biosynthetic gene cluster that governs the production of the ionophoric antibiotic polyether polyketide monensin in Streptomyces cinnamonensis, and the use of all or part of the cloned DNA first, in the specific detection of other polyether biosynthetic gene clusters; secondly in the engineering of mutant strains of S. cinnamonensis and of other actinomycetes which are suitable host strains for the high level production of novel recombinant polyketides; and thirdly in the provision of recombinant biosynthetic genes which lead to such novel polyketide products.

[0002] Polyketides are a large and structurally diverse class of natural products that includes many compounds possessing antibiotic or other pharmacological properties, such as erythromycin, tetracyclines, rapamycin, avermectin, monensin, epothilones and FK506. In particular, polyketides are abundantly produced by Streptomyces and related actinomycete bacteria. They are synthesised by the repeated stepwise condensation of acylthioesters in a manner analogous to that of fatty acid biosynthesis. The greater structural diversity found among natural polyketides arises from the selection of (usually) acetate or propionate as “starter” or “extender” units; and from the differing degree of processing of the β-keto group observed after each condensation. Examples of processing steps include reduction to β-hydroxyacyl-, reduction followed by dehydration to 2-enoyl-, and complete reduction to the saturated acylthioester. The stereochemical outcome of these processing steps is also specified for each cycle of chain extension. In addition, the biosynthetic pathways to many polyketides involve additional enzyme-catalysed modifications which may include: methylation by O- and C-methyltransferases, hydroxylation by cytochrome P450 enzymes, other oxidation or reduction processes, and the biosynthesis and attachment of novel sugars and/or deoxy sugars.

[0003] The biosynthesis of polyketides is initiated by a group of chain-forming enzymes known as polyketide synthases. Two classes of polyketide synthase (PKS) have been described in actinomycetes. One class, named Type I PKSs, represented by the PKSs for the macrolides erythromycin, oleandomycin, avermectin and rapamycin, consists of a different set or “module” of enzymes for each cycle of polyketide chain extension. (For examples see Cortés, J. et al. Nature (1990) 348:176-178; Donadio, S. et al. Science (1991) 252:675-679; Swan, D. G. et al. Mol. Gen. Genet. (1994) 242:358-362; MacNeil, D. J. et al. Gene (1992) 115:119-125; Schwecke, T. et al. Proc. Natl. Acad. Sci. USA (1995) 92:7839-7843.)

[0004] The term “extension module” as used herein refers to the set of contiguous domains, from a β-ketoacyl-ACP synthase (“KS”) domain to the next acyl carrier protein (“ACP”) domain, which accomplishes one cycle of polyketide chain extension. The term “loading module” is used to refer to any group of contiguous domains which accomplishes the loading of the starter unit onto the PKS and thus renders it available to the KS domain of the first extension module. The length of polyketide formed has been altered, in the case of erythromycin biosynthesis, by specific relocation using genetic engineering of the enzymatic domain of the erythromycin-producing PKS that contains the chain releasing thioesterase/cyclase activity (Cortés J. et al. Science (1995) 268:1487-1489; Kao, C. M. et al. J. Am. Chem. Soc. (1995) 117:9105-9106).

[0005] In-frame deletion of the DNA encoding part of the ketoreductase domain in module 5 of the erythromycin-producing PKS (also known as 6-deoxyerythronolide B synthase, DEBS) has been shown to lead to the formation of erythromycin analogues 5,6-dideoxy-3-α-mycarosyl-5-oxoerythronolide B, 5,6-dideoxy-5-oxoerythronolide B and 5,6-dideoxy,6-β-epoxy-5-oxoerythronolide B (Donadio, S. et al. Science (1991) 252:675-679). Likewise, alteration of active site residues in the enoylreductase domain of module 4 in DEBS, by genetic engineering of the corresponding PKS-encoding DNA and its introduction into Saccharopolyspora erythraea, led to the production of 6,7-anhydroerythromycin C (Donadio, S. et al. Proc. Natl. Acad. Sci. USA (1993) 90:7119-7123).

[0006] International Patent Application number WO 93/13663 describes additional types of genetic manipulation of the DEBS genes that are capable of producing altered polyketides. However many such attempts are reported to have been unproductive (Hutchinson, C. R. and Fujii, I. Annu. Rev. Microbiol. (1995) 49:201-238, at p. 231). The complete DNA sequence of the genes from Streptomyces hygroscopicus that encode the modular Type I PKS governing the biosynthesis of the macrocyclic immunosuppressant polyketide rapamycin has been disclosed (Schwecke, T. et al. (1995) Proc. Natl. Acad. Sci. USA 92:7839-7843). The DNA sequence is deposited in the EMBL/Genbank Database under the accession number X86780.

[0007] WO 98/01546 discloses that a PKS gene assembly (particularly of Type I) encodes a loading module which is followed by at least one extension module. The first open reading frame encodes the first multi-enzyme or cassette (DEBS1) which consists of three modules: the loading module (ery-load) and two extension modules (modules 1 and 2). The loading module comprises an acyltransferase and an acyl-carrier protein. This may be contrasted with FIG. 1 of WO 93/13663 (referred to above). This shows ORF1 as only two modules, the first of which is in fact both the loading module and the first extension module.

[0008] WO 98/01546 describes in general terms the production of a hybrid PKS gene assembly comprising a loading module and at least one extension module. It also describes (see also Marsden, A. F. A. et al. Science (1998) 279:199-202) construction of a hybrid PKS gene assembly by grafting the wide-specificity loading module for the avermectin-producing polyketide synthase onto the first multi-enzyme component (DEBS1) for the erythromycin PKS in place of the normal loading module. Certain novel polyketides can be prepared using the hybrid PKS gene assembly, as described for example in WO 98/01571.

[0009] WO 98/01546 further describes the construction of a hybrid PKS gene assembly by grafting the loading module for the rapamycin-producing polyketide synthase onto the first multi-enzyme component (DEBS1) for the erythromycin PKS in place of the normal loading module. The loading module of the rapamycin PKS differs from the loading modules of DEBS and the avermectin PKS in that it comprises a CoA ligase domain, an enoylreductase (“ER”) domain and an ACP, so that suitable organic acids including the natural starter unit 3,4-dihydroxycyclohexane carboxylic acid may be activated in situ on the PKS loading domain and, with or without reduction by the ER domain, transferred to the ACP for intramolecular loading of the KS of extension module 1 (Schwecke, T. et al. Proc. Natl. Acad. Sci. USA (1995) 92:7839-7843). WO 98/51695 and WO 98/49315 describe additional types of genetic manipulation of the DEBS genes that are capable of producing altered polyketides.

[0010] The second class of PKS, named Type II PKSs, is represented by the synthases for aromatic compounds. Type II PKSs contain only a single set of enzymatic activities for chain extension and these are re-used as appropriate in successive cycles (Bibb, M. J. et al. EMBO J. (1989) 8:2727-2736; Sherman, D. H. et al. EMBO J. (1989) 8:2717-2725; Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278-19290). The “extender” units for the Type II PKSs are usually acetate units, and the presence of specific cyclases dictates the preferred pathway for cyclisation of the completed chain into an aromatic product (Hutchinson, C. R. and Fujii, I. Ann. Rev. Microbiol. (1995) 49:201-238). Hybrid polyketides have been obtained by the introduction of cloned Type II PKS gene-containing DNA into another strain containing a different Type II PKS gene cluster, for example by introduction of DNA derived from the gene cluster for actinorhodin, a blue-pigmented polyketide from Streptomyces coelicolor, into an anthraquinone polyketide-producing strain of Streptomyces galileus (Bartel, P. L. et al. J. Bacteriol. (1990) 172:4816-4826).

[0011] The minimal number of domains required for polyketide chain extension on a Type II PKS when expressed in a Streptomyces coelicolor host cell (the “minimal PKS”) has been defined for example in WO 95/08548 as containing the following three polypeptides which are products of the actI genes: firstly KS; secondly a polypeptide termed the CLF with end-to-end amino acid sequence similarity to the KS but in which the essential active site residue of the KS, namely a cysteine residue, is substituted either by a glutamine residue or, in the case of the PKS for a spore pigment such as the whiE gene product (Davis, N. K. and Chater, K. F. Mol. Microbiol. (1990) 4:1679-1691) by a glutamic acid residue; and finally an ACP. The CLF has been stated (for example in WO 95/08548) to be a factor that determines the chain length of the polyketide chain that is produced by the minimal PKS. However it has been found (Shen, B. et al. J. Am. Chem. Soc. (1995) 117:6811-6821) that when the CLF for the octaketide actinorhodin is used to replace the CLF for the decaketide tetracenomycin in host cells of Streptomyces glaucescens, the polyketide product is not found to be altered from a decaketide to an octaketide, so the exact role of the CLF remains unclear. An alternative nomenclature has been proposed in which KS is designated KSα and CLF is designated KSβ, to reflect this lack of knowledge (Meurer, G. et al. Chemistry & Biology (1997) 4:433-443). The mechanism by which acetate starter units and acetate extender units are loaded onto the Type II PKS is not known, but it is speculated that the malonyl-CoA:ACP acyltransferase of the fatty acid synthase of the host cell can fulfil the same function for the Type II PKS (Revill, W. P. et al. J. Bacteriol. (1995) 177:3946-3952).

[0012] WO 95/08548 describes the replacement of actinorhodin PKS genes by heterologous DNA from other Type II PKS gene clusters, to obtain hybrid polyketides. It also describes the construction of a strain of Streptomyces coelicolor which substantially lacks the native gene cluster for actinorhodin, and the use in that strain of a plasmid vector pRM5 derived from the low-copy number vector SCP2* isolated from Streptomyces coelicolor (Bibb, M. J. and Hopwood, D. A. J. Gen. Microbiol. (1981) 126:427-442) and in which heterologous PKS-encoding DNA may be expressed under the control of the divergent actI/actIII promoter region of the actinorhodin gene cluster (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278-19290). The plasmid pRM5 also contains DNA from the actinorhodin biosynthetic gene cluster encoding the gene for a specific activator protein, ActII-orf4. The ActII-orf4 protein is required for transcription of the genes placed under the control of the actI/actIII bidirectional promoter and activates gene expression during the transition from growth to stationary phase in the vegetative mycelium (Hallam, S. E. et al. Gene (1988) 74:305-320).

[0013] Type II clusters in Streptomyces are known to be activated by pathway-specific activator genes (Narva, K. E. and Feitelson, J. S. J. Bacteriol. (1990) 172:326-333; Stutzman-Engwall, K. J. et al. J. Bacteriol. (1992) 174:144-154; Fernandez-Moreno, M. A. et al. Cell (1991) 66:769-780; Takano, E. et al. Mol. Microbiol. (1992) 6:2797-2804; Gramajo, H. C. et al. Mol. Microbiol. (1993) 7:837-845). The DnrI gene product complements a mutation in the actII-orf4 gene of S. coelicolor, implying that DnrI and ActII-orf4 proteins act on similar targets. A gene (srmR) has been described (EP 0 524 832 A2) that is located near the Type I PKS gene cluster for the macrolide polyketide spiramycin. This gene specifically activates the production of the macrolide antibiotic spiramycin, but no other examples have been found of such a gene. Also, no homologues of the ActII-orf4/DnrI/RedD family of activators have been described that act on Type I PKS genes. WO 98/01546 describes the use of the ActII-orf4 family of activators in conjunction with their cognate promoters (e.g actII-orf4 with the actI promoter) in a heterologous actinomycete to obtain high level expression of recombinant Type I polyketide synthase genes.

[0014] Although large numbers of therapeutically important polyketides have been identified, there remains a need to obtain novel polyketides that have enhanced properties or possess completely novel bioactivity. The complex polyketides produced by Type I PKSs are particularly valuable, in that they include compounds with known utility as anthelminthics, insecticides, immunosuppressants, antifungal agents or antibacterial agents. Because of their structural complexity, such novel polyketides are not readily obtainable by total chemical synthesis, nor by chemical modifications of known polyketides.

[0015] There is also a need to develop reliable and specific ways of deploying individual genes and portions of genes in practice so that all, or a large fractions of hybrid PKS genes that are constructed, are viable and produce the desired polyketide product. This includes the development of advantageous host strains for expression of such genes. For example many polyketides are rendered bioactive by the action of further enzymes other than the polyketide synthase, and host strains that contain and are able to express the genes for such enzymes are particularly convenient for the efficient synthesis of the bioactive material. In those cases where the construction of a known or a novel polyketide requires specialised precursors, host strains containing and able to express the genes for key enzymes that enhance the production of such specialised precursors are equally valuable and desirable. There is also a need to develop rational methods of increasing the expression level of all the genes required for production of a specific polyketide. Clearly also a host cell which is advantageous for the above reasons, and/or because of other favourable characteristics including but not limited to its speed of growth, excellent handling characteristics in fermentation, and ease of transformation with DNA by various techniques, can be made even more favourable by the cloning into that cell of such auxiliary genes for polyketide modification, or gene activation, or post-translational modification, or precursor supply.

[0016] The DNA sequences have been disclosed for several Type I PKS gene clusters that govern the production of 16-membered macrolide polyketides, including the tylosin PKS from Streptomyces fradiae (application EP 0 791 655 A2), the niddamycin PKS from Streptomyces caelestis (Kavakas, S. J. et al. J. Bacteriol. (1997) 179:7515-7522) and the spiramycin PKS from Streptomyces ambofaciens (application EP 0791 655 A2). DNA sequences have also been disclosed for Type I PKS gene clusters that govern the production of further complex polyketides, for example rifamycin from Amycolatopsis mediterranei (WO 98/07868), and soraphen from Sorangium cellulosum (U.S. Pat. No. 5,716,849), but so far no DNA sequence has been disclosed for one of the most widespread and important classes of complex polyketides, the polyethers.

[0017] Polyethers form an important group of complex polyketide antibiotics (Westley, J. W. in “Antibiotics IV. Biosynthesis” (Corcoran, J. W. Ed.), Springer-Verlag, New York (1981) p. 41-73). They are polyoxygenated carboxylic acids which act as selective ionophores transporting cations across the cell membrane of target cells and thereby causing depolarisation and cell death. Certain polyethers including monensin, lasalocid and tetronasin are in widespread use in animal husbandry as coccidiostats (principally targetted against Eimeria spp.) and as growth promoters. Polyethers have also been reported to be active in vitro and in vivo against the malarial parasite Plasmodium falciparum (Gumila, C. et al. Antimicrobial Agents and Chemotherapy (1997) 41: 523-529).

[0018] Polyethers contain multiple asymmetric centres and are characterised by the presence of tetrahydrofuran and tetrahydropyran rings, producing a characteristic shape which is non-polar on its outer surface and therefore well adapted for transport of material across bacterial membranes; and provides on its inner surface polar coordinating ligands for a centrally-bound metal ion. In addition to tetrahydrofuran and tetrahydropyran rings, other groups which are often present include spiroketal, dispiroketal, and substituted benzoic acid moieties and occasionally other groups for example a tetronic acid or a 6-membered carbocyclic ring

[0019] Monensins A and B are produced by the actinomycete Streptomyces cinnamonensis. Their structures are shown in FIG. 1. Monensin B differs from monensin A only in the presence of a methyl sidechain at C-16 rather than an ethyl sidechain. Monensin selectively binds and transports sodium ions. In addition to its antibacterial and antifungal properties monensin has some activity against protozoal parasites such as the malarial parasite Plasmodium falciparum. Although the structures of polyethers differ significantly from those of other complex polyketides such as the polyhydroxylated and polyene macrolides, their biosynthesis appears to take place by a metabolic pathway which has many common elements. Thus experiments using carbon 14-labelled precursors have shown that monensin A is synthesised from five acetate, one butyrate and seven propionate units (Day, L. E. et al. Antimicrob. Agents Chemother. (1973) 4:410-414). Similarly experiments using precursors doubly-labelled with carbon-13 and oxygen-18 have shown that oxygens (O)1, (O)3, (O)4, (O)5, (O)6 and (O)10 of monensin arise from the carboxylate oxygens of either propionate or acetate, while growth in the presence of oxygen-18 oxygen gas demonstrated that the three remaining ether oxygens (O)7, (O)8 and (O)9 are derived from molecular oxygen (Cane, D. E. et al., J. Am. Chem. Soc. (1981) 103:5962-5965; Cane, D. E. et al. J. Am. Chem. Soc. (1982) 104:7274-7281; Ajaz, A. A. and Robinson, J. A. J. Chem. Soc. Chem. Commun. (1983) 12:679-680). These findings have been rationalised by proposing that the biosynthesis of monensin proceeds via an acyclic triene intermediate (1) in which the geometry of all three carbon-carbon double bonds is E (entgegen) rather than Z (zusammen). The triene is then proposed to be subject to epoxidation to a tri-epoxide (2) and then ring opening is proposed to occur with concomitant sequential formation of the five ether rings as shown in FIG. 2A. Such a biosynthetic pathway, first mooted by Westley in 1974 (Westley J. W. et al., J. Antibiot. (1974) 27:597-604) accounts for the observed stereochemistry at the multiple asymmetric centres in monensin, (Cane, D. E. et al. J. Am. Chem. Soc. (1982) 104:7274-7281; Sood, G. R. et al. J. Chem. Soc. Chem. Commun. (1984) 21:1421-1424) and analogous schemes can be used to account for the biosynthesis of other known polyethers. such as lasalocid A (Hutchinson C. R. et al., J. Am. Chem. Soc. (1981) 103:5953-5956), tetronasin (ICI 139603) (Demetriadou, A. K. et al. J. Chem. Soc. Chem. Commun. (1985) 7:408-410) and narasin (Spavold, Z. et al. Tetrahedron Letters (1986) 27:3299-3302). The hydroxylation at C-26 and the introduction of an O-methyl group on oxygen 3-are proposed to occur as late steps in the biosynthesis, after formation of the polyether structure.

[0020] Unfortunately key aspects of the biosynthetic scheme shown in FIG. 2A have so far eluded experimental confirmation. No biosynthetic intermediates have been isolated from mutants of S. cinnamonensis that are blocked in early stages of monensin production. 26-deoxymonensin A has been isolated from a S. cinnamonensis mutant partially blocked in monensin production (Ashworth, D. M. et al. J. Antibiot. (1989) 42:1088-1099) and 3-0-demethylmonensins A and B have been recovered as minor components from the fermentation broth of a monensin-producing strain (Pospisil, S. et al. J. Antibiot. (1987) 40:555-557). When fed to cells of S. cinnamonensis in radio-labelled form, neither 26-deoxymonensin A, nor 3-0-demethylmonensin A, nor 3-0-demethyl, 26-deoxymonensin A were significantly incorporated into monensin A (Ashworth, D. M. et al. J. Antibiot. (1989) 42:1088-1099), either because they are actively excluded or because these modifications in fact occur earlier in the biosynthetic pathway so that these metabolites are shunt products not readily converted into the final antibiotic by the respective hydroxylase or methyltransferase. Similarly, the putative all (E)-triene precursor (1) has been synthesised and shown not to become incorporated into monensin when fed to growing cells of S. cinnamonensis (Holmes, D. S. et al. Helv. Chim. Acta (1990) 73:239-259). An alternative pathway has been proposed, as shown in FIG. 2B, based on the transition-metal-mediated oxidation of 1,5-dienes (Walba, D. M. and Edwards, P. D. Tetrahedron Lett. (1980) 21:3531-3534). The triene intermediate (4) would different from that of FIG. 2A (1) only in that each carbon-carbon double bond would have the (Z)-configuration (Townsend, C. A. and Basak, A. Tetrahedron (1991) 47:2591-2602) and not the (E)-configuration.

[0021] The genetic basis of secondary metabolite biosynthesis essentially exists in the genes which code for the individual biosynthetic enzymes and in the regulatory elements which control the expression of the biosynthetic genes. The genes encoding biosynthesis of polyketides in actinomycetes have hitherto been found as clusters of adjacent genes, ranging in size from 20 kilobasepairs (kbp) to over 100 kbp. The clusters often contain specific regulatory genes and genes conferring resistance of the producing strain to its own antibiotic.

