DE102008054660A1 - Polyketide biosynthesis genes - Google Patents

Abstract

The present invention provides a novel method for the targeted isolation of polyketide biosynthesis genes from metagenomes. Furthermore, for the first time, the nucleic acid and amino acid sequences of the PKS cluster of psymberin, as well as expression systems for the production of this polyketide are disclosed.

Description

The The invention relates to a method for the isolation of gene sequences, and the gene sequence and fragments isolated by this method same for peptides for biosynthesis of polyketides coded.

Sponges (Porifera) are the most important source of marine products with therapeutic potential ( Blunt et al., Marine natural products. Nat. Prod. Rep. 25, 35-94; 2008 ). Many of the drug candidates isolated from sponges are complex polyketides (PK) that are closely related to natural products from bacteria at the metabolic level ( Sipkema et al., Marine sponges as pharmacy. Mar. Biotechnol. 7, 142-144; 2005 ). This close relationship led to the assumption that the polyketides are not produced by the sponges themselves, but by prokaryotic symbionts. Investigations on the sponge Theonella swinhoei indicate that this assumption is correct ( fell et al., Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei. Proc. Natl. Acad. Sci. USA 101, 16222-16227; 2004 ). A sponge can house several hundred species of prokaryotic symbionts, each of which provides its own polyketide synthase (PKS) genes for various biosynthetic pathways. The resulting sponge metagenome may therefore contain hundreds of individual (prokaryotic) genomes, each providing its own PKS gene ( Kim et al., Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ. Microbiol. 8, 1460-1470; 2006 ).

A direct cultivation of individual symbionts resulted in the Most cases do not succeed, bringing the isolation of a specific, interesting PKS gene cluster for biosynthesis of polyketides from such a metagenome a big one Problem presents. Among other things, namely z. B. the Production and screening of extremely large DNA libraries required and the identification of the correct PKS gene cluster represents a big challenge.

The The present invention provides a solution to this problem ready and reveals a new approach to specific amplification of PKS gene regions based on the substrate specificity of Kethosynthasedomänen based.

In In a first aspect, the present invention relates to a Nucleic acid comprising at least one biosynthetic gene for a polyketide selected from the group consisting of psymberin or mycamide. In a specific embodiment the nucleic acid comprises a sequence selected 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, wherein the homolog has at least 75% sequence identity or under high salt conditions with the nucleic acid hybridized.

In In another aspect, the present invention relates to a peptide for the one of the invention Nucleic acids encoded. In a specific embodiment For example, the peptide has an amino acid sequence selected is selected from the group consisting of SEQ ID NO 20-38 or a Homolog of these sequences, wherein the homolog at least one sequence identity 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 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 from 96%, to SEQ ID NO 32 from 94%, to SEQ ID NO 33 from 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%.

In In another aspect, the present invention relates to one the nucleic acids according to the invention comprehensive vector and a recombinant host cell, the inventive Nucleic acids or the invention Vector includes.

In In another aspect, the present invention relates to a process for producing a polyketide comprising the step the cultivation of a recombinant according to the invention Host cell under conditions affecting the expression of the biosynthetic genes and allow production of the polyketide by the host cell.

In a further aspect, 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 pri merpaars from step a). In a specific embodiment, step b) comprises the amplification of the nucleic acid region enclosed by the primer pair. In further specific embodiments, the at least one primer pair is specific for a motif conserved in phylogenetically contiguous 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 one of in 3 . 4 or SEQ ID NO: 40-71 defined amino acid motifs. In another specific embodiment of the invention, the isolated polyketide biosynthesis gene is involved in the biosynthesis of psymberin or mycalamide.

1 Figure 4 shows a summary of some exemplary complex polyketides isolated from sponges, except for pederin, which are synthesized by symbionts or are suspected of being synthesized by symbionts. 1 psymberin (Irciniastatin 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 β branches.

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 7tream end. For example, 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, chivosazole; Dif, difficidin; Dz, disorazole; GU, uncharacterized PKS from Geobacter uraniumreducens; Lkc, lankacidin; Lm, Leinamycin; Mnn, macrolactin; Mmp, mupirocin; Onn, Onnamide; Ped, Pederin; Ta, myxovirescin.

3 shows the sequence alignment of KS protein sequences of individual clades. The numbering refers to the amino acid positions of PedI, which includes PedKS1. 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. V, sequences of the clade V; VI, sequences of the clade VIb; S, Sequences of SupA; BP17KS1 is from Burkholderia pseudomallei 1710b (BURPS1710b_A2618, first KS domain); KAKS1 is from Kordia algicida (KAOT1_04270, first CS); SupA.Ts1 is from Theonella swinhoei (GenBank accession ABE03935, first CS); SupA.Aa1 is from Aplysina aerophoba (ABE03915, first CS); SupA.Aa2 is from A. aeophoba (ABE03895, first CS); SupA.Pc1 is from Pseudoceratina clavata (ABB73286, first CS); SupA.PKSA is from Discodermia dissoluta (SA1_PKSA, second CS).

4A - 4I shows a sequence alignment of the clad-specific motifs which are 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 Nguyen, T., Ishida, K., Jenke-Kodama, H., Dittmann, E., Gurgui, C., Hochmuth, T., Taudien, S., Platzer, M., Hertweck, C. and Piel J. Nat. Biotechnol., 2008, 26, 225-233 (please refer 2 ). supA1: ABK01346.1, supA2: ABK01347.1 and supA4: ABK01355.1 were also added to the sequence alignment to avoid ubiquitous PKS sequences from sponges.

5A shows the genomic organization of the isolated PKS gene cluster for psymberin biosynthesis. 5B schematically illustrates the structure of the identified PsyA and PsyD proteins, illustrating the individual modules as well as the biochemical transformations occurring thereon. 5C compares the structures of the corresponding PKS proteins for pederin and onnamide biosynthesis.

