EP1212412A2

EP1212412A2 - Nucleic acids which code for the enzyme activities of the spinosyn biosynthesis

Info

Publication number: EP1212412A2
Application number: EP00956458A
Authority: EP
Inventors: Günther EBERZ; Volker MÖHRLE; Rita FRÖDE; Robert Velten; José A. SALAS
Original assignee: Bayer AG
Current assignee: Bayer CropScience AG
Priority date: 1999-08-27
Filing date: 2000-08-17
Publication date: 2002-06-12
Also published as: US7285653B1; AU6839300A; JP2003508050A; WO2001016303A3; WO2001016303A2

Abstract

The present invention relates to nucleic acids which code for the enzyme activities of the spinosyn biosynthesis. The invention also relates to the corresponding enzymes as such. The invention further relates to a method for producing spinosyn derivatives and spinosyn precursors.

Description

Nucleic acids that code for enzyme activities of spinosyn biosynthesis

The present invention relates to nucleic acids which code for enzyme activities of spinosyn biosynthesis, and the corresponding enzymes per se.

Spinosyns represent a new group of macrolide compounds that have been isolated from the Actinomycete Saccharopolyspora spinosa (Mertz and Yao, 1990). They are used to control insects (WO 97/00265, WO 94/20518, WO 93/09126, US 5670364, US 5362634, US 5227295, US 5202242). Spinosyne show a strong insecticidal, but no antibacterial

Activity which distinguishes them from conventional macrolides such as tylosin, spiramycin and erythromycin, which have no insecticidal but antimicrobial activity.

The structure of the Spinosyne consists of a tetracyclic polyketide backbone (aglycon) with a 12-membered macrolide ring and a 5,6,5-cis-anti-trans-tricyclic as well as a D-forosamine and a 2,3, 4-Tri-O-methyl-L-rhamnose sugar content (Kirst et al., 1991). More than 20 different natural spinosyn derivatives, the so-called A83543 complex, have been described so far (WO 97/00265, WO 94/20518, WO 93/09126). These derivatives vary in the substitution of one or a few methyl groups on the tetracyclic backbone, on the forosamine or on the tri-methyl-rhamnose sugar portion. A 17-pseudoaglycon, lacking the forosamine sugar portion, has also been isolated from S. spinosa culture broths.

The main components of the A83543 complex formed by S. spinosa are the variants Spinosyn A and Spinosyn D, which represent the essential components of the product Spinosad (see Pesticide Manual, British Crop Protection Council, ^1th ed., 1997, page 1272 and Dow Elanco trade maganzine Down to Earth, Vol. 52, NO :. 1, 1997 and the literature cited therein). Based on investigations into the incorporation of C-labeled acetate, propionate, butyrate or isobutyrate, it could be shown that the biosynthesis of A83543 follows a polyketide biosynthetic pathway (Nakatsukasa et al., 1990). Polyketides are built up from short-chain acid building blocks such as acetate, propionate or butyrate using multifunctional enzymes, the so-called polyketide synthases (PKS's). Similar to the related fatty acid synthases (FAS's), they catalyze decarboxylative polycondensation steps of the building blocks activated as CoA thioesters. While FAS's catalyze a complete reduction of the ß-oxoesters that are formed on the growing polyketide chain by ketoreduction, dehydration and enoyl reduction after each condensation step, PKSs can omit certain reduction steps. Modular type I PKSs consist of one or more large multifunctional proteins. Iterative Type II PKSs, on the other hand, represent a complex of largely monofunctional proteins.

The enzymatic activities of modular Type I PKS's can be summarized in so-called modules. Here, one module carries an arrangement of three enzyme-catalytically active domains, which lead to an extension of the growing polyketide chain by a biosynthetic extension unit. These domains are a β-ketoacyl: acyl carrier protein synthase domain, an acyltransferase domain and a β-ketoacyl: acyl carrier protein

Domain. A module can also carry a ketoreductase, a dehydratase, an enoyl reductase and a thioesterase domain. A so-called charge module, which is at the beginning of biosynthesis, can only carry an acyltransferase domain and a β-ketoacyl: acyl carrier protein domain from the domains mentioned, and also an enzymatically inactive β-ketoacyl: acyl carrier protein synthase domain. A

Polyketide synthase domain each includes one of these enzymatic activities.

Due to the potent insecticidal effects and the remarkable structure of the spinosyns, there is great interest in the genetic information for them

Decrypt biosynthesis. The invention relates to nucleic acids which comprise at least one region which codes for an enzyme activity which is involved in the biosynthesis of spinosyns.

The present invention provides a cluster of open reading frames (ORFs) whose translation products are involved in the biosynthesis of spinosyns. Furthermore, additional genes or ORFs are provided which are outside the approximately 120 kb large spinosyn biosynthesis cluster and whose translation products are involved in rhamnose sugar biosynthesis.

The nucleic acids according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). Preferred embodiments are fragments of genomic DNA and cDNA's.

The term "at least one region" as used herein means that the nucleic acid according to the invention can comprise one or more sequences which each code for individual activities which carry out steps in the synthesis of spinosyns. Accordingly, nucleic acids are also considered to be according to the invention which code only for a single enzyme activity of spinosyn biosynthesis.

The term “enzyme activity” as used in the present context means that, starting from the nucleic acids considered herein, at least that part of a complete enzyme which still exerts the catalytic properties of the enzyme can be expressed.

In particular, the nucleic acids according to the invention code for enzyme activities of polyketide synthases, methyl transferases, epimerases, glycosyl transferases, Aminotransferases, dimethyltransferases, reductases, dehydratases and / or cyclization enzymes.

The nucleic acids according to the invention are preferably DNA fragments which correspond to genomic DNA from S. spinosa.

The nucleic acids according to the invention particularly preferably comprise at least one sequence selected from

(a) the sequences according to SEQ ID NOS: 1, 2, 3, 4, 5, 7, 9, 11, 13, 15, 17,

19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52 or 54,

(b) at least 14 base pairs long partial sequences of the sequences defined under (a),

(c) sequences which hybridize to the sequences defined under (a),

(d) sequences which have at least 70%, preferably 80%, particularly preferably 90% identity to the sequences defined under (a),

(e) sequences which are complementary to the sequences defined under (a), and

(f) Sequences which, owing to the degeneracy of the genetic code, code for the same amino acid sequence as the sequences defined under (a) to (d). The term "hybridize" as used herein describes the process in which a single-stranded nucleic acid molecule with a complementary strand undergoes base pairing. In this way, for example, starting from genomic DNA from organisms which are phylogenetically related to S. spinosa and have the ability to biosynthesize spinosyns,

DNA fragments are isolated which have the same properties as the fragments isolated from S. spinosa.

Preferred hybridization conditions are given below: Hybridization solution: 5 x SSC; Blocking Reagens (Röche Diagnostics GmbH, Mannheim,

Germany), 1%; N-lauroylsarcosine, 0.1%; SDS (sodium dodecyl sulfate) 0.02%; Hybridization temperature: 60 ° C; first washing step: 2 x SSC at 60 ° C; second washing step: 2 x SSC at 60 ° C; preferably second washing step: 0.5 x SSC at 60 ° C; particularly preferred second washing step: 0.2 x SSC at 60 ° C.

The degree of identity of the nucleic acids is preferably determined with the aid of the GAP program from the GCG program package (Devereux et al., 1984), version 9.1 under standard settings.

Particular emphasis is placed on nucleic acids that

(1) either comprise all sequences coding for steps of forosamine and trimethyl rhamnose biosynthesis, in particular the sequences according to SEQ ID NOS: 4 and 51, or (2) comprise all sequences coding for steps of polyketide synthesis , in particular the sequences according to SEQ LD NOS: 5 and 6, or (3) all sequences which encode all steps of forosamine, trimethyl rhamnose and polyketide synthesis, in particular the sequences according to SEQ ID NOS: 1, 2, 3 and 51. All of the DNA sequences required for spinosyn biosynthesis or for the synthesis of precursors, as defined below, can thus be located on a single vector. However, these nucleic acids can also be present on two or more vectors and can be expressed simultaneously or in succession in a host cell.

All ORFs of the nucleic acids according to the invention can be switched on by their own promoters or by heterologous promoters.

The present invention also relates to the regulatory regions which naturally, i.e. in the original organism S. spinosa, control the transcription of the nucleic acids according to the invention.

The term "regulatory regions" as used herein refers to promoters, repressor or activator binding sites, repressor or activator sequences, and terminators. Furthermore, genetically mobile elements that are natural, i.e. occur in the original organism S. spinosa, also encompassed by this expression. Such genetically mobile elements can be transposable or mobilizable elements or functional parts thereof, IS elements or other insertion elements. There are also amplifiable DNA elements

(Amplifiable Units of DNA, AUD; Fishman and Hershberger, 1983), which naturally, i.e. occur in the original organism S. spinosa, encompassed by this expression. The invention also relates to any combination of these regulatory regions with one another or with heterologous DNA fragments, e.g. Promoters, repressors or activator binding sites, transposable, mobilizable or transducible elements.