[0022] In various of its aspects the invention provides the following:

[0023] (1) a DNA sequence encoding at least one-peptide necessary for the biosynthesis of monensin, preferably comprising one or more of the following genes: mon BI, mon BII, mon CI, mon CII, mon H, mon RI, mon RII, mon T, mon AIX and mon AX as depicted in the appended sequence data or an allele or mutation thereof;

[0024] (2) a DNA sequence according to the first aspect comprising all of the genes listed therein or an allele or mutation thereof;

[0025] (3) a DNA sequence according to the first aspect comprising the complete monensin gene cluster;

[0026] (4) a DNA sequence coding for one or more of the peptides set out below, said peptide having the amino acid sequence as set out in the appended sequence data or being a variant thereof having the specified activity:

1peptideactivitymon CIIepoxyhydrolase/cyclasemon ES-adenosylmethionine-dependent methyltransferasemon Tmonensin resistance genemon RIIrepressor proteinmon AIXthioesterasemon AIpolyketide synthase multienzymemon AIIpolyketide synthase multienzymemon AIIIpolyketide synthase multienzymemon AIVpolyketide synthase multienzymemon AVIpolyketide synthase multienzymemon AVIIpolyketide synthase multienzymemon AVIIIpolyketide synthase multienzymemon Hregulatory proteinmon CIflavin-dependent epoxidasemon BIIcarbon-carbon double bond isomerasemon BIcarbon-carbon double bond isomerasemon Dcytochrome P450 hydroxylasemon RIactivator proteinmon AXthioesterase

[0027] (5) a recombinant cloning or expression vector comprising a DNA sequence according to any of aspects 1-4;

[0028] (6) a transformant host cell which has been transformed to contain a DNA sequence according to any of aspects 1-4 and is capable of expressing a corresponding peptide;

[0029] (7) a hybridization probe comprising a polynucleotide which binds specifically to a region of the monensin gene cluster selected from mon BI, mon BII, mon CI, mon CII, mon H, mon RI, mon RII, mon T, mon AIX and mon AX;

[0030] (8) use of a probe according to aspect (7) in a method of detecting the presence of a gene cluster which governs the synthesis of a polyether, and optionally isolating a gene cluster detected thereby;

[0031] (9) Use of a probe comprising a polynucleotide which binds specifically to a gene responsible for levels of activity of the monensin gene cluster, preferably a regulatory gene, resistance gene or thioesterase gene, more preferably the regulatory gene mon RI, in a method of detecting an analogous gene in a gene cluster of another polyketide, preferably a polyether, and optionally manipulating the gene detected thereby to alter the level of expression of said other polyketide;

[0032] (10) a host cell, preferably Streptomyces cinnamonensis, containing a heterologous gene under the control of the mon RI gene and a monensin promoter;

[0033] (11) use of a portion of the monensin gene cluster having chain terminating activity, preferably comprising at least one of mon AIX and mon AX or a mutant or allele thereof having chain terminating activity, to effect chain release of a peptide other than one required for monensin biosynthesis;

[0034] (12) use of a portion of the monensin gene cluster having carbon-carbon double bond isomerase activity, preferably comprising at least one of mon BI and mon BII or a mutant or allele thereof having isomerase activity to provide a desired stereochemical outcome in the synthesis of a polyketide other than monensin;

[0035] (13) a polypeptide encoded by a portion of the monensin gene cluster, preferably comprising at least one of mon BI and mon BII or a mutant or allele thereof, having carbon-carbon double bond isomerase activity;

[0036] (14) an epoxidase enzyme encoded by mon CI or a derivative or variant thereof having epoxidase activity;

[0037] (15) a cyclase enzyme encoded by mon CII or a derivative or variant thereof having cyclase activity.

[0038] Some embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which:

[0039]
FIG. 1 shows the structure of monensins A and B;

[0040]
FIG. 2 illustrates proposed biosynthetic pathways;

[0041]
FIG. 3 illustrates the proposed organization of the monensin polyketide synthase (PKS) enzyme complex; and

[0042]
FIG. 4 illustrates the proposed organization of the monensin biosynthetic gene cluster.

[0043] The overall gene organization of the monensin biosynthetic gene cluster, as shown in FIG. 4, is similar to that previously found for many macrolide biosynthetic gene clusters, which have one or more open reading frames (ORFs) encoding large multifunctional PKSs flanked by other genes which encode functions required for the biosynthesis of the antibiotic. In the case of monensin, there is an unusually high number of distinct ORFs encoding PKS multi-enzymes (eight in total, labelled monAI to monAVIII) but there is again a separate module of enzymes for each cycle of polyketide chain extension, exactly as found for modular PKSs for macrolide biosynthesis (see FIG. 3). Thus there are 12 condensations predicted to be required for the production of the carbon skeleton of monensin, and in agreement with this there are found to be 12 extension modules of PKS enzymes distributed among the 8 PKS ORFs. However, as mentioned in detail below, the other genes in the monensin cluster include genes which have not previously been found in any other gene cluster for the biosynthesis of a complex polyketide, and which are not significantly similar to any genes in published sequence databases. The cloned DNA for these genes is useful to allow the diagnosis that a polyketide biosynthetic gene cluster in any actinomycete, uncovered previously by conventional hybridization against a PKS gene probe from (say) the DEBS or some other characterised PKS gene cluster, is one that governs the synthesis of a polyether; and these genes are also valuable either singly or in combination as specific hybridization probes for the specific detection and isolation of additional polyether biosynthetic gene clusters. Examples of these previously-unknown genes are the genes monBI, monBII, monCI and monCII. In addition the regulatory genes monH monRI, and monRII and the resistance gene monT and the thioesterase genes monAIX and monAX are all useful for the detection of analogous genes in other polyether clusters which are required for the rational manipulation of such genes in order to increase levels of the specific product.

[0044] The cloned and sequenced cluster of genes for monensin biosynthesis is useful secondly in the engineering of mutant strains of S. cinnamonensis and of other actinomycetes which are suitable strains for the high level production of either natural or novel recombinant polyketides. The sequence of the monensin cluster disclosed here shows the surprising fact, that the gene cluster contains a gene monRl whose gene product has an amino acid sequence highly similar to that of actII-orf4, the pathway-specific activator gene which activates the actI and other promoters of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The recognition of this aspect of the natural regulation of a Type I PKS cluster is important and valuable because first, it is possible to increase the yield of monensin by increasing the level of the activator MonRI, either by placing the gene monRI under the control of a powerful promoter or arranging for the presence within the cells of one or more additional copies of the monRI gene (as exemplified below); secondly, it will be possible to use the monRI gene as a specific hybridisation probe to locate similar genes in other complex PKS gene clusters, especially other polyether PKS gene clusters but also polyene and macrolide gene clusters and all other Type I modular PKS gene clusters; even in cases where (as for rapamycin and erythromycin) no such gene has been previously found within the currently accepted physical limits of the relevant biosynthetic gene cluster. In such cases the monRI gene probe might be expected to uncover the activator even if it resides on the chromosome at some distance from the main body of the gene cluster; and simple experiments would then show whether the activator(s) so uncovered are involved in regulation of the biosynthesis of those particular metabolites; thirdly, increasing the copy number of the monRI gene or of any of the activator genes uncovered will tend to increase the yield of a heterologous polyketide by “crosstalk” where the activator mimics the presence of the normal activator for the transcription of the genes for that heterologous polyketide synthase. It is clear from recently published work (Wietzorrek, A. and Bibb, M. Mol. Microbiol. (1997) 25:1181-1184) that the ActII-orf4 family of activators exert their effects by binding to promoter regions within the target gene cluster, so it will be possible to use the monRI gene together with monensin promoter regions to drive the high-level transcription and translation of heterologous genes in Streptomyces cinnamonensis, and perhaps in other host strains too; such genes need not be PKS genes or even involved in polyketide biosynthesis. Monensin promoter regions are found at the 5′ end of genes or groups of genes in the cluster and their location is clear from the sequence analysis disclosed here. Thus a useful vector would provide the monensin promoter and the ribosome binding site and continue up to the start of the open reading frame, after which the monensin ORF naturally found there would be replaced by the heterologous gene. The relative strength of the monensin promoters can be readily determined using any one of a number of known promoter probes, i.e. genes whose expression gives rise to readily measurable and quantifiable effects, such as Green Fluorescent Protein (GFP); or beta-galactosidase in the presence of a chromogenic substrate. It should be possible to mutate randomly the small region of the monensin promoters especially likely to interact with the MonRI activator (identified by the presence of tandem heptanucleotide repeats with a common consensus sequence between the various monensin promoters) (Wietzorrek, A. and Bibb, M. Mol. Microbiol. (1997) 25:1181-1184), and to determine the optimal DNA sequence for the maximal activation effect using either S. cinnamonensis (preferably—in case there are other unknown factors that make the activation function better in this strain than in other heterologous systems), or even in another host actinomycete strain. If the natural monensin promoters were mutated to have this optimal recognition sequence, then this would further increase the production of monensin. By extension, the use of this modified monensin promoter in conjunction with the monRI gene in heterologous systems could form the basis of further improvements in expression of polyketide synthases or other genes, either by appropriate chromosomal alterations to introduce the altered promoter and also the monRI gene; or by provision of vectors containing these optimised signals linked to specific genes and housed in suitable host cells.

[0045] The sequencing of the monensin cluster has uncovered another strategy for gene regulation in such Type I clusters. The previously-sequenced genes for the rapamycin biosynthetic pathway in Streptomyces hygroscopicus included a gene of unknown function (rapH). A closely similar gene has now been found in the monensin biosynthetic gene cluster (monH), and it is clear from this recurrence (and the comparison of the sequences with those of database proteins) that this gene is potentially an important DNA-binding sensor gene which acts to regulate the transcription of the cluster in concert with other regulatory signals. Simple experimentation is needed in order to define whether the gene is an activator, in which case putting in another copy or increasing its transcription will have the potential to increase polyketide biosynthesis; or alternatively the rapH gene product may be a negative regulator, whereupon deletion of this gene may release the biosynthetic pathway from this inhibitory effect and increase yields.

[0046] There is a continuing need to develop new methods of high-level production of bioactive metabolites and other valuable gene products in actinomycetes. Streptomyces cinnamonensis is a recognised and very valuable industrial strain for the production of very high levels of monensin, it is readily transformable with DNA by standard methods of conjugation or of protoplast transformation, it is a host for numerous known broad range plasmids including well-known expression plasmids of both high- and low-copy number, it also grows quickly relative to other actinomycete strains (for example about three times faster than wild type Saccharopolyspora erythraea the erythromycin producer, under comparable conditions) and sporulates relatively easily. Heterologous polyketides can be expressed in Streptomyces cinnamonensis using for example the low-copy number plasmid pCJR24 (which has no origin of replication active in actinomycetes so is maintained by integration into the chromosome) (Rowe, C. et al. Gene (1998) 216:215-223) or the related plasmid pCJR29 in which the polyketide synthase gene(s) are placed under the control of the acti promoter which is activated by the ActII-orf4 activator; or alternatively the monAI promoter can be substituted together with the MonRI activator; or some other pairing of activator and cognate promoter chosen from either a Type II or a Type I polyketide synthase gene cluster. As an example, the wild type strain of Streptomyces cinnamonensis has been used to express the plasmid pCJR29 (Rowe, C. et al. Gene (1998) 216:215-223) containing as insert the three ORFs for the PKS governing the production of 6-deoxyerythronolide B, the macrolide precursor of erythromycin A in Saccharopolyspora erythraea, these genes being placed under the control of the pathway-specific actI promoter from Streptomyces coelicolor together with its cognate activator gene actII-orf4. The transformed strain when cultivated in a suitable liquid medium produced 6-deoxyerythronolide B in good yield.

[0047] It is well known to the person skilled in the art that it is possible to use standard vectors unable to replicate in actinomycetes to introduce DNA into a Streptomyces cell, such DNA comprising two portions of contiguous DNA which are each identical to one of two portions of the cell's chromosome that are spaced up to 100 kbp apart; and that through recombination between the incoming DNA and the chromosome occurring in both portions of DNA the net result is that the chromosomal sequence is replaced by the defective sequence originally that of the incoming DNA. Such a procedure has been applied to the monensin-producing strain of S. cinnamonensis as described in detail below, and a strain of S. cinnamonensis has been obtained that carries a specific deletion in the monensin cluster and which is unable to produce the antibiotic. The use of such a strain facilitates the production of heterologous polyketides by removal of the background of monensin production.

[0048] The multiple uses of portions of the cloned and sequenced DNA from the monensin cluster will readily occur to the person skilled in the art. A surprising feature of the PKS of the monensin cluster is an unusual mechanism of polyketide chain initiation. We have found that the monensin PKS loading module has three domains, which from the amino-terminus of the protein are: a KSq domain, an acyltransferase domain and an ACP domain. We have uncovered this organisation in the PKS for the 14-membered macrolide oleandomycin as well as in the monensin PKS, an organisation of the loading module previously only found for the 16-membered macrolides and in which the KSq domain (which looks like a ketosynthase or condensation domain except that the active site cysteine residue is substituted by a glutamine for which the single letter notation is Q) had been previously speculated to have no function. It was realised that the acyltransferase of the loading module actually has malonyl-CoA and not acetyl-CoA as a substrate and that KSq is an active decarboxylase. It appears that a better discrimination can be achieved in the selection of the smaller acetate unit over propionate if the choice is made initially between methylmalonyl- and malonyl-CoA.

[0049] An unprecedented feature of the monensin PKS genes is that no integral chain-terminating domain is present as a C-terminal appendage of the PKS extension module that catalyzes the twelfth and final chain extension. Because the product of the monensin PKS is a carboxylic acid, it would have been firmly predicted that chain release would have been catalyzed by such a C-terminal domain containing a “thioesterase” activity. Previously sequenced PKS gene sets have been of two sorts: first, those macrolide PKSs typified by erythromycin, spiramycin, tylosin, niddamycin which have a readily recognisable C-terminal “thioesterase” domain, which in these enzymes functions as a specific cyclase rather than releasing the polyketide product as a free carboxylic acid; secondly, those macrolide PKSs typified by rapamycin, FK506, and rifamycin, where there is an alternative and recognised mode of chain termination by transfer of the polyketide chain to an acceptor moiety, catalyzed by a specific enzyme (eg pipecolate incorporating enzyme for rapamycin (Schwecke T. et al. Proc. Natl. Acad. Sci. USA (1995) 92:7839-7843) and FK506 (Mothamedi H. and Shafiee A, Eur. J. Biochemistry (1998) 256:528-534); arylamine synthetase for rifamycin (August P. R. et al. Chemistry & Biology (1998) 5:69-79).

[0050] The monensin PKS surprisingly falls into neither category, and therefore seems to be the first example of a novel mode of chain termination. It is novel and noteworthy in this connection that the monensin PKS gene cluster contains two small genes that encode discrete, monofunctional thioesterase enzymes. Although many PKS gene clusters have been previously shown to contain one such discrete thioesterase, none have been shown to have two. The role of such thioesterases is not known, although in the case of methymycin/pikromycin PKS, which has been reported to be responsible for the biosynthesis of both the 12-membered macrolide methymycin and the 14-membered macrolide pikromycin (Xue Y. Q. Proc. Natl. Acad. Sci. USA (1998) 95:12111-12116) the disruption of this thioesterase reportedly caused a ten-fold drop in the amount of both macrolides produced. A similar finding has been reported for the discrete thioesterase of the tylosin PKS gene cluster (Cundliffe E. et al. Chemistry & Biology in press). Additional copies of such thioesterases may therefore accelerate the production of specific polyketide, but this has not yet been demonstrated. However, the presence of the discrete thioesterase is not completely essential for polyketide production.

[0051] It is highly desirable to have a broadly effective method of catalysing the release of polyketide gene products from a PKS as the free acid. The well-studied integral thioesterase domain in the erythromycin PKS thioesterase has a broad specificity in cyclization to form a lactone (assuming that a hydroxy group is present in the growing polyketide chain at an appropriate position), but hydrolysis to form the free acid is very slow. The recognition of the unusual arrangement of the monensin PKS means that it is now possible to harness either the entire PKS module that catalyses the twelfth and final extension cycle in monensin biosynthesis, or the C-terminal portion of it, and graft it onto a different polyketide synthase by genetic engineering, so as to allow the release mechanism characteristic of monensin to operate in a different context. The use of this portion only of the monensin PKS suffices to allow the novel mechanism of chain release to operate successfully. The speed of the polyketide chain hydrolysis in a given case can depend on the additional presence of one or both of the discrete thioesterase genes (monAIX and monAX) from the monensin gene cluster. The use of this novel method of chain termination represents a valuable way of generating a large number of novel engineered polyketides that are currently inaccessible, and ensuring that the products have a specified chain length.

[0052] The genes monBI and monBII appear to encode very similar enzymes with significant amino acid sequence similarity to authentic ketosteroid isomerases which are known to catalyse the migration of an activated carbon-carbon double bond. The conservation of active site residues makes it very likely that these mon genes govern a reaction involving activated double bonds in the biosynthetic pathway to monensin and this surprising observation can be accommodated if the initial product of the polyketide chain growth on the monensin PKS is a linear precursor in which the double bonds were initially formed with a conventional trans or E (entgegen) geometry; but before the polyketide chain was extended by insertion of the next unit the monBI and/or the monBII gene product(s) catalyse the specific rearrangement of the newly-created double bond into the cis or Z (zusammen) geometry. This new view of the monensin biosynthetic pathway allows the deduction that the monBI and monBII genes, perhaps in combination with specific portions of the monensin modules where they normally exert their effects (namely modules 3, 5 and 7) might be used in order to achieve the extremely desirable targetted biosynthesis of novel polyketides containing double bonds with Z geometry at specified point(s) along the chain. Thus for example it should be possible to provide for the direct biosynthesis of C22-C23 cis or Z double bond in avermectins, thus avoiding tedious and expensive chemical conversion of an initial fermentation product into this important anthelminthic. Only limited experimentation is needed to see whether the monBI and/or monBII gene products are sufficient or whether the mon PKS at modules 3, 5 and 7 forms part of the specific docking site(s) for the isomerases and therefore must also be used in the creation of the hybrid PKS that will insert the cis or Z double bond at the desired position. The substrate specificity of the isomerases need not be limited to 2,3-unsaturated thioesters. The purified enzymes could also be used to effect such isomerisations in vitro, depending on the position of the equilibrium or whether further enzymes are used to achieve the further transformation of the product as it is formed (vide infra).

[0053] The product of the monCI gene is a novel oxidative enzyme with some sequence similarity to authentic examples of such enzymes in the databases; and with a clearly definable role in the monensin biosynthetic pathway, the epoxidation of the double bonds at three separate positions in the initially-formed acyclic intermediate in monensin biosynthesis. This epoxidase could therefore be used in conjunction with monBI/monBII gene products to effect oxidative reactions on suitable substrates in vitro and in vivo. Similarly the monCII gene product is a putative cyclase that opens the epoxides and causes the formation of ether rings in monensin.

[0054] Any or all of the monBI, monBII, monCI or monCII genes may be introduced into a heterologous strain containing the gene cluster for another polyether, in order to divert the biosynthetic pathway and produce a polyketide of altered structure. In these experiments the analogues of these monB genes could either be present or (once located and characterised using the mon genes as probes) they may be deleted prior to the introduction of the monB and monC genes into that strain. The converse experiment in which analogues of the monB and monC genes from other strains are introduced into S. cinnamonensis likewise has the potential to produce novel oxidised polyketides. Also, the monB and monC genes or their analogues may be introduced into a strain that normally produces a macrolide or a polyene or some other complex polyketide and expressed there, when they may effect the diversion of the growing polyketide chain on a heterologous modular PKS towards a new product, which may or may not have the structure of a polyether.

[0055] The availability of the monensin gene sequence allows the institution of domain swaps to alter the acyltransferase (AT) specificity of a given module, for example the ethylmalonyl-CoA specific extender found in one of the modules of the monensin PKS can be used to replace one of the other ATs to generate an ethyl side branch at that position in the chain, or the AT can be used to substitute in any other (e.g. macrolide) PKS, as described in WO 98/01571 and Wo 98/01546. Similarly the alteration of the level of reduction in a module, by manipulation of the reductive enzymes, can be applied to the monensin genes and here it will produce, depending on which module is affected, either an altered monensin, or a species which is only partly cyclised, or a polyether with an altered pattern of cyclisation, or even a linear polyketide.

[0056] In general the targetted alteration of the pattern of substitution of sidechains or reduction level along the polyketide chain produced by the monensin PKS will, like the disruption or deletion of the oxidative enzymes mentioned above, lead to non-polyether polyketide products. It should be possible, by introduction of the DEBS thioesterase at the C-terminus of one of the later modules of the monensin PKS, together with an appropriately placed hydroxy group earlier in the chain, to produce novel macrolide products from this polyether PKS system, or alternatively novel polyenes of defined chain length and chosen ring size.

