WO2010072600A1 - Gènes biosynthétiques de polykétide - Google Patents

Gènes biosynthétiques de polykétide Download PDF

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WO2010072600A1
WO2010072600A1 PCT/EP2009/067033 EP2009067033W WO2010072600A1 WO 2010072600 A1 WO2010072600 A1 WO 2010072600A1 EP 2009067033 W EP2009067033 W EP 2009067033W WO 2010072600 A1 WO2010072600 A1 WO 2010072600A1
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seq
polyketide
nucleic acid
gene
primer pair
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PCT/EP2009/067033
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German (de)
English (en)
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Jörn PIEL
Katja Fisch
Cristian Gurgui
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Rheinische Friedrich-Wilhelms-Universität Bonn
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin

Definitions

  • the invention relates to a method for the isolation of gene sequences, as well as the gene sequence isolated by this method and fragments thereof, which codes for peptides for the biosynthesis of polyketides.
  • the resulting sponge metagenome may therefore contain hundreds of individual (prokaryotic) genomes, each providing its own PKS genes (Kim et al., Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: eulture-dependent and eulture-independent approaches, Environ Microbiol 8, 1460-1470, 2006).
  • the present invention provides a solution to this problem and discloses a novel approach to specific amplification of PKS gene regions based on the substrate specificity of kethosynthase domains.
  • the present invention relates to a nucleic acid comprising at least one biosynthesis gene for a polyketide selected from the group consisting of psymberin or mycalamide.
  • the nucleic acid comprises a sequence selected from the group consisting of SEQ ID Nos. 1-19, 39, 75, a homolog of these sequences and the complement of the sequences or the homolog, the homolog having at least 75% sequence identity , or hybridized with the nucleic acid under high salt conditions.
  • the present invention relates to a peptide encoding one of the nucleic acids of the invention.
  • the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs 20-38 or a homolog of these sequences, wherein the homolog has at least one sequence identity to SEQ ID NO 20 of 36%, to SEQ ID NO 21 from 54%, to SEQ ID NO 22 from 44%, to SEQ ID NO 23 from 41%, to SEQ ID NO 24 from 52%, to SEQ ID NO 25 from 42%, to SEQ ID NO 28 from 44% to SEQ ID NO 29 of 94%, to SEQ ID NO 30 of 96%, to SEQ ID NO 31 of 96%, to SEQ ID NO 32 of 94%, to SEQ ID NO 33 of 98%, to SEQ ID NO 34 of 95%, to SEQ ID NO 35 of 97%, to SEQ ID NO 36 of 98%, to SEQ ID NO 37 of 96% or to SEQ ID NO 38 of 96%.
  • the present invention relates to a vector comprising the nucleic acids according to the invention and to a recombinant host cell which comprises the nucleic acids or the vector according to the invention.
  • the present invention relates to a process for producing a polyketide comprising the step of cultivating a recombinant host cell of the invention under conditions permitting expression of the biosynthetic genes and production of the polyketide by the host cell.
  • the present invention relates to a method of isolating at least one polyketide biosynthetic gene, comprising the steps of: a) providing at least one primer pair based on the substrate specificity of a ketosynthase domain of a trans-AT polyketide synthase; and b) isolating at least one polyketide biosynthetic gene using the at least one primer pair from step a).
  • step b) comprises the amplification of the nucleic acid region enclosed by the primer pair.
  • the at least one primer pair is specific for a motif conserved in phylogenetically adjacent kethosynthase domains of trans-AT polyketide synthases, or at least one primer of the primer pair is specific for at least 4 contiguous amino acid residues of any of those depicted in FIG. 3, FIG SEQ ID NO: 40-71 defined amino acid motifs.
  • the isolated polyketide biosynthesis gene is involved in the biosynthesis of psymberin or mycalamide.
  • the present invention provides a novel method for the targeted isolation of polyketide biosynthesis genes from metagenomes, taking advantage of the substrate specificity of the polyketide chain-extending ketosynthase (KS) domains.
  • KS ketosynthase
  • nucleic acid in the context of the present invention particularly, but not limited to, natural, preferably linear, branched or circular nucleic acids such as RNA, in particular mRNA, single-stranded and double-stranded viral RNA, siRNA, miRNA, snRNA, tRNA, hnRNA or Ribozymes, genomic, bacterial or viral DNA (single-stranded and double-stranded), chromosomal and episomal DNA, free-circulating nucleic acid and the like, synthetic or modified nucleic acids, for example plasmids or oligonucleotides, in particular primers used for PCR, - A -
  • nucleic acid often consists of two complementary strands Therefore, in a nucleic acid claimed by this invention, always the reverse complement of this nucleic acid to be considered as claimed.
  • high salt buffer in particular, but not limited to, a buffer having a high salt concentration (preferably chaotropic substances), preferably> 100 mM, more preferably> 500 mM and more preferably> 1 M.
  • high salt conditions is understood below to mean a medium which uses a high salt buffer, preferably a high salt buffer containing chaotropic salts
  • High salt preferably containing chaotropic salts
  • a polar surface as a hydrogen bond donor
  • the nucleic acids bind to this surface, where they undergo better stabilization than in water Water again a better hydrogen bond donor than the polar surface, and the nucleic acids can be detached again from the surface.
  • chaotropic substances or “chaotropic salts” are in particular - but not limited to - substances that alter the secondary, tertiary and / or quaternary structure of proteins and / or nucleic acids and leave at least the primary structure intact, the solubility reduce polar substances in water and / or enhance hydrophobic interactions, understood.
  • Preferred chaotropic substances are guanidine hydrochloride, guanidinium (iso) thiocyanate, sodium iodide, sodium perchlorate, potassium iodide, sodium (iso) thiocyanate and / or urea.
  • amplification or "amplification reaction” is understood to mean a process which makes it possible to increase the concentration of one or more analytes, preferably nucleated - to double at least.
