Artificial non-ribosomal peptide synthases and their use

Abstract

The present invention pertains to a novel architecture of non-ribosomal peptide synthases (NRPS). The invention provides artificial NRPS wherein the naturally occurring terminal condensation or thioesterase-domain is replaced by internal condensation or dual condensation/epimerization domains. Moreover, the present invention enables the portability of terminal condensation domains to unrelated NRPS in respect of peptide release of linear peptides. The replacement results in a product independent release of the synthesized product and therefore enables the rational design of NRPS. The invention provides the new NRPS, nucleic acids encoding them, methods for artificial NRPS generation, and methods for producing non-ribosomal peptides.

Claims

1. An artificial non-ribosomal peptide synthase (NRPS), comprising as C-terminal end in N- to C-terminal direction an adenylation (A) domain, a thiolation (T) domain, and a termination module, wherein the termination module releases a synthesized molecule selected from a non-ribosomal peptide, polyketide, or combination thereof from the NRPS, wherein the termination module comprises any one of a heterologous terminal condensation domain (C.sub.term), an internal condensation (C) domain, an internal condensation and epimerization (C/E)-didomain, a cyclization (Cy) domain or an epimerization (E) domain, wherein the termination module does not comprise a thioesterase (TE)-domain; and wherein in the NRPS, one or more of the following modification domains are inserted: E(epimerization)-, MT(methyltransferase)-, Ox(oxidase)-, and Re(reductase)-domain.

2. The artificial NRPS according to claim 1, wherein the C-terminal end of the NRPS is non-naturally occurring.

3. The artificial NRPS according to claim 1, comprising in N- to C-terminal direction an initiation module, and/or one or more elongation module(s), and the termination module.

4. The artificial NRPS according to claim 2, wherein the A-domain and/or T-domain of the C-terminal end of the NRPS are heterologous to the termination module.

5. The artificial NRPS according to claim 2, wherein the termination module comprises a heterologous C.sub.term-domain.

6. The artificial NRPS according to claim 1, wherein the termination module comprises the C/E didomain.

7. A nucleic acid construct, comprising a nucleic acid sequence encoding for a NRPS according to claim 1.

8. A library of nucleic acid constructs, wherein each nucleic acid construct in the library encodes one or more adjoining domains of the NRPS according to claim 1, and wherein the totality of nucleic acid constructs in the library encodes the complete NRPS according to claim 1.

9. A biological cell comprising a nucleic acid construct according to claim 7.

10. A method for generating an NRPS according to claim 1, comprising assembling the A domain, the T domain, and the termination module of the NRPS.

11. A method for the production of a non-ribosomal peptide, the method comprising assembling the A domain, the T domain, and the terminal module of the NRPS according to claim 1.

12. A method for the production of a non-ribosomal peptide, the method comprising expressing the nucleic acid construct according to claim 7.

13. A method for the production of a non-ribosomal peptide, the method comprising expressing at least one of the nucleic acid constructs of the library according to claim 8.

14. A method for the production of a non-ribosomal peptide, the method comprising expressing the nucleic acid construct included in the biological cell according to claim 9.

15. An artificial non-ribosomal peptide synthase (NRPS), comprising as C-terminal end in N- to C-terminal direction an adenylation (A) domain, a thiolation (T) domain, and a termination module, wherein the termination module releases a synthesized molecule selected from a non-ribosomal peptide, polyketide, or combination thereof from the NRPS, wherein the termination module comprises an internal condensation (C) domain or an internal condensation and epimerization (C/E)-didomain, wherein the termination module does not comprise a thioesterase (TE)-domain.

16. The artificial NRPS according to claim 15, wherein the C-terminal end of the NRPS is non-naturally occurring.

17. The artificial NRPS according to claim 16, wherein the A-domain and/or T-domain of the C-terminal end of the NRPS are heterologous to the termination module.