The present invention furthermore relates to DNA constructs which comprise at least one nucleic acid according to the invention and a heterologous promoter. The term "heterologous promoter" as used in the present context refers to a promoter which does not control the expression of the gene (ORF 's) in question in the original organism.

The choice of heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression. A preferred example of a heterologous promoter is the promoter of the mel gene from the vector pIJ702 (The John Innes Foundation, Norwich, UK 1985). The heterologous expression can e.g. can be used to increase the production of spinosyn compared to the natural spinosyn producer.

The invention further relates to vectors which contain at least one of the nucleic acids according to the invention. All phages, plasmids, phagmids, phasmids, cosmids, YACs, BACs, PACs, artificial chromosomes or particles used for a molecular biology laboratory can be used as vectors

Particle bombardment are suitable.

BAC vectors are preferred. BAC (Bacterial Artificial Chromosome) vectors have been developed for cloning large DNA fragments (Shizuya et al., 1992). They are "single-copy" plasmids with an F-factor origin that

Can carry DNA fragments with an average size of 120 kilobase pairs (kb). They are replicable in Escherichia coli. The BAC vector pBeloBACl l (Kim et al., 1996) carries a T7 and an SP6 promoter, which flank the cloning site and can be used as a starting area for sequencing primers and for generating RNA transcripts.

Particularly preferred are those on August 18, 1999 at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, in accordance with the provisions of the Budapest Treaty under the deposit numbers DSM 13010, DSM 13011 and DSM 13012 deposited B AC shuttle clones, which are the subject of the present invention. The deposited BAC shuttle clones P11 / G6, P8 / G11 and P11 / B10 each carry a DNA fragment from S. spinosa that is at least 100 kb in size. Clones P11 / G6 and P11 / B10 each carry part of the nucleic acid sequence according to SEQ ID NO: 4, as well as the adjacent complete nucleic acid sequences according to SEQ ID NOS:

5 and 6, and a DNA region adjacent to the nucleotide sequence according to SEQ ID NO: 6 3 '(FIG. 7). The clone P8 / G11 carries part of the nucleic acid sequence according to SEQ ID NO: 6, the complete nucleic acid sequences according to SEQ ID NOS: 5 and 4, and a DNA region adjacent to the sequence according to SEQ ID NO: 4 3 '(Fig. 7).

In the same way, PAC and all other functionally equivalent vectors which allow large DNA fragments, in particular those DNA fragments which are larger than 30 kb, preferably larger than 40 kb, particularly preferably larger than 60 kb, are also in to transmit heterologous host cells and there one

To ensure the establishment of foreign DNA, suitable for a spinosyn production. Preferably, such BAC, PAC and functionally equivalent vectors are used which are modified into a shuttle vector and e.g. allow plasmid replication both in Gram-negative bacteria, such as Escherichia coli, and in Gram-positive bacteria, such as Streptomyces. Such preferred

Shuttle vectors can carry DNA fragments of a size that cannot be cloned in conventional vectors, such as, for example, cosmid vectors, and cannot be transferred to heterologous hosts, such as Actinomycetes, for example Streptomycetes. The latter vectors may both by transformation, conjugation, electroporation, protoplast transformation, or other suitable methods are transmitted ^•. Such shuttle vectors can be transferred conjugatively within a heterologous population of Gram-negative or Gram-positive bacteria, between Gram-positive and Gram-negative bacteria, between bacteria and Archea, between pro- and eukaryotes. The BAC, PAC or functionally equivalent shuttle transferred to heterologous hosts such as Streptomycetes

Vectors can be replicated autonomously or integrated into the host's genome become. The latter integration can take place via homologous recombination, via a ΦC31 integration mechanism (Hopwood et al., 1985), via site-specific integration which depends on functions determined by pSAM2 (Smokvina et al., 1990; WO 95/16046) or via mini-circle Functions (Motamedi et al., 1995; WO 96/00282) take place.

Such shuttle vectors allow specific biosynthetic pathways of primary or secondary metabolites, which are determined by extraordinarily large DNA regions, to be heterologously expressed in particularly suitable host cells by transfer of a single recombinant vector. Thus the identified cluster can be used for the biosynthesis of spinosyn in organisms such as Actinomycetes, e.g. Streptomyces, can be expressed by transfer of a single recombinant shuttle vector. Because of the size of this biosynthesis cluster, this heterologous expression of the spinosyn biosynthesis with a single cosmid vector is not possible. The transfer of a recombinant BAC, PAC or functionally equivalent shuttle vector which carries the nucleic acids according to the invention can lead to a significant increase in the production of spinosyn compared to the spinosyn production of the S. spinosa strain or derived mutants with increased spinosyn formation to lead. In addition, such a shuttle vector coding for the spinosyn biosynthesis can be used to transfer the biosynthesis and transfer into heterologous host cells

To use modification power to achieve a significant modification of spinosyn or spinosyn biosynthesis precursors. This also makes it possible to produce new spinosyn derivatives by transferring a single recombinant vector to heterologous host cells.

Such shuttle vectors can also be used to genetically modify cloned biosynthetic pathways of secondary metabolites as part of a single recombinant shuttle vector. Such modifications can be carried out, for example, in an E. coli host, for example using recombination events with the participation of the recA gene product or the recE and recT gene products (Muyrers et al., 1999). Such vectors can also be used through in vitro experience, such as modifying the template generation system (Finnzymes, FLN-02201, Espoo, Finland) or the transposomics system (Epicenter Technologies, Biozym Diagnostika GmbH, Oldendorf, Germany). Such shuttle vectors coding for altered biosynthetic pathways can then be transferred into suitable host cells in order to arrive at the production of altered secondary metabolites. In an analogous manner, the shuttle vectors mentioned can be used to modify the nucleic acids according to the invention, in order then to use them after transfer to suitable host cells to produce modified spinosyns.

Parts of the nucleic acids according to the invention can also be part of two or more vectors, e.g. Determine cosmid vectors, a genetic information which, in combination with one another, for the biosynthesis of spinosyn or spinosyn precursors, e.g. Pseudoaglycon or Spinosyn-Aglycon are suitable. Such combinations of recombinant vectors can be used to achieve spinosyn production in organisms other than S. spinosa. When expressed in particularly suitable hosts, this can lead to a significant increase in spinosyn production compared to S. spinosa or derived production-enhanced mutants. Furthermore, it is possible to change nucleic acids according to the invention in individual recombinant vectors of this vector combination such that heterologous production of spinosyn derivatives in host cells is possible. Furthermore, such a combination of recombinant vectors, by transferring them into heterologous hosts, can be suitable for the formation of new spinosyn derivatives using the host's own enzyme system.

The present invention also relates to host cells which contain at least one of the nucleic acids according to the invention. Both prokaryotic cells, preferably actinomycetes, particularly preferably streptomycetes, and eukaryotic cells such as mammalian cells, plant cells or yeast cells are suitable as host cells. The nucleic acids according to the invention can be transferred and expressed in plant cells in a special way. In this way, transgenic plants can be produced which produce the plant-protecting, insecticidal spinosyn or derivatives thereof. The nucleic acids according to the invention can be transferred into the plant cells or plant cell cultures using conventional methods, including particle bombardment.

The present invention furthermore relates to the polypeptides which are encoded by the nucleic acids according to the invention. The polypeptides according to the invention can represent a complete enzyme which comprises a step of the spinosyn-

Catalyzed biosynthesis. However, the invention also covers those polypeptides which have only part of the complete amino acid sequence of the enzyme in question.

The term "partial sequence" as used herein thus refers to

Amino acid sequence of a polypeptide that can still exert the activity of the corresponding complete enzyme or an enzymatically active domain.

Preferred nucleic acids and polypeptides according to the invention are characterized in more detail below with reference to the corresponding SEQ ID NOS.

SEQ ID NOS: 7 and 8, ORF1:

Nucleotide position 828 to 1 of SEQ ID NO: 4,275 amino acids; the derivable gene product is a methyl transferase.

SEQ ID NOS: 9 and 10, ORF2:

Nucleotide positions 1,283 to 2,455 of SEQ ID NO: 4,390 amino acids; the derivable gene product is a glycosyltransferase. SEQ ID NOS: 11 and 12, ORF3:

Nucleotide positions 2,495 to 3,247 of SEQ ID NO: 4,250 amino acids; the derivable gene product is a methyl transferase.

SEQ ID NOS: 13 and 14, ORF4:

Nucleotide positions 4,440 to 3,253 of SEQ ID NO: 4,395 amino acids; the derivable gene product is a methyl transferase.

SEQ ID NOS: 15 and 16, ORF5: nucleotide positions 4,578 to 6,197 of SEQ ID NO: 4,539 amino acids; the derivable gene product is a C-C linking enzyme that performs cyclization reactions.