  • isothermal and thermocyclic amplification reactions In the former, the temperature remains the same throughout the process, while in the latter, thermal cycles are passed through which the reaction and amplification are controlled.
  • Preferred isothermal amplification reactions are e.g.
  • LAMP Loop mediated isothermal amplification
  • NASBA Nucleic Acid Sequence Based Amplification
  • thermocyclic amplification reactions are e.g.
  • Ligase chain reaction (LCR), and / or
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence based amplification
  • RNA RNA
  • a first primer binds to the complementary sequence in the region of The DNA strand complementary to the matrix is then polymerized with a reverse transcriptase, and the RNA matrix is then digested using RNase H (RNase H digests only RNA in RNA-DNA hybrids, not single-stranded)
  • RNase H RNase H digests only RNA in RNA-DNA hybrids, not single-stranded
  • a second primer is attached to the 5 'end of the DNA strand, which is used by the T7 RNA polymerase as a starting point for the synthesis of a DNA strand complementary RNA molecule, which then used again as the starting matrix NASBA is performed at a constant temperature of normally 41 ° C and under certain circumstances gives faster and better results than PCR.
  • TMA Transcription Mediated Amplification
  • rolling circle chain reaction (RCCR) or “rolling circle amplification” (RCA) refers to an amplification method that mimics the general nucleic acid replication according to the rolling circle principle and is described inter alia in US5854033.
  • nested PCR is understood to mean a process in which an already amplified DNA fragment (or a part thereof) is amplified a second time; this process is done with a second primer pair located within the primer pair used in the first reaction.
  • promoter is understood as meaning a nucleic acid sequence which permits the regulated expression of a gene, which is located upstream, ie 5 'to the RNA-coding region of the gene
  • An important property of a promoter is the specific interaction with particular DNA-binding proteins which mediate the initiation of transcription of the gene by RNA polymerase and are referred to as transcription factors
  • the most important prokaryotic transcription factors are the sigma factors. By far the most promoters in, for example, E.
  • coli are recognized by the factor Sigma-70 and have the following promoter elements: 1) an AT base-pair-rich UP element above the -35 region (which also corresponds directly to the ⁇ subunit of 2) the -35 region, with the consensus sequence: 5'-TTGACA-3 ', 3) the -10 region (Pribnow box), with the consensus sequence: 5'-TATAAT- 3 '.
  • promoter elements 1) an AT base-pair-rich UP element above the -35 region (which also corresponds directly to the ⁇ subunit of 2) the -35 region, with the consensus sequence: 5'-TTGACA-3 ', 3) the -10 region (Pribnow box), with the consensus sequence: 5'-TATAAT- 3 '.
  • strong promoters are rich in AT base pairs just before the start of transcription.
  • the consensus sequences give only an approximate guide to building a promoter.
  • a particular sigma-70 dependent promoter may differ from these sequences in several places.
  • the promoter is an inducible
  • an inducible promoter is the artificially generated tac promoter, which can be used to increase gene expression levels in bacterial cells (eg E. coli) and thus to overexpress recombinant proteins.
  • This synthetic promoter consists of the -35 region of the strong trp promoter from E. coli and the -10 region of the IPTG and lactose regulatable / inducible lac promoter.
  • the inducible tac promoter is about 3 times stronger than the trp promoter and about 10 times stronger than the lac promoter.
  • biosynthesis means the enzymatic production of a polyketide, preferably by means of one or more polyketide synthases, preferably a trans-AT polyketide synthase
  • biosynthesis gene thus encompasses any gene coding for a protein as part of one Polyketide synthase is.
  • biosynthesis gene also encompasses fragments of a biosynthetic gene coding for a truncated protein of a polyketide synthase which has the same function as the full-length protein.
  • the same function is understood here to mean the basically identical biochemical function, irrespective of whether it is carried out with an increased or decreased efficiency or speed, as an example of a biosynthesis gene is a gene for a KS Domain whose biochemical function is to lengthen ⁇ -saturated intermediates (see, for example, the KS of Klade V), or a gene whose gene product in a PKS has the biochemical function exercises to transfer a methyl residue (eg psyB).
  • biosynthetic gene any truncated version of such genes resulting in truncated gene products still performing the same biochemical function will also fall under the term “biosynthetic gene.”
  • biosynthetic genes and specific embodiments of the invention are psyA -psyN genes listed in Table 1.
  • metagenome is understood here to mean an amount of at least two genomes of different species, ie, a metagenome comprising the entire genomic information of at least two species
  • the metagenome comprises the genome of a sponge and that of at least one prokaryote
  • the prokaryote lives in symbiosis with the sponge.
  • a primer is considered to be "specific" for a given amino acid sequence if it hybridizes, preferably under stringent conditions, with or is identical to the sense or antisense strand of a nucleic acid which corresponds to the given amino acid sequence Since the amino acid code is degenerate, a number of suitable primers can be used for a given amino acid sequence It is known to those skilled in the art which base triplets encode which amino acid residues The primer should have a minimum length which allows it to react with a nucleic acid In a specific embodiment, the primer hybridizes to a nucleic acid comprising at least 5, 6, 7 or more than 7 contiguous amino acid residues of the given amino acid encoded sequence.
  • a primer is considered in the context of the invention as specific for the defined amino acid motifs, even if it is specific for an amino acid sequence containing at least 2, at least 3, or at least 4 contiguous amino acid residues one of the in Fig. 3 and Fig. 4 or SEQ ID NOs: 40-71 defined amino acid motifs and additionally at the 3'- or 5 'end further nucleotides for not in the defined amino acid motifs contained amino acid residues.
  • the number of nucleotides of the primer that are specific for amino acid residues that are not contained in the defined amino acid motif up to 30% of the total number of nucleotides of the primer can be.
  • a primer which is specific for the amino acid motif "YYQAGML" defined in FIG. 3 can also be prepared so that it is specific for the amino acid sequence "YYQ AGMLA", wherein the additional nucleotides for the alanine which is not contained in the defined amino acid motif 12.5% of the total primer sequence.