18. The artificial NRPS according to claim 15, comprising in N- to C-terminal direction an initiation module, and/or one or more elongation module(s), and the termination module.

19. The artificial NRPS according to claim 15, wherein in the NRPS one or more of the following modification domains are inserted: E(epimerization)-, MT(methyltransferase)-, Ox(oxidase)-, and Re(reductase)-domain.

Description

(1) The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

(2) FIG. 1: Schematic representation of a NRPS. The domains are colored: Adenylation (A, black), thiolation (T, light grey), condensation (C, grey), modification (M, dark grey), thioesterase (TE, dark grey). Donor (D) and acceptor (A) sites of the condensation domain.

(3) FIG. 2: Minimal NRPS (core domains). First, the A domain specifically recognizes a dedicated amino acid and catalyzes formation of the aminoacyl adenylate under consumption of ATP. Second, the activated aminoacyl adenylate is tethered to the free thiol group of the T domain bound phosphopantetheine (4′Ppan) cofactor. Third, the C-domain catalyzes peptide elongation.

(4) FIG. 3: Schematic diagram of NRPS adenylation and peptidyl carrier protein.

(5) FIG. 4: Schematic diagram showing termination by the thioesterase domain (TE).

(6) FIG. 5: A: Schematic representation of the NRPS involved in the experiments with GxpS and terminal C domains. B: Schematic representation of modified GxpS with a terminal C domain from RdpC (1), an internal C domain from GxpS (2) or Xenotetrapeptide NRPS (3), as well as an internal C/E domains from GxpS (4 and 5). GxpS without the TE is also shown (6). Additional extracted ion chromatograms show linear (1; m/z [M+H].sup.+=604.4) or cyclic (2; m/z [M+H]+=586.4) GameXPeptide.

(7) FIG. 6: A: Schematic representation of an artificial NRPSs with a TE (1), without a TE (2), and an internal Dual C/E domain from GxpS (3). The origin of EUs is indicated by their colour. B: Extracted ion chromatograms.

EXAMPLES

(8) Materials and Methods

(9) Cultivation of Strains:

(10) All E. coli, Photorhabdus and Xenorhabdus strains were grown in liquid or solid LB-medium (pH 7.5, 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl). Solid media contained 1% (w/v) agar. S. cerevisiae strain CEN.PK 113-7D and derivatives were grown in liquid and solid YPD-medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose). Agar plates contained 2% (w/v) agar. Kanamycin (50 μg/ml) and G418 (200 μg/ml) were used as selection markers. All strains were cultivated at 30° C.

(11) Molecular Biological Methods:

(12) Genomic DNA of selected Xenorhabdus and Photorhabdus strains were isolated using the Qiagen Gentra Puregene Yeast/Bact Kit. Polymerase chain reaction (PCR) was performed with oligonucleotides obtained from Sigma-Aldrich and Eurofins Genomics (Table X3). Fragments with homology arms were amplified in a two-step PCR program using Phire Hot Start II DNA polymerase (Thermo Scientific) and for all other applications Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Scientific) was used. Both polymerases were used according to the manufacturers' instructions. DNA purification was performed using MinE-lute PCR Purification Kit Qiagen). Plasmid isolation from E. coli was done by alkaline lysis.

(13) Overlap Extension PCR-Yeast Homologous Recombination (ExRec):

(14) Plasmids carrying the novel NRPS encoding gene(s) were constructured using ExRec cloning according to Schimming et al. (2014). Therefore, transformation of yeast cells was done according to the protocols from Gietz and Schiestl (2007). 100-2000 ng of each fragment was used for transformation. Successfully constructed plasmids were isolated from yeast transformants and retransformed in E. coli DH10B::mtaA by electroporation.

(15) Expression of NRPS Templates:

(16) For production of the biosynthetic NRPS templates 100 μl of an overnight culture in LB medium of E. coli DH10B::mtaA cells harboring the corresponding expression plasmids were used to inoculate 10 ml of LB medium containing 50 μg/mL kanamycin, 0.5 mg/ml L-arabinose, and 2% (v/v) XAD. The cells were grown at 37° C. up to 72 h.