EXAMPLE 1

Cloning of the Monensin A Biosynthetic Gene Cluster Using DNA Probes Derived From the Erythromycin-Producing Polyketide Synthase of Saccharopolyspora erythraea

[0057] A genomic library of the monensin A producing strain Streptomyces cinnamonensis ATCC 15413 was constructed using methods well-known in the art, namely, the production of high molecular weight genomic DNA, followed by the partial cleavage of this DNA using the frequent-cutting restriction enzyme Sau3A, fractionation of the fragments on a sucrose gradient and selection of fragments of average size 35-40 kbp, and the cloning of these fragments into the cosmid vector pWE15 (Evans, G. A. et al. Gene (1989) 79:9-20) which had been previously digested with BamHI and treated with shrimp alkaline phosphatase. The library was packaged and transfected into Escherichia coli XL-1 Blue MR cells. The library was plated out on 2×TY agar medium (10 g tryptone, 10 g yeast extract, 5 g NaCl, 15 g bactoagar per litre containing ampicillin 50 μg/ml) for cosmid selection and the colonies were allowed to grow overnight. The library was then screened by hybridisation using as a probe DNA encoding the ketosynthase domain of module 1 of the erythromycin-producing PKS (6-deoxyerythronolide B synthase, DEBS) of Saccharopolyspora erythraea. The colonies giving a positive hybridisation signal in the hybridisation were selected and the cosmid DNA from each colony was purified and mapped by restriction digestion. The presence of the target biosynthetic genes on a cosmid was verified by sequencing of the ends of the cosmid inserts using the commercially available T3 and T7 primers which hybridise specifically to the respective ends of each cosmid insert (Evans, G. A. et al. Gene (1989) 79:9-20).

EXAMPLE 2

Sequencing of the Biosynthetic Gene Cluster for Monensin A From Streptomyces cinnamonensis

[0058] Three cosmids obtained by screening of the genomic library of S. cinnamonensis were used to obtain the entire DNA sequence of the monensin biosynthetic gene cluster. These cosmids, MO.CN02, MO.CN11 and MO.CN33 between them contain the entire DNA sequence of the cluster and the adjacent regions of the chromosome. They have been deposited in NCIMB, 23 St Machair Drive, Aberdeen AB24 3RY, UK, under the NCIMB accession numbers 40956 (MO-CN11); 40957 (MO-CN33) and 40958 (MO-CN02) respectively.

[0059] The DNA of each cosmid was separately subjected to partial digestion with Sau3A and fragments of approximately 1.5-2.0 kbp were separated by agarose gel electrophoresis. The fragments were then ligated into the plasmid vector pUC18 (Messing, 1982), previously digested with BamHI and treated with shrimp alkaline phosphatase. The library was transformed into E. Coli strain XL1-Blue MR and plated on 2×TY agar medium containing ampicillin (100 μg/ml) to select for plasmid-containing cells. Plasmid DNA was purified from individual colonies and sequenced using the Sanger dye-terminator procedure on an ABI 377 automated sequencer (Sanger, F. Science (1981) 214:1205-1210). The sequence data obtained from single random subclones of a cosmid was assembled into a single continuous sequence and edited using GAP4.1 program of the STADEN gene analysis package (Staden, R. Molecular Biotechnology (1996) 5:233-241).

[0060] The sequence is set out in the appended sequence listing.

[0061] Tables I and II contain data about individual genes and gene products.

EXAMPLE 3

Inactivation of the Monensin A Biosynthetic Gene Cluster

[0062] A chromosomal gene disruption experiment was used to verify the identity of the cloned polyketide synthase gene cluster. Plasmid pMOB6314 is a pUC18 sequencing subclone of the presumed monensin A biosynthetic gene cluster prepared as described in Example 1, whose inserted DNA comprises the DNA sequence from nucleotide 9763 to nucleotide 10108 in SEQ ID 1, and which therefore contains a region of DNA wholly internal to orfE, a putative 3-O-methyltransferase. A HindIII fragment containing the thiostrepton resistance gene tsr from plasmid pIJ702 (Katz, E. et al. J. Gen. Microbiol. (1983) 129:2703-2714) was cloned into the HindIII site of plasmid pMOB6314 and the ligation mixture was used to transform E. coli cells. Transformants bearing the required plasmid pMOΔE01 were identified by isolation of plasmid DNA and analysis by restriction digestion. pMOΔE01. Plasmid pMOΔE01 was used to transform protoplasts of Streptomyces cinnamonensis as described by (Hopwood D. A. et al. (1985)). Since plasmid pMOΔE01 lacks an origin of replication that is active in Streptomyces, growth in the presence of thiostrepton (25 μg/ml) in the regeneration medium led to the isolation of stable integrants. Isolated putative integrants were tested for the presence of integrated pMOΔE01 sequences by Southern hybridisation. A clone of Streptomyces cinnamonensis identified by its restriction pattern in Southern hybridisation as bearing pMOΔE01 integrated in the region of monE of the monensin A biosynthetic gene cluster was designated S. cinnamonensis MO-DD01.

[0063] Detection of production of the monensin A related metabolites produced by S. cinnamonensis MO-DD01 was performed by GC-MS analysis of methanol extracts of the entire broth harvested in 72 hours of growth of the strain. No significant amounts of monensin A-related metabolite production were detectable.

EXAMPLE 4

Overproduction of Erythromycin Aglycone in Streptomyces cinnamonensis

[0064]

S. cinnamonensis
is a suitable system for overproduction not just of monensin A but also of other polyketide metabolites. Established techniques of genetic transformation allow fast introduction of foreign polyketide producing genes sets into this host. Fast growth of S. cinnamonensis in liquid culture and optimal precursor supply favour high yield of polyketide metabolites.

[0065] Construction of pIB061

[0066]

S. erythraea
NRRL2338 was transformed with pCJR30 (Rowe, C. J., et al. (1998) Gene 216:215-223) using a routine protoplast transformation technique as described by Hopwood et al. (1985). A stable integrant of S. erythraea [pCJR30] was identified and the production of 10 mg/L of the triketide lactone (delta lactone of (2S,3R,4R,5R)-2,4-dimethyl-3,5-dihydroxy-heptanoic acid) in addition to erythromycins was confirmed by MS analysis.

[0067] Total DNA of S. erythraea [pCJR30] was purified and approximately 200 ng was digested with EcoRI endonuclease. The digestion mixture was precipitated with isopropanol and the resulting DNA was treated with T4 DNA-ligase for 16 hours at 16° C. The ligation mixture was used to transform E.coli DH10B cells. The transformants were screened for the presence of the plasmid. A clone containing a 44.7kb plasmid was identified and confirmed by restriction analysis to contain three complete genes: eryAI, eryAII and eryAIII. The plasmid was named pIB061.

[0068] Transformation of S. cinnamonensis

[0069] Protoplasts of S. cinnamonensis were prepared by a modified procedure of Hopwood et al. (1985). Plasmid pIB061 was transformed into the protoplasts of S. cinnamonensis and stable thiostrepton resistant colonies were isolated. Individual colonies were checked for their plasmid content and the presence of plasmid pIB061 was confirmed by its restriction pattern. S. cinnamonensis (pIB061) was inoculated into 250 ml of M-C3 minimal production medium containing 10 μg/ml of thiostrepton and allowed to grow for 72 hours at 30° C. After this time the mycelia were removed by filtering. The broth was extracted with two volumes of ethyl acetate and the combined ethyl acetate extracts were washed with an equal volume of saturated sodium chloride, dried over anhydrous sodium sulphate, and the ethyl acetate was removed under reduced pressure to give about 200 mg of crude product. The product was analysed by LCQ and mass was confirmed to that of erythronolide B.

[0070] This example demonstrates the importance of S. cinnamonensis for production of high levels of foreign polyketide antibiotics. Introduction of the complete erythromycin gene cluster or other gene clusters into this system are likely to produce high levels of the corresponding metabolites.

EXAMPLE 5

Construction of Plasmid pCJW58 Containing the Monensin Activator Gene Under the ermE* Promoter

[0071] The ermE* promoter derived from the ermE resistance methyltransferase gene of S. erythraea (Bibb et al. Gene (1985) 38:215-226) was amplified by PCR as a SpeI-XbaI fragment using the following oligonucleotides 5′-CCACTAGTATGCATGCGAGTGTCCGTTCGAGT-3′ and 5′-TTGTATACACCTAGGATGGTTGGCCGTGC-3′ with pRH3 (Dhillon et al. Molecular Microbiology (1989) 3:1405-1414 as a template and cloned into SmaI-digested, phosphatase-treated pUC18, to produce plasmid pIB135. The integrative plasmid pSET152 (Bierman, M. et al. (1992) Gene 116:43-49)) was digested with XbaI and the backbone was dephosphorylated and ligated to the SpeI-XbaI fragment of pIB135 containing the ermE* promoter. The ligation mixture was used to transform E. coli DH10B and the orientation of the insert in the plasmids from individual clones was checked by using restriction analysis. A plasmid with the ermE* promoter oriented so that the NdeI and XbaI sites are adjacent to the apramycin resistance gene was selected and named pIB139.

[0072] The monR gene from the monensin biosynthetic gene cluster was amplified and NdeI and XbaI restriction sites introduced at 5′ and 3′ ends respectively, by PCR using as primers the following oligonucleotides: 5′-AGA TAC CAT ATG CTG GGC CCG CTC CGC AT-3′ and 5′-AAT GCT CTA GAC TGT CAG CGA CCG GAC AGG GCC AA-3′ and cosmid MO.CN11 as template. The PCR product was ligated into SmaI-treated and phosphatase-treated plasmid pUC18 and the ligation mixture was used to transform E. coli DH10B cells. Transformant colonies were analysed for the presence of plasmid and the identity of the plasmid inserts was verified by sequencing. A plasmid whose insert contained the monR gene flanked by NdeI and XbaI restriction sites was selected and designated pCJW57.

[0073] Plasmid pCJW57 was digested with NdeI and XbaI and the fragment containing the monR gene was ligated together with the backbone of plasmid pIB139 which had been digested with the same two restriction enzymes, and purified by gel elution. The ligation mixture was used to transform E. coli strain DH10B cells. Transformant colonies were analysed for the presence of plasmid and the identity of the plasmid inserts was verified by restriction analysis. One such recombinant was selected and named plasmid pCJW58.

[0074] Plasmid pCJW58 was used to transform the methylation-deficient E. coli strain ET 12567 (MacNeil D. J. et al. (1992) Gene 111:61-68) and the recovered, unmethylated plasmid was then used to transform the same E. coli strain ET12567 housing the plasmid pUB307, a derivative of RP4 which is mob and which contains a gene for kanamycin resistance (Piffaretti, J. C. et al. (1988) Mol. Gen. Genet. 212:215-218). Recombinants were plated on 2×TY agar medium containing apramycin and kanamycin at final concentrations of 50 micrograms per ml and 50 micrograms per ml respectively. The plasmid content of recombinants was checked isolation of plasmid DNA and checking of the identity of these plasmids by restriction analysis. One such clone which contained both pUB307 and plasmid pCJW58 was selected and used for further experiments.

[0075] Construction of Streptomyces cinnamonensis (pCJW58) and production of monensins

[0076] A single colony of E. coli ET12567 housing both pUB307 and pCJW58 was toothpicked into 3 ml of TY liquid medium, containing apramycin and kanamycin at 25 and 25 micrograms respectively, and grown overnight at 37° C. This culture was used to inoculate 25 ml of TY medium, supplemented with the same antibiotics at the same concentrations, and growth was continued until the absorbance at 600 nm (1 cm pathlength) was between 0.3-0.6. The cells were centrifuged (room temperature, 7 minutes, 2000×g), resuspended in TY liquid medium (10 ml) containing no added antibiotics, re-centrifuged as before, then resuspended in 2 ml of TSB medium and placed on ice. Meanwhile, 0.5 ml of TSB medium was added to 100 microL containing approximately 108 spores of S. cinnamonensis. After a brief heat shock, at 50° C. for 10 minutes, the suspension was briefly cooled, mixed with 0.5 ml of donor E. coli cells, and plated on solid A medium, which has composition as follows:

2A mediumSigma wheat starch 5 gCorn steep powder1.25 gYeast extract 1.5 gCaCO3 1.5 gFeSO4 6 mgDIFCO agar 10 gH2Oto 500 mlpH adjusted to pH 7 with KOH.

[0077] And to which in addition was added 10 mM MgCl2 to a final concentration of 10 mM.

[0078] The plates were allowed to dry overnight at room temperature, and were then allowed to incubate a further 18 hours at 30° C. After this time each 25 ml plate was overlaid with a solution of apramycin (final concentration 50 micrograms per ml) and nalidixic acid (final concentration 20 micrograms per ml), and the plates were allowed to incubate for four days at 30° C. At this time individual colonies were toothpicked onto solid A medium and allowed to grow. Four representative colonies from the A medium plate were grown up in liquid modified YEME medium, which has composition as follows:

3Modified YEME mediumSucrose100 gDIFCO Yeast extract 3 gBacto peptone 5 gOxoid Malt extract 3 gGlucose 10 gH2O to 1 LpH adjusted to pH 7.2 with NaOH.

[0079] These cultures were used to provide a 2% vol/vol inoculum for 30 ml of modified YEME which was grown for 7 days, and then transferred to SM16 medium, which has composition as follows:

4SM16 medium3-[N-Morpholino]-propane sulfonic acid 20.9 g(MOPS) bufferL-proline 10.0 gGlucose 20 gNaCl 0.5 gK2HPO4 2.1 gEthylenediaminetetraacetic acid, sodium 0.25 gsaltMgSO4.7H2O 0.49 gCaCl2.2H2O0.029 gTrace elements solution (Hopwood, D. A. 2 mlet al. (1985) Genetic Manipulationof Streptomyces - a Laboratory Manual,at p.235)0.5 M CoCl2 solution 2 microlitersH20 to 1 LpH adjusted to pH 7 with NaOH.

[0080] After growth for a further 7 days, mycelium was collected by centrifugation at 2000×g for 30 minutes, and the supernatant was extracted three times with 300 ml of ethyl acetate. The combined extracts were concentrated by evaporation under reduced pressure to an oil, which was mixed with 1 ml of methanol. Samples were applied to an LCQ liquid chromatograph fitted with a mass spectrometer detector unit. The column used was a C18 reversed phase column, equilibrated with a mixture of 80% 20 mM ammonium acetate/20% acetonitrile, and the column was eluted with a gradient of increasing acetonitrile, reaching 100% acetonitrile over 24 minutes. Monensins A and B emerged from the column with retention times respectively of 8.2 minutes and 9.2 minutes. The relative amounts of monensin produced by three independent clones (A-C) containing an additional copy of the monr gene were compared to a control fermentation of the wild type S. cinnamonensis strain, with the results shown in the Table below:

5Table showing increased monensin production in strainsbearing additional copy of monR genemonensin Amonensin BconcentrationconcentrationStrain(arbitrary units)(arbitrary units)Control188 861A4301 800B4501 300C2491 300

EXAMPLE 6

Construction of S. cinnamonensis M12AT5

[0081] A region lying immediately 5′ of the DNA encoding the acyltransferase (AT12) domain of module 12 of the monensin polyketide synthase in the mbnensin biosynthetic gene cluster was amplified with the following primers: 5′-GGTGGCCACGGAAACACCAACACCGGACCCGCGCC-3′, and 5′-CTCTCGGAGGCCCGGCGCAACGGCCACAA-3′, 3′ using casmid MO-CN11 as a template. The PCR product was ligated into SmaI digested and phosphatase-treated plasmid pUC18 and the ligation mixture was used to transform E. coli DH10B cells. Transformant colonies were analysed for the presence of plasmid and the identity of the plasmid inserts was verified by sequencing. A plasmid whose insert contained a fragment upstream of the AT12-encoding sequence from about 82.3kb to 83.2kb of the mon cluster was designated pMO81. Similarly a region lying immediately 3′ of the DNA encoding the acyltransferase (AT12) domain of module 12 of the monensin polyketide synthase in the monensin biosynthetic gene cluster was amplified with the following primers: 5′-GGCCTAGGGCTGCCTCGGGTGGTGGATCTGCCGA-3′ and 5′- TGGTCGGGCGCGGTGCGTGCGATACGT-3′, using cosmid MO-CN11 as a template. The PCR product was ligated into SmaI-treated and dephosphorylated pUC18 and the ligation mixture was used to transform DH10B E.coli cells. Transformant colonies were analysed for the presence of plasmid and the identity of the plasmid inserts was verified by sequencing. A plasmid whose insert contained a fragment downstream of the AT12-encoding sequence, from 80.5kb to 81.4kb of the mon cluster, was designated pMO82.

[0082] The DNA encoding AT of module 5 was amplified and MscI and AvrII restriction enzyme recognition sites were introduced at the ends by PCR using the following primers: 5′-CCTGGCCAGGGCGGCCAGTGGGTGGGCATG-3′ and 5′-GGCCTAGGGGTCGGCCGGGAACCAGCGCCGCCAGT-3′ and the cosmid MO-CN33 as a template. The PCR product was ligated into SmaI-treated and dephosphorylated pUC18 and the ligation mixture was used to transform DH10B E.coli cells. Transformant colonies were analysed for the presence of plasmid and the identity of the plasmid inserts was verified by sequencing. A plasmid whose insert DNA, with sequence from about 44.2kb to 45.2kb of the mon cluster, encoded the AT5 domain was designated pMO83.

[0083] pMO81 was digested with MscI and HindIII and ligated to the 0.9kb MscI-HindIII fragment of pMO82. A clone containing both fragments was designated pMO84. Plasmid pMO84 was cleaved with AvrII and HindIII, treated with phosphatase, and ligated together with the 1.0 kb AvrII-HindIII fragment of pMO83 to produce pMO85, which contains the DNA encoding the AT5 domain flanked by DNA from either side of the DNA encoding the AT12 domain of the monensin PKS. The thiostrepton resistance gene tsr, derived from plasmid pIJ702 (Katz, E. et al., J. Gen. Microbiol. 1983), was cloned into the HindIII site of pMO85. The resulting plasmid pMO86 was analysed by its restriction pattern and confirmed to contain all the desired elements.

[0084] Plasmid pMO86 was used to transform S. cinnamonensis protoplasts as described by Hopwood, D. A. (1985). Stable thiostrepton-resistant transformants were isolated and checked for the desired integration of the pMO85 into the AT12 flanking regions by Southern blot hybridisation. One such integrant, S. cinnamonensis MO-08, containing pMO85 integrated upstream of the AT12, was passed through 4 cycles of sporulation on a non-selective nutrient medium. Spores obtained after the fourth cycle were replica-plated onto media with and without thiostrepton. DNA of clones that had lost thiostrepton resistance was analysed by Southern blot hybridisation. Clones in which the DNA encoding the AT12 domain had been replace by the DNA encoding the AT5 domain was designated S. cinnamonensis M12-AT5. At this time individual colonies were toothpicked onto solid A medium and allowed to grow. Four representative colonies from the A medium plate were grown up in liquid modified YEME medium, which has composition as follows:

6Modified YEME mediumSucrose100 gDIFCO Yeast extract 3 gBacto peptone 5 gOxoid Malt extract 3 gGlucose 10 gH2O to 1 LpH adjusted to pH 7.2 with NaOH.

[0085] These cultures were used to provide a 2% vol/vol inoculum for 30 ml of modified YEME which was grown for 7 days, and then transferred to SM16 medium, which has composition as follows:

7SM16 medium3-[N-Morpholino]-propane sulfonic 20.9 gacid (MOPS) bufferL-proline 10.0 gGlucose 20 gNaCl 0.5 gK2HPO4 2.1 gEthylenediaminetetraacetic acid, 0.25 gsodium saltMgSO4.7H2O 0.49 gCaCl2.2H2O0.029 gTrace elements solution (Hopwood, D. A. 2 mlet al. (1985) GeneticManipulation of Streptomyces - aLaboratory Manual, at p. 235)0.5 M CoCl2 solution 2 microlitersH20 to 1 LpH adjusted to pH 7 with NaOH.

[0086] After growth for a further 7 days, mycelium was collected by centrifugation at 2000×g for 30 minutes, and the supernatant was extracted three times with 300 ml of ethyl acetate. To confirm presence of the C-2-ethyl substituents of both monensin A and B the combined extracts were concentrated by evaporation under reduced pressure to an oil, which was mixed with 1 ml of methanol. Samples were applied to an LCQ liquid chromatograph fitted with a mass spectrometer detector unit. The column used was a C18 reversed phase column, equilibrated with a mixture of 80% 20 mM ammonium acetate/20% acetonitrile, and the column was eluted with a gradient of increasing acetonitrile, reaching 100% acetonitrile over 24 minutes. Mass ions 14 mass units above those expected for both monensin A and B confirmed production of the respective C-2-ethyl substituents.