SEQ ID NOS: 17 and 18, ORF6: nucleotide positions 6,211 to 7,404 of SEQ ID NO: 4,397 amino acids; the derivable gene product is a methyl transferase.

SEQ ID NOS: 19 and 20, ORF7:

Nucleotide positions 7,401 to 8,300 of SEQ ID NO: 4,299 amino acids; the derivable gene product is a methyl transferase.

SEQ ID NOS: 21 and 22, ORF8:

Nucleotide positions 8,300 to 9,466 of SEQ ID NO: 4,388 amino acids; the derivable gene product is an enzyme that is involved in cyclization reactions.

SEQ ID NOS: 23 and 24, ORF9:

Nucleotide positions 10,572 to 9,562 of SEQ ID NO: 4,336 amino acids. the derivable gene product is a 2,3-reductase. SEQ ID NOS: 25 and 26, ORF10:

Nucleotide positions 12,029 to 10,569 of SEQ ID NO: 4,486 amino acids; the derivable gene product is a 2,3-dehydratase.

SEQ ID NOS: 27 and 28, ORF11:

Nucleotide positions 12,549 to 12,109 of SEQ ID NO: 4, 146 amino acids; the derivable gene product has homologies to a thioesterase.

SEQ ID NOS: 29 and 30, ORF12: nucleotide positions 13,865 to 12,546 of SEQ ID NO: 4,439 amino acids; the derivable gene product is a glycosyltransferase.

SEQ ID NOS: 31 and 32, ORF13:

Nucleotide positions 14,245 to 15,633 of SEQ ID NO: 4,462 amino acids; the derivable gene product is a 3,4-dehydratase.

SEQ ID NOS: 33 and 34, ORF14:

Nucleotide positions 15,671 to 16828 of SEQ ID NO: 4,385 amino acids; the derivable gene product is a 4-aminotransferase.

SEQ DP NO: 35 and 36, ORF15:

Nucleotide positions 16,831 to 17,580 of SEQ ID NO: 4,249 amino acids; the derivable gene product is an N-dimethyltransferase.

SEQ LP NOS: 37 and 38, ORF16:

Nucleotide positions 18,930 to 18,205 of SEQ LD NO: 4,241 amino acids; the derivable gene product is a 3,4-reductase.

SEQ ID NOS: 39 and 40, ORF17: nucleotide position 19.025-19.861 of SEQ ID NO: 4,278 amino acids; the derivable gene product is a transcription regulator. SEQ ID NOS: 41 and 42, ORF18:

Nucleotide positions 116-7903 of SEQ ID NO: 5, amino acid positions 1 to

2595: nucleotide positions 128-1402, amino acid positions 5-429 encode a β-

KetoacyhAcyl Carrier Protein Synthase Domain;

Nucleotide positions 1691-2656, amino acid positions 526-847 encode one

Acyltransferase domain;

Nucleotide positions 2798-3052, amino acid positions 895-979 encode a β-ketoacyhacyl carrier protein domain;

Nucleotide positions 3107-4372, amino acid positions 998-1419 encode a β-

Ketoacyl: acyl carrier protein synthase domain;

Nucleotide positions 4688-5662, amino acid positions 1525-1849 encode one

Acyltransferase domain; Nucleotide positions 6587-7138, amino acid positions 2158-2341 encode one

Keto reductase domain;

Nucleotide positions 7409-7666, amino acid positions 2432-2517 encode a β-

Ketoacyl: acyl carrier protein domain.

SEQ ID NOS: 43 and 44, ORF19:

Nucleotide positions 7921-14379 of SEQ ID NO: 5, amino acid positions 1 to

2152:

Nucleotide positions 8029-9318, amino acid positions 37-466 encode a β-

KetoacyhAcyl Carrier Protein Synthase Domain; Nucleotide positions 9634-10608, amino acid positions 572-896 encode one

Acyltransferase domain;

Nucleotide positions 10705-11259, amino acid positions 929-1113 encode one

Dehydratase domain;

Nucleotide positions 12043-13080, amino acid positions 1375-1720 encode an enoyl reductase domain; Nucleotide positions 13093-13635, amino acid positions 1725-1905 encode one

Keto reductase domain;

Nucleotide positions 13885-14142, amino acid positions 1989-2074 encode a β-ketoacyl: acyl carrier protein domain.

SEQ ID NOS: 45 and 46, ORF20:

Nucleotide positions 14424-23936 of SEQ ID NO: 5, amino acid positions 1 to

3170:

Nucleotide positions 14523-15824, amino acid positions 34-467 encode a β-ketoacyl: acyl carrier protein synthase domain;

Nucleotide positions 16110-17075, amino acid positions 563-884 encode one

Acyltransferase domain;

Nucleotide positions 17997-18536, amino acid positions 1192-1371 encode one

Keto reductase domain; Nucleotide positions 18795-19052, amino acid positions 1458-1543 encode a β-ketoacyl: acyl carrier protein domain;

Nucleotide positions 19107-20387, amino acid positions 1562-1988 encode a β-ketoacyl: acyl carrier protein synthase domain;

Nucleotide positions 20718-21692, amino acid positions 2099-2423 encode an acyltransferase domain;

Nucleotide positions 22620-23171, amino acid positions 2733-2916 encode one

Keto reductase domain;

Nucleotide positions 23436-23693, amino acid positions 3005-3090 encode a β-ketoacyl: acyl carrier protein domain.

SEQ ID NOS: 47 and 48, ORF21:

Nucleotide positions 23983-38757 of SEQ ID NO: 5, amino acid positions 1 to

4924:

Nucleotide positions 24082-25392, amino acid positions 34-470 encode a β-ketoacyk acyl carrier protein synthase domain; Nucleotide positions 25696-26661, amino acid positions 572-893 encode one

Acyltransferase domain;

Nucleotide positions 26761-27315, amino acid positions 927-1111 encode one

Dehydratase domain; Nucleotide positions 28231-28782, amino acid positions 1417-1600 encode one

Keto reductase domain;

Nucleotide positions 29035-29265, amino acid positions 1685-1761 encode a β-ketoacyl: acyl carrier protein domain;

Nucleotide positions 29329-30624, amino acid positions 1783-2214 encode a β-ketoacyl: acyl carrier protein synthase domain;

Nucleotide positions 30928-31902, amino acid positions 2316-2640 encode one

Acyltransferase domain;

Nucleotide positions 32827-33378, amino acid positions 2949-3132 encode one

Keto reductase domain; Nucleotide positions 33652-33900, amino acid positions 3224-3306 encode a β-ketoacyl: acyl carrier protein domain;

Nucleotide positions 33952-35262, amino acid positions 3324-3760 encode a β-ketoacyl: acyl carrier protein synthase domain;

Nucleotide positions 35554-36522, amino acid positions 3858-4180 encode an acyltransferase domain;

Nucleotide positions 37453-37998, amino acid positions 4491-4672 encode one

Keto reductase domain;

Nucleotide positions 38254-38511, amino acid positions 4758-4843 encode a β-ketoacyl: acyl carrier protein domain.

SEQ ID NOS: 49 and 50, ORF22:

Nucleotide positions 38808-50000 of SEQ ID NO: 5 and nucleotide positions 1 to 5574 of SEQ ID NO: 6, amino acid positions 1 to 5588:

Nucleotide positions 38907-40226 of SEQ ID NO: 5, amino acid positions 34-473 encode a β-ketoacyl: acyl carrier protein synthase domain; Nucleotide positions 40494-41453 of SEQ ID NO: 5, amino acid positions 563-

882 encode an acyltransferase domain;

Nucleotide positions 41556-42119 of SEQ LD NO: 5, amino acid positions 917-

1104 encode a dehydratase domain; Nucleotide positions 43017-43568 of SEQ ID NO: 5, amino acid positions 1404-

1587 encode a ketoreductase domain;

Nucleotide positions 43833-44090 of SEQ ID NO: 5, amino acid positions 1676-

1761 encode a ß-ketoacyl: acyl carrier protein domain;

Nucleotide positions 44151-45473 of SEQ JO NO: 5, amino acid positions 1782-2222 encode a β-ketoacyl: acyl carrier protein synthase domain;

Nucleotide positions 45765-46730 of SEQ ID NO: 5, amino acid positions 2320-

2641 encode an acyltransferase domain;

Nucleotide positions 46827-47459 of SEQ ID NO: 5, amino acid positions 2674-

2884 encode a dehydratase domain; Nucleotide positions 48378-48935 of SEQ ID NO: 5, amino acid positions 3191-

3376 encode a ketoreductase domain;

Nucleotide positions 49182-49412 of SEQ ID NO: 5, amino acid positions 3459-

3535 encode a β-ketoacyl: acyl carrier protein domain;

Nucleotide positions 49482-50000 of SEQ ID NO: 5 and nucleotide positions 1 to 759 of SEQ ID NO: 6, amino acid positions 3559-3984 encode a β-

KetoacykAcyl Carrier Protein Synthase Domain;

Nucleotide positions 1084-2049 of SEQ ID NO: 6, amino acid positions 4093-

4414 encode an acyltransferase domain;

Nucleotide positions 2146-2697 of SEQ ID NO: 6, amino acid positions 4447-4630 encode a dehydratase domain;

Nucleotide positions 3604-4155 of SEQ ID NO: 6, amino acid positions 4933-

5116 encode a ketoreductase domain;

Nucleotide positions 4420-4677 of SEQ ID NO: 6, amino acid positions 5205-

5290 encode a β-ketoacyl: acyl carrier protein domain; Nucleotide positions 4864-5538 of SEQ ID NO: 6, amino acid positions 5353-

5577 encode a thioesterase domain. SEQ ID NOS: 52 and 53, ORF23:

Nucleotide positions 344 to 1333 of SEQ ID NO: 51, 329 amino acids; the derivable gene product is a dNDP-glucose-4,6-dehydratase.