(17) Heterologous Expression and LC-MS Analysis:

(18) Constructed plasmids were transformed into E. coli DH10B::mtaA. Strains were grown overnight in LB medium containing kanamycin. Overnight cultures were used for inoculation of 10 ml cultures containing kanamycin and 2% XAD-16. After incubation for 72 h at 30° C. the XAD-16 was harvested. One culture volume methanol was added and incubated for 30 min. The organic phase was filtrated and evaporated to dryness under reduced pressure. The extract was diluted in 900 μl methanol and a 1:10 dilution was used for LC-MS analysis as described previously (Fuchs et al. 2013; Fuchs et al. 2014). Confirmation of synthetic products was carried out using a gradient from 5-95% ACN (0.1% formic acid) over 14 min. All measurements were carried out by using an Ultimate 3000 LC system (Dionex) coupled to an Ama ZonX (Bruker) electron spray ionization mass spectrometer.

(19) Experimental Examples:

(20) In general NRPS exhibit a TE domain at the end of the assembly line. However, some have a terminal C (C.sub.term) domain in place of a TE domain. C.sub.term domains release the synthesized peptides either by forming an amide-bond between the linear peptide and an amine from the environment or by cyclizing the peptide (Reimer et al. 2013, Fuchs et al. 2012, Gao et al. 2012). We constructed a recombinant GameXPeptide producing biosynthetic template (GxpS) (FIG. 5B-1) with a C.sub.term domain (GxpS_Cterm) from the rhabdopeptide producing biosynthetic cluster RdpA-C to determine the substrate range and applicability of its C.sub.term domain (RdpC) for NRPS regeneration. The recombinant NRPS GxpS_Cterm (FIG. 5B-1) is active but in contrast to our expectations the inventors observed a linear GameXPeptide (1) without C-terminal amidation. Thus it was shown that C.sub.term domains can be used for the production of artificial linear peptides.

(21) In further experiments with GxpS, internal C (GxpS_C.sub.int) and Dual C/E (GxpS_C/E.sub.int) domains were tested as TE domain alternatives (FIG. 5B). The inventors also deleted the GxpS TE domain without substitution (GxpS-TE). All recombinant NRPS were active. GxpS_C.sub.int produced linear (1), GxpS_C/E.sub.int linear (1) and/or cyclic (2) and GxpS-TE cyclic GameXPeptide (2). Thus the invention provides several ways of NRPS regeneration leading to the desired cyclic (or linear) peptide. A further improvement in comparison to naturally occurring TE domains seems to be the possibility of controlling cyclization or hydrolysis of the particular peptide.

(22) Therefore, cloning artificial NRPS with a library of internal C domains at the end of the assembly line is much more constructive, cheaper and timesaving than assaying a whole set of TE domains in vitro without guarantee of success.

(23) In the following, the inventors combined all gained insights like using the concept of EUs (EP application No. 1 500 234 0) and the applicability of internal C domains from elongation modules to create an artificial NRPS template capable of synthesizing a novel peptidic compound. For this purpose the building blocks from the gargantuanin synthase (GarS; unpublished data) and GxpS (FIG. 6a) were rebuilt. The artificial NRPS was designed in three variants to explore different peptide releasing strategies: (i) with a common TE domain from GxpS (FIG. 6A-1); (ii) without any peptide release catalyzing enzyme (NRPS ends with a T domain) (FIG. 6A-2); and (iii) with an internal C/E domain of module three from GxpS (FIG. 6A-3). According to our expectations, no product formation was observed for NRPS variants (i) and (ii). Yet, we observed the production of three novel linear lipo-penta-peptides with fatty acids varying in length (FIG. 6B).