  • Polyketide synthases can be divided into three classes based on their structure and function.
  • Type I enzymes are multifunctional, often modular proteins.
  • Bacterial modular type I PKSs produce a large amount of therapeutically important natural products, e.g. Erythromycin, Epothilone and FK506 (Fischbach & Walsh Assembly-line enzymology for polypeptides and nonribosomal peptide antibiotics: logic, machinery and mechanisms, Chem. Rev. 106, 3468, 2006).
  • Fig. 1 shows some exemplary polyketides which, as far as their gene clusters are known, are formed by Type I PKS.
  • Each of these enzymes comprises at least one ⁇ -ketosynthase (KS), an acyltransferase (AT) and an acyl carrier protein (ACP), which select the next building block for the formation of the polyketide, and activate to form a ⁇ -ketoacyl-S ACP intermediates, catalyze a decarboxylative Claisen condensation between the next building block and the growing polyketide chain.
  • KS ⁇ -ketosynthase
  • AT acyltransferase
  • ACP acyl carrier protein
  • trans-AT-PKSs A special and evolutionarily delimited enzyme family of PKSs are the trans-AT-PKSs, which use free-standing AT to trans select the building blocks needed to make the polyketide (Cheng et al., Type I polyketide synthase requiring a discrete acetyltransferase for polyketide biosynthesis Proc. Natl. Acad., USA 100, 3149-3154, 2003).
  • ice-AT PKS have integrated AT domains.
  • the previously published studies on complex polyketides have shown that all previously cloned from symbionts PKS clusters belong to this family of trans-AT-PKS.
  • the present invention is based on the surprising discovery that the KS of trans AT-PKS have a unique phylogenetic pattern in which the closest related KS domains are specific for the same or similar substrates.
  • different substrates eg .beta.-hydroxylated and olefinic
  • the phylogenetic analysis of one or more CSs thus provides useful information about the structure of the intermediates involved.
  • the reverse approach is adopted, in which, due to the chemical structure of the polyketide, conclusions can be drawn about the KS involved. It has surprisingly been found that this approach allows the identification of conserved sequence patterns / motifs within these substrate-specific KS domains. Because of these conserved motifs, it is then possible in accordance with the invention to generate primers which are specific for KS domains having a specific, desired substrate specificity. These primers then allow the amplification of such KS domains that are specific to a particular substrate. These amplicons may then be used to identify and isolate one or more polyketide biosynthetic genes, e.g. in gene clusters, e.g. by PCR, screening of fosmid libraries or genome walking.
  • One of the main advantages of the present invention thus lies in the fact that, starting from the polyketide, a KS domain of a trans-AT PKS involved in its biosynthesis can be identified in a targeted manner in order to obtain, in a next step, its nucleotide sequence and the nucleotide sequence of the polyketide biosynthesis complex comprising it. To identify and isolate gene clusters.
  • the present invention is therefore based on the surprising discovery that, based on the chemical structure, ie the functional groups of a polyketide, conserved motifs can be identified within KS domains involved in the biosynthesis of this polyketide. Based on these conserved motifs, primers can then be deduced which can be used to amplify and isolate the genes involved in the biosynthesis of this polyketide.
  • nucleic acid comprising at least one gene coding for a polypeptide involved in the biosynthesis of the polyketides psymberin (also known as ircinamin A), or mycamide.
  • the nucleic acid is in isolated form.
  • nucleic acid is the complement of a nucleic acid comprising at least one gene coding for a polypeptide involved in the biosynthesis of the polyketides psymberin or mycalamide.
  • the gene is a prokaryotic gene and / or part of a polycistronic gene segment or gene cluster that encodes at least one protein involved in the biosynthesis of a polyketide.
  • the gene belongs to the type I PKS; In a preferred embodiment, the gene belongs to the evolutionarily delimited enzyme family of trans-ATP PKS.
  • TransAT PKSs use free-standing acetyltransferases (AT) to trans select the building blocks needed to make the polyketide (Cheng et al., Type I polyketide synthase requiring a discrete acetyltransferase for polyketide biosynthesis., Proc. Natl. Acad 100, 3149-3154, 2003), while cis AT PKS has integrated AT domains.
  • the nucleic acid according to the invention was amplified from the metagenome of a sponge.
  • Eukaryotic sponges enter into symbiosis with various prokaryotes, although these prokaryotes can often not be cultured in isolation.
  • the resulting sponge and prokaryote metagenome includes the genome of the eukaryotic sponge and the prokaryotic genomes of the individual symbionts.
  • the sponge belongs to the Demospongiae, the Dictyoceratida or the Irciniidae.
  • the sponge belongs to Psammocinia sp .; more preferably, the sponge is Psammocinia äff. bulbosa. In a preferred embodiment, the sponge belongs to the Demospongiae. In further preferred embodiments, the sponge belongs to the family of Spongiidae, Thorectidae, Halichondriidae, Callyspongiidae, Chalinidae, Mycalidae, Coelosphaeridae, Tedaniidae, Plakinidae, or Triaenosina. For the purposes of this invention, preferred representatives of these families are Spongia spp., Cacospongia spp., C.
  • the amplification of the nucleic acid from the metagenome can be carried out by means of any method of nucleic acid amplification known to the person skilled in the art.
  • the amplification of the nucleic acid according to the invention takes place by means of an isothermal or a thermocyclic amplification reaction.
  • the amplification is preferably carried out by means of ligase chain reaction (LCR), and / or polymerase chain reaction (PCR); more preferably, the PCR is a nested PCR.
  • the nucleic acid according to the invention comprises at least one sequence which is selected from the group consisting of SEQ ID NO 1-19, 39, 75, a homolog of these sequences and the complement of the sequences or the homolog.
  • the nucleic acid according to the invention comprises at least one sequence which is selected from the group consisting of SEQ ID NO 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 (psy A, psyB, psyC, psyD, psyE, psyF, psyG, psyH, psyl, psyJ, psyK, psyL, psyM and psyN), a homolog of these sequences and / or the complement of the sequences or the homolog.