EXAMPLE 7

Construction of pSGK005 and Its Use in the Production of C-13 Propyl-Erythromycin

[0087] Plasmid pSGK005 is a pCJR24 based plasmid containing a PKS gene comprising a loading module plus the first and second extension modules and the chain terminating thioesterase of the PKS responsible for the production of erythromycin (DEBS). The loading module comprises the KS and ethyl-malonyl CoA specific AT from module 5 of the monensin PKS linked to the DEBS loading ACP domain. In addition, the active site cysteine of this module 5 KS has been mutated to glutamine to convert an extender di-domain to a loading di-domain. Plasmid pSGK005 was constructed as follows.

[0088] A 2769 bp DNA segment of the monensin cluster of S. cinnamonensis extending from nucleotide 42438 to 45207 was amplified by PCR using the following oligonucleotide primers. 5′-GTGACGTCATATGTCGAGTGCTGAAGAGTCG-3′ and 5′-GGGGTCGCCTAGGAACCAGCGCCGCCAGTCGA-3′

[0089] The design of these primers introduced Nde I and Avr II sites at the ends of the amplifed fragment. Monensin cosmid 05 was used as a template for the reaction. The resulting 2769 bp fragment was digested with Nde I and Xho I and a 656 bp fragment (Fragment A) purified by preparative gel electrophoresis.

[0090] A second PCR reaction was used with the same template to amplify DNA from nucleotide 43098 to 45207. The primers used were

85′-CGGCCTCGAGGGCCCGTCGGTCAGTGTCGACACGGCGCAGTCCTCCTCGC-3′and5′-GGGGTCGCCTAGGAACCAGCGCCGCCAGTCGA-3′

[0091] The design of the upstream oligonucleotide primer incorporated a change of the codon specifying the KS active site cysteine (nucleotides 43135-43137, TGC) to glutamine (CAG). The resulting 2109 bp DNA fragment (Fragment B) was digested with Xho I and Avr II and purified by preparative gel electrophoresis.

[0092] Plasmid pCJW80 is derived from pCJR24 and DEBS1-TE in which Msc I and Avr II sites have been introduced to flank the AT of the DEBS loading module. This plasmid was digested with Nde I and Avr II and the larger fragment (Fragment C) purified by preparative gel electrophoresis.

[0093] The three fragments (Fragments A, B, C) were ligated together using T4 DNA ligase and the ligation mixture used to transform electrocompetent E. coli DH10B cells. Individual clones were checked for the presence of the desired plasmid pSGK005. The identity of pSGK005 was confirmed by restriction pattern and sequence analysis.

[0094] Plasmid pSGK005 was used to transform S. erythraea NRRL2338 using a routine protoplast transformation technique. Thiostrepton resistant colonies were selected on R2T20 media containing g/ml thiostrepton. Further analysis confirmed that pSGK005 had integrated into the S. erythraea NRRL2338 chromosome by Southern blot hybridisation of their genomic DNA with DIG-labelled DNA containing the actII orf4 promoter. The culture S. erythraea NRRL2338 (pSGK005) was inoculated into 5 ml tap water medium in a 30 ml flask. After three days incubation at 29° C. this flask was used to inoculate 30 ml of Ery-P medium in a 300 ml flask. The broth was incubated at 29° C. at 200rpm for 6 days. After this time the whole broth was adjusted to pH8.5 with NaOH, and then extracted twice with an equal volume of ethyl acetate. The ethyl acetate extract was evaporated to dryness at 45° C. under a nitrogen stream using a Zymark Turbovap LV evaporator. The product identities were confirmed by LC/MS. A peak was observed with a m/z value of 734 (M+H)+ required for erythromycin A. A second peak was observed with a m/z value of 748 (M+H)+, required for 13-propyl erythromycin A.

REFERENCES

[0095] 1. Ajaz, A. A. and Robinson, J. A. (1983) The utilization of oxygen atoms from molecular oxygen during the biosynthesis of Monensin-A. Journal of the Chemical Society-Chemical Communications, 12, 679-680.

[0096] 2. Ashworth, D. M., Holmes, D. S., Robinson, J. A., Oikawa, H. and Cane, D. E. (1989) Selection of a specifically blocked mutant of Streptomyces cinnamonensis—isolation and synthesis of 26-deoxymonensin-A. Journal of Antibiotics, 42, 1088-1099.

[0097] 3. August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R., Yu, T. W., Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X. H., Hutchinson, C. R. and Floss, H. G. (1998) Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chemistry & Biology, 5, 69-79.

[0098] 4. Bartel, P. L., Zhu, C. B., Lampel, J. S., Dosch, D. C., Connors, N. C., Strohl, W. R., Beale, J. M. and Floss, H. G. (1990) Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in Streptomycetes—clarification of actinorhodin gene functions. Journal of Bacteriology, 172, 4816-4826.

[0099] 5. Bibb, M. J., Biro, S., Motamedi, H., Collins, J. F. and Hutchinson, C. R. (1989) Analysis of the nucleotide sequence of the Streptomyces glaucescens Tcml genes provides key information about the enzymology of polyketide antibiotic biosynthesis. EMBO Journal, 8, 2727-2736.

[0100] 6. Bibb, M. J. and Hopwood, D. A. (1981) Genetic studies of the fertility plasmid SCP2 and its SCP2* variants in Streptomyces coelicolor A3(2). Journal of General Microbiology, 126, 427-442.

[0101] 6a. Bibb M. J., Janssen G. R. and Ward J. M. (1985) Cloning and analysis of the promoter region of the erythromycin resistance gene (ErmE) of Streptomyces erythraeus. Gene, 38, 215-226.

[0102] 6b. Bierman, M., Logan, R., O'Brien, K., Seno, E. T., Rao R. N. and Schoner B. E. (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 116, 43-49.

[0103] 7. Cane, D. E., Liang, T. C. and Hasler, H. (1981) Polyether biosynthesis—origin of the oxygen atoms of Monensin-A. Journal of the American Chemical Society, 103, 5962-5965.

[0104] 8. Cane, D. E., Liang, T. C. and Hasler, H. (1982) Polyether biosynthesis 2. Origin of the oxygen atoms of monensin A. Journal of the American Chemical Society, 104, 7274-7281.

[0105] 9. Cortés, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. and Leadlay, P. F. (1990) An unusually large multifunctional polypeptide in the erythromycin producing polyketide synthase of Saccharopolyspora erythraea. Nature, 348, 176-178.

[0106] 10. Cortés, J., Wiesmann, K. E. H., Roberts, G. A., Brown, M. J. B., Staunton, J. and Leadlay, P. F. (1995) Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage. Science, 268, 1487-1489.

[0107] 11. Davis, N. K. and Chater, K. F. (1990) Spore color in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics. Molecular Microbiology, 4, 1679-1691.

[0108] 12. Day, L. E. (1973) Antimicrobial Agents and Chemotherapy, 4, 410-414.

[0109] 13. Demetriadou, A. K., Laue, E. D., Staunton, J., Ritchie, G. A. F., Davies, A. and Davies, A. B. (1985) Biosynthesis of the polyketide polyether antibiotic ICI-139603 in Streptomyces longisporoflavus from O-18-labeled acetate and propionate. Journal of the Chemical Society Chemical Communications, 7, 408-410.

[0110] 13a. Dhillon, N., Hale, R. S., Cortes, J. and Leadlay P. F. (1989) Molecular characterization of a gene from Saccharopolyspora erythraea (Streptomyces erythraeus) which is involved in erythromycin biosynthesis. Molecular Microbiology 3, 1405-1414.

[0111] 14. Donadio, S., McAlpine, J. B., Sheldon, P. J., Jackson, M. and Katz, L. (1993) An erythromycin analog produced by reprogramming of polyketide synthesis. Proceedings of the National Academy of Sciences of the United States of America, 90, 7119-7123.

[0112] 15. Donadio, S., Staver, M. J., McAlpine, J. B., Swanson, S. J. and Katz, L. (1991) Modular organization of genes required for complex polyketide biosynthesis. Science, 252, 675-679.

[0113] 16. Evans, G. A., Lewis, K. and Rothenberg, B. E. (1989) High efficiency vectors for cosmid microcloning and genomic analysis. Gene, 79, 9-20.

[0114] 17. Fernandez-Moreno, M. A., Caballero, J. L., Hopwood, D. A. and Malpartida, F. (1991) The Act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the blda transfer-RNA gene of Streptomyces. Cell, 66, 769-780.

[0115] 18. Fernandez-Moreno, M. A., Martinez, E., Boto, L., Hopwood, D. A. and Malpartida, F. (1992) Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. Journal of Biological Chemistry, 267, 19278-19290.

[0116] 19. Gramajo, H. C., Takano, E. and Bibb, M. J. (1993) Stationary phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated. Molecular Microbiology, 7, 837-845.

[0117] 20. Gumila, C., Ancelin, M. L., Delort, A. M., Jeminet, G. and Vial, H. J. (1997) Characterization of the potent in vitro and in vivo antimalarial activities of ionophore compounds. Antimicrobial Agents and Chemotherapy, 41, 523-529.

[0118] 21. Hallam, S. E., Malpartida, F. and Hopwood, D. A. (1988) Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor. Gene, 74, 305-320.

[0119] 22. Holmes, D. S., Sherringham, J. A., Dyer, U. C., Russell, S. T. and Robinson, J. A. (1990) Synthesis of putative intermediates on the monensin biosynthetic pathway and incorporation experiments with the monensin-producing organism. Helvetica Chimica Acta, 73, 239-259.

[0120] 23. Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. and Schrempf, H. (1985) Genetic manipulation of Streptomyces, a laboratory manual. John Innes Institution, Norwich, UK.

[0121] 24. Hutchinson, C. R. and Fujii, I. (1995) Polyketide synthase gene manipulation—a structure function approach in engineering novel antibiotics. Annual Review of Microbiology, 49, 201-238.

[0122] 25. Hutchinson, C. R., Sherman, M. M., Vederas, J. C. and Nakashima, T. T. (1981) Biosynthesis of macrolides.5. Regiochemistry of the labeling of lasalocid a by C-13, O-18-labeled precursors. Journal of the American Chemical Society, 103, 5953-5956.

[0123] 26. Kakavas, S. J., Katz, L. and Stassi, D. (1997) Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. Journal of Bacteriology, 179, 7515-7522.

[0124] 27. Kao, C. M., Luo, G. L., Katz, L., Cane, D. E. and Khosla, C. (1995) Manipulation of macrolide ring size by directed mutagenesis of a modular polyketide synthase. Journal of the American Chemical Society, 117, 9105-9106.

[0125] 28. Katz, E., Thompson, C. J. and Hopwood, D. A. (1983) Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. Journal of General Microbiology, 129, 2703-2714.

[0126] 29. MacNeil, D. J., Occi, J. L., Gewain, K. M., Macneil, T., Gibbons, P. H., Ruby, C. L. and Danis, S. J. (1992) Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase. Gene, 115, 119-125.

[0127] 29a. MacNeil, D. J., Gewain, K. M., Ruby, C. L., Dezeny, G., Gibbons, P. H. and MacNeil, T. (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61-68.

[0128] 30. Marsden, A. F. A., Wilkinson, B., Cortés, J., Dunster, N. J., Staunton, J. and Leadlay, P. F. (1998) Engineering broader specificity into an antibiotic-producing polyketide synthase. Science, 279, 199-202.

[0129] 31. Meurer, G., Gerlitz, M., Wendt Pienkowski, E., Vining, L. C., Rohr, J. and Hutchinson, C. R. (1997) Iterative type II polyketide synthases, cyclases and ketoreductases exhibit context-dependent behavior in the biosynthesis of linear and angular decapolyketides. Chemistry & Biology, 4, 433-443.

[0130] 32. Motamedi, H. and Shafiee, A. (1998) The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. European Journal of Biochemistry, 256, 528-534.

[0131] 33. Narva, K. E. and Feitelson, J. S. (1990) Nucleotide sequence and transcriptional analysis of the RedD locus of Streptomyces coelicolor A3(2). Journal of Bacteriology, 172, 326-333.

[0132] 33a. Piffaretti J. C., Arini A. and Frey J. (1988) pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae. Mol Gen Genet. 212, 215-218.

[0133] 34. Pospisil, S., Sedmera, P., Vokoun, J., Vanek, Z. and Budesinsky, M. (1987) 3-O-Demethylmonensin-A and 3-O-demethylmonensin-B produced by Streptomyces cinnamonensis. Journal of Antibiotics, 40, 555-557.

[0134] 35. Revill, W. P., Bibb, M. J. and Hopwood, D. A. (1995) Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant. Journal of Bacteriology, 177, 3946-3952.

[0135] 36. Rowe, C. J., Cortés, J., Gaisser, S., Staunton, J. and Leadley, P. F. (1998) Construction of new vectors for high-level expression in actinomycetes. Gene, 216, 215-223.

[0136] 37. Sanger, F. (1981) Determination of nucleotide sequences in DNA. Science, 214, 1205-1210.

[0137] 38. Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw, L. E., Haydock, S. F., Oliynyk, M., Caffrey, P., Cortés, J., Lester, J. B., Böhm, G. A., Staunton, J. and Leadlay, P. F. (1995) The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proceedings of the National Academy of Sciences of the United States of America, 92, 7839-7843.

[0138] 39. Shen, B., Summers, R. G., Wendtpienkowski, E. and Hutchinson, C. R. (1995) The Streptomyces glaucescens TcmKl polyketide synthase and TcmN polyketide cyclase genes govern the size and shape of aromatic polyketides. Journal of the American Chemical Society, 117, 6811-6821.

[0139] 40. Sherman, D. H., Malpartida, F., Bibb, M. J., Kieser, H. M. and Hopwood, D. A. (1989) Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tu22. EMBO Journal, 8, 2717-2725.

[0140] 41. Sood, G. R., Robinson, J. A. and Ajaz, A. A. (1984) Biosynthesis of the polyether antibiotic Monensin-A—incorporation of [2-2-2H-2]-propionate, (R)-[2-2H-1]-propionate and (S)-[2-2H-1]- propionate. Journal of the Chemical Society-Chemical Communications, 21, 1421-1423.

[0141] 42. Spavold, Z., Robinson, J. A. and Turner, D. L. (1986) Biosynthesis of the polyether antibiotic narasin origins of the oxygen atoms and the mechanisms of ring formation. Tetrahedron Letters, 27, 3299-3302.

[0142] 43. Staden, R. (1996) The Staden sequence analysis package. Molecular Biotechnology, 5, 233-241.

[0143] 44. Stutzman-Engwall, K. J., Otten, S. L. and Hutchinson, C. R. (1992) Regulation of secondary metabolism in Streptomyces Spp and overproduction of daunorubicin in Streptomyces peucetius. Journal of Bacteriology, 174, 144-154.

[0144] 45. Swan, D. G., Rodriguez, A. M., Vilches, C., Mendez, C. and Salas, J. A. (1994) Characterization of a Streptomyces antibioticus gene encoding a type I polyketide synthase which has an unusual coding sequence. Molecular & General Genetics, 242, 358-362.

[0145] 46. Takano, E., Gramajo, H. C., Strauch, E., Andres, N., White, J. and Bibb, M. J. (1992) Transcriptional regulation of the redD transcriptional activator gene accounts for growth phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2). Molecular Microbiology, 6, 2797-2804.

[0146] 47. Townsend, C. A. and Basak, A. (1991) Experiments and speculations on the role of oxidative cyclization chemistry in natural product biosynthesis. Tetrahedron, 47, 2591-2602.

[0147] 48. Walba, D. M. and Edwards, P. D. (1980) Tetrahedron Letters, 21, 3531-3534.

[0148] 49. Westley, J. W. (1974) Journal of Antibiotics, 27, 597-604.

[0149] 50. Westley, J. W. (1981) Antibiotics IV. Biosynthesis. Springer-Verlag, New York.

[0150] 51. Wietzorrek, A. and Bibb, M. (1997) A novel family of proteins that regulates antibiotic production in Streptomycetes appears to contain an OmpR-like DNA-binding fold. Molecular Microbiology, 25, 1181-1184.

[0151] 52. Xue, Y. Q., Zhao, L. S., Liu, H. W. and Sherman, D. H. (1998) A gene cluster for macrolide an antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity. Proceedings of the National Academy of Sciences of the United States of America, 95, 12111-12116.

9TABLE IgenefunctionstartendgdhAglutamate dehydrogenase (partial)10380dapAdihydrodipicolinate synthase21401220orf3putative transcriptional activator22113152orf4hypothetical protein32643680orf5hypothetical protein43073684orf6hypothetical protein45704758orf7hypothetical protein50585612acpXacyl carrier protein60105693ksXketoacyl synthase85316045monCIprobable epoxihydrolase/cyclase95428643monEmethyltransferase104269596monTmonensin resistance gene (ABC-1065612191monRIprobable repressor1220512780monAIthioesterase1382913023monAIpolyketide synthase loading &1412123198KS-L1417215486AT-L malonate specific1577716880ACP-L1701917276KS11735818626AT1 methylmalonate specific1896019976DH1 (potential)2001920519KR1 (inactive)2163622241ACP12253622793monAIpolyketide synthase module 22320529921KS22330724569AT2 methylmalonate specific2489125913DH22595326369ER22760028463KR22848529042ACP22931329570monAIpolyketide synthase modules 3 & 42997442372KS33007631347AT3 malonate specific3179832838DH33288433465KR33469235181ACP33555335811KS43589937170AT4 methylmalonate specific3748938511DH43855738982ER44012340986KR44100541562ACP44184842105monAIpolyketide synthase modules 5 & 64244854564KS54262843890AT5 ethylmalonate specific4422145243DH54528945744KR54678547337ACP54759347850KS64794749218AT6 malonate specific4957950601DH65064451075ER65222253102KR65310153661ACP65405254306monApolyketide synthase modules 7 & 85461466934KS75471655978AT7 methylmalonate specific5630057319DH75735857802KR75904859608ACP75986760124KS86018561453AT8 malonate specific6180862839DH86288263316ER86457765437KR86545666016ACP86640466661monApolyketide synthase module 96695272054KS96707568340AT9 malonate specific6869869729KR9 (potential)7073571262ACP97153671783monHprobable regulator7205174993monCIFAD containing epoxidase7654175051monBIdouble bond isomerase7696076538monBIdouble bond isomerase7745077016monApolyketide synthase modules 11 &8870877447KS118861287344AT11 methylmalonate specific8702285993KR118511184562ACP118429284035KS128396282694AT12 methylmalonate specific8235481335DH12 (potential) delta8128680855ER12 (potential)7961878914KR127889578337ACP127807077812monApolyketide synthase module 109374188816KS109363692368AT10 methylmalonate specific9204091021KR109013289584ACP108932289068monDP450 oxygenase9408195273monRIprobable activator9614195338monAthioesterase9694196138orf29cell wall biosynthesis capK9758098953lipBlipase B9998398991orf31ion pump101433100507orf32membrane structural protein102581101490amtAglycine amidinotransferase102924103450

[0152]