(24) In summary, the invention demonstrates how novel biosynthetic templates can be designed de novo, without any need of evolutionary optimized peptide releasing enzymes. Furthermore, it is confirmed that internal C and C/E domains easily can be recruited for the biotechnological production of novel peptides according to the invention, whereas naturally occurring TE domains are not applicable if the assembled peptide does not meet its substrate specificity range.

(25) TABLE-US-00001 TABLE X1 Strains used and constructed in this work. Strain Genotype Reference E. coli DH10B F_mcrA (mrr-hsdRMS- Hanahan, mcrBC), 80lacZ .Math., 1983 M15, .Math. lacX74 recA1 endA1 araD 139 .Math. (ara, leu)7697 galU galK .Math. rpsL (Strr) nupG E. coli DH10B::mtaA DH10B with mtaA Schimming, from pCK_mtaA Δ entD 2014 S. cerevisiae CEN. MATα, MAL2-8.sup.c, SUC2 Euroscarf PK 113-7D P. asymbiotica DSMZ P. luminescens TT01 DSMZ X. nematophila DSMZ X. bovienii SS2004 DSMZ E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GxpS_C.sub.term pFF1_GxpS_C.sub.term, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GxpS_C2.sub.int pFF1_GxpS_C2.sub.int, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GxpS_C/E1.sub.int pFF1_GxpS_C/E1.sub.int, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GxpS_C/E3.sub.int pFF1_GxpS_C/E3.sub.int, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GxpS_ohne_TE pFF1_GxpS_ohne_TE, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GarS_GxpS_ohne_TE pFF1_GarS_GxpS_ohne_TE, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GarS_GxpS_mit_TE pFF1_GarS_GxpS_mit_TE, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GarS_GxpS_C/E3.sub.int pFF1_GarS_GxpS_C/E3.sub.int, Kan.sup.R E. coli DH10B::mtaA E. coli DH10B::mtaA this work pFF1_GxpS_Xcn1_C2.sub.int pFF1_GxpS_Xcn1_C2, Kan.sup.R

(26) TABLE-US-00002 TABLE X2 Plasmids constructed in this work. Plasmid Genotype Reference pFF1 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work MCS pFF1_GxpS_C.sub.term 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work rdpC (from base 4597 to 5997) was inserted downstream of gxpS (from base 1 to 14625) pFF1_GxpS_C2.sub.int 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work gxpS from P. luminescens (from base 5177 to 6637 ) was inserted downstream of gxpS from P. asymbiotica (from base 1 to 14625) pFF1_GxpS_C/E1.sub.int 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work gxpS from P. asymbiotica (from base 1831 to 3336) was inserted downstream of gxpS from P. asymbiotica (from base 1 to 14625) pFF1_GxpS_C/E3.sub.int 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work rdpC from P. asymbiotica (from base 8200 to 9705) was inserted downstream of gxpS from P. asymbiotica (from base 1 to 14625) pFF1_GxpS_ohne_TE 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work gxpS from P. asymbiotica (from base 1 to 14784) pFF1_GarS_GxpS_ohne_TE 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work gxpS from P. luminescens TT01 (from base 13034 to 14944) was inserted downstream of garS (from base 1 to 14241) pFF1_GarS_GxpS_mit_TE 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work gxpS from P. luminescens TT01 (from base 13034 to 15699) was inserted downstream of garS (from base 1 to 14241) pFF1_GarS_GxpS_C/E3.sub.int 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work gxpS (nt) from P. luminescens (from base 13034 to 14785) followed by gxpS from P. asymbiotica (from base 8200 to 9705 ) was inserted downstream of garS (from base 1 to 14241) pFF1_GxpS_Xcn1_C2.sub.int 2 μ ori, kanMX4, P.sub.BAD promoter, pCOLA ori, Kan.sup.R, this work rdpC from P. asymbiotica (from base 5119 to 6567) was inserted downstream of gxpS from P. asymbiotica (from base 1 to 14625)