  • the homolog has at least 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, preferably 97%, 98%, 99%. , 99.5%, 99.7%, 99.9% sequence identity with the nucleic acid being compared, and / or hybridizes with the nucleic acid under high salt conditions.
  • the nucleic acid according to the invention codes for a protein which has at least one identity to SEQ ID NO 23 of 41%, to SEQ ID NO 24 of 52%, to SEQ ID NO 25 of 42%, to SEQ ID NO 28 of 44 %, to SEQ ID NO 29 of 94%, to SEQ ID NO 30 of 96%, to SEQ ID NO 31 of 96%, to SEQ ID NO 32 of 94%, to SEQ ID NO 33 of 98%, to SEQ ID NO 34 of 95%, to SEQ ID NO 35 of 97%, to SEQ ID NO 36 of 98%, to SEQ ID NO 37 of 96% or to SEQ ID NO 38 of 96%.
  • the nucleic acid according to the invention encodes a protein, which has at least one identity to SEQ ID NO 23 of 50%, to SEQ ID NO 24 of 55%, to SEQ ID NO 25 of 50% or to SEQ ID NO 28 of 50%.
  • Another aspect of the present invention is a peptide, polypeptide, protein or enzyme encoded by one of the nucleic acids of the invention.
  • the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs 20-38.
  • the peptide has at least one identity to SEQ ID NO 20 of 36%, to SEQ ID NO 21 of 54%, to SEQ ID NO 22 of 44%, to SEQ ID NO 23 of 41%, to SEQ ID NO 24 from 52%, to 42% SEQ ID NO 25, 44% to SEQ ID NO 28, 94% to SEQ ID NO 29, 96% to SEQ ID NO 30, 96% to SEQ ID NO 31, to SEQ ID NO 32 of 94%, to SEQ ID NO 33 of 98%, to SEQ ID NO 34 of 95%, to SEQ ID NO 35 of 97%, to SEQ ID NO 36 of 98%, to SEQ ID NO 37 of 96% or to SEQ ID NO 38 of 96%.
  • the peptide has at least one identity to SEQ ID NO 23 of 50%, to SEQ ID NO 24 of 55%, to SEQ ID NO 25 of 50% or to SEQ ID NO 28 of 50%.
  • the peptide has a sequence identity of at least 60%, 70%, 75%, 80%, 90%, 92%, 95%, 97%, 98%, 99% or 99.5%.
  • the invention relates to a vector comprising the nucleic acid according to the invention.
  • this vector is a cosmid, a YAC, BAC or a virus, more preferably the vector is a plasmid.
  • the vector further comprises a promoter under the control of which the expression of the nucleic acid according to the invention is.
  • this promoter is an inducible promoter.
  • the nucleic acid according to the invention is under the control of an inducible expression system.
  • the inducible expression system is the tetracycline operator / repressor (TetO2 / TetR) system.
  • the invention relates to a recombinant host cell which comprises one of the nucleic acids or vectors according to the invention.
  • the present invention also provides an expression / production system for polyketides.
  • this recombinant host cell is able to synthesize polyketides.
  • the recombinant host cell is an E. coli cell, in further embodiments, the recombinant host cell is a host cell of Pseudomonas spp., Bacillus spp., Streptomyces spp. or Acinetobacter baylyi.
  • a further aspect of the invention relates to the use of the nucleic acids, vectors or recombinant host cells according to the invention in a process for the preparation of a polyketide.
  • an expression system is used which allows the expression of the nucleic acids according to the invention.
  • the expression system is a recombinant host cell comprising the nucleic acids or vectors of the invention and capable of expressing the gene products of the nucleic acids of the invention.
  • the expressed gene products can then be used to synthesize the polyketide in vivo or in vitro.
  • the polyketide produced is psymberin or mycalamide.
  • the method or the expression system comprises the use of at least one nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO 4, 5, 6, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 (psyA, psyB, psyC, psyD, psyE, psyF, psyG, psyH, psyl, psyJ, psyK, psyL, psyM and psyN) or a homologue of these sequences.
  • the cultivation of a recombinant host cell according to the invention takes place under conditions which allow expression of the biosynthetic genes and production of the polyketide by the host cell. Such conditions are well known to those skilled in the art or can be obtained by simple routine experimentation for a given polyketide.
  • the recombinant host cell is a host cell of Pseudomonas spp., Bacillus spp., Streptomyces spp., Corynebacterium spp., Acinetobacter baylyi, Pseudomonas tripe, Bacillus subtilis or Bacillus amyloliquefaciens.
  • the expression of the gene product can be specifically induced by addition of the respective inducing substance.
  • an inducible expression system eg the tetracycline operator / repressor (TetO2 / TetR) system
  • the genes of the gene cluster are integrated into the genome of the expression hosts. The integration takes place successively by homologous recombination of shorter (in about 40 kb) sections of the gene cluster.
  • transformable bacteria such as Pseudomonas, Acinetobacter baylyi, Bacillus subtilis or Bacillus amyloliquefaciens, which can receive linearized DNA.
  • the natural promoters of the gene cluster are replaced by their own promoters (eg the baeB- ⁇ xovaotox in B. amyloliquefaciens).
  • genes on or distributed on several compatible plasmids can be transferred into the expression hosts by conjugation, protoplast transformation or electroporation. The expression then takes place of freely replicating plasmids or again after integration into the genome.
  • the method further comprises the step of isolating the produced polyketide.
  • this isolation is carried out by means of HPLC and / or preparative column chromatography.
  • the isolation of the at least one polyketide biosynthesis gene using the at least one primer pair comprises, in one embodiment, the amplification of the nucleic acid region enclosed by the respective primer pair by means of the primer pair.
  • a nucleic acid region of the kethosynthase domain is amplified.
