SYSTEM FOR THE ASSEMBLY AND MODIFICATION OF NON-RIBOSOMAL PEPTIDE SYNTHASES

Abstract

The present invention pertains to a system for the assembly and modification of non-ribosomal peptide synthases (NRPS). The system uses novel well defined building blocks (units) comprising condensation subdomains. This strategy allows for the efficient combination of assembly units referred to as eXchange Units (XU2.0) independent on their natural occurring specificity for the subsequent NRPS adenylation domain. The system of the invention allows for the easy assembly of NRPS having any amino acid sequence of choice, without any restrictions due to natural occurring NRPS units. The system also allows the exchange of natural NRPS building blocks with the inventive XU2.0 leading to the production of modified peptides. The invention provides the system, their individual exchange units, nucleic acids encoding these units, as well as methods and uses thereof.

Claims

1. A system for the production of a non-ribosomal peptide synthases (NRPS), wherein the system comprises at least one, preferably two, NRPS eXchange Units (XU.sub.2.0) each specific for a different or identical amino acid X for assembling an NRPS, and wherein the XU.sub.2.0 comprises at least one partial condensation (C)- or partial condensation/epimerization (C/E)-domain selected from the group consisting of a condensation-domain acceptor site subdomain (C.sub.Asub) specific for a given amino acid X, a condensation/epimerization-domain acceptor site subdomain (C/E.sub.Asub) specific for a given amino acid X, a condensation-domain donor site subdomain (C.sub.Dsub) specific for a given amino acid X and a condensation/epimerization-domain donor site subdomain (C/E.sub.Dsub) specific for a given amino acid X.

2. The system according to claim 1, wherein at least one XU.sub.2.0 comprises a C- or C/E acceptor domain and a C- or C/E donor domain separated by one or more NRPS domains other than C or C/E domains.

3. The system according to claim 1 or 2, wherein at least a first and a second XU.sub.2.0 of the system, when connected to each other, form a functional assembled C or C/E domain composed of one partial C or C/E domain of the first XU.sub.2.0 and one partial C or C/E domain of the second XU.sub.2.0.

4. The system according to any one of claims 1 to 3, comprising at least two XU.sub.2.0 of which each has a different amino acid specificity X, preferably wherein each X is selected from a specificity to any one or two, three, four or more natural or non-natural amino acid.

5. The system according to any one of claims 1 to 7, the system further comprising a XU.sub.2.0 termination and/or initiation unit, wherein the XU.sub.2.0 initiation unit comprises only a C or C/E domain donor subdomain, a domain structure C-A.sup.X-T-C.sub.Dsub.sup.X or C-A.sup.X-T-C/E.sub.Dsub.sup.X, specific for the incorporation of acyl units (fatty acids and their derivatives) as starting units, and wherein the termination module comprises any one of a terminal condensation domain (Cterm), an internal condensation (C) domain, an internal condensation and epimerization (C/E)-didomain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (R), an oxidation (Ox) or a thioesterase (TE) domain.

6. The system according to any one of claims 1 to 7, the system further comprising a XU2.0 initiation unit, preferably selected from the formulas: C.sub.Asub-A.sup.X-T-C.sub.Dsub.sup.X, C/E.sub.Asub-A.sup.X-C.sub.Dsub.sup.X, C.sub.Asub-A.sup.X-T-E/E.sub.Dsub.sup.X, or C/E.sub.Asub-A.sup.X-T-C/E.sub.Dsub.sup.X

7. The system according to any one of claims 1 to 9, wherein at least two, preferably three, four, or more, XU.sub.2.0 when put into sequence provide the NRPS.

8. The system according to any one of claims 1 to 7, further comprising at least one XU.sub.2.0 having a modification domain, such as an E, MT or Ox or other modification domain.

9. An isolated nucleic acid, comprising a partial non-ribosomal peptide synthases (NRPS) condensation (C)-domain sequence or partial NRPS condensation/epimerization (C/E)-domain sequence, wherein said sequence encodes for at least a partial C or C/E domain comprising a C or C/E domain donor or acceptor site, but does not encode for a partial or full-length C or C/E domain comprising a functionally connected donor and acceptor site.

10. A library of nucleic acid molecules, wherein the library comprises at least two or more nucleic acid constructs each encoding a NRPS eXchange Unit (XU2.0) having an amino acid specificity X, and wherein the XU.sub.2.0 comprises at least one partial condensation (C)- or partial condensation/epimerization (C/E)-domain selected from the group consisting of a condensation-domain acceptor site subdomain (C.sub.Asub) having an amino acid specificity X, a condensation/epimerization-domain acceptor site subdomain (C/E.sub.Asub) having an amino acid specificity X, a condensation-domain donor site sub-domain (C.sub.Dsub) having an amino acid specificity X and a condensation/epimerization-domain donor site subdomain (C/E.sub.Dsub) having an amino acid specificity X.