10

TABLE II

GdhA, glutamate dehydrogenase (partial coding

sequence) Length: 346 amino acids

1
LTTRPDTKTA LSQKTALSQL LTEIEHRNPA QPEFHQAARE VLETLAPVIA

51
ARPEYAEAGL IERLCEPERQ IVFRVPWQDD HGRVRVNRGF RVEFNSALGP

101
YKGGLRFHPS VNLGVIKFLG FEQIFKNALT GLGIGGGKGG SDFDPRGRSD

151
AEVMRFCQSF MTELYRHIGE HTDVPAGDIG VGGREIGYLF GQYRRITNRW

201
EAGVLTGKGR NWGGSLIRPE ATGYGNVLFA AAMLRERGET LEGRTAVVSG

251
SGNVAIYTIQ KLAALGANAV TCSDSSGYVV DEKGIDLDLL KQVKEVERAR

301
VDTYAQRRGA SARFVPGRRV WEVPADIALP SATQNELDAD DATALI

DapA, dihydrodopicolinate synthase Length: 307 amino acids

1
MTLASSLEPT TEPLFNGLYV PLVTPFTDDL RLAPEALARL ADEALSAGAS

51
GLVALGTTAE AATLTAEERE TVIRVCSAAC RAHGAPLIVG VGTNDTATAI

101
TALRELAARG DVAAALVPAP PYIRPGEAGT LAHFAALAEH GGLPLVVYDI

151
PYRTGQTLGA GTITALGRLP EVVGIKHATG SIDPTTMELL DSPLPGFAVL

201
GGDDIVLSPL VAAGAHGGIV ASANLRTADY AEMIALWRRG SAAPARALGA

251
DLARLSAALF TEPNPTVIKG VLHAQNRIPS PAVRMPLLAA SADSVRRAAP

301
LAASRK*

ORF3, putative transcriptional activator protein

Length: 314 amino acids

1
MLDVRRLHLL RELDRRGTIA AVAEALTFTA SAVSQQLGVL EREAGVPLLE

51
RSGRRVVLTP AGRSLVAHAD AVLNRLEQAV AELAGARDGI GGPLRIGTFP

101
SGGHTIVPGA LAELASRHPA LEPMVREIDS ARVSDGLRAG ELDVALVHDY

151
DFVPATPDTT VDEVPLLEEP MYLVTHAADT ATDSGSGSTL AALLGPCAEV

201
PWITARDGTT GHAMAVRACQ AAGFQPRIRH QVNDFRTVLA LVAAGQGAGF

251
VPRMAAEPSP AGVVLTKLPL FRRSKVAFRA GGGAHPAIAA FVAAATTAVE

301
RMAGSRGPAG GSE*

ORF4, hypothetical protein Length: 139 amino acids

1
MADDAYLFLL PDRHPRLGAA LAAVGALECT ETPAVHAWLQ AHEASVSSEQ

51
VRILPADAET LIPKDAERLP VPLSEEEALK VEQECAPQTV TDMESELLAF

101
RETTQDWQAL VHRALTAGIP AQRIARLTGL DPEEIGRL*

ORF5, hypothetical protein Length: 208 amino acids

1
LAVAACAAVV LPIDAVVRIS AADVGVLVFF AYLLPYLAIT MTVFVSVAPE

51
QVRSWARREA RGTFLQRYVL GTAPGPGGSL FIAAAALVVA VLWLPGHLST

101
TFSALPRTLV ALALVVAAWI CVVVAFAVTF QADNLVENER ALEFPGERSP

151
AWADYVYFAL AAMTTFGTTD VDVTSRDMRR TVAANTVIAF VFNTVTVAIL

201
VSALGGR*

ORF6, hypothetical protein Length: 63 amino acids

1
MTVMDKLKQM LKGHEDKAGQ GIDKAGDFVD GKTQGKYSGQ VDTAQDKLRD

51
QFGSDQQEPP QR*

ORF7, hypothetical protein Length: 185 amino acids

1
MGTAQSQEQA AAPGACAAFV RFVLCGGGVG LASSFAVVAL ASWVPWALAN

51
ALVAVVSTVV ATELHARFTF GAGGRATWRQ HAQSAGSAAA AYAVTCVAMF

101
VLQQLVAAPG AVLEQVVYLS ASALAGVARF VVLRLVVFAR NRSLPAAAAV

151
RTARPVRRVP APVPATVAHA ASRPAGPAAL CPAA*

AcpX, acyl carrier protein (ACP) Length: 106 amino acids

1
MTSTDHTSGQ DATELEKQLA AATPEEREKL LTDTIRTQAG TLLNTTLSDD

51
SNFLENGLNS LTALELTKTL MTLTGMEIAM VAIVENPTPA QLAHHLGQEL

101
AHTTA*

KsX, ketoacyL-ACP synthase Length: 829 amino acids

1
VANEEKLVEY LKWTTAELHQ AQQQLRELKA AQHEPIAVVS MACRLPGKTR

51
TPDDLWDLVS EGRDAVTGFP DDRAWELPEE RPYAELGGFL DDAAGFDAGF

101
FDISDTEAVA TEPLQRLMLH LAWETVERGH IAPHTLRSTL TGVYVGATGH

151
DYATRLETAP DELLPYLGGG TSGSLVSGRI AYALGLEGPA ISVDTACSSS

201
LVALHLLACQA LRRGECGLAL AGGGTVMSTP HTFHAFAHQK SLAQDGRCKP

251
FAAAADGMGL GEGVGLVLLE RLGDARKNGH PVLAVIRGSA VNQDGAGYGL

301
AAPNGPSQQH VTRAALADAG LTPDQIDAVE AHGTGTPIGD AIEVQALLAT

351
YGADRSPDRP LWLGSVKSNT GHTQGAAGAA ALIKMVQAFR HGTLPPTLHV

401
DRPTPLAAWK KGAVRLLTEA VDWPRREEPR RVGISAFATS GTNAHLILEE

451
PPVDEAPVPD AARDQTSPVA PELPVAWSLS ARTPEALRAQ AKALVTHLAA

501
TDPAPSPAEV AYSLAATRSP LEHRAVLTGT DHTELLAAAR ALAAGEDHPD

551
LVRSTPGAGP KKIAWHFDGR PADGVTTGAA PGAKPGATFG ATFGAAFGGA

601
EFHSAFPLFA SAFDEARALL DTHLPTPLPT PHSELARFAV HTALARLLLE

651
TGVRPHTLTG DGVGHIAAAY AAGILTLDDA CRLAAAHAAA AQAAEGEQPA

701
PPDAYEPVLK QLTFQRATLT LTSTAPADTP IASADYWHHH LTSPAPTAPP

751
TPETHTLLHL GALSPEGTQT SAVSALLTAL ARLHTTGGTV DWTPLVRRTP

801
HPRTIDLPTY SFQATRYWLH DHTAHAAV*

MonCII, probable epoxyhydrolase/cyclase

Length: 300 amino acids

1
VKNLRIPVSQ TVSLNVRYRP ADGPGAPGRP FLLLHGMLSN ARMWDEVAAR

51
LAAAGHPAYA VDHRGHGESD TPPDGYDNAT VVTDLVAAVT ALDLSGALVA

101
GHSWGAHLAL RLAAEHPDLV AGLALIDGGW YEFDGPVMRA FWERTADVVR

151
RAQQGTTSAA DMRAYLRATH PDWSPTSIEA RLADYRVGPD GLLIPRLTST

201
QVMSIVAGLQ REAPADWYPK VTVPVRLLPL IPAIPQLSDQ VRAWVAAAEA

251
ALEQVSVRWY PGSDHDLHAG APDEIAADLL LLARSCEAMP GGKAGVRPA*

MonE, S-adeonosylmethionine-dependent methyltransferase

Length: 277 amino acids

1
VNKTVAPEPS DIGHYYDHKV FDLMTQLGDG NLHYGYWFDG GEQQATFDEA

51
MVQMTDEMIR RLDPAPGDRV LDIGCGNGTP AMQLARARDV EVVGISVSAR

101
QVERGNRRAR EAGLADRVRF EQVDAMNLPF DDGSFDHCWA LESMLHMPDK

151
QQVLTEAHRV VKPGARMPIA DMVYLNPDPS RPRTATVSDT TTYAALTDIG

201
DYPDIFRAAG WTVLELTDIT RETAKTYDGY VEWIRAHRDE YVDIIGVEGY

251
ELFLHNQAAL GKMPELGYIF ATAQRP*

MonT, putative monensin resistance gene (ABC-transporter)