  • this amplification is performed by PCR, more preferably by nested PCR.
  • the present invention is directed to a method for isolating at least one polyketide biosynthetic gene, and comprises the steps of providing at least one primer pair which, due to the substrate specificity of a Kethosynthasedomäne a trans-AT polyketide synthase has been created, and the isolation of at least one polyketide Biosynthesegen using this at least one primer pair.
  • the inventive provision, i. the "design" of the primers is based on the surprising discovery that the KS of trans-AT PKS have a unique phylogenetic pattern in which the closest related KS domains are specific for the same or similar substrates, thus starting from the functional groups of a polyketide to identify a conserved motif within KS domains on the basis of which primers can be produced that are specific for such a conserved motif - and thus for a KS domain involved in the biosynthesis of the polyketide KS domain and thus the KS domain comprising PKS and other genes involved in the biosynthesis of the polyketide, which are often present in a gene cluster, is thus made possible according to the invention.
  • Whether a given KS or KS domain is similar or phylogenetically adjacent to a second KS or KS domain can be determined, for example, by sequence comparisons in which mutually more similar sequences correspond to phylogenetically more closely adjacent KS or KS domains.
  • a phylogenetic analysis is used for this purpose.
  • a cladogram is created for the phylogenetic analysis.
  • the cladogram is generated by the Bayesian method and as described in Example 3.
  • the domain architecture of the N-terminal adjacent module is analyzed according to the phylogenetic analysis for KS and derived from the polyketide structure, the substrate of the KS domain.
  • domain architecture is meant the sequence of domains in a module, such as e.g. KS DH KR ACP.
  • the method according to the invention is directed to the directed isolation of at least one gene involved in the polyketide biosynthesis, ie is / are based on the functional groups of a polyketide / s necessary for the preparation of this polyketide gene (s) isolated.
  • the polyketide biosynthesis gene is involved in the biosynthesis of psymberin.
  • Psymberin Figure 1, 1 does not carry a fully reduced residue but an acetyl-derived starter unit, which is usually extended by a very particular type of KS.
  • FIG. 2 it was found that KS with such a substrate specificity belongs to clade VI.
  • Klade VI can be further differentiated into KS domains that follow a module with a GCN5-related N-acetyltransferase (GNAT) domain (clade VIa) and those that do not have a GNAT domain (clade VIb).
  • GNAT GCN5-related N-acetyltransferase
  • clade VIa GCN5-related N-acetyltransferase domain
  • clade VIb GNAT domain
  • a sequence alignment of the Klade VIa contained KS domains together with all known sequences of Klade VI revealed two conserved motifs: the EDAGY motif, as well as the Klade VIa motif YYQ / KAGML, which is a module with a GCN5-related N-acetyltransferase (GNAT) domain followed (Figure 3). These conserved motifs did not appear in the sequences of the other clades.
  • a tyrosine residue was identified in the EDAGY motif, which is conserved in 84% of trans-AT KS, but does not occur in the ubiquitous sup KS. Therefore, due to the identified conserved motifs, primers that are specific for the EDAGY motif in the forward orientation and specific for the YYQ / KAGML motif in reverse orientation can now be generated to isolate a gene involved in psymberin biosynthesis.
  • the amplicons generated using these primers can then be used to isolate the nucleic acid sequence for the KS domain, the nucleic acid sequence for the KS domain-containing PKS, and the nucleic acid sequence of the entire gene cluster necessary for polyketide synthesis. Methods for isolating such nucleic acid sequences are well known to those skilled in the art and include, for example, genome walking or screening of cosmid or fosmid libraries. The isolated nucleic acids can then be identified / identified by sequencing.
  • At least one primer is specific for 2, 3, 4, 5, 6, or more than 6 contiguous amino acid residues of one of the defined in Fig. 3 or Fig. 4 ten kladpezif ⁇ schen amino acid motifs, which are also shown in SEQ ID NO 40-71.
  • the at least one primer of the primer pair at the 3 'end of the primer is specific for the said amino acid residues.
  • both primers of the primer pair are specific for at least 2, 3, 4, 5, 6 or more than 6 contiguous amino acid residues of the amino acid motifs of a clade defined in FIG. 3 or 4, wherein the two primers are based on the nucleic acid with which they hybridize, keep a sufficient distance from each other to allow amplification.
  • the primer pair used has the nucleotide sequence: 5'-GCN HTN GAR GAY GCN GGN TAY GC-3 '(EDAGY; SEQ ID NO: 72) and 5'-C NAR CAT NCC NGC YTK RTA RTA- 3 '(YYQAGMLA; SEQ ID NO: 73).
  • a second primer pair is used for the amplification of the nucleic acid region of the KS domain enclosed by the at least one primer pair in addition to the at least one primer pair according to the invention, wherein the first and the second primer pair are nested, i. one pair of primers lies completely within the nucleic acid region to be amplified by the other primer pair.
  • the second primer pair is a primer pair prepared according to the invention and is therefore likewise based on the substrate specificity of a ketosynthase domain of a trans-AT polyketide synthase.
  • the second primer pair is a degenerate primer pair.
  • this primer pair is specific for the amino acid sequence KSDPQQF (forward) or KSHGTGR (reverse).
  • the second primer pair has the nucleotide sequence 5'-MGN GAR GCN NWN SMN ATG GAY CCN CAR CAN MG-3 'and 5'-GGR TCN CCN ARN SWN GTN CCN GTN CCR TG-3', respectively.
  • amplification with the two primer pairs is performed simultaneously or in two stages.
  • the outer primer pair is used in the first round of amplification.
  • the amplificates from the first round of amplification are amplified with the internally interleaved primers.
  • the amplification to be carried out with the at least two primer pairs is a nested PCR.
  • the isolation of the at least one polyketide biosynthesis gene from a metagenome takes place.
  • the metagenome comprises the genome of at least one eukaryote and at least one prokaryote.
  • eukaryotic and prokaryotic are in symbiosis.