11. The library according to claim 10, comprising nucleic acid constructs encoding at least one XU.sub.2.0 termination and/or initiation unit, wherein the XU.sub.2.0 initiation unit comprises only a C or C/E domain donor subdomain, a domain structure C-A.sup.X-T-C.sub.Dsub.sup.X or C-A.sup.X-T-C/E.sub.Dsub.sup.X, specific for the incorporation of acyl units (fatty acids and their derivatives) as starting units, or A.sup.X-T-C.sub.Dsub.sup.X or A.sup.X-T-C/E.sub.DSub.sup.X, and wherein the termination module comprises any one of a terminal condensation domain (Cterm), an internal condensation (C) domain, an internal condensation and epimerization (C/E)-didomain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (R), an oxidation (Ox) or a thioesterase (TE) domain

12. The library according to claim 10 or 11, wherein each XU.sub.2.0 is encoded by a separate nucleic acid construct, preferably by a nucleic acid according to claim 9.

13. The library according to any one of claims 10 to 12, wherein the library comprises the XU.sub.2.0 of the system according to any one of claims 1 to 8.

14. A method for producing a NRPS, the method comprising a step of assembling at least two NRPS eXchange Units (XU.sub.2.0) each having an identical or different amino acid specificity X, and wherein the XU.sub.2.0 comprises at least one partial condensation (C)- or partial condensation/epimerization (C/E)-domain selected from the group consisting of a condensation-domain acceptor site subdomain (CAsub) having an amino acid specificity X, a condensation/epimerization-domain acceptor site subdomain (C/E.sub.Asub) having an amino acid specificity X, a condensation-domain donor site subdomain (C.sub.Dsub) having an amino acid specificity X and a condensation/epimerization-domain donor site subdomain (C/E.sub.Dsub) having an amino acid specificity X.

15. A method for the production of non-ribosomal peptides having a specific sequence, the method comprising assembling a NRPS according to the method of claim 14, wherein the NRPS is composed of a sequence of XU.sub.2.0 having specificity according to the non-ribosomal peptide to be produced.

Description

[0081] 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:

[0082] FIG. 1: Modulation of C-domain substrate specificity. (a) C-domain excised from the T-C bidomain TycC 5-6 from tyrocidine syntethase (TycC) of Brevibacillus brevis (PDB-ID: 2JGP) with N-terminal (yellow) and C-terminal (blue) subdomains depicted in ribbon representation (top). Boxed: enlarged representation of the C.sub.Dsub-C.sub.Asub linker with contributing linker AAs in stick representation and fusion site marked in red. Bottom: sequence logo of C.sub.Dsub-C.sub.Asub linker sequences from Photorhabdus and Xenorhabdus. (b) Schematic representation of WT GxpS, recombinant NRPS-1 and -2 as well as corresponding peptide yields as obtained from triplicate experiments. For peptide nomenclature the standard one letter AA code with lowercase for D-AA is used. (c) Schematic representation of BicA with modules and eXchange Units (XU and XU.sub.2.0) highlighted. Specificities are assigned for all A-domains. For domain assignment the following symbols are used: A (large circles), T (rectangle), C (triangle), C/E (diamond), TE (C-terminal small circle).

[0083] FIG. 2: De novo design of recombinant NRPS for peptide production. (a) Generated recombinant GxpS (NRPS-3-5) and corresponding amounts of GameXPeptide derivatives 1, 3, 6, and 7 as determined in triplicates. (b) Recombinant NRPS-6 synthesizing 8. Building blocks are of Gram-positive origin. Bottom: Color code of NRPS used as building blocks (for details see FIG. 8). For assignment of domain symbols see FIG. 1.

[0084] FIG. 3: Exchange of NRPS starter units. Schematic representation of recombnant GxpS (NRPS-7-9) and corresponding peptide yields as obtained from triplicate experiments. For peptide nomenclature the standard one letter AA code with lowercase for D-AA is used. For assignment of domain symbols see FIG. 1. Bottom: Color code of NRPS used as building blocks (for details see FIG. 8).

[0085] FIG. 4: Creation of functionalized xenotetrapeptide derivatives. Schematic representation of WT XtpS, recombinant NRPS-10 and corresponding peptide yields as obtained from triplicate experiments. For peptide nomenclature the standard one letter AA code with lowercase for D-AA is used. For assignment of domain symbols see FIG. 1. Bottom: Color code of NRPS used as building blocks (for details see FIG. 8).

[0086] FIG. 5: Targeted randomization of GxpS at position three. Schematic representation of all possible recombinant NRPSs (top left) and corresponding A domain specificity (bottom left). Detected peptides (solid line) and corresponding peptide yields (top right) as obtained from triplicate experiments. Dashed lines indicate not determined GxpS derivatives. For peptide nomenclature the standard one letter AA code with lowercase for D-AA is used. For assignment of domain symbols see FIG. 1. Bottom: Color code of NRPS used as building blocks (for details see FIG. 8).

[0087] FIG. 6: The creation of a library with randomization of position one and three from GxpS. Schematic representation of all possible recombinant NRPSs (top left) and corresponding A domain specificity (bottom left). Detected peptides and corresponding peptide yields (right) as obtained from triplicate experiments. For peptide nomenclature the standard one letter AA code with lowercase for D-AA is used. For assignment of domain symbols see FIG. 1. Bottom Color code of NRPS used as building blocks (for details see FIG. 8).