The The present invention provides a new method for targeted isolation of polyketide biosynthesis genes from metagenomes, where the substrate specificity of the polyketide chain-extending Ketosynthase (KS) domains is exploited.

definitions

The term "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, probes or standards used with the PCR, with digoxigenin, Biotin or fluorescent dyes labeled nucleic acids or so-called PNAs ("peptide nucleic acids") understood. A nucleic acid often consists of two complementary strands. Therefore, in the case of a nucleic acid claimed by this invention, the reverse complement of this nucleic acid should generally also be considered as being claimed.

Under The term "high-salt buffer" is especially - but not limited to this - having a buffer a high salt concentration (preferably chaotropic substances), preferably ≥ 100 mM, more preferably ≥500 mM, and more preferably ≥1 M, understood.

Under the term "high salt conditions" is hereafter understood a medium that uses a high salt buffer, preferably a high salt buffer containing chaotropic salts. By high salt, preferably having chaotropic salts, the solubility becomes of nucleic acids in water decreased. Reason is the break up of hydrogen bonds and associated reduction the stabilization of secondary and tertiary Structures of nucleic acids in water. Will now be a polar Surface offered as a hydrogen bond donor, bind the nucleic acids to this surface, because they experience better stabilization there than in water. When the salt concentration is reduced, water becomes better again Hydrogen bond donor as the polar surface, and the nucleic acids are released from the surface peel off.

Under the term "chaotropic substances" or "chaotropic Salts "are in particular - but not on it limited - substances that are the secondary, Tertiary and / or quaternary structure of proteins and / or nucleic acids change and at least leave the primary structure intact, the solubility reduce polar substances in water and / or hydrophobic interactions reinforce, understood. Preferred chaotropic substances are guanidine hydrochloride, guanidinium (iso) thiocyanate, sodium iodide, Sodium perchlorate, potassium iodide, sodium (iso) thiocyanate and / or Urea.

The Terms "protein", "peptide", "polypeptide" and "enzyme" used synonymously unless the contrary is described.

Under the term "amplification" or "amplification reaction" understood a method that allows the concentration one or more analytes - preferably nucleic acids - at least to double. A distinction is made here between isothermal and thermocyclic Amplification reactions. In the former, the temperature remains during the same throughout the procedure, while in the latter case undergo thermal cycles, which help the reaction and the amplification is controlled.

• • Loop mediated isothermal amplification (LAMP),
• • Nucleic Acid Sequence Based Amplification (NASBA),
• • Rolling Circle Chain Reaction (RCCR), oder Rolling Circle Amplification (RCA), und/oder
• • Transcription mediated amplification (TMA) Bevorzugte thermocyclische Amplifikationsreaktionen sind z. B.
• • Ligase-Kettenreaktion (LCR), und/oder
• • Polymerase Ketttenreaktion (PCR)

Preferred isothermal amplification reactions are, for. B.

• Loop mediated isothermal amplification (LAMP),
• Nucleic Acid Sequence Based Amplification (NASBA),
• • Rolling Circle Chain Reaction (RCCR), or Rolling Circle Amplification (RCA), and / or
• Transcription mediated amplification (TMA) Preferred thermocyclic amplification reactions are e.g. B.
• Ligase chain reaction (LCR), and / or
• Polymerase chain reaction (PCR)

Under the term "polymerase chain reaction" (PCR) a method for in vitro amplification of nucleic acids understood how it z. As described in Bartlett & Stirling (2003).

Under the term "ligase chain reaction" (LCR) becomes Detection method for smallest amounts of nucleic acids understood, similar to the polymerase chain reaction works, only using a different enzyme (instead the polymerase a ligase). Two samples per DNA strand become a sample ligated. The resulting, often only 30-50 bp long amplificates of one cycle serve in the following cycles themselves again as a starting point for the supplemented primer.

Under the term "loop-mediated isothermal amplification" (LAMP) becomes a method for isothermal nucleic acid amplification understood in which 6 different primers are used, recognize the specific regions on the target sequence and it tie. LAMP utilizes a DNA polymerase with strand-displacement activity and runs at a constant temperature of about 65 ° C from. Amplification and detection of the target sequence takes place in one single step instead.

Under the term "nucleic acid sequence based amplification" (NASBA), is a process for the amplification of RNA understood (Compton 1991). An RNA matrix is added to a reaction mixture, and a first primer binds to the complementary sequence in the region of the 3'-end of the matrix. Subsequently, with a reverse transcriptase complementary to the matrix DNA strand polymerized. With the help of RNase H then the RNA matrix Digested (RNase H digested only RNA in RNA-DNA hybrids, not single-stranded RNA). Subsequently, a second primer is attached to the 5 'end bound to the DNA strand. This is from the T7 RNA polymerase as a starting point for the synthesis of a DNA strand complementary to RNA molecule, which is then used again as the starting matrix can be used. NASBA is at a constant temperature of normally 41 ° C. performed and delivered under certain circumstances faster and better results as a PCR.

Under the term "Transcription Mediated Amplification" (TMA) becomes an isothermal amplification process developed by the US company Gen-Probe understood, which is similar to the NASBA and in which also RNA Polymerase and reverse transcriptase are used (Hill, 2001)

The term "rolling circle chain reaction" (RCCR) or "rolling circle amplification" (RCA) refers to an amplification process that mimics the general nucleic acid replication according to the rolling-circle principle and inter alia in the US 5854033 is described.

Under The term "Nested PCR" is understood to mean a method in which an already duplicated DNA fragment (or part of it) is amplified a second time; this process takes place with a second primer pair, which is within the first Reaction used primer pair is arranged.