(27) TABLE-US-00003 TABLE X3 Oligonucleotides used for primer construction in this work Plasmid Oligonucleotide Sequence (5′.fwdarw.3′) Template pFF1_GxpS_C.sub.term KB_Pau-P1 TTATCGCAACTCTCTACT P. asymbiotica GTTTCTCCATACCCGTTT TTTTGGGCTAACAGGAG GAATTCCATGAAAGAGA GCATCGTGAG (SEQ ID Nr. 1) KB_Pau-P2 ATAATGCCACAGGCGAC CTG (SEQ ID Nr. 2) KB_Pau-P3 ATACGTCTGGCTCTACCG P. asymbiotica G (SEQ ID Nr. 3) KB_Pau-P4 GATTTCTGCTACCAGTTC AGCC (SEQ ID Nr. 4) KB-Rdp3-FW ATTTGCACATTGAATAAT X. nematophila CTGTTCCAATTCCCTGTG TTGGCTGAACTGGTAGC AGAAATCCGTAGCGCTC AAGACCATG (SEQ ID Nr. 5) KB-Rdp3-RV AAACAGTTCTTCACCTTT GCTCATGAACTCGCCAG AACCAGCAGCGGAGCCA GCGGATCCGTCATAAAA GTAACTGATATTTTC (SEQ ID Nr. 6) pFF1_GxpS_C2.sub.int KB_Pau-P1 TTATCGCAACTCTCTACT P. asymbiotica GTTTCTCCATACCCGTTT TTTTGGGCTAACAGGAG GAATTCCATGAAAGAGA GCATCGTGAG (SEQ ID Nr. 7) KB_Pau-P2 ATAATGCCACAGGCGAC CTG (SEQ ID Nr. 8) KB_Pau-P3 ATACGTCTGGCTCTACCG P. asymbiotica G (SEQ ID Nr. 9) KB_Pau-P4 GATTTCTGCTACCAGTTC AGCC (SEQ ID Nr. 10) KB-PluC2-FW ATTTGCACATTGAATAAT P. luminescens TTO1 CTGTTCCAATTCCCTGTG TTGGCTGAACTGGTAGC AGAAATCTGCGCACAGA TCTGTGCAC (SEQ ID Nr. 11) KB-PluC2-RV AAACAGTTCTTCACCTTT GCTCATGAACTCGCCAG AACCAGCAGCGGAGCCA GCGGATCCATGGACACA TACCTGAGTAGG (SEQ ID Nr. 12) pFF1_GxpS_C/E1.sub.int KB_Pau-P1 TTATCGCAACTCTCTACT P. asymbiotica GTTTCTCCATACCCGTTT TTTTGGGCTAACAGGAG GAATTCCATGAAAGAGA GCATCGTGAG (SEQ ID Nr. 13) KB_Pau-P2 ATAATGCCACAGGCGAC CTG (SEQ ID Nr. 14) KB_Pau-P3 ATACGTCTGGCTCTACCG P. asymbiotica G (SEQ ID Nr. 15) KB_Pau-P4 GATTTCTGCTACCAGTTC AGCC (SEQ ID Nr. 16) KB-Pau-CE1-FW ATTTGCACATTGAATAAT P. asymbiotica CTGTTCCAATTCCCTGTG TTGGCTGAACTGGTAGC AGAAATCGAGCACCATC AGTCTTTCG (SEQ ID Nr. 17) KB-Pau-CE1-RV AAACAGTTCTTCACCTTT GCTCATGAACTCGCCAG AACCAGCAGCGGAGCCA GCGGATCCATGGATACA CAACGAATCAGG (SEQ ID Nr. 18) pFF1_GxpS_C/E3.sub.int KB_Pau-P1 TTATCGCAACTCTCTACT P. asymbiotica GTTTCTCCATACCCGTTT TTTTGGGCTAACAGGAG