SEQ ID NOS: 54 and 55, ORF24:

Nucleotide positions 1330 to 2247 of SEQ ID NO: 51, 305 amino acids; the derivable gene product is a dNDP-4-keto-6-deoxyglucose-3,5-epimerase.

The products of the ORF 5 (SEQ

ID NO: 16) and ORF 8 (SEQ JD NO: 22) are of particular interest due to the unusual cyclization reactions. The present invention therefore also includes, in particular, homologous nucleic acids or homologous gene products. These homologous gene products preferably show at least a 50%, preferably a 60% and particularly preferably a 70% identity at the amino acid level.

The invention furthermore relates to antibodies which bind specifically to the abovementioned polypeptides. Such antibodies are produced in the usual way. These antibodies can be used to express clones e.g. to identify a gene bank carrying the nucleic acids according to the invention.

The present invention also relates to methods for producing the nucleic acids according to the invention. The nucleic acids according to the invention can be prepared in the usual way. For example, the nucleic acid molecules can be completely chemically synthesized. It is also possible to chemically synthesize short pieces of the nucleic acids according to the invention and to label such oligonucleotides radioactively or with a fluorescent dye. The labeled oligonucleotides can also be used to generate libraries of

Search organisms. Clones to which the labeled oligonucleotides hybridize are selected to isolate the DNA in question. After characterizing the isolated DNA, the nucleic acids according to the invention are obtained in a simple manner. The nucleic acids according to the invention can also be produced by means of PCR methods using chemically synthetic oligonucleotides.

The present invention furthermore relates to processes for producing the polypeptides according to the invention. To produce the polypeptides which are encoded by the nucleic acids according to the invention, host cells which contain at least one of the nucleic acids according to the invention can be cultivated under suitable conditions. The desired polypeptides can then be isolated from the cells or the culture medium in a conventional manner. The polypeptides can also be produced in in vz ^' tro systems.

The isolated and characterized gene cluster and neighboring or associated DNA

Regions represent a target for increasing spinosyn biosynthesis through genetic manipulation, over- or under-expression of genes or regulatory sequences directly or indirectly involved in biosynthesis. These manipulations can occur both in natural spinosyn-producing organisms and in genetically engineered spinosyn-producing organisms Organisms are carried out. For example, selected ORFs can be placed under the control of customary strong promoters such as the me / promoter of the plasmid pIJ702 (John Innes Foundation, Norwich, UK, 1985).

By cloning and identifying the spinosyn biosynthesis genes, the present invention creates the genetic basis for producing new spinosyn precursors and derivatives by means of molecular genetic methods.

The term "spinosyn precursors" as used herein refers to all detectable and all postulated spinosyn precursors. The term "spinosyn derivatives", as used herein, refers to structural derivatives of all previously known spinosyns.

The present invention thus also relates to a method for producing spinosyn precursors and derivatives.

The nucleic acids according to the invention can be used, for example, to produce new spinosyn derivatives with changes in the spinosyn aglycone by combinatorial biosynthesis. This can be achieved, for example, by replacing the acyltransferase domain encoded by ORF 19 and incorporating an acetate unit with an acyltransferase domain incorporating a propionate unit. In the same way, the acyltransferase domain of the ORF 18 incorporating an acetate unit can be exchanged for an acyltransferase domain incorporating a propionate unit. It is furthermore possible to inactivate or delete both or in each case one ketoreductase domain which is encoded by the two ORFs mentioned by an inactive ketoreductase domain, as a result of which a hydroxyl group at the corresponding position in the macrocycle can be produced biosynthetically. All acyltransferase, ketoreductase, dehydratase, enoyl reductase, ß-ketoacyl: acyl carrier protein and thioesterase domains can be replaced individually or in any combination by appropriate polyketide synthase domains with a different substrate or reaction specificity, in any combination with one another fused, mutagenized, deleted or duplicated individually or in any combination. Module-coding sequences can also be exchanged. It is conceivable that the DNA sequence coding module 2 (Fig. 6) is against the DNA sequence coding module 1 or module 3, 4, 5, 6, 7, 8 or module 9 (Fig. 6) replace and express functionally. It is also conceivable that the module 2-coding DNA sequence or any other module-coding DNA sequence of the spinosyn-polyketide synthase gene cluster against another module-coding DNA sequence of the spinosyn-polyketide synthase gene cluster, the other built-in biosynthetic extension unit. In addition, any other module coding DNA Sequence of the Spinosyn polyketide synthase gene cluster can be exchanged for a different module coding DNA sequence of a different polyketide synthase nucleic acid sequence from S. spinosa or an organism other than S. spinosa, such as Saccharopolyspora erythraea. These changes can be carried out using ET recombination (WO 99/29837; Muyrers et al., 1999) or other cloning and recombination techniques.

The invention thus also relates to all nucleic acids coding for modules or domains which are a natural or genetically engineered component of spinosyn polyketide synthase.

The term "module" as used herein means that there is an arrangement of three enzyme-catalytically active domains which lead to an extension of the growing polyketide chain by a biosynthetic extension unit. These domains are a ß-ketoacyl: acyl carrier protein

Synthase domain, an acyltransferase domain and a β-ketoacyl: acyl-Caπier protein domain. A module can also carry a ketoreductase, a dehydratase, an enoyl reductase and a thioesterase domain. A so-called charge module, which is at the beginning of biosynthesis, can only carry an acyltransferase domain and a β-ketoacyl: acyl carrier protein domain from the domains mentioned, and also an enzymatically inactive β-ketoacyl: acyl carrier protein synthase domain. A polyketide synthase domain each comprises one of these enzymatic activities.

The nucleic acids of the invention can also be used to

In the course of combinatorial biosynthesis by rearrangement and expression of spinosyn polyketide synthase nucleic acid sequences or by combination and expression together with nucleotide acid sequence encoding polyketide synthase nucleic acid sequences from another polyketide synthase from S. spinosa or another organism, such as, for example, Saccharopolyspora erythaea, libraries of recombinant nucleotide sequences recombinant polyketide synthase proteins or recombinantly produced polyketides. These polyketides can be glycosylated by using the nucleic acids according to the invention or by using other nucleic acids, the derivable products of which are involved in the biosynthesis of other sugars and coupling to the aglycon. Glycosylation of the aglycone is known to be crucial

Plays a role in the biological activity at the site of action. These changes can occur both in natural and in genetically engineered spinosyn-producing organisms, in particular bacteria. Furthermore, these changes can be carried out using ET recombination (WO 99/29837; Muyrers et al., 1999) or other cloning and recombination techniques.

The nucleic acids, vectors and regulatory or genetically mobile regions according to the invention can also be used to find genes which code for polypeptides which code functionally similar polyketide synthases or functionally similar products which are involved in sugar biosynthesis.

Since the nucleic acids according to the invention make up a large part of the S. spinosa genome, the nucleic acids according to the invention can be used as markers in the sequencing of the S. spinosa genome, which considerably facilitates the arrangement of partial sequences of a genome sequencing project.

Thus, the nucleic acids according to the invention provide data which are within the scope of a

Genome sequencing project and a metabolic engineering based on it can be used to increase spinosyn production. Explanations of the pictures;

Figure 1: Model for the biosynthesis of the spinosyn sugar D-forosamine and 2, 3, 4-tri-O-methyl-L-rhamnose.

Figure 2: Location of DNA regions 1 (SEQ ID NO: 4) and DNA region 2 (SEQ ED NOS: 5 and 6) directly or indirectly involved in spinosyn biosynthesis. The black bars in the lower part of the figure schematically indicate the positions of the cosmid DNA inserts relative to each other and in relation to DNA regions 1 and 2. The cosmid inserts shown were used for the sequencing of SEQ ID NOS: 1 to 3.

Figure 3: Schematic representation of the position of the insert DNA (black bars in the lower part of the figure) of the named cosmids, which were used to couple a forosamine residue or a trimethyl rhamnose residue by biotransformation of the spinosyn aglycone and spinosyn pseudoaglycone are.