Under The term "promoter" in the present case is a nucleic acid sequence understood that allows the regulated expression of a gene. It is upstream, d. H. 5 'to the RNA coding region of the gene. An important property of a promoter is the specific interaction with certain DNA-binding proteins that initiate transcription of the gene through the RNA polymerase and as transcription factors be designated. The most important prokaryotic transcription factors are the sigma factors. By far the most promoters at z. B. E. coli are known and know about the factor Sigma-70 the following promoter elements: 1) an AT-base pair-rich UP element above the -35 region (which is also directly with the α subunit the bacterial RNA polymerase), 2) the -35 region, with the consensus sequence: 5'-TTGACA-3 ', 3) the -10 region (Pribnow box), with the consensus sequence: 5'-TATAAT-3 '. Furthermore, strong promoters right before the start point of transcription, rich in AT base pairs. The consensus sequences give only approximate clues for the construction of a promoter. A specific sigma-70 dependent Promoter may differ from these sequences in several places. In In a preferred embodiment, the promoter is a inducible promoter. An example of an inducible Promotor is the artificially generated tac promoter used to Increase gene expression level in bacterial cells (e.g. B. E. coli) and thus for overexpression recombinant Proteins can be used. This synthetic promoter exists from 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.

Under the term "biosynthesis" is used herein understood enzymatic preparation of a polyketide, preferably by means of one or more polyketide synthases, preferably one trans-AT-polyketide synthase. The term "biosynthesis gene" includes so that any gene coding for a protein, the part such polyketide synthase.

Farther The term "biosynthesis gene" also includes fragments a biosynthetic gene coding for a truncated protein encode a polyketide synthase that has the same function like the full-length protein. The skilled person is sufficient known that truncation (i.e., deletion of individual regions, preferably the ends) of a protein increased or decreased Enzyme activity can be brought about. Under same function here is the same principle biochemical Function understood, regardless of whether this with a increased or decreased efficiency or speed is performed. As an example of a biosynthesis gene be a gene for a KS domain called whose it is biochemical function, αβ-saturated Extend intermediates (see, for example, the KS clade V), or a gene whose gene product in a PKS biochemical Function to transfer a methyl radical (z. PsyB). Any truncated version of such genes that were too truncated Gene products that still perform the same biochemical function thus also fall under the term "biosynthesis gene". Further examples of biosynthetic genes and specific embodiments of the invention are the genes psyA-psyN listed in Table 1.

Under The term "metagenome" is used in the present context understood by at least two genomes of different species. One Metagenome thus includes the entire genomic information of this at least two species. In a specific embodiment The metagenome includes the genome of a sponge and that of at least a prokaryote. In another embodiment lives the prokaryote in symbiosis with the sponge.

One For the purposes of the present application, primer is then termed "specific" for a given amino acid sequence, if he - preferred under stringent conditions - with the sense or antisense strand a nucleic acid hybridized or identical to this is that for the given amino acid sequence or encoded a part of it. Because the amino acid code degenerates can, for a given amino acid sequence several suitable primers are used. The person skilled in the art is aware which base triplets for which amino acid residues encode. The primer should have a minimum length, which allows it to interact with a nucleic acid to hybridize for at least 2, at least 3, or at least 4 contiguous amino acid residues of encoded amino acid sequence. In a specific embodiment the primer hybridizes with a nucleic acid suitable for at least 5, 6, 7 or more than 7 contiguous amino acid residues encoded the given amino acid sequence.

For the purposes of the invention, a primer is also considered to be specific for the defined amino acid motifs if it is specific for an amino acid sequence which contains at least 2, at least 3, or at least 4 contiguous amino acid residues of one of the amino acid sequences 3 and 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. Here, 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. As an example, a primer specific for the in 3 defined amino acid motif "YYQAGML" also be prepared so that it is specific for the amino acid sequence "YYQAGML A ", 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 PKS produce a large amount of therapeutically important natural products, such. Erythromycin, epothilones and FK506 ( Fischbach & Walsh. Assembly-line enzymology for poliketide and nonribosomal peptide antibiotics: logic, machinery and mechanisms. Chem. Rev. 106, 3468; 2006 ). 1 Figure 4 shows some exemplary polyketides which, as far as their gene clusters are known, are formed by Type I PKS.

each these enzymes comprise at least one β-ketosynthase (KS), an acyltransferase (AT) and an acyl carrier protein (ACP), that select the next building block to build the polyketide, activate and form a β-ketoacyl-S-ACP intermediate, a decarboxylative Claisen condensation between the next Block and the growing polyketide chain catalyze. While of the synthesis process transfers an AT domain the corresponding acyl-CoA building block on an ACP domain and a KS domain prolongs the polyketide chain with this acyl moiety, with additional domains further modify the resulting β-oxo intermediate can. The order of the modules in the PKS is given here the order of biosynthesis steps mandatory. Thus, can by variation of the domains / modules a high structural Diversity can be generated in the resulting polyketides.

A special and evolutionarily delimited enzyme family of PKSs are the trans-AT-PKSs, which use free-standing AT to select in trans the building blocks required for the production of the polyketide ( Cheng et al., Type I polyketide synthase requiring a discrete acetyltransferase for polyketide biosynthesis. Proc. Natl. Acad. Sci. USA 100, 3149-3154; 2003 ). In contrast, cis-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 is a unique phylogenetic Pattern in which the closest related KS domains specific for the same or similar substrates are. In contrast, different substrates, eg. B. β-hydroxylated and olefinic, by evolutionary clearly distinguishable, d. H. not closely related, KS processed. The phylogenetic analysis of one or more KS thus provides useful information about the structure of the involved Intermediate.

According to the invention, the reverse path is taken, in which due to the chemical Struk Polyketide conclusions can be drawn on 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. B. be used in gene clusters, for. By PCR, screening of Fosmid libraries or genome walking.

one The main advantages of the present invention are therefore that that starting from the polyketide targeted one in its biosynthesis involved KS domain of a trans AT PKS identified in a next step their nucleotide sequence, and the nucleotide sequence of the polyketide biosynthetic gene cluster comprising it to identify and isolate. The present invention is Accordingly, based on the surprising discovery that starting from the chemical structure, d. H. the functional groups of a Polyketides, conserved motifs within KS domains can be identified in the biosynthesis of this Polyketides are involved. Based on these conserved motifs then can be derived primers for amplification and isolation used the genes involved in the biosynthesis of this polyketide can be.