Length: 512 amino acids

1
MSADLGARRW WAVGALVLAS MVVGFDVTIL SLALPAMADD LGANNVELQW

51
FVTSYTLVFA AGMIPAGMLG DRFGRKKVLL TALVIFGIAS LACAYATSSG

101
TFIGARAVLG LGAALIMPTT LSLLPVMFSD EERPKAIGAV AGAAMLAYPL

151
GPILGGYLLN HFWWGSVFLI NVPVVILAFL AVSAWLPESK AKEAKPFDIG

201
GLVFSSVGLA ALTYGVIQGG EKGWTDVTTL VPCIGGLLAL VLFVMWEKRV

251
ADPLVDLSLF RSARFTSGTM LGTVINFTMF GVLFTMPQYY QAVLGTDAMG

301
SGFRLLPMVG GLLVGVTVAN KVAKALGPKT AVGIGFALLA AALFYGATTD

351
VSSGTGLAAA WTAAYGLGLG IALPTAMDAA LGALSEDSAG VGSGVNQSIR

401
TLGGSFGAAI LGSILNSGYR GKLDLDGVPE QAHGAVKDSV FGGLAVARAI

451
KSNGLADSVR SAYVHALDVV LVVSGGLGLL GVVLAVVWLP RHVGQSTAKT

501
AESEHEAADA V*

MonRII, probable repressor protein Length: 192 amino acids

1
VPGLRERKKA RTKAAIQREA VRLFREQGYT ATTIEQIAEA AEVAPSTVFR

51
YFATKQDLVF SHDYDLPFAM MVQAQSPDLT PIQAERQAIR SMLQDISEQE

101
LALQRERFVL ILSEPELWGA SLGNIGQTMQ IMSEQVAKRA GRDPRDPAVR

151
AYTGAVFGVM LQVSMDWAND PDMDFATTLD EALHYLEDLR P*

MonAIX, thioesterase Length: 269 amino acids

1
MDRGTAARAP QIGDEFGAAT GNGVWLRRYH AAAEAPVRLV CFPFAGGSAS

51
YYFGLSGLLA PGVEVLAVQY PCRQDRHAEP CLASVAELAD GVVPHLPCDG

101
KPFALFGHSL GAIVAFEVAR RLRGPAGPGL PVHLFVSGGL ARPYRPAGRS

151
GAFGDADILA HLRAMGGTDE RFFRSPELQE LVLPALRADY RAVATYEAPG

201
PGRLDCPITA LIGDADERTS PEQAATWRER TGAAFDLRVL PGGHFYLDGC

251
QEQVAAVVTE ALTAGPGV*

MonAI, polyketide synthase multi-enzyme MONS1, housing

loading module and extension module 1

Length: 3026 amino acids

1
MAASASASPS GPSAGPDPIA VVGMACRLPG APDPDAFWRL LSEGRSAVST

51
APPERRRADS GLHGPGGYLD RIDGFDADFF HISPREAVAM DPQQRLLLEL

101
SWEALEDAGI RPPTLARSRT GVFVGAFWDD YTDVLNLRAP GAVTRHTMTG

151
VHRSILANRI SYAYHLAGPS LTVDTAQSSS LVAVHLACES IRSGDSDIAF

201
AGGVNLICSP RTTELAAARF GGLSAAGRCH TFDARADGFV RGEGGGLVVL

251
KPLAAARRDG DTVYCVIRGS AVNSDGTTDG ITLPSGQAQQ DVVRLACRRA

301
RITPDQVQYV ELHGTGTPVG DPIEAAALGA ALGQDAARAV PLAVGSAKTN

351
VGHLEAAAGI VGLLKTALSI HHRRLAPSLN FTTPNPIAPL ADLGLTVQQD

401
LADWPRPEQP LIAGVSSFGM GGTNGHVVVA AAPDSVAVPE PVGVPERVEV

451
PEPVVVSEPV VVPTPWPVSA HSASALRAQA GRLRTHLAAH RPTPDAARVG

501
HALATTRAPL AHRAVLLGGD TAELLGSLDA LAEGAETASI VRGEAYTEGR

551
TAFLFSGQGA QRLGMGRELY AVFPVFADAL DEAFAALDVH LDRPLREIVL

601
GETDSGGNVS GENVIGEGAD HQALLDQTAY TQPALFAIET SLYRLAASFG

651
LKPDYVLGHS VGEIAAAHVA GVLSLPDASA LVATRGRLMQ AVRAPGAMAA

701
WQATADEAAE QLAGHERHVT VAAVNGPDSV VVSGDRATVD ELTAAWRGRG

751
RKAHHLKVSH AFHSPHMDPI LDELRAVAAG LTFHEPVIPV VSNVTGELVT

801
ATATGSGAGQ ADPEYWARHA REPVRFLSGV RGLCERGVTT FVELGPDAPL

851
SAMARDCFPA PADRSRPRPA AIATCRRGRD EVATFLRSLA QAYVRGADVD

901
FTRAYGATAT RRFPLPTYPF QRERHWPAAA CVGQQPETPE LPESSESSEQ

951
AGHEREEGAR AWGGPEGRLA GLSVNDQERV LLGLVTKHVA VVLGDASGTV

1001
QAARTFKQLG FDSMAAAELS ERLGTETGLP LPATLTFDYP TPLAVAAHLR

1051
AELTGTPAPA GSAPATGALG AGDLGTDEDP VAIVAMSCRY PGGAGTPEDL

1101
WRLVAGGADA IGDFPTDRGW DLARLFHPDP DRSGTSCTRQ GGFLYDAADF

1151
DAEFFDISPR EALAVDPQQR LLLECAWEAF ERAGLDPRAL KGSPTGVFVG

1201
MTGQDYGPRL HEPSQATDGY LLTGSTPSVA SGRLSFSFGL EGPALTVDTA

1251
CSSSLVTLHL AAQALRRGEC DLALAGGATV LATPGMFTEF SRQRGLAPDG

1301
RCKPFAAGAD GTGWAEGVGL VLLERLSEAR RKGHAVLAVI RGSAINQDGA

1351
SNGLTAPNGP SQQRVIRAAL AAARLTADEV DVVEAHGTGT TLGDPIEAQA

1401
LLATYGQGRS AERPLWLGSV KSNIGHTQAA AGVAGVIKMV MAMRHDLLPA

1451
TLHVDEPSGH VDWSTGAVRL LTEPVVWPRG ERPRRAAVSS FGISGTNAHL

1501
VLEEAGQDEY VAGAADDAGP VDGAVLPWVV SGRTGAALRE QARRLRELVT

1551
GGSADVSVSG VGRSLVTTRA VFEHRAVVVG RDRDTLIGGL EALAAGDASP

1601
DVVCGVAGDV GPGPVLVFPG QGSQWVGMGA QLLGESAVFA ARIDACEQAL

1651
SPYVDWSLTE VLRGDGRELS RVDVVQPVLW AVMVSLAAVW ADHGVTPAAV

1701
VGHSQGEIAA VVVAGALTLE DGAKIVALRS RALRQLSGGG AMASLGVGQE

1751
QAAELVEGHP GVGIAAVNGP SSTVISGPPE QVAAVVADAE ARELRGRVID

1801
VDYASHSPQV DAITDELTHT LSGVRPTTAP VAFYSAVTGT RIDTAGLDTD

1851
YWVTNLRRPV RFADAVTALL ADGHRVFIEA SSHPVLTLGL QETFEEAGVD

1901
AVTVPTLRRE DGGRARLARS LAQAFGAGCA VRWENWFPAT GTSTVELPTY

1951
AFQRRRYWLE APTGTQDAAG LGLAAAGHPL LGAATEIAIDG DIRLLTGRIS

2001
RHSHPWLAQH TLFGAAVVPA SVLAEWALRA ADEAGCPRVD DLTLRTPLVL

2051
PETAGVQVQI VVGPADARDG HRDFHVYARP DGKDASEGEG IAEGEGASEG

2101
EGASGGTDAP WTCHADGRLV AEPTGTASED SPDTVWPPPG AEPVDLGDFY

2151
ERAAATGVGY GPVFTGLRAL WRRDGELFAE AVLPQEAPET AGFGMHPALL

2201
DAALHPALLG ERPAEEDKVW LPFTLTGVTL WATGATSVRV RLTPLDDDPD

2251
ASADGRAWRV GVSDPTGAEV LTCEALVAVA AGRRELRAAG ERVSDLYAVE

2301
WVPVPGPGPV GEGADFSGWA GLGECGERWE CVGRVERWYE DLDALGAAVE

2351
GGASVPSVVL ATAAAAPGGA GDGAADALSA VRWTGALLDQ WLADARFADA

2401
RLVVITSGAV ATGDDFLPDP AAAAVRGLVE QAQVRHPGRI LLVDTEAGAG

2451
LGVGAGVDDA LLEQAVAMAL GADEPQLALR AGRVLAPRLT APQDAAVTEA

2501
ARPLDPDGTV LITGPAGAPV ADLAEHLVRT GQCRHLLLLP GDGELEEMAE

2551
ELRGLGATVD LSTADPADPT ALAEVVAAVE GDHPLTGVIH ATGVVDAFDP

2601
GDSASDLMID SASDSFAEAW SSRAGVTAAL HTATAHLPLD LFAVLSPAGA

2651
DLGIARSAAA AGADAFSAAL ALRRHTTVTT DTTAPPRTTA PPRTTASPRT

2701
TALSSSRTTG VALAYGPPTA PRPGIKGTAP GRIPVLLDAA RAHGGGSPLL

2751
GARLAARALA AESAAEGVAG LPAPLRALAV AAAAAGAPTR RTAADRKPPA

2801
DWPARLAPLS APEQLRLLID AVRTHAAAVL GRTDPEALRG DATFKQLGLD

2851
SLTAVELRNR LVEDTGLRLP TALVFRYPTP AAIAAHLRER LTSPSETTAT

2901
QRSGGQTPAA GQASSALAPG GSAAGPPAAD TVLSDLTRME NTLSVLAAQL

2951
PHTETGEITT RLEALLTRWK TTNATANDSG DGNGGDDDAA ERLKAASADQ

3001
IFDFIDNELG VGHGTSRVTP TPKAG*

MonAII, polyketide synthase multi-enzyme MONS2, housing

extension module 2 Length: 2239 amino acids

1
MASEEQLVEY LRRVTTELHD TRRRLVQEED RRQEPVALVG MACRFPGGVA

51
SPEDLWDLVA AGKDAIEDFP TDRGWDLEAL YDPDPAAYGT SYVRHGGFVD

101
DAGSFDADFF GISPREALAM DPQQRLMLET SWELFERAGI EPVSLKGSRT

151
GVYAGVSSED YMSQLPRIPE GFEGHATTGS LTSVISGRVA YNYGLEGPAV

201
TVDTACSASL VAIHLASQAL RQRECDLALA GGVLVLSSPL MFTEFCRQRG

251
LAPDGRCKPF AAAADGTGFS EGIGLLLLER LSDARRNGHK VLAVIRGSAV

301
NQDGASNGLT APNDAAQEQV IRAALDNARL TPSEVDAVEA HGTGTKLGDP

351
IEAGALLATY GQHRARPLLL GSLKSNIGHT HATAGVAGVI KTVMAIRNGL

401
LPATLHVEEL SPHVDWDAGA VEVVTEPTPW PETGHPRRAG VSAFGISGTN

451
AHLILEEAPP EEDVPAPVVV ESGGVVPWVV SGRTPEALRE QARRLGEFVA

501
GDTDALPNEV GWSLATTRSV FEHRAVVVGR DRDALTAGLG ALAAGEASAG

551
VVAGVAGDVG PGPVLVFPGQ GAQWVGMGAQ LLDESAVFAA RIAECERALS

601
AHVDWSLSAV LRGDGSELSR VEVVQPVLWA VMVSLAAVWA DYGVTPAAVI

651
GHSQGEMAAA CVAGALSLED AARIVAVRSD ALRQLQGHGD MASLSTGAEQ

701
AAELIGDRPG VVVAAVNGPS STVISGPPEH VAAVVADAEA RGLRARVIDV

751
GYASHGPQID QLHDLLTERL ADIRPTNTDV AFYSTVTAER LTDTTALDTD

801
YWVTNLRQPV RFADTIEALL ADGYRLFIEA SAHPVLGLGM EETIEQADMP

851
ATVVPTLRRD HGDTTQLTRA AAHAFTAGAD VDWRRWFPAD PAPRTIDLPT

901
YAFQRRRYWL ADTVKRDSGW DPAGSGHAQL PTAVALADGG VVLNGRVSAE

951
RGGWLGGHVV AGTVLVPGAA LVEWVLRAGD EAGCPSLEEL TLQAPLVLPE

1001
SGGLQVQVVV GAADEQGGRR DVHVYSRSEQ DASAVWQCHA VGELGRASVA

1051
RPVRQAGQWP PAGAEPVEVG GFYEGVAAAG YEYGPAFRGL RAMWRHGDDL

1101
LAEVELPEEA GSPAGFGIHP ALLDAALHPL LAQRSRDGAG AGAHGGQVLL

1151
PFSWSGVSLW ASEATTVRVR LTGLGGGDDE TVSLTVTDPA GGPVVDVAEL

1201
RLRSTSARQV RGSAGPGADG LYELRWTPLP EPLPVPAPAN GRDVAADLSG

1251
CAVLGELVAE PGPGIDLEGC PCYPGVGALA DNASPPSMIL APVHSDTTGG

1301
DGLALTERVL RVIQDFLAAP SLEQKQTRLA FVTRGAADTG STTGGSAAPA

1351
EAVDPAVAAV WGLVRSAQSE NPGRFVLLDT DAPLDQASVA PLVDAVRSAV

1401
EADEPQVALR GGRLLVPRWA RAGEPVELAG PAGARAWRLV GGDSGTLEAV

1451
VAEACDDIVL RPLAPGQVRV AVHTAGVNFR DVLIALGMYP DPDALPGTEA

1501
AGVVTEVGPG VTRLSVGDRV MGMMDGAFGP WAVADARMLA PVPPGWGTRQ

1551
AAAAPAAFLT AWYGLVELAG LKAGERVLIH AATGGVGMAA VQIARHVGAE

1601
VFATASPGKH AVLEEMGIDA AHRASSRDLA FEDAFRQATD GRGVDVVLNS

1651
LTGELLDASL RLLGDGGRFV EMGKSDPRDP ELVALEHPGV SYEAFDLVAD

1701
AGPERLGLML DRLGELFAGG SLVPLPVTAW PLGRAREALR HMSQARHTGK

1751
LVLDVPAPLD PDGTVLVTGG TGTIGAAVAE HLARTGESKH LLIVSRSGPA

1801
AHGAEELVSR IAEFGAEATF VAADVSEPDA VAALIEGIDP AHPLTGVVHA

1851
AGVLDNALIG SQTTESLTRV WAAKAAAAQQ LHEATRESRL GLFVMFSSFA

1901
STMGTPGQAN YSAANAYCDA LAALRRAEGL AGLSVAWGLW EATSGLTGTL

1951
SAADRARIDR YGIRPTSAAR GCALLAAARA HGRPDLLAMD LDARVPAASD

2001
APVPAVLRTL AAAGAPATAR PTAAAAADGA TDWSGRLAGL TEEARLELLT

2051
ELVCTHAAGV LGHADAGAVQ VDAPFKELGF DSLTAVELRN RIAAATGLKL

2101
PAALVFDYPQ ARVLAAHLAE RLVPEGAGAM GGVSGAEGVR DAYGAGGPGG

2151
DMTAQVLLEV ARVEHTLSAA VPHGLDRAAV AARLEALLAR CTATTAATGA

2201
AGAAVEGDGD SDGDGAVDQL ETATAEQVLD FIDNELGV*

MonAIII, polyketide synthase multi-enzyme MONS3, housing

extension modules 3 and 4 Length: 4133 amino acids

1
MVSEEKLVDY LKRVSADLHA TRQRLREAEE RGQEPVAVVE AACRYPGGIR

51
TPEDLWDLVA AGGNALGAFP DNRGWDLRRL FHPDPDHPGT TYAREGGFLH

101
DADLFDPEFF GISPREAAVL DPQQRLLLEC AWEALERAGI DPRSLQGSRT

151
GVYAGAALPG FGTPHIDPAA EGHLVTGSAP SVLSGRLAYT FGLEGPAVTI

201
DTACSSSLVA VHLAAHALRQ RECDLALAGG VTVMTTPYVF TEFSRQRGLA

251
ADGRCKPFAA AADGTAFSEG AGLLVLERLS DARRAGHRVL AVIRGSAVNQ

301
DGASNGLTAP NGPAQQRVIR AALAGARLSP AEVDAVEAHG TGTRLGDPIE

351
ADALLATYGQ ERHGGRPLWL GSVKSNIGHT QGAAGAAGLI KMVQALRHET

401
LPATLYADEP TPHADWESGA VRLLSAPVAW PRGEHGEHTR RAGISSFGIS

451
GTNAHLILEE APAADAEGAG GDGDGDGGGV RPVVRVGATG PREEQGQGQG

501
QEQHQQQRQQ RQRSSMMPTP HLPWLLSARS PAALRAQADA LANHVAHADH

551
SIADIGGTLL RRTLFEHRAV VLGTDRDERA AALAALAAGR AHPALTRAAG

601
PARNGGTAFL FTGQGSQRPG MGRQLYDTFD VFAESLDETC ARLDPLLEQP

651
LKPVLFAPAD TAQAAVLHGT GMTQAALFAL EVALYRQVTS FGIAPSHLTG

701
HSVGEIAAAH VAGVFSLADA CTLVAARGRL MQALPAGGAM LAVQAAEDDV

751
LPLLAGQEER LSLAAVNGPT AVVVSGEAAA VGEVEKALRG RGLKTKRLNV

801
SHAFHSPLIE PMLDDFREVA RGLTFHAPTL PVVSNLTGRL ADAELMADAE

851
YWVRHVRRPV RFHDGLRALS EQGVVRYLEL GPDPVLATMV QDGLPAPAEG

901
EEPEPVVAAA LRSKHDEGRT LLGAVAALHT DGQPADLTAL FPADAGQVPL

951
PTYRFQRRRY WRVAPDAAAP ARAAGLQETG HPLLPAVIRQ ADGGILLAGR

1001
LSLRTHPWLA DHTIAGGVPL PATAFVELAL LAGRHAACDT IDDLTLETPL

1051
LLDDTGTGVG AAVGAGADAL VDAIEVQLAL GAPDGSGRRA LTVHSRPADD

1101
AADDGDAADA ADAAGRGGPG GSGDLGDPGD PGDLGDGGGS RGWRRHATGI

1151
LSAGPAAEPA APDAAPWPPA DATALDVDAL YARLDAQGYS YGPAFRAVHA

1201
AWRHGDDLYA DVRLADEQRA EADAFALHPA LLDAALHAVD ELYRGSEGRC

1251
QEQGQGGQEP EQGRGDADAP VRLPFSFSDI RHHATGATRL WVRLSPQGDD

1301
RLRLSLTDGE GGQVATVDAL QLRLIPADRW RAARPTTAAP LYHLDWHELP

1351
LPEPAETDPA AHSWAVLGAH DAGLAPAAHY PDLAALKAAV EAGEPVPDIV

1401
FAPFPAQGTE TDVPAQVRAH ARHALELLRD WLTTEAFAAA RLVVLTTGAV

1451
TARPEDGPAD LATAPVWGLV RAAQAEQPDH VVLVDIDKDI DKDTDEETDQ

1501
ATDAGTASRH ALPAALAAAA AQAETQLALR AGTVLVPRLA VVPPRTDTPA

1551
LHATAPESTT DTVDSTGIAG AAESGGTVLI TGGTGGLGQA VARHLAAAHG

1601
ARHLLLVSRR GDAAEGVAEL RADLADDGVD VRVAACDITD RDALAGLLAD

1651
IPAAHPLTAV VHTAGVIDDS LITAMTPERL DAVLAPKADA AWHLHELTRD

1701
KDLSAFVLFS SGASVLGNGG QANYAAANTF LNTLAEHRRA AGLAATSVAW

1751
GLWESASGGM AARLGDADA RIHRTGVTGL TDEQALALFD AALTAEIIPTV

1801
LATRFDRAVL RGQAAARTLQ PALRGLVRTP RPTASAGAIG STAATGSATD

1851
ENAPSSWAAR LARLSAADRD RALNELIREQ IATVLAHPSP DTIELGRAFQ

1901
ELGFDSLTAL ELRNRLSTAT GIRLPATLVF DHPSPTALVR HLHSHLPDEA

1951
QHTSPTAPGA SAEGTAATAT GIDDDPIAIV GMACRYPGGV TSPEQLWQLV

2001
ATGTDAIGPF PEDRGWDTAG LFDPDPDQVC HSYTREGGFL YDAARFDAGF

2051
FGISPREAAA TDPQQRLLLE TAWQAFEHAG IDPAALRGTP CGVITGIMYD

2101
DYGSRFLARK PDGFEGRIMT GSTPSVASGR VAYTFGLEGP AITVDTACSS

2151
SLVAMHLAAQ ALRQGECELA LAGGVTVMAT PNTFVEFSRQ RGLAPDGRCK

2201
PFAAAADGTG WGEGAGLVVL ERLSDARRKG HRVLALLRGS AVNQDGASNG

2251
MTAPNGPSQE RVIRTALAGA GRGPEDIDVV EAHGTGTTLG DPIEAQALLA

2301
TYGQGRPEDR PLWLGSVKSN IGHTQAAAGV AGVIKMVMAL RHEQLPTTLH

2351
ADEPTPHVQW DGGGVRLLTE PVPWSRGERT RRAGVSSFGI SGTNAHLILE

2401
EPPEEDLPEP VAAEPGGVVP WVVSGRTPDA LREQARRLGE FVVGAGDVSA

2451
AEVGWSLATT RSVFEHRAVV AGRDRDDLVA GMQALAAGET PTDVVSGAAA

2501
SSGAGPVLVF PGQGSQWVGM GAQLLDESPV FAARIAECEQ ALSAYVDWSL

2551
SDVLRGDGSE LSRVEVVQPV LWAVMVSLAA VWADYGVTPA AVVGHSQGEM

2601
AAACVAGALS LEDAARIVAV RSDALRQLQG HGDMASLGTG AEQAAELIGD

2651
RPGVVVAAVN GPSSTVISGP PEHVAAVVAE AEARGLRARV IDVGYASHGP

2701
QIDQLHDLLT EGLADIRPAN TDVAFYSTVT AERLTDTTAL DTDYWVTNLR

2751
QPVRFADTIE ALLADGYRLF IEASAHPVLG LGMEETIEQA DIPATVVPTL

2801
RRDHGDTTQL TRAAAHAFTA GADVDWRRWF PADPTPRTVD LPTYAFQHQH

2851
YWLEEPSGLT GDAADLGMVA AGHPLLGACV ELAESDSYLF TGRLSRRAPS

2901
WLAEHVVAGT VLVPGAALVE WVLRAGDEAG CPTIEELTLQ APLVLPESGG

2951
LQVQVVVGAT DEQSGRRDVH VYSRSEQDAS AVWVCHAVGV VSSEMPEAAA

3001
ELSGQWPPAG AEAVDVEDFY ARAAEAGYAY GPAFQGLRAL WRHGTELFAE

3051
VVLPEQAGGH DGFGIHPALL DAALHPLMLL DRPADGQMWL PFAWSGVSLN

3101
ADRATHVRVR LSPRGEAAER DLRVVIADAT GAPVLTVDAL TLRAADPGRL

3151
GAAARGGVDG LYTVDWTPLP LPQPLPLPRT DAGGSADWVI LSDNSSAALA

3201
DAVSSATAAG GGAPWALLAP VGGGSADDGL PVVRRTLSLV QEFLAAPELT

3251
ESRLVIVTRG AVATDADGDV AASAAAVWGL IRSAQSENPG RFVLLDVEEE

3301
HLHPDGGELP YAALRHAVEE LDEPQLALRS GKFLVPRMTP AAAPEELVPP

3351
VGTSGWRLGT SGTATLENLS VIDAPEAFAP LEPGQVRISV RAAGMNFRDV

3401
LIALGMYPDK GTFAGSEGAG HVTEVGPGVT HLSVGDRVMG LFEGAFAPLA

3451
VADARMVVPI PEGWSFQEAA AVPVVFLTAW YGLVDLGRLR AGESLLIHAG

3501
TGGVGMAATQ IARHLGAEVF ATASPAKHGV LDGMGIDAAH RASSRDLDFE

3551
ETLRAATGGR GMDVVLNSLA GEFTDASLRL LAEGGRMVDM GKTDKRDPDR

3601
VAAEHAGAWY RAFDLVPHAG PDRIGEMLAE LGELFASGAL APLPVQTWPL

3651
GRAREAFRFM SQAKHTGKLV LEIPPALDPD GTVLITGGTG VLAAAVAEHL

3701
VREWGVRHLL LAGRRGSEAP GSSELAEELT ELGAEVTFAA ADVSDPDAVA

3751
ELVGKTDPAH PLTGVIHAAG VLDDAVVTAQ TPESLARVWA AKATAAHLLH

3801
EATREARLGL FLVFSSAAAT LGSPGQANYA AANAYCDALV RQRREAEGLAG

3851