  • the eukaryote is a sponge, preferably this sponge belongs to the Demospongiae, the Dictyoceratida or the Irciniidae.
  • the sponge belongs to Psammicinia sp .; more preferably, the sponge is Psammocinia äff. bulbosa.
  • the method according to the invention further comprises the step of isolating the entire gene region required for the polyketide synthesis based on the sequence information of the isolated at least one polyketide biosynthesis gene.
  • this isolation of the entire gene region is done using a library.
  • the library is a cosmid, YAC, BAC, or fosmid library.
  • the library was generated from a metagenome.
  • the metagenome is the metagenome of a sponge.
  • Figure 1 shows an overview of some exemplary complex polyketides isolated from sponges, except for pederin, which are synthesized by symbionts or suspected of being synthesized by symbionts.
  • psymberin iriniastatin A
  • 2 onnamides A 3 pederin
  • 4 myclamide A 5 laulimalide (Fijianolide B)
  • 6 latrunculin A 7 mycothiazole
  • 8 pateamine A 9 spongistatin 1 (altohyrtin A).
  • the zodiac sign (*) indicates the position of the ß branches.
  • Figure 2 is a Bayesian cladogram of full-length KS domains from trans AT-PKS.
  • the KS numbering refers to the position within the gene cluster starting from the upstream end.
  • LkcKS3 is the third lankacidin KS domain.
  • the cis-AT KS4 of erythromycin PKS was used as an outgroup. Probability values> 0.6 are given at the nodes.
  • Clade types are specified in Roman numerals along with the main substratum type. Bae, Bacillaene; BT, uncharacterized PKS from B.
  • thailandensis Chi
  • Chi chivosazole
  • Dif difficidin
  • Dz disorazole
  • GU uncharacterized PKS from Geobacter uraniumreducens
  • Lkc lankacidin
  • Lm Leinamycin
  • MIn Macrolactin
  • Mmp mupirocin
  • Onn Onnamide
  • Ped Pederin
  • Ta myxovirescin.
  • FIG. 3 shows the sequence alignment of KS protein sequences of individual clades.
  • the numbering refers to the amino acid positions of Pedl, which includes PedKSl.
  • the arrows indicate regions to which primers were prepared according to the invention, which were used for the amplification of the PKS gene fragments. Clade-specific amino acid residues within these regions are shown in bold.
  • the zodiac marks the conserved cysteine of the active site.
  • SupA is from Burkholderia pseudomallei 1710b (BURPS 1710b_A2618, first KS domain);
  • KAKS 1 comes from Kordia algicida (KAOT 1 04270, first KS);
  • SupA.Tsl is from Theonella swinhoei (GenBank accession ABE03935, first CS);
  • SupA.Aal is from Aptysina aerophoba (ABE03915, first CS);
  • SupA.Aa2 is from A.
  • SupA.Pcl is from Pseudoceratina clavata (ABB73286, first CS); SupA.PKSA is from Discodermia dissoluta (SA1_PKSA, second CS).
  • Figures 4A-4I show a sequence alignment of the clad-specific motifs suitable for a group-specific primer design according to the invention.
  • the group specific amino acids are shown as white letters against a black background; Generally conserved amino acids are grayed out (80% treshold) and amino acids conserved in some but not all groups are shown in bold.
  • the division into the clades is based on a phylogenetic analysis according to Nguyen, T., Ishida, K., Jenke-Kodama, H., Dittmann, E., Gurgui, C, Hochmuth, T., Taudien, S., Platzer, M ., Hertweck, C. and Piel J. Nat.
  • supAl ABK01346.1
  • supA2 ABK01347.1
  • supA4 ABK01355.1 were also added to the sequence alignment to avoid ubiquitous PKS sequences from sponges.
  • Figure 5A shows the genomic organization of the isolated PKS gene cluster for psymberin biosynthesis.
  • Figure 5B schematically illustrates the structure of the identified PsyA and PsyD proteins, illustrating the individual modules as well as the biochemical transformations occurring thereon.
  • Figure 5C compares the structures of the corresponding PKS proteins for pederin and onnamide biosynthesis.
  • the amino acid sequences of 138 known trans-AT PKS modules were taken from the GenBank database and ordered and aligned ("alignment") under manual control using ClustalX, thereby excluding deletions and unalignable sequence regions.
  • a phylogenetic analysis of the full-length KS domains of the processed amino acid sequences was performed using the MrBayes software (v3) using "WAG replacement mode” and a four-category gamma distribution.
  • the Marko vketten Monte Carlo analysis ran for 2.5 million Cycles were sampled every 100 generations and the convergence was assessed by plots of the maximum likelihood values and a standard deviation of ⁇ 0.05
  • the consensus tree and the later plaque probabilities were calculated after discarding the trees that had died before
  • the phylogenetic analysis based on the Neighborhood Joining technique was performed using the Seqboot, Protdist, Neighbor, and Consens programs of the PHYLIP software, and the bootstrap analysis was performed with 1000 pseudo-replicate sequences aximum parsi
  • the derived cladogram shown in Figure 2 clearly shows that individual clades contain KS of different bacteria. Subsequently, the domain architecture of the N-terminal neighboring module was analyzed for all CSs and, together with biosynthetic considerations (predicted from the polyketide structure), the substrates of all KS domains were derived. Phylogenetically clearly defined groups of KS with the same substrates were found, ie a distribution of the domains of the individual biosynthetic pathways to individual clades.
  • FIG. 3 and FIG. 4 show the thus detected consensus sequences from which primers can be derived according to the invention which are each specific only for that KS domain which is involved in the biosynthetic pathway of a particular group found in a polyketide. Furthermore, a tyrosine residue was identified in an EDAGY motif that is conserved in 84% of all trans AT-KS, but does not occur in the ubiquitous sup KS.
  • the total DNA from the samples was isolated by a method known to those skilled in the art to remove the high level of polysaccharides (Murray, MG and WF Thompson (1980) Rapid Isolation of High-Molecular-Weight Plant DNA. Nuc. Acids Res 8: 4321-4325).