Nucleic acids and Peptides of the present invention

One aspect of the present invention is a 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. In one embodiment, the nucleic acid is in isolated form. In a further embodiment, the 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. In another embodiment, 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. In one embodiment, 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. Trans-AT 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. Sci. USA 100, 3149-3154; 2003 ), while cis-AT PKS have integrated AT domains.

In In a preferred embodiment, the inventive Nucleic acid amplified from the metagenome of a sponge. Eukaryotic sponges (Porifera) go with various prokaryotes Symbioses, these prokaryotes often not yet isolated could be cultivated. The resulting sponge and prokaryotes The resulting metagenome includes the genome of the eukaryotic sponge as well as the prokaryotic genomes of the individual symbionts. In a Embodiment, the sponge belongs to the Demospongiae, the Dictyoceratida or the Irciniidae. In a preferred embodiment the sponge belongs to Psammocinia sp .; even more preferred is the sponge Psammocinia aff. bulbosa. In a preferred embodiment the sponge belongs to the Demospongiae. In further preferred Embodiments include the sponge to the family Spongiidae, Thorectidae, Halichondriidae, Callyspongiidae, the Chalinidae, the Mycalidae, the Coelosphaeridae, the Tedaniidae, the Plakinidae, or the Triaenosina. For the purposes of this invention preferred representatives of these families are Spongia spp., Cacospongia spp., C. mycofijiensis, Halichondria spp., Callyspongia spp., Haliclona spp., Mycale spp., M. hentscheli, Lyssodendoryx spp., Tedania spp., Plakortis spp., Theonella swinhoei, and Discodermia spp.

The Amplification of the nucleic acid from the metagenome can by means of any method of nucleic acid amplification known to those skilled in the art be performed. In one embodiment the amplification of the invention Nucleic acid by means of an isothermal or a thermocyclic Amplification reaction. The amplification preferably takes place by means of Ligase chain reaction (LCR), and / or polymerase chain reaction (PCR); more preferably, the PCR is a nested PCR.

In one embodiment, the nucleic acid according to the invention comprises at least one sequence selected from the group consisting of SEQ ID NO 1-19, 39, a homolog of these sequences and the complement of the sequences or the homolog. In a preferred embodiment, 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 (psyA, psyB, psyC, psyD, psyE, psyF, psyG, psyH, psyI, psyJ, psyK, psyL, psyM and psyN), a homolog of these sequences and / or the complement of the sequences or the homolog. In a further embodiment, 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.

In Another embodiment encodes the invention Nucleic acid for a protein containing 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 from 94%, to SEQ ID NO 30 from 96%, to SEQ ID NO 31 from 96%, too 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%. In a preferred Embodiment encodes the invention Nucleic acid for a protein containing 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%.

One Another aspect of the present invention is a peptide, polypeptide, Protein or enzyme for which one of the inventive Nucleic acids encoded.

In In one embodiment, the peptide has an amino acid sequence which is selected from the group consisting of SEQ ID NO 20-38.

In In another embodiment, the peptide is at least an identity to SEQ ID NO 20 of 36%, to SEQ ID NO 21 from 54%, to SEQ ID NO 22 of 44%, to SEQ ID NO 23 of 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 from 96%, to SEQ ID NO 32 from 94%, to SEQ ID NO 33 from 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%. In a preferred embodiment, the peptide at least an 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%. In a further preferred embodiment, the Peptide has a sequence identity of at least 60%, 70%, 75%, 80%, 90%, 92%, 95%, 97%, 98%, 99% or 99.5%.

In In another embodiment, the invention relates to a Vector containing the nucleic acid according to the invention includes. Preferably, this vector is a cosmid, a YAC, BAC or a virus, more preferably the vector is a plasmid. In a Embodiment, the vector further comprises a promoter under whose control the expression of the invention Nucleic acid is. Preferably, this promoter is an inducible Promoter. In a further embodiment, the inventive Nucleic acid under the control of an inducible expression system. In a specific embodiment, the inducible expression system is the tetracycline operator / repressor (TetO2 / TetR) system.

In In another embodiment, the invention relates to a recombinant host cell, which is one of the inventive Nucleic acids or vectors. Thus represents the present Invention also an expression / and production system for Polyketides ready. Preferably, this recombinant host cell after transformation / transfection able to synthesize polyketides. In one embodiment, this is the recombinant host cell an E. coli cell, in other embodiments is the recombinant host cell, a host cell from Pseudomonas spp., Bacillus spp., Streptomyces spp. or Acinetobacter baylyi.

invention Process for the preparation of polyketides

One Another aspect of the invention relates to the use of the invention Nucleic acids, vectors or recombinant host cells in a process for producing a polyketide. This is used an expression system which expresses the expression of the invention Nucleic acids allowed.

In In one embodiment, the expression system is a recombinant Host cell containing the nucleic acids according to the invention or vectors and for expression of the gene products of the nucleic acids according to the invention be able to. The expressed gene products can then be used for the synthesis of the polyketide in vivo or in vitro. In a preferred embodiment, the produced Polyketide Psymberin or Mycalamide. In a further preferred Embodiment includes the method or the expression system comprising the use of at least one nucleic acid 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 (psy A, psyB, psyC, psyD, psyE, psyF, psyG, psyH, asylum, psyJ, psyK, psyL, psyM and psyN) or a homolog of these sequences.