LSIGWGLWQT ASGMTGHLGE TDLARMKRTG FTPLTTEGGL ALLDAARAHG

3901
RPHVVAVDLD ARAVAAQPAP SRPALLRALA AGATPGARTA RRTAAAGSVA

3951
PAGGLADRLA GLPHPERRRL LLDLVRGNVA GVLGHSDHDA VRPDTSFKEL

4001
GFDSLTAVEL RNRLAAATGL KLPAALVFDY PESATLVDHL LERLSPDGAP

4051
PPVKDAADPV LNDLGRIESS LDALALDADA RSRVTRRLNT LLSKLNGAAT

4101
AGSPADVTDL DALDALDDVS DDEMFEFIDR EL*

MonAIV, polyketide synthase multi-enzyme MONS4, housing

extension modules 5 and 6 Length: 4039 amino acids

1
MSSAEESSPD VSGTGVSGTG ESATGTSSTE AKLRQYLKRV TVDLGQARRR

51
LREVEERAQE PIAIVSMACR FPGDTRTPEA LWDLVAEGGD AIDDFPTNRG

101
WDLESLYHPD PDHPGTSYVR RGGFLYDAPA FDASFFGISP REALAMDPQQ

151
RVLMETAWQL LERAGIDPAS LKLSATGVYI GAGVLGFGGA QPDKTVEGHL

201
LTGSALSVLS GRISFTLGLE GPSVSVDTAC SSSLVSMHLA AQALRQGECD

251
LALAGGVTVM STPGAFTEFS RQGALSPDGR SKAFAASADG TGFSEGAGLL

301
LLERLSDARR NGHKVLAVIR GSAVNQDGAS NGLTAPNGPS QERVIRAALA

351
NAGLGAAEVD AVEAHGTGTK LGDPIEAGAL LATYGRDRDE DRPLWLGSVK

401
SNIGHPQGAA GVAGVIKMVM ALQRELLPAT LYVDEPTPHV DWSSGSVRLL

451
TEPVPWTRGE RPRRAGVSAF GMSGTNAHVI LEEAPPEEAA AAETPAEGTG

501
AVVPWVVSGR GEEALRAQAA QLAEHVRDDD QRPASPLEVG WSLATTRSVF

551
ENRAVVVGDD RDALLDGLRS LAAGEASPDV VSGAVGPTGP GPVMVFPGQG

601
GQWVGMGARL LDESPVFAAR IAECEQALSA YVDWSLTDVL RGDGSELARI

651
DVVQPVLWAV MVALAAVWAD QGIEPAAVVG HSQGEIAAAC VVGAISLDEA

701
ARIVAVRSVL LRQLSGRGGM ASLGMGQEQA ADLIDGHPGV VVAAVNGPSS

751
TVISGPPEGI AAVVADAQER GLRARAVASD VAGHGPQLDA ILDQLTEGLA

801
GIRPAATDVA FYSTVTAGHL TDTTELDTAY WVRNVRRTVR FADTIDALLA

851
DGYRLFIEVS PHPVLNLALE GLIERAAVPA TVVPTLRRDH GDTTQLARAA

901
AHAFAAGADV DWRRWFPADP APRTVDLPTY APQRQDFWPA PAGGRSGDPA

951
GLGLAASGHP LLGASVGLAS GDVHLLSGRV SRQSAAWLDD HVVAGQALVP

1001
GAAQVEWVLR AGDDAGCSAL EELTLQTPLV LPDTGGLRIQ VVVEAADAHG

1051
RRDVRLFSRP DDDDAFASTH PWTCHATGVL APAPTDGTNG TRDAADTLDG

1101
AWPPADAEPV PADDLYAQAD RTGYGYGPAF RGVRALWRHG KDVLAEVTLP

1151
KEAGDPDGFG IHPALLDAVL QPAALLLPPT DAEQVWLPFA WNDVALHAVR

1201
ATTVRVRLTP LGERIDQGLR ITVADAVGAP VLTVRDLRSR PTDTGRLAAA

1251
ATRDRHGLFD LEWIAPENAA ENAAGPARDA SEGWVTLGED AASLADLLAS

1301
VEAGAPAPQL VAAPVEPDRT DDGLALATHV LDLVQTWLAS PLHDSRLVLV

1351
TRGAVTDADV DVAAAAVWGL VRSAQSEHPG RFTLIDLGPD DTLAAAMQAA

1401
HLEEPQLAVH GGEIRVPRLV RATTDPTAPN GTPEADRTAD PSEGLHRNGT

1451
VLITGGTGVL GRLVAEHLVT EWGVRHLLLA SRRGDQAPGS AELRARLSEL

1501
GASVEIAPAD VGDAEAVAAL IASVDPAHPL TGVIHAAGVL DDAVITAQTP

1551
ESLARVWATK ATAARHLHEA TRETPLDFFV VFSSAAASLG SPGQANYAAA

1601
NAYCDALVQH RRAQGLAGLS IAWGLWQATS GMTGQLSETD LARMKRTGFA

1651
ALTDEGGLAL LDAARAHDRA YVVAADLDPR AVTDGLSPLL RALTAPATRR

1701
RVASEGLADG ALATRLAGLD ADGRLRLLTD VVREYVAAVL GHGSAARVGV

1751
DIAFKDLGFD SLTAVELRNR LSAACDVRLP ATLIFDHPTP QALATHLVDR

1801
LAGSTSATTT VNATAPAAAH VAAGADVDAD TDDPVAIVAM TCRFPGGVAS

1851
PDDLWDLLDA RKDAMGAFPT DRGWDLERLF HPDPDHPGTS YTDQGGFLPD

1901
AGDFDAAFFG INPREALAMD PQQRLLLEAS WEVLERAGID PTTLKGTPTG

1951
TYVGLMYHDY AKSFPTADAQ LEGYSYLAST GSMVSGRVAY TLGLEGPAVT

2001
VDTACSSSLV SIHLATQALR HGECDLALAG GVTVMADPDM FAGFSRQRGL

2051
SPDGRCKAYA AAADGVGFSE GVGVLLLERL SDARRHGRRV LGVVRGSAVN

2101
QDGASNGLTA PNGPSQERVI RQALASGGLS SVDVDVVEGH GTGTTLGDPI

2151
EAQALLATYG QGRPEDRPLW LGSVKSNIGH TQAAAGVAGV IKMVMANRHG

2201
VVPASLHVDV PSPHVEWDSG AVRLAVESVP WPQVEGRPRR AGVSSFGASG

2251
TNAHVIVESV PDGLEEDSVS VGGEALETET DGRLVPWVVS ARSPQALRDQ

2301
ALRLRDFASD ASFRAPLADV GWSLLKTRAL HEHRAVVVGA ERAELIAALE

2351
ALATGEPHAA LVGPACSQAR VGGDDVVWLF SGQGSQLVGM GAGLYERFPV

2401
FAAAFDEVCG LLEGPLGVEA GGLREVVFRG PRERLDHTVW AQAGLFALQV

2451
GLARLWESVG VRPDVVLGHS IGEIAAAHVA GVFDLADACR VVGARARLMG

2501
GLPEGGAMCA VQATPAELAA DVDGSAVSVA AVNTPDSTVI SGPSDEVDRI

2551
AGVWRERGRK TKALSVSHAF HSALMEPMLA EFTEAIRGVK FRQPSIPLMS

2601
NVSGERAGEE ITDPEYWARH VRNAVLFQPA IAQVADSAGV FVELGPAPVL

2651
TTAAQHTLDE SDSQESVLVA SLAGERPEES AFVEAMARLH TAGVAVDWSV

2701
LFAGDRVPGL VELPTYAFQR ERFWLSGRSG GGDAATLGLV AAGHPLLGAA

2751
VEFADRGGCL LTGRLSRSGV SWLADHVVAG AVLVPGAALV EWALRAGDEV

2801
GCVTVEELML QAPLVVPEAS GLRVQVVVEE AGEDGRRGVQ IYSRPDADAV

2851
GGDDSWICHA TGVLSPESAR LDTELGGVWP PAGAEPLDVD GFYAQAGEAG

2901
YGYGPAFRGL RAVWRHGQDL LAEVVLPEAA GAHDGYGIHP ALLDATLHPL

2951
LAARFMDGSE DDQLYVPFGW AGVSLRAVGA TTVRVRLRPV GESVDQGLSV

3001
TVTDATGGPV LSVDSLQTRP VKPSQLAAAQ QPDVRGLFTV EWTPLPQTDA

3051
DGEADWVVLS DGVGRLADVV SAAGGEAPWA VVAPVDASVG DGREGLDGRL

3101
VVERVLSLVQ EFLALPELAE SRLLVVTRGA VATGVDGDGD VDASAAAVWG

3151
LVRSAQSENP GRFILLDVDG DGDDQGPDLN GRHLPHATLR HAAEELDEPQ

3201
LALREGTLYV PRLTQARQSA ELVVPPGEPA WRLRMVHDGS LDALAAVACP

3251
EALEPLAPGQ VRIAVHAAGI NFRDVLVALG MVPAYGANGG EGAGVVTEVG

3301
PEVTHVSVGD RVMGVFEGAF GPVVIAEARM VTPVPQGWDM REAAGIPAAF

3351
LTAWYGLVEL AGLKAGERVL VHAATGGVGM AAVQIARHVG AEVFATASPG

3401
KHAVLEEMGI DAAHRASSRD LAFEGTFREA TGGRGMDVVL NSLAGEFIDA

3451
SLRLLGDGGR FLEMGKTDVR AAEEVAAEHA DVSYTAYDLV GDAGPDRISN

3501
MLDKLVELFA SERLKPLPVR SWPLDKAQEA FRFMSQAKHT GKLVLEIPPA

3551
LDPEGTVLVT GGTGALGQVV AEHLVREWGV RHLLLASRRG PEAPGSDELA

3601
SKLTGLGAEV TIVAADVSDP ASVVELVGKT DPSHPLTGVV HAAGVLEDGV

3651
VTAQTPEGLA RVWAAKAAAA ANLHEATREM RLGLFVVFSS AAATLGSPGQ

3701
ANYAAANAYC DALMQHRRAV GQVGLSVGWG LWEAPDAKPG VAADAKASAA

3751
TVGKASALSD GTNGSAPQDT TGTAPQGMTG GLTDTDVARM ARIGVKGMSN

3801
AHGLALFDAA HRHGRPHLVG FNLDLRTLAT HPLHTRPALL RGLATPTAGG

3851
ASRPTATAGG QPADLAGRLA ALSPSDRHHT LVRLIREQAA TVLGHHPDSL

3901
TTGSTFKELG FDSLTAVELR NRLSAATGLR LPAGLVFDHP DADILAEHLG

3951
AQLAPDGDTP AGAEATDPVL RDLAKLENAL SSTLVEHLDA DAVTARLEAL

4001
LSNWKAASAA PGSGSTKEQL QVATTDQVLD FIDKELGV*

MonAV, polyketide synthase multi-enzyme MONS5, housing

extension modules 7 and 8 Length: 4107 amino acids

1
MASEEELVDY LKRVAAELHD TRQRLREVED RRQEPVAVVG MACRFPGGIE

51
TPEGLWELVA AGDDAIEPFP TDRGWDLEGI YHPDPDHPGT CYVREGGFLA

101
APDRFDSDFF CPSPREALAS SPQLRLLLET SWEALERAGI NPASLKGSPT

151
GVYVGAATTG NQTQGDPGGK ATEGYAGTAP SVLSGRLSFT LGLEGPAVTV

201
ETACSSSLVA MHLAANALRQ GECDLALAGG VTVMSTPEVF TGFSRQRGLA

251
PDGRCKPFAA AADGTGWGEG AGLILLERLS DARRKGHKVL AVIRGSAINQ

301
DGASNGFTAP NGPSQRRVIR QALSSAHLST SEIDVVEAHG TGTRLGDPIE

351
AEALIATYGK EREDDRPLWL GSVKSNIGHT QAAAGVAGVI KMVMALQREL

401
LPATLNVDEP TPHVQWEGGG VRLLTEPVPW SRGERPRRAG ISSFGISGTN

451
AHVVLEEAPP EEDVPGPVAA EPEGVVPWVV SARTEEALSE QARRLGEFVA

501
DTDPSTADVG WSLTTSRAIL EHRAVVVGRD RDALTAGLAA LAAGEESADV

551
VAGVAGDVGP GPVLVFPGQG SQWVGMGAQL LDESPVFAAR IAECEQALSA

601
YVDWSLSAVL RGDGSELSRV EVVQPVLWAV MVSLAAVWAD YGVTPAAVIG

651
HSQGEMAAAC VAGALSLEDA ARVVAVRSDA LRQLMGQGDM ASLGASSEQA

701
AELIGDRPGV CIAAVNGPSS TVISGPPEHV AAVVADAEER GLRARVIDVG

751
YASHGPQIDQ LHDLLTDRLA DIRPATTDVA FYSTVTAERL TDTTALDTDY

801
WVTNLRQPVR FADTIDALLA DGYRLFIEAS AHPVLGLGME ETIEQADIPA

851
TVVPTLRRDH GDTTQLTRAA AHAFTAGATV DWRRWFPADP TPRTIDLPTY

901
AFQRRSYWLP VDGVGDVRSA GLRRVEHSLL PAALGLADGA LVLTGRLAAS

951
GGGGGWLADH AVAGTTLVPG AALVEWALRA ADEAGCPSLE ELTLQAPLVL

1001
PGSGGLQVQV VVGPADGQGG RREVRVFSRV DSDDEAAGQD EGWSCHATGV

1051
LSPEPGAVPD GLSGQWPPTG AEPLEISDLY EQAASAGYEY GPSPRGLRSV

1101
WRHGHNLLAE VELPEQAGAH DDFGIHPVLL DAALHPALLL DQNAPGEEQE

1151
PAQPALRLPF VWNGVSLWAT GAATVRVRLA PHGGGETDDS AGLRVTVADA

1201
TGAPVLSVDS LALRPADPEL LRTAGRAGSC TNGLFTVEWT ALPPADVADH

1251
AAGDGWAVLG QDVPDWAGAD MPRHPDMASL SAALDEGTQA PAAVFVETTA

1301
TSHATPNTAA DVTLDASGRA VAERTLHLLR DWLAEPRLAE TRLVLITHHA

1351
VTTPADDDVN AAPLDVPAAA LWGLIRSAQA EHPDRFVLLD TDAKANTDPG

1401
PDTSTDHSTA SGTYRTVIAR ALATGEPQLA VRAGELLAPR LAPAATPTPE

1451
TPTPETQPDT GSGSEAGAGS GSGPGATLDP DGTVTLIAGGT GMMGGLVAEH

1501
LVRAWSVRHL LLVSRQGPDA PDARDLADRL VGLGATVRIV AADLTDGRAT

1551
ADLVASVDPA HPLTGVIHAA GVLDDAVVTA QTSDQLARVW AAKASVAANL

1601
DAATSELPLG LFLMFSSAAG VLGNAGQAGY AAANAFVDAL VGRRRATGLP

1651
GLSIAWGLWA RGSAMTRHLD DADLARLRAG GVKPLLDEQC LALLDAARAT

1701
AAHTSLVVAA GIDVRGLNRD DVPAILRDLA GRTRRRAAAD STVDQAALER

1751
RLTGLDEAER RAVVTDVVRE CVAAVLGHRS AADVRTEANF KDLGFDSLTA

1801
VQLPNRLSAA SGLRLPATLA FDHPTPQALA AYLGTRLSGR TATPVAPVAP

1851
SAAATDEPVA IVAMACKYPG GATSPEGLWD LVAEGVDAVG AFPTGRGWDL

1901
ERLFHPDPDH PGTSYADEGA FLPDAGDFDA AFFGINPREA LAMDPQQRLL

1951
LEASWEVLER AGIDPTTLKG TPTGTYVGVM YHDYAAGLAQ DAQLEGYSML

2001
AGSGSVVSGR VAYTLGLEGP AVTVDTACSS SLVSIHLAAQ ALRQGECTLA

2051
LAGGVTVMAT PEVFTGFSRQ RGLAPDGRCK PFAAAADGTG WGEGVGVLLL

2101
ERLSDARRHG RRVLGVVRGS AVNQDGASNG LTAPNGPSQE RVIRQALASG

2151
GLSSVDVDVV EGHGTGTTLG DPIEAQALLA TYGQGRPVDR PLWLGSVKSN

2201
IGHTQAAAGV AGVIKMVMAM RHGVVPASLH VDVPSPHVEW DSGAVRLAVE

2251
SVPWPEVEGR PRRAGVSSFG ASGTNAHVIV ESVPDGLGED SVSVSGEAPE

2301
TETDGRLVPW VVSARSPQAL RDQALRLRDA VAADSTVSVQ DVGWSLLKTR

2351
ALFEQRAVVV GRERAELLSG LAVLAAGEEH PAVTRSREDG VAASGAVVWL

2401
FSGQGSQLVG MGAGLYERFP VFAAAFDEVC GLLEGPLGVE AGGLREVVFR

2451
GPRERLDHTM WAQAGLFALQ VGLARLWESV GVRPDVVLGH SIGEIAAAHV

2501
AGVFDLADAC RVVGARARLM GGLPEGGAMC AVQATPAELA ADVDDSGVSV

2551
AAVNTPDSTV ISGPSGEVDR IAGVWRERGR KTKALSVSHA FHSALMEPML

2601
AEFTEAIREV KFTRPKVSLI SNVSGLEAGE EIASPEYWAR HVRQTVLFQP

2651
GIAQVASTAG VFVELGPGPV LTTAAQHTLD DVTDRHGPEP VLVSSLAGER

2701
PEESAFVEAM ARLHTAGVAV DWSVLFAGDR VPGLVELPTY AFQRERFWLS

2751
GRSGGGDAAT LGLVAAGHPL LGAAVEFADR GGCLLTGRLS RSGVSWLADH

2801
VVAGAVLVPG AALVEWALRA GDEVGCVTVE ELMLQAPLVV PEASGLRVQV

2851
VVEEAGEDGR RGVQIYSRPD ADAVSGDDSW ICHATGTLTP QHTDAPNDGL

2901
AGAWPAAGAV PVDLAGFYER VADAGYAYGP GFQGLRAVWR HGQDLLAEVV

2951
LPEAAGAHDG YGIHPALLDA TLHPALLLDW PGEVQDDDGK VWLPFTWNQV

3001
SLRAAGAATV RVRLSPGEHD EAEREVQVLV ADATGTDVLS VGSVTLRPAD

3051
IRQLQAVPGH DDGLFSVDWT PLPLSRTDVS QTDADGDADW VVLSDGVGSL

3101
ADVVSAAGGE APWAVVAPVG ASAGGGLAGF DRREGLDGRL VVERVLSLVQ

3151
EFLAAPELAE SRLLVLTRGA VATGGDGDGD VDASAAAVWG LVRSAQSENP

3201
GRFILLDVDM DVDVDVDMDV DVDVDVDVDV DGDGNGSDLD PDLNGRRLPH

3251
ATLRHAAEEL DEPQLALRDG QLLVPRLVRA TGGGLVVAPT DRAWRLDKGS

3301
AETLESVAPV AYPGVMEPLG PGQVRLGIHA AGINFRDVLV SLGMVPGQVG

3351
LGGEGAGVVT ETGPDVTHLS VGDRVMGVLH GSFGPTAVAD TRMVAPVPQG

3401
WDMRQAAAMP VAYLTAWYGL VELAGLKAGE RVLIHAATGG VGMAAVQIAR

3451
HLGAEVFATA SAAKHVVLEE MGIDAAHRAS SRDLAFEDTF RQATDGRGMD

3501
VVLNSLTGEF IDASLRLLGD GGRFLEMGKT DVRTPEEVAA EYPGVTYTVY

3551
DLVTDAGPDR IAVMMSELGE RFASGALDPL PVRSWPLDKA REAFRFMSQA

3601
KHTGKLVLDV PAPLDPDGTV LITGGTGALG QVVAEHLVRE WGVRELLLAS

3651
RRGLDAPGSG ELADRLSDLG AEVTVAAADV SDPASVVELV GKTDPSHPLT

3701
GVVHAAGVLE DGIVTAQTPE GLARVWAAKA AAAANLHEAT REMRLGLFVV

3751
FSSAAATLGS PGQANYAAAN AYCDALMQRR RAAGQVGLSV GWGLWEAPDA

3801
KPGVAADAKP DVAADAKTGV AADGTPQGMT GTLSGTDVAR MARIGVKAMT

3851
SAHGLALLDA AHRHGRPHLV AVDLDTRVLA HKPAPALPAL LRAFAGDQGG

3901
QGGGRGGGRG GGPARPAAAT TRQNVDWAAK LSVLTAEEQH RTLLDLVRTH

3951
AAAVLGHAGT DAVRADAAFQ DLGFDSLTAV ELRNRLSAST GLRLPATFIF

4001
RHPTPSAIAD ELRAQLAPAG ADPAAPLFGE LDKLETVITG HAHDESTRTR

4051
LAARLQNLLW RLDDTSARSD HAAGASDADG DAVENRDLES ASDDELFELI

4101
DRELPS*

MonAVI, polyketide synthase multi-enzyme MONS6, housing

extension module 9 Length: 1701 amino acids

1
MPGTNDMPGT EDKLRHYLKR VTADLGQTRQ RLRDVEERQR EPIAIVAMAC

51
RYPGGVASPE QLWDLVASRG DAIEEFPADR GWDVAGLYHP DPDHPGTTYV

101
REAGFLRDAA RFDADFFGIN PREALAADPQ QRVLLEVSWE LFERAGIDPA

151
TLKDTLTGVY AGVSSQDHMS GSRVPPEVEG YATTGTLSSV ISGRIAYTFG

201
LEGPAVTLDT ACSASLVAIH LACQALRQGD CGIAVAGGVT VLSTPTAFVE

251
FSRQRGLAPD GRCKPFAEAA DGTGFSEGVG LILLERLSDA RRNGHQVLGV

301
VRGSAVNQDG ASNGLTAPND VAQERVIRQA LTNARVTPDA VDAVEAHGTG

351
TTLGDPIEGN ALLATYGKDR PADRPLWLGS VKSNIGHTQA AAGVAGVIKM

401
VMAMRHGELP ASLHIDRPTP HVDWEGGGVR LLTDPVPWPR ADRPRRAGVS

451
SFGISGTNAH LIVEQAPAPP DTADDAPEGA ATPGASDGLV VPWVVSARSP

501
QALRDQALRL RDFAGDASRA PLTDVGWSLL RSRALFEQRA VVAGRERAEL

551
LAGLAALAAG EEHPAVTRSR EEAAVAASGD VVWLFSGQGS QLVGMGAGLY

601
ERFPVFAAAP DEVCGLLEGE LGVGSGGLRE VVFWGPRERL DHTVWAQAGL

651
FALQVGLARL WESVGVRPDV VLGHSIGEIA AAHVAGVFDL ADACRVVGAR

701
ARLMGGLPEG GAMCAVQATP AELAADVDGS SVSVAAVNTP DSTVISGPSG

751
EVDRIAGVWR ERGRKTKALS VSHAFHSALM EPMLGEFTEA IRGVKFRQPS

801
IPLMSNVSGE RAGEEITSPE YWARHVRQTV LFQPGVAQVA AEARAFVELG

851
PGPVLTAAAQ HTLDHITEPE GPEPVVTASL HPDRPDDVAF AHAMADLHVA

901
GISVDWSAYF PDDPAPRTVD LPTYAFQGRR FWLADIAAPE AVSSTDGEEA

951
GFWAAVEGAD FQALCDTLHL KDDEHRAALE TVFPALSAWR RERRERSIVD

1001
AWRYRVDWRR VELPTPVPGA GTGPDADTGL GAWLIVAPTH GSGTWPQACA

1051
RALEEAGAPV RIVEAGPHAD RADMADLVQA WRASCADDTT QLGGVLSLLA

1101
LAEAPATSSD TTSHTSTSCG TGSLASHGLT GTLTLLHGLL DAGVEAPLWC

1151
ATRGAVSCGD ADPLVSPSQA PVWGLGRVAA LEHPELWGGL VDLPADPESL

1201
DASALYAVLR GDGGEDQVAL RRGAVLGRRL VPDATPDVAP GSSPDVSGGA

1251
AHADATSGEW QPHGAVLVTG GVGHLADQVV RWLAASGAEH VVLLDTGPAN

1301
SRGPGRNDDL AAEAAEHGTE LTVLRSLSEL TDVSVRPIRT VIHTSLPGEL

1351
APLAEVTPDA LGAAVSAAAR LSELPGIGSV ETVLFFSSVT ASLGSREHGA

1401
YAAANAYLDA LAQRAGADAA SPRTVSVGWG IWDLPDDGDV ARGAAGLSRR

1451
QGLPPLEPQL ALGALRAALD GGKGHTLVAD IEWERFAPLF TLARPTRLLD

1501
GIPAAQRVLD ASSESAEASE NASALRRELT ALPVRERTGA LLDLVRKQVA

1551
AVLRYEPGQD VAPEKAFKDL GFDSLVVVEL RNRLRAATGL RLPATLVYDY

1601
PTPRTLAAHL LDRVLPDGGA AELPVAAHLD DLEAALTDLP ADDPRRKGLV

1651
RRLQTLLWKQ PDAMGAAGPA DEEEQAAPED LSTASADDMF ALIDREWGTR

1701
*

MonH, probable regulatory protein Length: 981 amino acids

1