Figure 4: Schematic representation of the open reading frames (ORF 's) of DNA region 3, which corresponds to SEQ ID NO: 51, on Cosmid 16-2-2.

Figure 5: Schematic representation of open reading frames (ORF 's) of DNA regions 1 and 2. The ORFs are numbered from 1 to 22 according to SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49.

Figure 6: Schematic representation of open reading frames (ORF 's) of DNA region 2 (SEQ ED NOS: 5 and 6) and derivable modules and domains. SM, start module; Ml to MIO, module 1 to module 10; KS, β-ketoacyl: acyl carrier protein synthase; AT, acyltransferase; ACP, β-ketoacyl: acyl carrier protein; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; TE, thioesterase. Figure 7: Schematic representation of the position of BAC shuttle clone insert DNA as black bars in the lower part of the figure. The size of the insert DNA is at least 100 kb. Solid bars: DNA sequence is identical to parts of DNA region 1 and all of DNA region 2 (P11 / G6 and Pl I / BlO) or with all of DNA region 1 and parts of DNA region 2 (P8 / G11). Dashed bars: DNA sequence is outside the sequenced area.

Examples

Bacterial strains and plasmids

Escherichia coli XLl-Blue MRF 'and the cosmid vectors SuperCosl (Stratagene, Europe) and pOJ446 (Biermann et al., 1992) were used to create

S. spinosa gene banks ATCC49460 (American Type Culture Collection, U.S.A., EP-A 0 375 316). E. coli JM110 (Stratagene, Europe) was used to propagate plasmids which were transferred to Streptomyces by transformation. Streptomyces albus J1074 (Chater and Wilde, 1980; John Innes Institute in Norwich, UK) was used for the heterologous expression of and for the biotransformation

Spinosyn biosynthetic genes used.

Plasmid pBeloBACl 1 (Kim et al., 1996) and pOJ446 (Biermann et al, 1992) were used to produce an E. coli - Streptomyces BAC shuttle vector.

Molecular biological methods

Molecular biological methods such as DNA restriction, agarose gel electrophoresis of DNA, ligation of restriction fragments, cultivation and transformation of E. coli were carried out as described in Sambrook et. al (1989).

Plasmids were isolated using the Qiagen Plasmid Kit (Qiagen, Hilden, Germany). The enzymes used came from Röche Diagnostics GmbH (Mannheim, Germany).

Cultivation conditions and molecular genetic methods with S. spinosa and

Streptomycetes are described in (Hopwood et al., 1985). All liquid cultures of S. spinosa or Streptomycetes were grown aerobically in Erlenmeyer flasks at 28 ° C. The DNA-DNA hybridizations were carried out using the DIG-High Prime DNA Labeling and Detection Kit according to the manufacturer (Röche Diagnostics GmbH, Mannheim, Germany).

Growth media:

LB Sambrook et. al., 1989

TS Difco order number 0 370-17-3 (Difco Detroit, MI, USA)

R5A Illing et al., 1985

Production of a Cosmid gene bank from S. spinosa

To obtain a gene bank from S. spinosa, chromosomal DNA from S. spinosa ATCC49460 was partially cut with Mbol and separated by centrifugation in a glucose density gradient. According to the manufacturer, the cosmid DNA (SuperCosl, Stratagene Europe) was prepared, ligated to the S. spinosa DNA fragments between 35 and 45 kb and packed into phage particles using the Gigapack packaging system (Stratagene Europe). The transfection took place in

E. coli XL-1 blue MRF '. This method was also used to create a second S. spinosa library using the E. coli Streptomyces Shuttle Cosmids pOJ446.

Sequencing of the spinosyn biosynthesis gene cluster and a DNA fragment that lies outside this cluster, but whose products are involved in the biosynthesis of spinosyn

The insert DNA of the SuperCosl Cosmide 16-1-8, 16-59-1, and 16-59-8 were sequenced. An approximately 4 kb gap between cosmids 16-59-1 and 16-1-8 was closed by pimer walking sequencing a corresponding portion of Cosmid 16-59-6.

An approximately 2.3 kb DNA sequence on the SuperCosl Cosmid 16-2-2 was sequenced.

Identification and characterization of chromosomal DNA fragments from a BAC shuttle vector gene bank from S. spinosa. that carry spinosyn biosynthetic gene sequences

To generate the BAC shuttle vector, which can be transmitted and propagated not only in E. coli but also in actinomycetes such as Streptomyces, the vector pBeloBACl 1 was linearized with Xhol and smooth DNA ends were produced using Klenow polymerase. An approximately 6 kb Dral - EcoRV DNA fragment of the cosmid vector pOJ446, which carries the replication origin of the plasmid SCP2 *, the apramycin resistance gene and the oriT for conjugative transfer, was ligated with the linearized BAC vector. The resulting vector was named pΕBZ333.

A BAC gene library was created on the basis of genomic DNA of the strain S. spinosa ATCC49460 partially cut with Mbol and the vector pEBZ333 cut with BamΑI.

Analysis and annotation of open reading frames directly or indirectly of DNA sequences involved in the spinosyn biosynthesis

Starting from the sequence according to SEQ JD NOS: 1 to 3, open reading frames (ORF 's) were identified that are directly or indirectly involved in the biosynthesis of spinosyn. These ORFs were divided into two DNA regions, which are designated as DNA region 1 and DNA region 2 (FIGS. 2 and 5) and which

Wear sequences according to SEQ ID NO: 4 or 5 and 6. The DNA region 1 carries open reading frames, the products of which are involved in the modification and tricyclization of the spinosyn aglycone, while DNA region 2 (FIGS. 2, 5 and 6) comprises open reading frames, the products of which encode the spinosyn polyketide synthase. The two first nucleotides of each of these DNA regions lie directly next to each other (Fig. 2, 3 and 5).

Another DNA region 3 (SEQ ID NO: 51) lies outside of this cluster of DNA sequences and carries open reading frames, the products of which are also involved in the biosynthesis of the spinosyn sugar trimethyl rhamnose.

Production of the Spinosyn aglycone and 17-pseudoaglycone from Tracer ^®

Starting from 18.7 g of the commercially available product Tracer ^® , after freeze drying and column chromatography on silica gel, 8.92 g of Spinosyn A and D were obtained in a ratio of 82:18.

The hydrolysis of the amino sugar forosamine was achieved under reflux with 2.7 N sulfuric acid in ethanol. The majority of the resulting 17-pseudo-aglycon from Spinosyn A / D failed. In addition to additional 17-pseudoaglycon, small to medium amounts of the spinosyn-

Aglycons found.

Complete hydrolysis to the aglycone was achieved under somewhat more drastic conditions (7.2 N sulfuric acid in methanol under reflux). The Aglycon fraction contained only Aginoscon from Spinosyn A. This is very good with the

Literature agree (Creemer et al., 1998), which describes a complete decomposition of the pseudoaglycone of Spinosyn D under appropriate reaction conditions. The authors assume that the protonation of the 5,6 double bond in Spinosyn D is easier, with the formation of a tertiary carbocation and subsequent rearrangements. Thus, starting from 18.7 g of commercially available tracer, 3.0 g of Spinosyn A aglycon could be produced.

Tracer (Dow AgroScience) 18.7 g

Freeze-drying

gray solid 10.0 g

Spinosyn A / D (A, R = H, 82%; D, R = CH ₃ , 18%)

8.92 g

2.7 NH ₂ S0 ₄ / EtOH 3 h reflux

69% 16%

17-pseudo aglycon from Spinosyn A / D aglycon from Spinosyn A

(A, R = H, 82%; D, R = CH ₃ , 18%)

7.2 NH ₂ S0 ₄ / MeOH 3 h reflux Extraction of Spinosyn A / D from Tracer ^®

Freeze drying of 18.7 g tracer gave 10.0 g gray solid. After column chromatography of this solid on 800 cm ^{3 of} silica gel (eluent: dichloromethane / methanol 95: 5), 8.92 g of pure Spinosyn A / D (82% A, 18% D) were obtained.

TLC: R _{ (SiO ₂ , dichloromethane / methanol 9: 1) = 0.46. - 1H NMR: CDC1 ₃ , δ = 6.77 (s, 13-H); 5.88 (d, 5-H from Spinosyn A); 5.80 (m, 6-H from Spinosyn A); 5.49 (m, 5-H from Spinosyn D); 4.87 (d, l '-H); 4.67 (m, 21-H); 4.43 (d, 1 "-H); 4.31 (m, 9-H) and others - LC / MS: electrospray positive; peak at RT 44.0 min: m / z = 733 (100%) [M + H] ⁺ (Spinosyn A); peak at 44.7 min: mz = 747 (100%) [M + H] ⁺ (Spinosyn D).