In a specific embodiment finds the cultivation a recombinant host cell according to the invention under conditions that control the expression of the biosynthetic genes and allow production of the polyketide by the host cell. Such Conditions are well known to those skilled in the art or can be integrated in the course of simple routine experiments given polyketide. In a specific embodiment the recombinant host cell is a Pseudomonas host cell spp., Bacillus spp., Streptomyces spp., Coryne bacterium spp., Acinetobacter baylyi, Pseudomonas stutzeri, Bacillus subtilis or Bacillus amyloliquefaciens. If that is at least one gene of the invention, which is in the host cell under the control of a inducible expression system (eg of the tetracycline operator / repressor (TetO2 / TetR) Systems) can be induced by adding the respective inducing Substance expression of the gene product can be specifically induced.

to Production of the polyketide are the genes of the gene cluster in the Integrated genomes of expression hosts. The integration takes place successively by homologous recombination shorter (approximately up to 40 kb) sections of the gene cluster. To keep the genes efficient can preferably transform transformable bacteria such as Pseudomonas stutzeri, Acinetobacter baylyi, Bacillus subtilis or Bacillus amyloliquefaciens which are linearized DNA can record. If necessary, the natural Replaced promoters of the gene cluster against host promoters (eg the baeB promoter in B. amyloliquefaciens). Alternative to Integration into the genome can spread or distribute genes on several compatible plasmids by conjugation, protoplast transformation or electroporation into the expression hosts. Expression is then carried out by freely replicating plasmids or again after integration into the genome.

In In another embodiment, the method further comprises the step of isolating the produced polyketide. In a Embodiment, this isolation is carried out by means of HPLC and / or preparative column chromatography.

invention Process for the isolation of polyketide biosynthesis genes

The Isolation of the at least one polyketide biosynthetic gene using of the at least one primer pair comprises in one embodiment the amplification of the trapped by the respective primer pair Nucleic acid region by means of the primer pair. Thus, will a nucleic acid region of the kethosynthase domain amplified. In a preferred embodiment this amplification is performed by PCR, more preferably by means of a nested PCR.

In In another aspect, the present invention is a method for isolating at least one polyketide biosynthesis gene and comprises the steps of providing at least one primer pair, due to the substrate specificity of a kethosynthase domain a trans-AT polyketide synthase has been created, and isolation at least one polyketide biosynthesis gene using this at least one primer pair.

The inventive provision, d. H. the "design" of Primer is based on the surprising discovery that the KS of trans-AT PKS a unique phylogenetic pattern exhibit, in which the closest related KS domains specific for the same or similar substrates are. Thus, starting from the functional groups of a Polyketide, a conserved motif within KS domains be identified on the basis of which primers are produced can, for such a conserved motive - and thus for a KS domain involved in the biosynthesis of Polyketide is involved - are specific. The amplification and isolation of the KS domain and thus of the KS domain comprehensive PKS and others in the biosynthesis of the polyketide involved genes that are often present in a gene cluster, is thus made possible according to the invention.

If a given KS or KS domain to a second KS or KS domain similar or phylogenetically adjacent can, for example, be determined by sequence comparisons, in which mutually similar sequences phylogenetic closely adjacent KS or KS domains. In a preferred embodiment of this is a used phylogenetic analysis. In a specific embodiment a cladogram is created for the phylogenetic analysis. In a further specific embodiment, the Kladogram with the Bayesian method and as described in Example 3 created.

In a further embodiment, according to the phylogenetic analysis for the KS, the domain architecture of the N-terminal adjacent module is analyzed and from the polyketide structure the substrate of the Derived KS domain. Domain architecture refers to the sequence of domains in a module, such as a domain. B. KS DH KR ACP.

In a preferred embodiment, the method according to the invention is directed to the directional isolation of at least one gene involved in the polyketide biosynthesis, ie, starting from the functional groups of a polyketide, the gene (s) necessary for the production of this polyketide is / are isolated. In an exemplary but preferred embodiment, the polyketide biosynthesis gene is involved in the biosynthesis of psymberin. Psymberin ( 1 , 1) does not carry a completely reduced residue, but an acetyl-derived starter unit, which is usually extended by a very specific type of KS. In a phylogenetic analysis ( 2 ) showed that KS with such a substrate specificity belongs to Klade 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). 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 ( 3 ). These conserved motifs did not appear in the sequences of the other clades. Furthermore, 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 to isolate the nucleic acid sequence for the KS domain, the nucleic acid sequence for the KS domain comprehensive PKS, as well as the nucleic acid sequence of the entire gene cluster required for polyketide synthesis be used. Method for isolating such nucleic acid sequences are well known to those skilled in the art and include, for example Genome walking or the screening of cosmid or fosmid libraries one. The isolated nucleic acids can then be determined / identified by means of sequencing.

In a specific embodiment, at least one primer is specific for 2, 3, 4, 5, 6, or more than 6 contiguous amino acid residues of any of the amino acid residues described in U.S. Pat 3 or 4 defined kladpezifischen amino acid motifs, which are also shown in SEQ ID NO 40-71. In a further specific embodiment, the at least one primer of the primer pair at the 3 'end of the primer is specific for the said amino acid residues.

In a further specific embodiment, 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 in 3 or 4 defined amino acid motifs of a clade, wherein the two primers based on the nucleic acid with which they hybridize, maintain a sufficient distance from each other to allow amplification.

In Another specific embodiment has the one used Primer pair 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).

In Another embodiment is for the amplification the nucleic acid region enclosed by the at least one primer pair the KS domain in addition to the at least one invention Primer pair used a second primer pair, wherein the first and the second primer pair are nested, d. H. the one primer pair completely within that to be amplified by the other primer pair Nucleic acid range is located. The second primer pair is in a preferred embodiment of a manufactured according to the invention Primer pair and thus also based on the substrate specificity a kethosynthase domain of a trans-AT polyketide synthase. In another embodiment, the second primer pair a degenerate primer pair. In a particularly preferred embodiment this primer pair is specific for the amino acid sequence KSDPQQF (forward) or KSHGTGR (reverse). In an even more preferred Embodiment, 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, respectively CCN ARN SWN GTN CCN GTN CCR TG-3 '.

