EP1212412A2 - Nucleinsäuren, die für enzymaktivitäten der spinosyn-biosynthese codieren - Google Patents

Nucleinsäuren, die für enzymaktivitäten der spinosyn-biosynthese codieren

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Publication number
EP1212412A2
EP1212412A2 EP00956458A EP00956458A EP1212412A2 EP 1212412 A2 EP1212412 A2 EP 1212412A2 EP 00956458 A EP00956458 A EP 00956458A EP 00956458 A EP00956458 A EP 00956458A EP 1212412 A2 EP1212412 A2 EP 1212412A2
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EP
European Patent Office
Prior art keywords
nucleic acid
spinosyn
seq
acid sequence
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00956458A
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German (de)
English (en)
French (fr)
Inventor
Günther EBERZ
Volker MÖHRLE
Rita FRÖDE
Robert Velten
José A. SALAS
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Bayer CropScience AG
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Bayer AG
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Priority claimed from DE19957268A external-priority patent/DE19957268A1/de
Application filed by Bayer AG filed Critical Bayer AG
Publication of EP1212412A2 publication Critical patent/EP1212412A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • 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
  • 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).
  • 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).
  • modules The enzymatic activities of modular Type I PKS's can be summarized in so-called modules.
  • 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 ⁇ -ketoacyl: acyl carrier protein synthase domain, an acyltransferase domain and a ⁇ -ketoacyl: acyl carrier protein
  • 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 ⁇ -ketoacyl: acyl carrier protein domain from the domains mentioned, and also an enzymatically inactive ⁇ -ketoacyl: acyl carrier protein synthase domain.
  • Polyketide synthase domain each includes one of these enzymatic activities.
  • 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.
  • ORFs open reading frames
  • nucleic acids according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • Preferred embodiments are fragments of genomic DNA and cDNA's.
  • nucleic acid 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.
  • enzyme activity 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.
  • 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.
  • nucleic acids according to the invention particularly preferably comprise at least one sequence selected from
  • DNA fragments are isolated which have the same properties as the fragments isolated from S. spinosa.
  • Hybridization solution 5 x SSC; Blocking Reagens (Röche Diagnostics GmbH, Mannheim,
  • 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.
  • 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.
  • regulatory regions refers to promoters, repressor or activator binding sites, repressor or activator sequences, and terminators.
  • 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
  • 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.
  • heterologous promoter refers to a promoter which does not control the expression of the gene (ORF 's) in question in the original organism.
  • heterologous promoters depend 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
  • 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.
  • 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:
  • 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).
  • 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
  • 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.
  • Gram-negative bacteria such as Escherichia coli
  • Gram-positive bacteria such as Streptomyces
  • 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.
  • Vectors can be replicated autonomously or integrated into the host's genome become.
  • the latter integration can take place via homologous recombination, via a ⁇ C31 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.
  • 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.
  • 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.
  • a shuttle vector coding for the spinosyn biosynthesis can be used to transfer the biosynthesis and transfer into 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).
  • 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.
  • 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.
  • 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.
  • 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-
  • the invention also covers those polypeptides which have only part of the complete amino acid sequence of the enzyme in question.
  • 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.
  • ORF12 nucleotide positions 13,865 to 12,546 of SEQ ID NO: 4,439 amino acids; the derivable gene product is a glycosyltransferase.
  • nucleotide positions 128-1402, amino acid positions 5-429 encode a ⁇ -
  • Nucleotide positions 2798-3052, amino acid positions 895-979 encode a ⁇ -ketoacyhacyl carrier protein domain
  • Ketoacyl acyl carrier protein synthase domain
  • Ketoacyl acyl carrier protein domain.
  • KetoacyhAcyl Carrier Protein Synthase Domain Nucleotide positions 9634-10608, amino acid positions 572-896 encode one
  • 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
  • Nucleotide positions 13885-14142, amino acid positions 1989-2074 encode a ⁇ -ketoacyl: acyl carrier protein domain.
  • Keto reductase domain Nucleotide positions 18795-19052, amino acid positions 1458-1543 encode a ⁇ -ketoacyl: acyl carrier protein domain;
  • Nucleotide positions 19107-20387, amino acid positions 1562-1988 encode a ⁇ -ketoacyl: acyl carrier protein synthase domain;
  • Nucleotide positions 20718-21692, amino acid positions 2099-2423 encode an acyltransferase domain
  • Nucleotide positions 23436-23693, amino acid positions 3005-3090 encode a ⁇ -ketoacyl: acyl carrier protein domain.
  • Nucleotide positions 29035-29265, amino acid positions 1685-1761 encode a ⁇ -ketoacyl: acyl carrier protein domain;
  • Nucleotide positions 29329-30624, amino acid positions 1783-2214 encode a ⁇ -ketoacyl: acyl carrier protein synthase domain;
  • Keto reductase domain Nucleotide positions 33652-33900, amino acid positions 3224-3306 encode a ⁇ -ketoacyl: acyl carrier protein domain;
  • Nucleotide positions 33952-35262, amino acid positions 3324-3760 encode a ⁇ -ketoacyl: acyl carrier protein synthase domain;
  • Nucleotide positions 35554-36522, amino acid positions 3858-4180 encode an acyltransferase domain
  • Nucleotide positions 38254-38511, amino acid positions 4758-4843 encode a ⁇ -ketoacyl: acyl carrier protein domain.
  • Nucleotide positions 44151-45473 of SEQ JO NO: 5, amino acid positions 1782-2222 encode a ⁇ -ketoacyl: acyl carrier protein synthase domain;
  • Nucleotide positions 2146-2697 of SEQ ID NO: 6, amino acid positions 4447-4630 encode a dehydratase domain
  • 5116 encode a ketoreductase domain
  • 5290 encode a ⁇ -ketoacyl: acyl carrier protein domain; Nucleotide positions 4864-5538 of SEQ ID NO: 6, amino acid positions 5353-
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • the present invention creates the genetic basis for producing new spinosyn precursors and derivatives by means of molecular genetic methods.
  • spinosyn precursors refers to all detectable and all postulated spinosyn precursors.
  • spikenosyn derivatives 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.
  • 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.
  • 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.
  • ET recombination WO 99/29837; Muyrers et al., 1999
  • other cloning and recombination techniques 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.
  • module 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
  • nucleic acids of the invention can also be used to generate nucleic acids of the invention.
  • 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
  • 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.
  • 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.
  • 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.
  • ORF 's open reading frames
  • 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 ⁇ -ketoacyl: acyl carrier protein synthase
  • AT acyltransferase
  • ACP ⁇ -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.
  • Escherichia coli XLl-Blue MRF and the cosmid vectors SuperCosl (Stratagene, Europe) and pOJ446 (Biermann et al., 1992) were used to create
  • Plasmid pBeloBACl 1 (Kim et al., 1996) and pOJ446 (Biermann et al, 1992) were used to produce an E. coli - Streptomyces BAC shuttle vector.
  • Plasmids were isolated using the Qiagen Plasmid Kit (Qiagen, Hilden, Germany). The enzymes used came from Röche Diagnostics GmbH (Mannheim, Germany).
  • 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).
  • chromosomal DNA from S. spinosa ATCC49460 was partially cut with Mbol and separated by centrifugation in a glucose density gradient.
  • 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.
  • the vector pBeloBACl 1 was linearized with Xhol and smooth DNA ends were produced using Klenow polymerase.
  • the resulting vector was named p ⁇ BZ333.
  • 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 Bam ⁇ I.
  • ORF 's open reading frames
  • 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).
  • SEQ ID NO: 51 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.
  • 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).
  • 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.
  • the culture supernatant from S. albus contained no compounds with MW 543 and no Spinosyn A.
  • microorganisms and plasmids are at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg lb, D-38124
  • 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.