Representation of the 17-pseudo-aglycon by Spinosyn A / D:

8.65 g (11.81 mmoi) of Spinosyn A / D were dissolved in 61 ml of ethanol and mixed with 104 ml of water and 208 ml of 4 NH ₂ SO ₄ . After heating under reflux for 3 h, the precipitated solid (A) was filtered off and worked up separately from the filtrate (B). The solid (A) was washed with 1 NH ₂ SO ₄ , taken up in 140 ml dichloromethane, successively with sat. NaHCO solution and sat. Washed NaCl solution, dried over Na ₂ SO ₄ and concentrated in vacuo. Recrystallization from ethanol gave 3.03 g of 17-pseudoaglycon from Spinosyn A / D and mother liquor (C).

The filtrate (B) was extracted several times with dichloromethane. The extracts were washed with sat. NaHCO solution and sat. Washed NaCl solution, dried over Na ₂ SO ₄ and concentrated in vacuo. The residue was combined with the mother liquor (C), which was concentrated in vacuo, and separated by column chromatography on 650 cm ^{3 of} silica gel (eluent: cyclohexane / ethyl acetate 1: 1, then 100% ethyl acetate). In addition to a further 1.76 g of 17-pseudoaglycon from Spinosyn A / D, 0.78 g (16%) of aglycon from Spinosyn A was obtained. The overall yield of 17-pseudoaglycon from Spinosyn A / D was 4.79 g (69%). - a) 17-pseudoaglycon from Spinosyn AD (82% A, 18% D): TLC: R _{ (SiO ₂ , ethyl acetate) = 0.48. 1 H NMR: CDC1 ₃ , δ = 6.78 (s, 13-H); 5.88 (d, 5-H from Spinosyn A); 5.80 (m, 6-H from Spinosyn A); 5.49 (m, 5-H from Spinosyn D); 4.86 (d, l'-H); 4.70 (m, 21-H); 4.32 (m, 9-H) inter alia - LC / MS: electrospray positive; Peak at RT 40.7 min: m / z = 609 (100%) [M + NH ₄ ] ⁺ , m / z = 641 (10%) [M + NH., + CH ₃ OH] ⁺ (pseudoaglycon from Spinosyn A); Peak at RT 41.4 min: m / z = 623 (100%) [M + NIL ^, m / z = 655 (8%) [M + NH ₄ + CH ₃ OH] ⁺ (pseudoaglycon from Spinosyn D). - b) Spinosyn A aglycon: TLC: R _{ (SiO ₂ , ethyl acetate) = 0.29. - 1H NMR: CDC1 ₃ , δ = 6.80

(s, 13-H); 5.89 (d, 5-H); 5.80 (m, 6-H); 4.70 (m, 21-H); 4.44 (m, 9-H) inter alia - LC / MS: electrospray positive; Peak at RT 36.8 min: m / z = 420 (100%) [M + NIL;] *, m / z = 452 (10%) [M + NH ₄ + CH ₃ OH] ⁺ .

Representation of the Aglycon by Spinosyn A / D

4.30 g (7.29 mmol) of pseudoaglycon from Spinosyn A / D were dissolved in 190 ml of methanol and 285 ml of 7.2 NH ₂ SO _{4 were} added. After heating under reflux for 3 h, the cooled reaction mixture was carefully added to 1700 ml of saturated NaHCO ₃ solution. It was extracted with diethyl ether, the extracts were washed successively with sat. NaHCO ₃ solution and sat. NaCl solution, dried over Na ₂ SO ₄ and concentrated in vacuo. After column chromatography of this solid on 650 cm of silica gel (eluent: cyclohexane / ethyl acetate 1: 2, then 100% ethyl acetate), 1.88 g (64%) of aglycone from Spinosyn A. - TLC: i? _f (SiO ₂ , ethyl acetate) = 0.29. - 1H NMR: CDC1 ₃ , δ = 6.80 (s, 13-H); 5.89 (d, 5-H);

5.80 (m, 6-H); 4.70 (m, 21-H); 4.44 (m, 9-H) inter alia - LC / MS: electrospray positive; Peak at RT 36.6 min: m / z = 420 (100%) [M + NEU, mz = 452 (14%) [M + NH + CH ₃ OH] ⁺ (aglycon from Spinosyn A).

Forosaminylation of the spinosyn aglycone and attachment of a trimethyl

Rhamnose sugar to the spinosyn aglycon by biotransformation with a recombinant Streptomyces strain which heterologously expresses spinosyn sugar biosvthese genes

20 ml of R5A medium (Illing et al., 1989) with 5 μg apramycin / ml was inoculated with mycelium from the recombinant strain S. albus (165-1) or S. albus (165-8) and Incubated aerobically for 24 h at 28 ° C. This culture was 50 ug / ml of the produced spinosyn aglycone (100 ul of a 1% stock solution in methanol; preparation see section "preparation of the spinosyn aglycone and 17-pseudoaglycone of tracer ^®", "recovery of Spinosyn A / D from Tracer ^® "and" Representation of the Aglycon from Spinosyn A / D ") were added and incubated aerobically for approx. 120 h at 28 ° C. As

Control was cultivated in the same way S. albus (pEBZ340; pOJ446 vector with a DNA fragment from Cosmid 16-1-8 carrying about 1.8 kb large Spinosyn PKS) and spinosyn aglycon was added. After incubation, the cultures were centrifuged to separate cell mycelium and 25 ml of methanol were added to the supernatant (20 ml).

35 ml of the culture supernatant mixed with methanol were lyophilized, taken up with 15 ml of water and extracted twice with 10 ml of ethyl acetate. The combined organic phases were evaporated to dryness and taken up in 350 μl of methanol. An aliquot of these extracts was examined using LC / MS with electrospray positive ionization.

The culture supernatant from the cultivation of S. albus (165-1) contained a compound with the molecular weight of a forosaminylated aglycone of Spinosyn A and Spinosyn A.

Peak 1: RT = 41.0 min: m / z = 544 (100%) [M + H] ⁺ , m / z = 576 (16%) [M + H + CH ₃ OH] ⁺ (forosaminylated aglycon from Spinosyn A); LC / MS / MS: m / z = 142 (38%) (forosamine fragment).

Forosaminylated aglycon of Spii riosyn A molecular weight = 543 empirical formula = C ₃₂ H _4g N0 ₆

Peak 2: RT = 44.2 min: m / z = 733 (100%) [M + H] ⁺ (Spinosyn A); LC / MS / MS: m / z = 142 (21%) (forosamine fragment).

The culture supernatant from S. albus (165-8) contained a compound with the molecular weight of a forosaminylated aglycone from Spinosyn A. Peak 1: RT = 40.9 min: m / z = 544 (100%) [M + H] ⁺ , m / z = 576 (16%) [M + H + CH ₃ OH] ⁺ (forosaminylated aglycon from Spinosyn A); LC / MS / MS: mz = 142 (39%) (forosamine fragment).

The culture supernatant from S. albus (pEBZ340) contained no compounds with MW 543 and no Spinosyn A.

Forosaminylation of the spinosyn-17 pseudoaglycone by biotransformation with a recombinant Streptomyces strain which heterologously expresses spinosyn sugar biosynthesis genes

20 ml of R5A medium (Illing et al., 1989) with 5 μg apramycin / ml was inoculated with mycelium from the recombinant strain S. albus (165-1) or S. albus (165-8) and

Incubated aerobically for 24 h at 28 ° C. This culture was 50 ug / ml of the prepared 17-pseudoaglycone of spinosyn (100 ul of a 1% stock solution in methanol; preparation see section "preparation of the spinosyn aglycone and 17-pseudoaglycone of tracer ^®", "recovery of Spinosyn A / D from Tracer ^® "and" Dar- position of the 17-pseudoaglycon from Spinosyn A / D ") and incubated aerobically for about 120 h at 28 ° C. After incubation, the cultures were centrifuged to separate cell mycelium and the supernatant (20 ml) was mixed with 25 ml of methanol.

The culture supernatant from S. albus (165-1) contained traces of Spinosyn A and D. Peak 1: RT = 44.2 min: mz = 733 (100%) [M + H] ⁺ (Spinosyn A); LC / MS / MS: m / z = 142 (8%) (forosamine fragment). Peak 2: RT = 44.7 min: m / z = 747 (100%) [M + H] ⁺ (Spinosyn D); LC / MS / MS: m / z = 142 (37%) (forosamine fragment).

The culture supernatant from S. albus (165-8) contained traces of Spinosyn A and D. Peak 1: RT = 44.1 min: m / z = 733 (100%) [M + H] ⁺ (Spinosyn A). Peak 2: RT = 44.7 min: m / zz = 747 (100%) [M + H] ⁺ (Spinosyn D).