In specific embodiments, the amplification with the two primer pairs carried out simultaneously or in two stages.

In the two-stage amplification in the first round of amplification is the outer primer few used. Thereafter, in a second round of amplification, the amplificates from the first round of amplification are amplified with the internal, nested primers. In a further preferred embodiment, the amplification to be carried out with the at least two primer pairs is a nested PCR.

In In another embodiment, the isolation of the at least one polyketide biosynthetic gene from a metagenome. In a preferred embodiment comprises the metagenome the genome of at least one eukaryote and at least one prokaryote. Preferably, eukaryotic and prokaryotic are in symbiosis. In another embodiment, the eukaryote is a Sponge, preferably this sponge belongs to the Demospongiae, the Dictyoceratida or the Irciniidae. In a preferred embodiment the sponge belongs to Psammicinia sp .; even more preferred is the sponge Psammocinia aff. bulbosa.

In In another preferred embodiment, this is to be isolated Polyketide Biosynthesis Gene in the Biosynthesis of Psymberin, the Mycalamides, Onnamids, Theopederines, Cadadamids, Pederin, Laulimalides (Fijianoliden), the Latrunculinen, Mycothiazol, Pateamin A, the Spongistatins (altohyrtins), discodermolide, dictyostatin, tedanolide and / or calyculin, d. H. Part of a polyketide synthase, which synthesizes one of these polyketides.

In In another embodiment, the invention comprises Procedure continues the step of isolating the whole for the polyketide synthesis required gene region based on the sequence information of the isolated at least one polyketide biosynthetic gene. In a specific embodiment, this isolation takes place of the entire gene region using a library. In further specific embodiments, the library is a cosmid, YAC, BAC or Fosmid library. In another specific embodiment the library was created from a metagenome. In more specific Embodiments of the invention is the metagenome the metagenome a sponge.

EXAMPLES

example 1

The Amino acid sequences of 138 known trans-AT PKS modules were taken from the GenBank database and under manual control ordered and aligned by means of ClustalX ("Alignment"). in this connection Deletions and non-alignable sequence regions were excluded.

hereafter was a phylogenetic analysis of full-length KS domains of the processed amino acid sequences by Mr Bayes Software (v3) using the "WAG replacement model" and a Gamma distribution performed with four categories. The Markov chains-Monte Carlo's analysis ran for 2.5 million cycles, with all 100 generations a sample was taken. The convergence was Plots of the maximum likelihood values and a standard deviation of <0.05. The consensus tree and the later cladding probabilities were calculated after discarding those trees before had been taken as a sample of convergence.

The phylogenetic based on the Neighborhood Joining method Analysis was carried out by means of the programs Seqboot, Protdist, Neighbor and consens of the PHYLIP software. The bootstrap analysis was performed with 1000 pseudoreplicase sequences. For the maximum parsimony analysis became the heuristic search option the software PAUP * 4.0b10 used.

This in 2 The depicted derived cladogram 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.

All Clades given in this cladogram have been further detailed analyzed separately to detect sequence patterns which the provision of primers specific to these motifs allow.

3 and 4 show the thus detected consensus sequences from which primers can be derived according to the invention, each of which is specific only for that KS domain involved in the biosynthetic pathway of a particular group found in a polyketide.

Farther a tyrosine residue was identified in an EDAGY motif that was expressed in 84% of all trans-AT-KS is conserved, but in the ubiquitous sup KS does not occur.

Example 2: Isolation of Metagenomic DNA and providing a library

insulation

rehearse sponges P. aff. bulbos and M. hentscheli were collected and frozen at -80 ° C. The metagenome of P. aff. bulbos was used to isolate the psymberin PKS gene cluster, while the M. hentscheli metagenome is the source of provided the myclamide biosynthetic gene clusters.

The total DNA from the samples was isolated by a method known to those skilled in the art to remove the high polysaccharide content ( Murray, MG and WF Thompson (1980). Rapid isolation of high molecular weight plans DNA. Nuc. Acids Res. 8: 4321-4325 ). Here, the frozen sponge material was thawed and a small piece of about 1 gram 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 lysis reaction was carried out for 15 min at 60 ° C, the mixture was carefully mixed every 5 min.

The isolated DNA was then extracted twice with 10 ml phenol / CHCl ₃ / isoamyl alcohol (25: 24: 1, v: v: v) and once with 10 ml CHCl ₃ . Each extraction was followed by a centrifugation step at 11,000 rpm for 5 min.

Around To remove polysaccharides, the upper aqueous phase, containing the DNA, with a 10% (w / v) aqueous Cetyltrimethylammonium bromide (CTAB) solution with a final concentration treated by 0.5%. The mixture was at 60 ° C for 15 min incubated and the precipitate was centrifuged at 11,000 rpm for 15 min.

The Nucleic acids were pre-cooled with 2 volumes Ethanols (98%) and 1/10 volume of a 3 M sodium acetate solution (pH 7) like. This after a 30 minute centrifugation step at 11,000 rpm at 4 ° C resulting precipitate was washed twice with cold 70% ethanol. The crude DNA (approx. 5 μg) was left in the air for 10-15 min and resuspended in Tris buffer (10 mM, pH 8.5).