GAATTCCATGAAAGAGA GCATCGTGAG (SEQ ID Nr. 19) KB_Pau-P2 ATAATGCCACAGGCGAC CTG (SEQ ID Nr. 20) KB_Pau-P3 ATACGTCTGGCTCTACCG P. asymbiotica G (SEQ ID Nr. 21) KB_Pau-P4 GATTTCTGCTACCAGTTC AGCC (SEQ ID Nr. 22) KB-Pau-CE3-FW ATTTGCACATTGAATAAT P. asymbiotica CTGTTCCAATTCCCTGTG TTGGCTGAACTGGTAGC AGAAATCGAGCAACATC GTGAAATCAG (SEQ ID Nr. 23) KB-Pau-CE3-RV AAACAGTTCTTCACCTTT GCTCATGAACTCGCCAG AACCAGCAGCGGAGCCA GCGGATCCATGAATGCA CAATTGGTCAG (SEQ ID Nr. 24) pFF1_GxpS_ohne_TE KB_Pau-P1 TTATCGCAACTCTCTACT P. asymbiotica GTTTCTCCATACCCGTTT TTTTGGGCTAACAGGAG GAATTCCATGAAAGAGA GCATCGTGAG( SEQ ID Nr. 25) KB_Pau-P2 ATAATGCCACAGGCGAC CTG (SEQ ID Nr. 26) KB_Pau-P3 ATACGTCTGGCTCTACCG P. asymbiotica G (SEQ ID Nr. 27) KB-Pau-TE-RV AAACAGTTCTTCACCTTT GCTCATGAACTCGCCAG AACCAGCAGCGGAGCCA GCGGATCCTAACGCATA AATCGGGTAATC (SEQ ID Nr. 28) pFF1_GarS_GxpS_ohne_ LH 6 P1 CGGATCCTACCTGACGCT X. bovienii SS2004 TE TTTTATCGCAACTCTCTA CTGTTTCTCCATACCCGT TTTTTTGGGCTAACAGGA GGAATTCCATGCCTATGT CATGCAATCGTATC (SEQ ID Nr. 29) LH 6 P2 GTTGCGCCAGTGCTAAC G (SEQ ID Nr. 30) LH 6 P3 CGTCTGGGTGTCAGTCCG X. bovienii SS2004 (SEQ ID Nr. 31) LH 6 P4 CTCTACCAGCAGTTGTTG TCGC (SEQ ID Nr. 32) LH 6 P5 CCCTGACCCGAGATCCG P. luminescens TT01 CAACAATTGATCCGGGA TGTATCCATCTTACCGCC GACAGAGCGACAACAAC TGCTGGTAGAGGGCAAT GGCCCGCAAACG (SEQ ID Nr. 33) LH 6 P7 AGAATCGGAACAACACC GGTAAACAGTTCTTCACC TTTGCTCATGAACTCGCC AGAACCAGCAGCGGAGC CAGCGGATCCTAGCGCA TAAATCGGGTAATCC (SEQ ID Nr. 34) pFF1_GarS_GxpS_mit_ LH 6 P1 CGGATCCTACCTGACGCT X. bovienii SS2004 TE TTTTATCGCAACTCTCTA CTGTTTCTCCATACCCGT TTTTTTGGGCTAACAGGA GGAATTCCATGCCTATGT CATGCAATCGTATC (SEQ ID Nr. 35) LH 6 P2 GTTGCGCCAGTGCTAAC G(SEQ ID Nr. 36) LH 6 P3 CGTCTGGGTGTCAGTCCG X. bovienii SS2004 (SEQ ID Nr. 37) LH 6 P4 CTCTACCAGCAGTTGTTG TCGC (SEQ ID Nr. 38) LH 6 P5 CCCTGACCCGAGATCCG P. luminescens TT01 CAACAATTGATCCGGGA TGTATCCATCTTACCGCC GACAGAGCGACAACAAC TGCTGGTAGAGGGCAAT GGCCCGCAAACG (SEQ ID