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EP00956458A 1999-08-27 2000-08-17 Nucleinsäuren, die für enzymaktivitäten der spinosyn-biosynthese codieren Withdrawn EP1212412A2 (de)

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DE19940596 1999-08-27
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DE19957268 1999-11-29
DE19957268A DE19957268A1 (de) 1999-08-27 1999-11-29 Nucleinsäuren, die für Enzymaktivitäten der Spinosyn-Biosynthese kodieren
PCT/EP2000/008013 WO2001016303A2 (de) 1999-08-27 2000-08-17 Nucleinsäuren, die für enzymaktivitäten der spinosyn-biosynthese codieren

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US6143526A (en) * 1998-03-09 2000-11-07 Baltz; Richard H. Biosynthetic genes for spinosyn insecticide production
TWI237802B (en) 2000-07-31 2005-08-11 Semiconductor Energy Lab Driving method of an electric circuit
AR033022A1 (es) * 2001-03-30 2003-12-03 Dow Agrosciences Llc Genes biosinteticos para la produccion de insecticida de butenilespinosina
DE10135550A1 (de) 2001-07-20 2003-01-30 Bayer Cropscience Ag Verfahren zum Herstellen von neuen Spinosyn-Derivaten
IL163529A0 (en) 2002-02-19 2005-12-18 Dow Agrosciences Llc Novel spinosyn-producing polyketidesynthases
DE10301519A1 (de) 2003-01-17 2004-07-29 Bayer Cropscience Ag 9-Ketospinosyn-Derivate
CA2737875C (en) * 2008-09-22 2015-11-24 Christine Kritikou Spinosyn antifouling compositions, methods of use thereof and articles protected from attachment of biofouling organisms
EP2520658B1 (en) * 2011-05-03 2015-10-07 Dow AgroSciences LLC Enhancing spinosyn production with oxygen binding proteins
US9404107B2 (en) 2011-05-03 2016-08-02 Dow Agrosciences Llc Integration of genes into the chromosome of Saccharopolyspora spinosa
JP2018011518A (ja) * 2016-07-19 2018-01-25 国立大学法人 筑波大学 ストレプトマイセス属微生物用ベクター
CN113755364B (zh) * 2021-08-10 2023-05-09 湖北省生物农药工程研究中心 产多杀菌素的放线菌及其在制备杀虫剂中的应用

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US5227295A (en) 1991-11-08 1993-07-13 Dowelanco Process for isolating A83543 and its components
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EP0837870B1 (en) 1995-06-14 2002-07-24 Dow AgroSciences LLC Synthetic modification to spinosyn compounds
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CN102382848A (zh) * 2005-12-09 2012-03-21 三得利控股株式会社 新型脂肪酶
CN102382848B (zh) * 2005-12-09 2013-07-31 三得利控股株式会社 新型脂肪酶

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US7285653B1 (en) 2007-10-23
AU6839300A (en) 2001-03-26
WO2001016303A3 (de) 2001-08-02
JP2003508050A (ja) 2003-03-04

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