Library

to Production of the library was the CopyControl Fosmid Library Production Kit (Epicenter Biotechnologies), the protocol of the Manufacturer was modified. The DNA was low-melting in a 1% (w / v) Pointed agarose gel and fragments of one size of about 35 kb were isolated by gel digestion with GELase. in this connection 1 U of the GELase enzyme solution was added to 300 μl each time molten agarose, followed by incubation from 2 h at 45 ° C. After inactivation of the enzyme (70 ° C, 10 min) and removal of undigested agarose by centrifugation for 20 min at 11,000 rpm, the DNA was at room temperature with 2.5 volumes of 98% ethanol and 1/10 volume of 3M sodium acetate (pH 5.2). After a centrifugation step for 30 min at 11,000 rpm and two washes with 70% ethanol at 4 ° C, the pelleted DNA was in 50 μl Tris buffer (10 mM, pH 8.5). This metagenomic DNA was used with Isopropanol and 3 M Nariumacetat (pH 5.2) like.

Approximately 250 ng of blunt-ended DNA (eluted with Tris buffer, 10 mM, pH 8.5) was used in a ligation step with the pCC1FOS vector overnight at 16 ° C according to the manufacturer's instructions, but using no T4 DNA ligase. The DNA was then packed into the Lamdaphagen and used to transfect the EPI300-T1 ^R E. coli cells according to the manufacturer's used.

For the production of libraries of large size All steps were taken from DNA preparation to plating the library in one day (and without freezing the samples) performed.

Example 3: Isolation of an psymberin KS gene fragments

In a first step, the complexity of the DNA sample was first reduced by means of a first PCR amplification. Here, degenerate primers were used which had previously been used for the isolation of the genes of pederin and omnamide ( fell et al., antitumor polyketide biosynthesis by an unculti vated bacterial symbiont od 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 ).

These primers used were for the amino acid motifs KSDPQQF or KSHGTGR specific. The forward primer had the sequence 5'-MGNGARGCNNWNSMNATG GAYCCNCARCANMG-3 '; had the reverse primer the sequence 5'-GGRTCNCCNA RNSWNGTNCCNGTNCCRTG-3 '.

The PCR took place under the following conditions: 96 ° C for 45 s, 55 ° C for 45 s, 72 ° C for 1 min (40x), then 72 ° C for 10 min. used became Taq Polymerase (NEB).

The Amplificate was separated by agarose gel and at 700 bp running DNA gel digestion as described above. 1 μl of this purified PCR amplificate was used as a template used for a second round of PCR.

For the second round of PCR, the target was the acetyl-derived starter unit, which is usually extended by KS belonging to Klade VI. In an additional alignment that contained all published Klade VI sequences, the unique sequence motif YYQ / KAGML (Klade VIa motif, 3 ) were found in all CSs 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 used primers were specific for the EDGAY motif, as well as for the YYQAGML motif. The forward primer had the Sequence 5'-GCNHTNGARGAYG CNGGNTAYGC-3 '(SEQ ID NO: 72); the reverse primer the sequence had 5'-CANRCATNCCNGCY TKRTARTA-3 '(SEQ ID NO: 73).

The Reaction conditions were: 95 ° C for 300 s, 95 ° C for 60 s, 52 ° C for 60 s and 74 ° C for 100 s (35X), 74 ° C for 10 min. It Taq polymerase (NEB) was used.

The Amplification gave a single PCR product with the expected Size, whose sequencing revealed that it was in indeed belonged to the acetyl-specific clade VIa.

These Results prove that by means of the invention Procedure single PKS genes in complex metagenomes with a exceptional accuracy selected and can be amplified.

Example 4: Isolation of the psymberin PKS gene cluster

A fosmid library of total DNA of the psymberin-positive sponge P. aff. bulbosa was produced (410,000 clones) and according to a previously known strategy ( Hrvatin & Piel, Rapid isolation of rare clones from highly complex DNA libraries. J. Microbiool. Methods 68, 434-436; 2007 ) screened for positive clones.

The Screening was performed by PCR and using specific Primers performed from the amplicon of the clade VI were derived.

Within three rounds of primer walking, five positive fosmids could be isolated. 5A and Table 1 represent the result of sequencing (SEQ ID NO: 39) of these five fosmids. The isolated gene cluster 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. Table 1: ORF protein size Proposed function SEQ ID NO ORF1 302 Zn-dependent hydrolase 1.20 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 ORF4 367 transposase 7.26 ORF5 491 transposase 8.27 PsyD 12644 PKS NRPS 9.28 psyE 571 Phosphoenolpyruvate synthase β + γ subunit 10.29 psyF 497 Phosphoesterase-like 11,30 psyG 309 Phosphoenolpyruvate synthase subunit 12.31 psyH 369 acyltransferase 13.32 PsyI 440 HMG-CoA synthase 14.33 psyJ 254 Crotonase superfamily 15.34 Psyk 367 Flavin-dependent oxygenase 16.35 psyL 81 ACP 17,36 Psym 429 3-oxoacyl ACP synthase 18.37 PSYN 709 Cation transport ATPase 19.38 ORF6 Unknown (partially)

As from z. B. 5A it can be seen, the isolated gene region (SEQ ID NO: 39) in its central region comprises a psy gene cluster (psyA to psyN) of 62238 bp length. The large genes psyA and psyD encode PKS with three and ten modules, the latter terminating with a thioesterase domain (see 5B ). This reveals for the first time the complete sequence information of a polyketide biosynthetic cluster.

With regard to the architecture of the PKS domains, PsyA and the first five modules of PsyD are almost identical to the omnamide (OmnB and OnnI) and pederin (PedI and PedF) biosynthetic pathways ( 5B , C).

Example 5: Isolation of a Mycalamide KS gene fragments

Again In a first step, complexity first became apparent the DNA sample is lowered by means of a first PCR amplification, where the primers used are also the sequences 5'-MGNGARGCNNWNSMNATGGAYCCNCARCANMG-3 ' (forward) and 5'-GGRTCNC CNARNSWNGTNCCNGTNCCRTG-3 '(reverse).

The first round of PCR took place under the following conditions: 96 ° C for 45 s, 55 ° C for 45 s, 72 ° C for 1 min (40x), then 72 ° C for 10 min. Taq polymerase (NEB) was used.