  • the frozen sponge material was thawed and a small piece of ca. Ig was cut off which contained both the sponge surface and the interior of the sponge.
  • the tissue was ground under liquid nitrogen using pre-chilled mortar and pestle.
  • the resulting powder was transferred immediately thereafter into a 50 ml Falcon tube containing 10 ml lysis buffer (8 M urea, 2% sarcosyl, 1 M NaCl, 50 mM EDTA and 50 mM Tris-HCl pH 7.5).
  • the Lysere force was carried out at 60 0 C for 15 min, the mixture was carefully mixed every 5 min.
  • the isolated DNA was then extracted twice with 10 ml of phenol / CHCl 3 / isoamyl alcohol (25: 24: 1, v: v: v) and once with 10 ml of C HC I 3 . Each extraction was followed by a centrifugation step at 11,000 rpm for 5 min. To remove polysaccharides, the upper aqueous phase containing the DNA was treated with a 10% (w / v) aqueous cetyltrimethylammonium bromide (CTAB) solution at a final concentration of 0.5%. The mixture was incubated at 60 ° C for 15 min and the precipitate was removed by centrifugation at 11,000 rpm for 15 min.
  • CTAB cetyltrimethylammonium bromide
  • the nucleic acids were precipitated with 2 volumes of precooled ethanol (98%) and 1/10 volume of a 3 M sodium acetate solution (pH 7).
  • the precipitate obtained after a 30 minute centrifugation step at 11000 rpm at 4 ° C was washed twice with cold 70% ethanol.
  • the crude DNA (approximately 5 ⁇ g) was air-dried for 10-15 min and resuspended in Tris buffer (10 mM, pH 8.5).
  • the CopyControl Fosmid Library Production Kit (Epicenter Biotechnologies) was used, whereby the manufacturer's protocol was modified.
  • the DNA was separated in a 1% (w / v) low-melting point agarose gel and fragments about 35 kb in size were isolated by gel digestion with GELase.
  • 1 U of the GELase enzyme solution was added to each 300 ul of molten agarose, followed by incubation for 2 h at 45 0 C.
  • a first step the complexity of the DNA sample was first reduced by means of a first PCR amplification.
  • degenerate primers were used which had previously been used for the isolation of the genes of pederin and omnamide (Piel et al., Anititumor polyketide biosynthesis by an uncultivated bacterial symbiont or the marine sponge Theonella swinhoei., PNAS 101, 16222-16227; 2004; Piel, A polyketide synthase peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles, PNAS 99, 14002-14007, 2002).
  • the forward primer had the sequence 5 '-MGNGARGCNNWNSMNATG GAYCCNCARCANMG-3'; the reverse primer had the sequence 5'-GGRTCNCCNA RNA WNGTNCCNGTNCCRTG-3 '.
  • the PCR took place under the following conditions: 96 0 C for 45 s, 55 0 C for 45 s, 72 0 C for 1 min (40 x), then 72 0 C for 10 min.
  • Taq polymerase (NEB) was used.
  • the amplificate was separated by agarose gel and the DNA running at 700 bp purified by gel digestion as described above. 1 ⁇ l of this purified PCR amplificate was used as template for a second round of PCR.
  • the target was the acetyl-derived starter unit, which is usually extended by KS belonging to Klade VI.
  • the unique sequence motif YYQ / KAGML was found in all KSs following a module with a GCN5-related N-acetyltransferase (GNAT) domain. This motif was chosen for the primer design, as it could be shown that GNATs incorporate decarboxylation of malonyl-CoA acetyl starter units specifically into polyketides.
  • the primers used were specific for the EDGAY motif as well as for the YYQ AGML motif.
  • the forward primer had the sequence 5'-GCNHTNGARGAYG CNGGNTAYGC-3 '(SEQ ID NO: 72); the reverse primer had the sequence 5'-CANCATNCCNGCY TKRT ART A-3 '(SEQ ID NO: 73).
  • reaction conditions were: 95 0 C for 300 s, 95 0 C for 60 s, 52 0 C for 60 s and 74 0 C for 100 s (35 X), 74 0 C for 10 min.
  • Taq polymerase NEB was used.
  • the amplification yielded a single PCR product of the expected size, the sequencing of which revealed that it indeed belonged to the acetyl-specific clade VIa.
  • a fosmid library of total DNA from the psymberin-positive sponge P. äff. bulbosa was generated (410,000 clones) and assayed for positivity according to a previously known strategy (Hrvatin & Piel, Rapid Isolation of rare clones from highly complex DNA pools, J. Microbiool., Methods 68, 434-436, 2007) Clones screened out.
  • the screening was performed by PCR and using specific primers derived from the clade VI amplicon.
  • five positive fosmids were isolated, and Fig. 5A and Table 1 show the result of sequencing (SEQ ID NO: 39) for these five fosmids, which showed a typical bacterial architecture No introns were detected, Shine-Dalgarno-like sequences were located in front of the individual genes and the close proximity of the genes suggests a polycistronic mRNA.
  • ORF2 426 adenylosuccinate synthetase 2, 21
  • ORF3 367 transposase 3, 22 psyA 3297 PKS 4, 23 psyB 331 methyltransferase 5, 24 psyC 343 PedK-like 6, 25
  • the isolated gene region (SEQ ID NO: 39) in its central region comprises a psy-Gznc chill (psyA to psyN) of 62238 bp length.
  • the large genes psyA and psyD encode PKS with three and ten modules, respectively, with the latter ending with a thioesterase domain (see Figure 5B). This reveals for the first time the complete sequence information of a polyketide biosynthetic cluster.
  • PsyA and the first five modules of PsyD are almost identical to the omnamide (OmnB and OnnI) and pederin (Pedl and PedF) biosynthetic pathways ( Figure 5 B, C).