Deposit of microorganisms

The following microorganisms and plasmids are at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg lb, D-38124

Braunschweig, in accordance with the provisions of the Budapest Treaty. Microorganism and plasmid deposit date number

E. coli XLl-Blue MRF'mit Cosmid 16-1-8 DSM 12961 1999-08-02 E. coli XL 1 -blue MRF'mit Cosmid 16-2-2 DSM 12962 1999-08-02

E. coli XLl-Blue MRF 'with Cosmid 16-59-1 DSM 12963 1999-08-02

E. coli XLl-Blue MRF 'with Cosmid 16-59-6 DSM 12964 1999-08-02

E. coli XLl-Blue MRF 'with Cosmid 16-59-8 DSM 12965 1999-08-02

E. coli XLl-Blue MRF'mit Cosmid 165-1 DSM 13005 1999-08-18 E. coli XLl-Blue MRF'mit Cosmid 165-8 DSM 13007 1999-08-18

E. coli DH10B with the BAC shuttle clone P8 / Gl 1 DSM 13012 1999-08-18

E. coli DH10B with the BAC shuttle clone Pl 1 / BIO DSM 13011 1999-08-18

E. coli DH10B with the BAC shuttle clone Pl 1 / G6 DSM 13010 1999-08-18

literature

Biermann M., Logan R., O'Brien K, Seno E.T., Nagaraja R., Schoner B.E. (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116: 43-49

Chater K., Wilde L. (1980) Streptomyces albus G Mutants defective in the SalGl restriction-modification system. J. Gen. Microbiol. 116: 323-334.

Devereux J., Haeberli P., Smithies (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12: 387-395.

Mertz F., Yao R.C. (1990) Saccharopolyspora spinosa sp. nov. isolated from soil collected in a sugar mill rum still. Int. J. Syst. Bacteriol. 40: 34-39.

Creemer LC, Kirst HA, Paschal JW (1998) Conversion of spinosyn A and spinosyn D to their respective 9- and 17-pseudoaglycones and their aglycones. J. Antibiotics 51: 795-800. Fishman SE, Hershberger CL (1983) Amplified DNA in Streptomyces fr adiae. J. Bacteriol. 155: 459-466.

Hopwood DA, Bibb MJ, Chater KF, Kieser T., Bruton CJ, Kieser HM, Lydiate DJ., Smith CP, Ward JM, Schrempf H. (1985) Gentic manipulation of streptomyces a laboratory manual, The John Innes Foundation, Norwich, 1985th

Illing G.T., Normansell I.D., Peberdy J.F. (1989) Protoplast isolation and regeneration in Streptomyces clavuligerus. J. Gen. Microbiol. 135: 2289-2297.

Kim U.J. Birren B.W. Slepak T. Mancino: V. Boysen C. Kang H.L. Simon MI. Shizuya H. (1996), Construction and characterization of a human bacterial artificial chromosome library. Genomics 34: 213-218.

Kirst H.A., Michel K.H., Martin J.W., Creemer L.C., Chao E.H., Yao R.C.,

Nakatsukasa W.M., Boeck L.D., Occolowitzh J.L., Paschal J.W., Deeter J.B., Jones N.D., Thompson GD. (1991) Tetrahedron Lext. 32: 4839-4842.

Motamedi H., Shafiee A., Sheng-Jian C. (1995) Integrative vectors for heterologous gene expression in Streptomyces spp. Gene 160: 25-31.

Muyrers J.P.P., Zhang Y., Testa G., Stewart A.F. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Research 27: 1555-1557.

Nakatsukasa WM, Mabe JA, Michel KH, Martin JW, Paschal JW, Elzey TK (1990) Abstracts of 2 ^nd Int. Conf. on Biotechnology of Microbial Prod .: P-21.

Sambrook J., Fritsch EF, Maniatis T. (1989) Molecular cloning - A laboratory manual. Second edition. Cold Spring Harbor Laboratory Press. Shizuya H., Birren B., Kim U.-J., Mancino V., Slepak T., Tachiiri Y., Simon MI (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Be. USA 89: 8794-8797.

Smokvina T., Mazodier P., Boccard F., Thompson C.J., Guerineau M. (1990) Construction of a series of pSAM2-based integrative vectors for use in actinomycetes. Gene 94: 53-59.

Claims

claims

1. Nucleic acid which comprises at least one region which codes for an enzyme activity which is involved in the biosynthesis of spinosyns.

2. Nucleic acid according to claim 1, characterized in that it is single-stranded or double-stranded DNA or RNA.

3. Nucleic acid according to claim 2, characterized in that it is a DNA fragment.

4. Nucleic acid according to claim 3, characterized in that it comprises all regions coding for enzyme activities which are involved in the biosynthesis of spinosyns.

5. Nucleic acid according to one of claims 1 to 4, characterized in that it is enzyme activities of polyketide synthases, methyl transferases, glycosyl transferases, epimerases, aminotransferases, dimethyl transferases, reductases, dehydratases and / or cyclization enzymes.

6. Nucleic acid according to one of claims 1 to 5, characterized in that it comes from Saccharopolyspora spinosa.

7. The nucleic acid according to claim 1, comprising at least one sequence selected from

(a) the sequences according to SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 52 or 54, (b) at least 14 base pairs long partial sequences of the sequences defined under (a),

(c) Sequences which hybridize to the sequences defined under (a)

(d) sequences which have at least 70% identity to the sequences defined under (a),

(e) sequences which are complementary to the sequences defined under (a), and

(g) Sequences which, owing to the degeneracy of the genetic code, code for the same amino acid sequence as the sequences defined under (a) to (d).

8. Nucleic acid according to claim 7, characterized in that it comprises the sequence according to SEQ ID NOS: 1 to 6.

9. Nucleic acid according to claim 7, characterized in that it is the

Sequence according to SEQ ID NO: 4 comprises.

10. Nucleic acid according to claim 7, characterized in that it comprises the sequence according to SEQ ID NOS: 5 and 6.

11. Nucleic acid according to claim 7, characterized in that it has at least one sequence according to SEQ ID NOS: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39 includes.

12. Nucleic acid according to claim 7, characterized in that it comprises at least one sequence according to SEQ JD NOS: 41, 43, 45, 47 or 49.

13. Regulatory region which controls the transcription of a nucleic acid according to one of claims 1 to 7 in Saccharopolyspora spinosa.

14. DNA construct comprising a nucleic acid according to one of claims 1 to 12 and at least one heterologous promoter.

15. Vector comprising at least one nucleic acid according to one of claims 1 to 12, a regulatory region according to claim 13 or a DNA construct according to claim 14.

16. Vector according to claim 15, characterized in that the nucleic acid is functionally linked to regulatory sequences which ensure the expression of the coding regions of the nucleic acid in pro- or eukaryotic cells.

17. Vector according to claim 15 or 16, characterized in that it is a BAC vector, PAC vector or a vector which is functionally equivalent to BAC or PAC vectors.

18. Vector according to claim 17, characterized in that it is a vector which corresponds to the BAC clones with the deposit numbers DSM 13010, DSM 13011 or DSM 13012.

19. Vector according to one of claims 15 to 18, characterized in that it is a shuttle vector which can be transmitted both in prokaryotes and in eukaryotes.

20. Vector according to one of claims 15 to 19, characterized in that it is a shuttle vector that is both in Gram-negative and in

Gram-positive bacteria is also transferable to Archea.

21. Vector according to one of claims 15 to 19, characterized in that it is a shuttle nector which can be transmitted both in Escherichia coli and in Actinomycetes.

22. Vector according to claim 21, characterized in that it is a shuttle vector which can be transmitted both in Escherichia coli and in Streptomyces.

23. Vector according to one of claims 15 to 22, characterized in that it can be replicated autonomously in a prokaryote.

24. Vector according to one of claims 15 to 22, characterized in that it can be integrated into the genome in a prokaryote with the participation of the phage ΦC31, pSAM2 or the mini-circle integration mechanism.

25. Vector according to one of claims 15 to 22, characterized in that it can be integrated into the genome in a prokaryote by RecA-mediated recombination.

26. Vector according to one of claims 15 to 22, characterized in that it can be integrated into the genome in a prokaryote by RecE- and RecT-mediated recombination.

27. Host cell containing a nucleic acid according to one of claims 1 to 12, a regulatory region according to claim 13, a DNA construct according to claim 14 or at least a vector according to one of claims 15 to 26.

28. Host cell according to claim 27, characterized in that it is a pro- or eukaryotic cell.

29. Host cell according to claim 28, characterized in that the prokaryotic cell belongs to the group of actinomycetes, preferably to the group of streptomycetes.

30. Host cell according to claim 28, characterized in that the eukaryotic cell is a plant cell.

31. A polypeptide which is encoded by a nucleic acid according to any one of claims 1 to 7.

32. Polypeptide according to claim 31, characterized in that it has the activity of a methyl transferase.

33. Polypeptide according to claim 32, characterized in that it has the amino acid sequence according to SEQ ID NOS: 8, 12, 14, 18 or 20, or a partial sequence thereof.