The Amplificate was separated by agarose gel and at 700 bp running DNA gel digestion as described above. 1 μl of this purified PCR amplificate was used as a template used for a second round of PCR.

The target for the second round of PCR was a motif detected in KS that prolongs αβ-saturated intermediates (Klade V). The identified motif has the consensus sequence EPI / VE / DTAC (s. 4A ).

The used primers were specific for the EDGAY motif, as well as for the EPI / VE / DTAC motif. The forward primer had the sequence 5'-GCNHTNGARGAYG CNGGNTAYGC-3 '(SEQ ID NO: 72); of the reverse primer had the sequence 5'-RCANGCNGTNTCDATNGGTTC-3 '(SEQ ID NO: 74).

The Reaction conditions were: 95 ° C for 105 s, 95 ° C for 60 s, 51 ° C for 60 s and 74 ° C for 100 s (35X), 74 ° C for 10 min. It Hot Start Taq Polymerase (Jena Biosciences) was used.

The Amplification again gave a single PCR product with the expected Size of 211 bp. Furthermore, one showed afterwards performed sequencing also that only one single KS gene fragment had been amplified, which was perfect fit into the predicted clade (SEQ ID NO: 75).

These Results prove again that by means of the invention Procedure single PKS genes in complex metagenomes with a exceptional accuracy selected and can be amplified.

Example 6: Isolation of the Mycalamide PKS Gene Cluster

The The screening method described above under Example 4 Fosmid library from mycamide-positive samples of the sponge M. hentscheli is repeated with primers similar to those obtained in Example 5 KS gene fragment are specific.

Out the detected positive fosmids will become the gene sequence of the whole PKS gene cluster determined for mycamide biosynthesis is needed.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• US 5854033 [0028]

Cited non-patent literature

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• - Kim et al., Diversity of polyketide synthase genes from bacteria associated with the marine sponge Pseudoceratina clavata: culture-dependent and culture-independent approaches. Environ. Microbiol. 8, 1460-1470; 2006 [0002]
• Nguyen, T., Ishida, K., Jenke-Kodama, H., Dittmann, E., Gurgui, C., Hochmuth, T., Taudien, S., Platzer, M., Hertweck, C. and Piel J Nat. Biotechnol., 2008, 26, 225-233 [0013]
• - Fischbach & Walsh. Assembly-line enzymology for poliketide and nonribosomal peptide antibiotics: logic, machinery and mechanisms. Chem. Rev. 106, 3468; 2006 [0036]
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• - Hrvatin & Piel, Rapid isolation of rare clones from highly complex DNA libraries. J. Microbiool. Methods 68, 434-436; 2007 [0097]

Claims (17)

Nucleic acid comprising at least one Biosynthesis gene selected for a polyketide the group consisting of psymberin or mycamide. Nucleic acid according to claim 1, wherein the nucleic acid comprises a sequence selected is selected from the group consisting of SEQ ID NO 1-19, 39, 75, a homolog of these sequences and / or the complement of the sequences or the homolog, wherein the homolog has at least 75% sequence identity or under high salt conditions with the nucleic acid hybridized. Nucleic acid according to claim 1-2, wherein the nucleic acid is for a protein encodes 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 from 44%, to SEQ ID NO 29 from 94%, to SEQ ID NO 30 from 96%, to SEQ ID NO 31 of 96%, to SEQ ID NO 32 of 94%, to SEQ ID NO 33 from 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%. Peptide for one of the nucleic acids encodes the preceding claims. Peptide selected from the group consisting from SEQ ID NO 20-38 or a homolog of these sequences, wherein the homolog is at least one sequence identity identity to SEQ ID NO 20 of 36%, to SEQ ID NO 21 of 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 from 94%, to SEQ ID NO 30 of 96%, to SEQ ID NO 31 of 96%, to SEQ ID NO 32 from 94%, to SEQ ID NO 33 from 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%. Vector comprising a nucleic acid according to claim 1-3. Recombinant host cell comprising a nucleic acid according to claim 1-3 or a vector according to claim 6. A process for producing a polyketide comprising the step of cultivating a recombinant host cell according to claim 7 under conditions involving the expression of biosynthetic genes and Allow production of the polyketide by the host cell. The method of claim 8, further comprising the step of isolating the produced polyketide. Method for isolating at least one polyketide biosynthetic gene, comprising the steps: a) providing at least one Primer pair based on the substrate specificity of a kethosynthase domain of a trans-AT polyketide synthase based; and b) isolation of at least a polyketide biosynthetic gene using the at least one Primer pair from step a). The method of claim 10, wherein step b) the Amplification of the enclosed by the primer pair nucleic acid region includes. The method of claim 10 or 11, wherein the at least a primer pair is specific for a motif that is phylogenetic adjacent kethosynthase domains of trans-AT polyketide synthases conserved. The method of claim 10-12, wherein at least one primer of the primer pair is specific for at least 4 contiguous amino acid residues of any of the amino acid residues in 3 . 4 or SEQ ID NO: 40-71 defined amino acid motifs. The method of claim 11-13, wherein, for the amplification of the nucleic acid region enclosed by the primer pair in addition to the at least one primer pair from step a) another Primer pair is used, with the two primer pairs nested are. The method of claim 10-14, wherein the polyketide biosynthesis gene is isolated from a metagenome becomes. The method of claim 10-15, wherein the Polyketide Biosynthesis Gene in the Biosynthesis of Psymberin, the Mycalamides, the Onnamids, Theopederinene A, the Cadamides, Pederin, the Laulimaliden, the Latrunculinen A, Mycothiazol, Pateamin A, the Spongistatins 1, discodermolide, dictyostatin, tedanolide and / or Calyculin is involved. Process according to claims 10-16, further comprising the step of isolating the whole for the polyketide synthesis required gene region based on the sequence information of at least in step b) isolated a polyketide biosynthetic gene.