  • the complexity of the DNA sample was first reduced by means of a first PCR amplification, the primers used also having the sequences 5 '-MGNGARGCNNWNSMNATGGAYCCNCARCANMG-S' (forward) and 5'-GGRTCNCCNARNSWNGTNCCNGTNCCRTG-S '(reverse). had.
  • the first round of PCR took place under the following conditions: 96 0 C for 45 s, 55 0 C for 45 s, 72 0 C for 1 min (40 x), then 72 0 C for 10 min.
  • Taq polymerase (NEB) was used.
  • the amplificate was separated by agarose gel and the DNA running at 700 bp purified by gel digestion as described above. 1 ⁇ l of this purified PCR amplificate was used as template for a second round of PCR.
  • the target for the second round of PCR was a motif detected in KS that prolongs ⁇ -saturated intermediates (Klade V).
  • the identified motif has the consensus sequence EPI / VE / DTAC (see Fig. 4A).
  • the primers used were specific for the EDGAY motif as well as for the EPI / VE / DTAC motif.
  • the forward primer had the sequence 5'-GCNHTNGARGAYG CNGGNT AYGC-3 '(SEQ ID NO: 72); the reverse primer had the sequence 5'-RCANGCNGTNTCDATNGGTTC-3 '(SEQ ID NO: 74).
  • the reaction conditions were: 95 0 C for 105 s, 95 0 C for 60 s, 51 0 C for 60 s and 74 0 C for 100 s (35 X), 74 0 C for 10 min. Hot Start Taq Polymerase (Jena Biosciences) was used.
  • the gene sequence of the entire PKS gene cluster required for mycalamide biosynthesis is determined.
  • Bacillus amyloliquefaciens (strain FZB42 J NCBI Accession Number CP000560) is one of the richest bacterial resources for bioactive natural products, such as polyene anti- biotika bacillaene and difficidin. Therefore, this organism was used for heterologous expression.
  • Bacillaene PKS gene cluster has been replaced by several steps of double crossover recombination by that of the psymberin PKS gene cluster.
  • the Bacillaene PKS gene cluster was deleted in the first step and instead a psymberin recombination cassette was integrated together with a chloramphenicol marker at the beginning of the gene cluster.
  • the plasmid used for this was pBBaeKO (SEQ ID NO: 76). This generated the mutant strain FZB42 Bae (see Fig. 6).
  • a ⁇ -red double crossover was performed to modify the first positive fosmid, pPSKFl, for the integration of the first psymberin regions into the genome of strain FZB42bae.
  • the plasmid used for this was pBPsyI20 (SEQ ID NO: 77).
  • a bacillaene recombination cassette was introduced into the fosmid together with a spectomycin selection marker, immediately downstream of the end of the psymberin sequence. This resulted in pPSKFlBae20 (see Fig. 7, SEQ ID NO: 78).
  • this construct was then transformed into a FZB42 Bae to generate FZB42 Bae-Psy + ( Figure 8).
  • the further downstream psymberin gene regions can be integrated into the genome of FZB42 Bae-Psy + in a similar fashion until the gene cluster is completed.
  • the chloramphenicol resistance cassette (CamR) was obtained from the vector pDG268 by EcoRI and SphI digestion, while the spectinomycin resistance cassette (SpeR) was obtained by digesting the vector pIC333 with XbaI and BamHI.
  • the constructs used for the bacillaene deletion and the modification of the positive fosmids, namely pBBaeKO and pBPsyI20, respectively, were performed by means of two 3-point ligation steps Cloning of the abovementioned fragments into the pBluescript SK II (-) vector (see Figs. 9A and 9B)
  • the pBBaeKO construct was linearized by KpnI and purified from the gel.
  • competent B. amyloliquefaciens cells were prepared by the method of Art and Rapoport (J Bacteriol 1995; 177 (9): 2403-7) with minor modifications introduced (Idris et al., Mol Plant Microbe Interact. 2007; 20 (6): 619-26).
  • Approximately 5 ⁇ g of the linearized pBBaeKO construct was transformed and the recombiinant clones were selected by chloramphenicol (5 ⁇ g / ml).
  • the new mutant strain FZB42bae thus generated was checked by means of the following primer pairs (see PCR 1 and PCR 2 in FIG. 6):
  • PCRl BaeHKO for: 5'-TAGACCATCATACCGGAAGC-3 '(SEQ ID NO: 91) and PsyAKO rev: 5'AATGGAGCGAAGATAGCCG-S' (SEQ ID NO: 92);
  • PCR2 CatKO for: 5'-ACAGCTCCAGATCCTCTACG-S '(SEQ ID NO: 93) and nucBKOrev: 5'-GACTTGCCGCCCATTTCATA-3' (SEQ ID NO: 94).
  • the pPSKF! Positive fosmid can be appropriately modified by the incorporation of a bacillaene recombinant cassette (BaeS) together with a spectinomycin selection marker (SpeR).
  • BaeS bacillaene recombinant cassette
  • SpeR spectinomycin selection marker
  • the resulting construct pPSKFlBae20 was then naturally transformed in circular form into the strain FZB42 Bae.
  • the DNA concentrations ranged from 5 to 20 ⁇ g.
  • the recombinant clones were selected on spectinomycin (100 ⁇ g / ml).
  • the new mutants thus generated were checked by means of the following primer pairs (see PCR in FIG. 8):
  • pPSKFIKanBae SEQ ID NO: 97, Figure 11
  • pPSKFIKanBae SEQ ID NO: 97, Figure 11

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Abstract

La présente invention porte sur un nouveau procédé d'isolation ciblée de gènes biosynthétiques de polykétide issus de métagénomes. En outre, on a proposé pour la première fois les séquences d'acides nucléiques et d'acides aminés du cluster PKS de la psymbérine, ainsi que des systèmes d'expression pour la production de ce polykétide.
PCT/EP2009/067033 2008-12-15 2009-12-14 Gènes biosynthétiques de polykétide WO2010072600A1 (fr)

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