Nr. 39) LH 6 P6 AGAATCGGAACAACACC GGTAAACAGTTCTTCACC TTTGCTCATGAACTCGCC AGAACCAGCAGCGGAGC CAGCGGATCCCAGCGCC TCCGCTTCACAATTC (SEQ ID Nr. 40) pFF1_GarS_GxpS_C/E3.sub.int LH 6 P1 CGGATCCTACCTGACGCT X. bovienii SS2004 TTTTATCGCAACTCTCTA CTGTTTCTCCATACCCGT TTTTTTGGGCTAACAGGA GGAATTCCATGCCTATGT CATGCAATCGTATC (SEQ ID Nr. 41) LH 6 P2 GTTGCGCCAGTGCTAAC G (SEQ ID Nr. 42) LH 6 P3 CGTCTGGGTGTCAGTCCG X. bovienii SS2004 (SEQ ID Nr. 43) LH 6 P4 CTCTACCAGCAGTTGTTG TCGC (SEQ ID Nr. 44) LH 6 P5 CCCTGACCCGAGATCCG P. luminescens TT01 CAACAATTGATCCGGGA TGTATCCATCTTACCGCC GACAGAGCGACAACAAC TGCTGGTAGAGGGCAAT GGCCCGCAAACG (SEQ ID Nr. 45) LH 6 P8 AACGGTAACATCGCCGG CGTCAGTACAACCGTAT CCAGTGTAATGCTGTTGT CAGGCACCCTGATTTCAC GATGTTGCTCGATCTCTG CCACCAGTTCCG (SEQ ID Nr. 46) LH 3 P13 GAGCAACATCGTGAAAT P. asymbiotica CAG (SEQ ID Nr. 47) LH 3 P14 AGAATCGGAACAACACC GGTAAACAGTTCTTCACC TTTGCTCATGAACTCGCC AGAACCAGCAGCGGAGC CAGCGGATCCATGAATG CACAATTGGTCAG (SEQ ID Nr. 48) pFF1_GxpS_Xcn1_C2.sub.int KB_Pau-P1 TTATCGCAACTCTCTACT P. asymbiotica GTTTCTCCATACCCGTTT TTTTGGGCTAACAGGAG GAATTCCATGAAAGAGA GCATCGTGAG (SEQ ID Nr. 49) KB_Pau-P2 ATAATGCCACAGGCGAC CTG (SEQ ID Nr. 50) KB_Pau-P3 ATACGTCTGGCTCTACCG P. asymbiotica G (SEQ ID Nr. 51) KB_Pau-P4 GATTTCTGCTACCAGTTC AGCC (SEQ ID Nr. 52) KB_XcnC2_FW ATTTGCACATTGAATAAT X. nematophila CTGTTCCAATTCCCTGTG TTGGCTGAACTGGTAGC AGAAATCTGCGTACAAC GTCATGCG (SEQ ID Nr. 53) KB_XcnC2_RV AAACAGTTCTTCACCTTT GCTCATGAACTCGCCAG AACCAGCAGCGGAGCCA GCGGATCCATGAATACA TAACGATTCAGG (SEQ ID Nr. 54) R. D. Gietz, R. H. Schiestl, Nat Protoc. 2007; 2(1), 35-7. S. W. Fuchs, K. A. J. Bozhüyük, D. Kresovic, F. Grundmann, V. Dill, A. O. Brachmann, N. R. Waterfield, H. B. Bode, Angew. Chem. Int. Ed. 2013, 52, 4108-4112. S. W. Fuchs, F. Grundmann, M. Kurz, M. Kaiser, H. B. Bode, Chembiochem 2014, 15, 512-516. O. Schimming, F. Fleischhacker, F. I. Nollmann, H. B. Bode, Chembiochem 2014, 15(9), 1290-4.