34. Polypeptide according to claim 31, characterized in that it has the activity of a glycosyltransferase.

35. Polypeptide according to claim 34, characterized in that it has the amino acid sequence according to SEQ ID NOS: 10 or 30, or a partial sequence thereof.

36. Polypeptide according to claim 31, characterized in that it has the activity of a C-C linking enzyme which carries out cyclization reactions.

37. Polypeptide according to claim 36, characterized in that it has the amino acid sequence according to SEQ ID NO: 16 or a partial sequence thereof.

38. Polypeptide according to claim 31, characterized in that it has the activity of an enzyme which is involved in cyclization reactions.

39. Polypeptide according to claim 38, characterized in that it has the amino acid sequence according to SEQ ID NO: 22 or a partial sequence thereof.

40. Polypeptide according to claim 31, characterized in that it has the activity of a 2,3-reductase.

41. Polypeptide according to claim 40, characterized in that it has the amino acid sequence according to SEQ ID NO: 24 or a partial sequence thereof.

42. Polypeptide according to claim 31, characterized in that it has the activity of a 2,3-dehydratase.

43. Polypeptide according to claim 42, characterized in that it has the amino acid sequence according to SEQ ID NO: 26 or a partial sequence thereof.

44. Polypeptide according to claim 31, characterized in that it has the activity of a thioesterase.

45. Polypeptide according to claim 44, characterized in that it has the amino acid sequence according to SEQ ID NO: 28 or a partial sequence thereof.

46. Polypeptide according to claim 31, characterized in that it has the activity of a 3,4-dehydratase.

47. Polypeptide according to claim 46, characterized in that it has the amino acid sequence according to SEQ JD NO: 32 or a partial sequence thereof.

48. Polypeptide according to claim 31, characterized in that it has the activity of a 4-aminotransferase.

49. Polypeptide according to claim 48, characterized in that it has the amino acid sequence according to SEQ ID NO: 34 or a partial sequence thereof.

50. Polypeptide according to claim 31, characterized in that it has the activity of an N-dimethyltransferase.

51. Polypeptide according to claim 50, characterized in that it has the amino acid sequence according to SEQ JD NO: 36 or a partial sequence thereof.

52. Polypeptide according to claim 31, characterized in that it has the activity of a 3,4-reductase.

53. Polypeptide according to claim 52, characterized in that it has the amino acid sequence according to SEQ ID NO: 38 or a partial sequence thereof.

54. Polypeptide according to claim 31, characterized in that it has the activity of a transcription regulator.

55. Polypeptide according to claim 54, characterized in that it has the amino acid sequence according to SEQ ID NO: 40 or a partial sequence thereof.

56. Polypeptide according to claim 31, characterized in that it has the activity of a polyketide synthase.

57. Polypeptide according to claim 56, characterized in that it has the amino acid sequence according to SEQ ID NOS :: 42, 44, 46, 48 or 50, or a partial sequence thereof.

58. Polypeptide according to claim 31, characterized in that it has the activity of a glucose dehydratase.

59. Polypeptide according to claim 58, characterized in that it has the amino acid sequence according to SEQ ID NO: 53.

60. Polypeptide according to claim 31, characterized in that it has the activity of a 3,5-epimerase.

61. Polypeptide according to claim 60, characterized in that it has the amino acid sequence according to SEQ ID NO: 55.

62. Enzymes which are involved in cyclization reactions, characterized in that they comprise the amino acid sequence according to SEQ ID NO: 15 or 22, or a partial sequence thereof, which can still carry out at least one partial reaction, or have at least 50% identity to it Have amino acid level.

63. Antibody which specifically reacts with a polypeptide according to one of claims 31 to 62.

64. A method for producing a nucleic acid according to any one of claims 1 to 7, comprising the following steps:

(a) Complete chemical synthesis in a manner known per se or

(b) chemical synthesis of oligonucleotides, labeling of the oligonucleotides, hybridization of the oligonucleotides to DNA of a genomic or cDNA library, which was prepared from S. spinosa genomic DNA or mRNA, selecting positive cloning and isolating the hybridizing DNA from positive clones or

(c) Chemical synthesis of oligonucleotides and amplification of the target DNA using PCR.

65. A method for producing a polypeptide according to any one of claims 31 to 62, comprising the following steps:

(a) cultivating a host cell according to any one of claims 27 to 30 under conditions which ensure expression of the nucleic acid according to any one of claims 1 to 7, or

(al) expressing a nucleic acid according to one of claims 1 to 7 in an in v / tro system, and

(b) recovering the polypeptide from the cell, the culture medium or the in vz ^' tro system.

66. Method for producing spinosyn, spinosyn precursors or spinosyn

Derivatives include the following steps:

(a) culturing a host cell according to any one of claims 27 to 30 under conditions which ensure the expression of the nucleic acid according to any one of claims 1 to 7 and

(b) recovering the spinosyn, the spinosyn precursor or the spinosyn derivative from the cell or the culture medium.

67. Methods of making spinosyn derivatives, including spinosyn

Precursors, comprising the following steps: (a) replacing at least one module-coding nucleic acid sequence according to claim 7 by at least one other module-coding nucleic acid sequence according to claim 7, or

(b) exchanging at least one module-coding nucleic acid sequence according to claim 7 for at least one other module-coding nucleic acid sequence from S. spinosa, or

(c) exchanging at least one module-coding nucleic acid sequence according to claim 7 for at least one module-coding nucleic acid sequence from an organism other than S. spinosa, or

(d) exchanging at least one domain-coding nucleic acid sequence according to claim 7 for at least one other domain-coding nucleic acid sequence according to claim 7, or

(e) replacing at least one domain-coding nucleic acid sequence according to claim 7 with at least one other domain-coding nucleic acid sequence from S. spinosa, or

(f) exchanging at least one domain-coding nucleic acid sequence according to claim 7 for at least one other domain-coding nucleic acid sequence from an organism other than S. spinosa, or

(g) replacing a first acyltransferase-coding nucleic acid sequence according to claim 7 with a second acyltransferase-coding nucleic acid sequence according to claim 7, wherein the second Acyltransferase has a different substrate specificity than the first acyltransferase, or

(h) replacing a first acyltransferase-coding nucleic acid sequence according to claim 7 with a second acyltransferase-coding nucleic acid sequence from S. spinosa, the second

Acyltransferase has a different substrate specificity than the first acyltransferase, or

(i) replacing a first acyltransferase-coding nucleic acid sequence according to claim 7 with a second acyltransferase-coding nucleic acid sequence from an organism other than S. spinosa, the second acyltransferase having a different substrate specificity than the first acyltransferase, or

(j) deleting at least one domain coding nucleic acid sequence according to claim 7, or

(k) integrating at least one domain-coding nucleic acid sequence according to claim 7 into a module-coding nucleic acid sequence according to claim 7, or

(1) mutagenizing at least one domain-coding nucleic acid sequence according to claim 7,

and expressing the recombined nucleic acid sequence in a host cell under conditions that allow synthesis of a spinosyn derivative or precursor.

68. Use of a nucleic acid according to one of claims 1 to 7 for identifying, inactivating and / or modifying genes of the spinosyn biosynthesis.

69. Use of a nucleic acid according to any one of claims 1 to 7 for generating a library of polyketide synthases.

70. A method for adding a forosamine sugar residue to the spinosyn aglycon or to the spinosyn 17 pseudoaglycon or to a polyketide aglycon, comprising the following steps:

(a) transferring a nucleic acid according to SEQ ID NOS: 23, 25, 29, 31, 33, 35 and 37 into a host cell which can produce the spinosyn aglycon or the spinosyn 17 pseudoaglycon or the polyketide aglycon, or

(al) transfer of a nucleic acid according to SEQ ID NOS: 23, 25, 29, 31,

33, 35 and 37 into a host cell which is the spinosyn aglycon or the

Spinosyn 17-pseudoaglycon or the polyketide aglycon cannot produce and adding the spinosyn aglycon or the spinosyn

17-pseudoaglycon or the polyketide aglycon to the culture medium, and

(b) culturing the host cell under conditions that lead to active cell metabolism.

71. A method for attaching a trimethyl rhamnose sugar residue to the spinosyn aglycon or to the spinosyn 9 pseudoaglycon or to a polyketide aglycon, comprising the following steps: (a) transferring a nucleic acid according to SEQ ID NO: 7, 9, 11, 13, 17 and / or 19 into a host cell which can produce the spinosyn aglycon or the spinosyn 9 pseudoaglycon or the polyketide aglycon, or

(al) transferring a nucleic acid according to SEQ ID NO: 7, 9, 11, 13, 17 and / or 19 into a host cell which the spinosyn aglycon or the spinosyn 9 pseudoaglycon or the polyketide aglycon cannot produce and adding of the Spinosyn aglycon or the Spinosyn 9 pseudoaglycon or the polyketide aglycon to the culture medium, and

A method according to claim 71, characterized in that in step (a) nucleic acids according to SEQ ID NOS: 9, 11, 13 and 17 are transferred.