VSGVERGVGS AGPVEQGDGL AGLVERAEAL AALRGAFDGS PGTGGSLVVL

51
SGAVGTGKTA LLRAWADRIG ADADALVLTA TACRAERDLP LGVLEQLVRS

101
PGLPPASAER ALAWWDEEAS ATPGKTDANG TSANGTDANG TGAGQTGAGQ

151
AGVGQTGVGG EPVLAASALR QLCEVLRDLL AERPVVVAVD DAHHADAASL

201
QCLLSVVRRL RSARLHVLFT EYAHQKAQNA LLSSEFLHEP ALRRIRLEPL

251
SKAGVEALLA RHLDERTAQD LTPVVHGMSA GHPLLVRALA EDHRAAGGAG

301
EAYGRAVLSF LYRHETPVTQ VARAIAALGA HAGPGQVGRL LDVDAASVER

351
AVRQLTVAEV LHEGRLCHPA FAAAVLDGMP PEERRALHGR VADLLHEEGA

401
PATEVAAHLV AADRSDAPWA VPVFQEAAQL ALDEDQVETG VDYLRAAHQR

451
CRGAAQRAAV VGALADAEWR LDPAKVLRHL PDPAAMAPQT DPAALAPHTD

501
PAPTAAPTAA PTPTPIPTTP PLPTHLLWHG RVEEGLDAIG TLTGPGPNPA

551
GAPPMNPADL DTPWLWGAYL YPGHVKERLG SGALSPQRST PPAVTPELQG

601
AGTLMNDLLH GGERDATEAA ERALNRYRLG PRTIAVQTAA LAALTYRDRP

651
HRAAAWCDGL VAQADERNSP TWRALFTAWR ALLHLRQGDP AAAEQRAETA

701
LALLGSKGWG AAIGLPLAAA VQAKAALGDV DGAAALLERP VPQAVFQTRT

751
GLHYLAARGR YHLATGCHYA ALCDFYACGT RMSSWGVDLP ALEPWRLGAA

801
EAYLALGEGL LARQLVDGQL PLPTPDDGRT WGMTLRLRAA TSPAPARAEL

851
LDEAVAVLRE SGDTFELARA VADQAVAVRE GGEAERARLL ARKAELLARR

901
WGSAPAPATV PEPPERPGPA TPDAELTSAE RRVAELAAEG FTNREISRKL

951
CVTVSTVEQH LTRIYRKLDV RRLDLQAALG *

MonCI, flavin-dependent epoxidase Length: 496 amino acids

1
VTTTRPAHAV VLGASMAGTL AAHVLARHVD AVTVVERDAL PEEPQHRKGV

51
PQARHAHLLW SNGARLIEEM LPGTTDRLLA AGARRLGFPE DLVTLTGQGW

101
QHRFPATQFA LVASRPLLDL TVRQQALGAD NITVRQRTEA VELTGSGGGS

151
GGRVTGVVVR DLDSGRQEQL EADLVIDATG RGSRLKQWLA ALGVPALEED

201
VVDAGVAYAT RLFKAPPGAT THFPAVNIAA DDRVREPGRF GVVYPIEGGR

251
WLATLSCTRG AQLPTHEDEF IPFAENLNHP ILADLLRDAE PLTPVFGSRS

301
GANRRLYPER LEQWPDGLLV IGDSLTAFNP IYGHGMSSAA RCATTIDREF

351
ERSVQEGTGS ARAGTRALQK AIGAAVDDPW ILAATKDIDY VNCRVSATDP

401
RLIGVDTEQR LRFAEAITAA SIRSPKASEI VTDVMSLNAP QAELGSNRFL

451
MAMRADERLP ELTAPPFLPF ELAVVGLDAA TISPTPTPTP TAAVRS

MonBII, carbon-carbon double bond isomerase

Length: 141 amino acids

1
MPDEAARKQM AVDYAERINA GDIEGVLDLF TDDIVFEDPV GRPPMVGKDD

51
LRRHLELAVS CGTHEVPDPP MTSMDDRFVV TPTTVTVQRP RPMTFRIVGI

101
VELDEHGLGR RVQAFWGVTD VTMDDPAGPA DTTHPEGIRA *

MonBI, carbon-carbon double bond isomerase

Length: 144 amino acids

1
MNEFARKKRA LEHSRRINAG DLDAIIDLYA PDAVLEDPVG LPPVTGHDAL

51
RAHYEPLLAA HLREEAAEPV AGQDATHALI QISSVMDYLP VGPLYAERGW

101
LKAPDAPGTA RIHRTAMLVI RMDASGLIRH LKSYWGTSDL TVLG

MonAVIII, polyketide synthase multi-enzyme MONS8, housing

extension modules 11 and 12 Length: 3754 amino acids

1
MSNEEKLLDH LKWVTAELRQ ARQRLHDKES TEPVAIVGMA CRYPGGARSA

51
EDLWELVRDG GDAVAGFPDD RGWDLESLYH PDPEHPATSY VRDGAFLYDA

101
GHFDAEFFGI SPREATAMDP QQRLLLETAW EAIEHAGMNP HALKGSDTGV

151
FTGVSAHDYL TLISQTASDV EGYIGTGNLG SVVSGRISYT VGLEGPAVTV

201
DTACSSSLVA IHLASQALRQ GECSLALAGG STVMATPGSF TEFSRQRGLA

251
PDGRCKPFAA AADGTGWGEG AGVVALELLS EARRRGHKVL AVIRGSATNQ

301
DGTSNGLAAP NGPSQERVIR AALANARLSA EDIDAVEAHG TGTTLGDPIE

351
AQALIATYGQ GRPEDRPLWL GSVKSNIGHT QAAAGVAGVI KMVMAMRNGL

401
LPTSLHIDAP SPHVQWEQGS VRLLSEPVDW PAERTRRAGI SAFGISGTNA

451
HLILEEAPPE EDAPGPVAAE PGGVVPWVVS GRTPDALREQ ARRLGEFAAG

501
LADASVSEVG WSLATTRALF DQRAVVVGRD LAQAGASLEA LAAGEASADV

551
VAGVAGDVGP GPVLVFPGQG SQWVGMGAQL LDESPVFAAR IAECEQALSA

601
HVDWSLSDVL RGDGSELSRV EVVQPVLWAV MVSLAAVWAD YGITPAAVIG

651
HSQGEMAAAC VAGALSLEDA ARIVAVRSDA LRQLQGHGDM ASLSTGAEQA

701
AELIGDRPGV VVAAVNGPSS TVISGPPEHV AAVVADAEAQ GLRARVIDVR

751
YASHGPQIDQ LHDLLTDRLA DIQPTTTDVA FYSTVTAERL DDTTALDTAY

801
WVTNLRQPVR FADTIEALLA DGYRLFIEAS PHPVLNLGIQ ETIEQQAGAA

851
GTAVTIPTLR RDHGDTTQLT RAAAHAFTAG APVDWRRWFP ADPTPRTVDL

901
PTYAFQHKHY WVEPPAAVAA VGGGHDPVEA RVWQAIEDLD IDALAGSLEI

951
EGQAESVGAL ESALPVLSAW RRRHREQSTV DSWRYQVTWK HLPDVPAPEL

1001
SGAWLLLVPA AHADHPAVLA TAQTLTAHGG EVRRHVVDAR AMERTELAQE

1051
LRVLMDGAAF AGVVNLLALD EEPHPEHSAV PAGLAATTAL VQALADNGAD

1101
IAVRTLTQGA VSTSAGDALT HPVQAQVWGL GRVAALEYPR LWGGLVDLPA

1151
RIDHQTLARL AAALVPQDED QISIRPSGVH ARRLAHAPAN TVGSGLGWRP

1201
DGTTLITGGT GGIGAVLARW LARAGAPHLL LTSRRGPDAP GAQELAAELT

1251
ELGAAVTVTA CDVGDREQVR RLIDDVPAEH PLTAVIHAAG VPNYIGLGDV

1301
SGAELDEVLR PKALAAHHLH ELTRELPLSA FVMFSSGAGV WGSGQQGAYG

1351
AANHFLDALA EHRRAEGLPA TSIAWGPWAE AGMAADQAAL TFFSRFGLHP

1401
LSPELCVKAL QQALDAGETT LTVANFDWAQ FTSTFTAQRP SPLLADLPEN

1451
RRASAPAAQQ EDATEASSLQ QELTEAKPAQ QRQLLLQHVR SQAAATLGHS

1501
DVDAVPATKP FQELGFDSLT AVELRNRLNK STGLTLPTTV VPDHPTPDAL

1551
TDVLRAELSG DAAASADPVR AAGASRGAAD DEPIAIVGMA CRYPGDVRSA

1601
EELWDLVAAG KDAMGAFPDD RGWDLETLYD PDPESRGTSY VREGGFLYDA

1651
GDFDAGFFGI SPREAVAMDP QQRLLLETAW EAIERAGLDR ETLKGSDAGV

1701
FTGLTIFDYL ALVGEQPTEV EGYIGTGNLG CVASGRVSYV LGLEGPAMTI

1751
DTGCSSSLVA IHQAAHALRQ GECSLALAGG ATVMATPGSF VEFSLQRGLA

1801
KDGRCKPFAA AADGTGWAEG VGLVVLERLS EARRNGHNVL AVIRCSAINQ

1851
DGTSNGLTAP NGQAQQRVIR QALANARLSA EDVDAVEAHG TGTMLGDPIE

1901
ASALVATYGK ERPADRPLWL GSIKSNIGHA QASAGVAGVI KMVMALRNEQ

1951
LPASLHIDAP TPHVDWDGSG VRLLSEPVSW PRGERPRRAG VSAFGISGTN

2001
AHLILEQAPD APEPVTAPAE DAAAPAGVVP WVVSARGEEA LRAQARLLAD

2051
RATADPRLAS PLDVGWSLVK TRSVFENPAV VVGKDRQTLL AGLRSLAAGE

2101
PSPDVVEGAV QGASGAGPVL VFPGQGSQWV GMGAQLLDES PVFAARIAEC

2151
ERALSAHVDW SLSAVLRGDG SELSRVEVVQ PVLWAVMVSL ASVWADYGIT

2201
PAAVIGHSQG EMAAACVAGA LSLEDAARIV AVRSDALRQL MGQGDMASLG

2251
AGSEQVAELI GDRPGVCVAA VNGPSSTVIS GPPEHVAAVV ADAEARGLRA

2301
RVIDVGYASH GPQIDQLHDL LTERLADIRP TTTDVAFYST VTAERLDDTT

2351
TLDTDYWVTN LRQPVRFADT IEALLADGYR LFIEASPHPV LNLGMEETIE

2401
RADMPATVVP TLRRDHGDAA QLTRAAAQAF GAGAEVDWTG WFPAVPLPRV

2451
VDLPTYAFQR ERFWLEGRRG LAGDPAGLGL ASAGHPLLGA AVELADGGSH

2501
LLTGRISPRD QAWLAEHRVM DTVLLPGSAF VELALQAAVR AGCAELAELT

2551
LHTPLAFGDE GAGAVDVQVV VGSVAEDGRR PVTVHSRPTG EGEEAVWTRH

2601
AAGVVAPPGP DAGDASFGGT WPPPGATPVG EQDPYGELAS YGYDFGPGSQ

2651
GLVSAWRLGD DLFAEVALPE AESGRADRYQ VHPVLLDATL HALILDAVTS

2701
SADTDQVLLP FSWSGLRVHA PGAEKLRVRI ARTAPDQLAL TAVDGGGGGE

2751
PVLTLESLTV RPVAANQIAG ARAADRDALF RLVWMEVAAR AEETGGGAPR

2801
AAVLAPVESG PMGGTSAGAL ADALSDALAA GPVWDTFGAL RDGVAAGGEA

2851
PDVVLAVCAA PGAGAGAVAD ADGRGGDPAG YARLATVSLL SLLKEWVDDP

2901
AFAATRLVVV TRGAVAARPG ETAGDLAGAS LWGLVRSAQA ENPGRLTLLD

2951
VDGLESSPAT LTGVLASGEP ELALRDGRAY VPRLVRDDAS VRLVPPVGSL

3001
TWRLARCQEA GGGQQLSLVD APEAGRALEP HEVRVAVRAA APGPLTAGQV

3051
EGAGVVTEVG GEVGSVAVGD RVMGLFDAVG PVAVTDAALL MPVPAGWSWA

3101
QAAGSLGAYV SAYHVLADVV APRGGETLLV GEETGSVGRA VLRLALAGRW

3151
RVEAVDGAST ADDSGAERAA DVTLRHEGAL VVHRAGGRPD EGQAVVPPEP

3201
GRVREILAEL TELTELAEIT ESAEPGLPAE RGDSRALTPL DITVWDIRQA

3251
PAAMAAPPSA GTTVFSLPPA FDPEGTVLVT GGTGALGSLT ARHLVERYGA

3301
RHLLLSSRRG ADAPGALELA ADLSALGARV TFAACDPGDR DEAAALLAAV

3351
PSDHPLTAVF HCAGTVNDAV VQNLTAEQVE EVMRVKADAA WHLHELTRDA

3401
DLSAFVLYSS VAGLLGGPGQ CSYTAANAFL DALARHRHDG GAAATSLAWG

3451
YWELASGMSG RLTDADRARH ARAGVVGLGA DEGLALLDAA WAGGLPLYAP

3501
VRLDLARMRR QAQSHPAPAL LRDLVRGGSK SGGGAVSAGA AALLKSLGAM

3551
SDPEREEALL DLVCTHIAAV LGYDAATPVN ATQGLRELGF DSLTAVELRN

3601
RLSAATGLKL PATFVFDHPN PAELAAQLRQ ELAPRAADPL ADVLAEFERI

3651
EDSLLSVSSK DGSARAELAG RLRATLARLD APQDTAGEVA VATRTRIQDA

3701
SADEIFAFID RDLGRDGASG QGNGQPTGQG NGHGNGNGNG NGNGHGQAVE

3751
GQR*

MonAVII, polyketide synthase multi-enzyme MONS7, housing

extension module 10 Length: 1642 amino acids

1
MAHTEEKLLE YLKRVTADLR QTERRLQDVE SAGHEPVAVI GMACRLPGGV

51
RSPEEFWELV STGGDAVAPL PGNRNWDLDS LYDPDPESTG TSYVREGGFV

101
YDAGDFDPTF FGIGPTEAAA MAPQQRLALE TAWEAIERAG IDPLSLRSSD

151
TSTFIGCDGL DYALGASEVP EGTAGYFTIG NSGSVTSGRV AYTLGLEGPA

201
VTVDTACSSS LVSLHLATQA LRTQECSLAL AGGTYVMSSP APLIGFSELR

251
GLAPDGRCKP FSASSDGMGM AEGTGVVLLE RLSDARRKGH KVLAVIRGSA

301
INQDGASNGL TAPNGPAQER VIRAALANAR LAPEDIDAVE AHGTGTTLGD

351
PIEAGALISA YGRERPEDRP LWVGAVKSNI GHTQIAAGVA GVIKMVLALR

401
HDLLPAILHV DAPSPHVEWD GSGLRLLTDP VKWPRGERPR RAGVSSFGFS

451
GTNAHLILEE APPEEEDVPG SVAEEPGGVV PWVVSGRTPD ALRAQARRLG

501
EFAAGPADAS AADVGWSLTT TRSVFEHRAV VVGRDRDALT AGLGALAAGE

551
ASAGVVAGVA GDVGPGPVLV FPGQGSQWVG MGAQLLDESP VFAARIAECE

601
RALSAYVDWS LSAVLRGDGS ELSRVEVVQP VLWAVMVSLA AVWADYGVTP

651
AAVIGHSQGE MAAACVAGAL SLEDAARIVA VRSDALRRLQ GHGDMASLST

701
GAEQAAELIG DRPGVVVAAV NGPSSTVISG PPEHVAAVVA DAEARGLRAR

751
VIDVGYASHG PQIDQLHDLL TERLADIRPA NTDVAFYSTV TAERLTDTTA

801
LDTDYWVTNL RQPVRFADTI EALLADGYRL FIEASAHPVL GLGMEETIEQ

851
ADIPATVVPT LRRDHGDTTQ LTRAAAHAFT AGAPVDWRRW FPADPTPRTV

901
DLPTYAFQHQ HYWLERSASA SGAVSGEQSA AEAQLWHAVE ELDLGLLAET

951
LGSEEGSEEA VRALEPALPV LKGWRRRHQD QATIDSWRYR VTWKQRSDGP

1001
APELGGDWLL FVPADKAEHP AVRATAEALS EHGAAAVRLH PVETGRAGRQ

1051
ELAAVDTAGL AGIVNLLALD EEPHPEHPAV PAGLAATTAL LQALGDNGTT

1101
APLHTVTQGA VSTGATDPLT HPLQAHVWGL GRVAALEHPR LWAGLVDLPA

1151
RIDRHTLPRL AAALLPQDDE DQTAVRPTGI HHRRLTHAVG SIQNPVHSEA

1201
TWRPRGTTLI TGGTGGIGAV LARWLARQGA PRLHLTSRRG PDAPGARELA

1251
AELDGLGTAV TITACDVSDP RQLSGLIDDM PAEHPLTAVI HAAGMTDLTA

1301
IGDLTTARLG EVLGSKSDAA WNLHELTRDL DLSAFVMFSS GAGVWGSGQQ

1351
GAYGAANHFL DALAEHRRAQ GLPATSIAWG PWAEAGMSAD PESLTYFKRF

1401
GLLPIAPDLC VKALHQAVDA GDATLTVANF DWAKFTPTFT AQRPSPFLDD

1451
LPENQREAEQ TGTAAETSAF REELAKTPAS QRLGFLVQQV RTYAAATLGR

1501
TVEDIPAAKP FQELGFDSLT AVQLRNQLNT TTGLSLPATV IFDHPTPEAL

1551
ATHLRGQLGD GAEVAGEGDV LAALDKWDTA FGAAEVDEAA RRRIVGRLQV

1601
LVSKWSPAQD GPEGTDSAHA DLEAASADDI FDLISSEFGK S*

MonD, cytochrome P450 hydroxylase Length: 431 amino acids

1
VGLTVGPDNA KRGIVPITDS KPAATFPDLV DPSFWARPHA ERVALFEEMR

51
GLPRPAFIRQ NMPGVPWTFG YHALVKYADI VEVSRRPQDF SSNGATTIIG

101
LPPELDEYYG SMINMDNPEH SRLRRIVSRS FGRNMIPEFE AVATRTARRI

151
IDELIARGPG DFIRPVAAEM PIAVLSDMMG IPAEDHDFLF DRSNTIVGPL

201
DPDYVPDRAD SERAVIEASR ELGDYIAGLR AERLAAPGND LITKLVQVQA

251
DGEQLTRQEL VSFFILLVIA GMETTRNAIS HALVLLTEHP EQKQLLLSDF

301
DTHAPNAVEE ILRVSTPINW MRRVATRDCD NNGHRFRRGD RIFLFYWSGN

351
RDESVFPDPY RFDITRGTNA HVTFGAVGPH VCLGAHLARM EITVLYRELL

401
AALPQIHAVG QPRRLDSSFI EGIKHLHCAF *

MonRI, probable activator protein Length: 268 amino acids

1
VRYEMLGPLR IKDGNDYATI NAQKVEIVLT VLLIRADRVV SLEQLMREIW

51
GEDLPRRATA GLHVYISQLR KFLKVPGSAG NPVETRAPGY VLHKRDDDQI

101
DAQIFPELVD VGRSLLREKR PDEAASCFGQ ALALWRGPIL GQGGNGPGTN

151
GPIIDGFSTW LTEIRLECQE MLVECQLQLG RHREAVGMLY ALTAENPMCE

201
AFYRQLMLAL YRSERQADAL KVYQSVRKTL NDELGLEPGR PLQELQRAIL

251
AGDMHLMSPP PLALSGR*

MonAX, thioesterase Length: 278 amino acids

1
LSAFLAKGKI LSAFPPPDMS DPWIRRFRPR PEAVVRLVCF PHAGGSASYY

51
HPLAQSPTLP TDSEVLAVQY PGRQDRRRER LLDDIGELAD LITDALGPFD

101
DRPLAFFGHS MGAVLAYEVA QRLRERTGKQ PCRLFVSGRR APSRFRRGTV

151
HLLDDTELAA ELRRAGGTDP RFLDDEELLA EIIPVVRNDY RAVELYRWNP

201
SPPLSCPITA LVGDRDPQAP LDEVEAWQQH TEGPFDLKVF AGGHFYLNTN

251
QQGVTEVISK ALADSAQQRA TARGNAR*

ORF29, a homologue of CapK involved in cell wall

biosynthesis Length: 428 amino acids

1
LADLVAHARS ASPYYRELYH GLPERIEDPT LLPVTDKKQL MDHFDDWPTD

51
RDITFEKVRA FTDDPELIGR RFLGRYLVAT TSGTSGRRGL FVLDDRYMNV

101
SSAVSSRVLA SWLGPLGIAR AVVHGGRFAQ LVATEGHYVG FAGYSRLRQD

151
GEARSKLVRA FSVHEPMSRL VAELNEYRPA FVIGYASTIM LFTAEQEAGR

201
LHIDPVLVEP AGETMTESDT DRIAAAFGAK VRTMYSATEC TYLSHGCAEG

251
WYHVNDDWAV LEPVDADHRP TPPGEFSHTT LISNLANRVQ PFLRYDLGDS

301
VMLRPDPCPC GTPSPAIRVQ GRSGDILTFP SGRGDDVSLA PLAFSSLFDR

351
MPGVELFQIE QTAPSTLRVR VVQAPGADAD HVWQRAHDGL THLLADNKLD

401
NVTVERGEEP PRQASGGKYR TIIPLAA*

LipB, lipase B Length: 338 amino acids

1
VKVPVEVTVR LSSWLGGLVA AVLAATVLPA SAASAADVSS PPLEIPAAEL

51
AKALHCGTEL GDLRDAGDKP TVLFVPGTGL KGEENYAWNY MAELKKKGYQ

101
SCWVDSPGRG LRDMQESVEY VVYATRAIQE ATGRKVDLVG HSQGGLLTAW

151
ALRFWPDLPG KVDDMVTLGS PFQGTRLASP CRPIAEVAGC PASVLQFARD

201
SMWSKALGAD GTPMPAGPSY TTIYSYADES VVADGEAPSL PGAHRIGVQD

251
ICPGRPWPTH IAMVVDQVSY DLVADAIEHP GPADTSRIDR AHCAKPVMPL

301
NSQEAVDALP GLLNFPIELL THSQPWVDEE PPLRPYAR

ORF31, putative ion pump Length: 309 amino acids

1
MGHDHGPSAG AAGGTLSGTY RKRLLWTIGI SGSITVIQVV GALLSGSLAL

51
LADAAHSLTD AVGVSLALGA ITLAQRAPTP RRTFGFCRVE IFSAVLNALL

101
LVVIFAWVLW SAIGRFSEPV EVKGGLMFVV ALGGLAANLV GLWLLRDAKE

151
KSLNLRGAYL EVLGDALGSV AVIVGGLVIL LTGWQAADPI ASIVTGLLIV

201
PRAYGLLRDS LHVLLEATPQ DVDLGEVRRH LLEERGVVAV HDLHGWTVTS

251
GMPVLTAHVV VTEEALASGY GELLGRLQRC VGGHFDVAHS TIQLEPEGHV

301
EEDGALHT*

ORF32, hypothetical membrane protein Length: 364 amino acids

1
MTRALTLHDW IVAGIAVVAG VVAGLLLRAL LRWLGERASK TRWSGDDVIV

51
DALRTLVPCA AITAGLAAAA GALPLTPRTG RNVTMTLTAL LILAATLTAA

101
RIVTGLVKAV AQSRSGVAGS ATIFVNITRV VVLAMGFLIV LQTLGISIAP

151
LLTALGVGGL AVALALQDTL ANLFAGVHIL AAKTVQPGDY IQLSSGEEGY

201
VVDINWRNTT VRQLSNNLVI IPNAKLAGTN MTNYSRPEQE LSIMVQVGVS

251
YDSDLEQVEK VTTEVVDEVM AEITGAVPDH EAAIRFHTFG DSRISFTVIL

301
GVGEFSDQYR IKHEFIKRLH QRYRAEGIRV PAPVRTVRVQ QGELPPPLGI

351
PHQRDTSTQA RLH*

AmtA, glycine amidinotransferase (partial coding sequence)

Length: 131 amino acids

1
MSPVNSHNEW DPLEEIIVGR LEGATIPSSH PVVACNIPTW AARLQGLAAG

51
FEYPQRLIEP AQQELDQFIA LLQSLDVTVR RPAAVDHKHR FGTPDWQSRG

101
FCNSCPRDSM LVVGDEIIET PMAWPCRCFE T

[0153]

Polyketides and their synthesis

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