[0088] FIG. 7: Randomization of adjacent positions. (a) Crystal structure of TycC6 (PDB-ID: 2JGP), subdivided into N terminal subdomain (grey) and C terminal subdomain (light red). The subdomain linker is highlighted in red and the targeted area (I253-F265) for homologues recombination in yeast is highlighted in green (39 nucleotides). The Consensus sequence used to generate library three is shown bottom right. (b) Schematic representation of all possible recombinant NRPSs (top left) and corresponding A domain specificity (bottom left). Detected peptides and corresponding peptide yields (right) as obtained from triplicate experiments. For peptide nomenclature the standard one letter AA code with lowercase for D-AA is used. For assignment of domain symbols see FIG. 1. Bottom right: Color code of NRPS used as building blocks (for details see FIG. 8).

[0089] FIG. 8: Schematic overview of all NRPS used in this work. GxpS, BicA, XtpS, HCTA, PaxB, KolS, AmbS.sub.mir from X. miraniensis and AmbS.sub.ind from X. indica have been described previously. For GarS producing gargantuanin see Genbank accession number PRJNA224116. For Xenolindicin-like syntethase see Genbank accession number PRJNA328553. For XeyS producing xindeyrin see Genbank accession number PRJNA328572.

EXAMPLES

Example 1: C-Domains Have Acceptor Site Substrate Specificity

[0090] To verify the influence of the C-domains acceptor site (C.sub.Asub) proof reading activity, GameX-Peptide producing NRPS GxpS of Photorhabdus luminescens TT01 was chosen as a model system (Bode et al. 2012; Nollmann et al. 2014). A recombinant GxpS was constructed, not complying with the rules of the XU concept (WO 2017/020983). Here, XU2 of GxpS (FIG. 1b, NRPS-1) was exchanged against XU2 of the bicornutin producing NRPS (BicA, FIG. 1c) (Fuchs et al. 2012). Although both XUs are Leu specific, they are differentiated by their C.sub.Asub specificitiesPhe for XU2 of GxpS and Arg for XU2 of BicA. Therefore, no peptide production was observed as expected. This experiment confirmed previously published scientific results from in vitro experiments (Belshaw et al. 1999, Clugston et al. 2003, Samel et al. 2007, Rausch et al. 2007), and illustrates that C-domains indeed are highly substrate specific at their C.sub.Asub (WO 2017/020983).

Example 2: Modulation of C-Domain Substrate Specificity

[0091] In developing a new and C-domain specificity independent and/or evading strategy, the inventors strived to determine the structural basis for this purpose by reviewing available structural data of C-domains (Samel et al. 2007, Tanovic et al. 2008). As C-domains have a pseudo-dimer configuration (Keating et al. 2002, Samel et al. 2007, Tanovic et al. 2008, Bloudoff et al. 2013), and the catalytic center, including the HHXXXDG motif, has two binding sitesone for the electrophilic donor substrate and one for the nucleophilic acceptor substrate (Rausch et al. 2007) (FIG. 1a)the inventors concluded that the four AA long conformationally flexible loop/linker between both subdomains might be the ideal target to reconfigure C-domain specificities, by engineering C-domain hybrids (FIG. 1a). For this purpose the Arg specific C.sub.Asub of the GxpS-BicA hybrid NRPS (FIG. 1b, NRPS-1) was re-exchanged to the Leu specific C.sub.Asub of GxpS, restoring the functionality of the hybrid NRPS (NRPS-2) and leading to the production of GameXPeptide A-D (1-5) in 217% yield compared to the WT GxpS (FIG. 1b) confirmed by MS/MS analysis and comparison of the retention times.

Example 3: The eXchange Unit 2.0

[0092] From above mentioned results in conjunction with bioinformatics analysis, the inventors concluded that C-domains acceptor and donor site (C.sub.Dsub) mark a self-contained catalytically active unit C.sub.Asub-A-T-C.sub.Dsub (XU.sub.2.0)without interfering major domain-domain interfaces/-actions during the NRPS catalytic cycle (Marahiel 2015). In order to validate the proposed XU.sub.2.0 building block (FIG. 1c) and to compare the production titers with a natural NRPS, the inventors reconstructed GxpS (FIG. 1b) in two variants (FIG. 2, NRPS-3 and -4). Each from five XU.sub.2.0 building blocks from four different NRPSs (XtpS, AmbS, GxpS, GarS, HCTA) (FIG. 8):

[0093] NRPS-3: although leading to a mixed C/E.sub.Dsub-C.sub.Asub-domain between XU.sub.2.03 and XU.sub.2.04 (FIG. 2), XU.sub.2.0 building blocks from XtpS (XU.sub.2.01), AmbS (XU.sub.2.04), GxpS (XU.sub.2.03 and 5), and GarS (XU.sub.2.04) were used, to reveal if C and C/E domains can be combined.

[0094] NRPS-4: to prevent any incompatibilities, XU.sub.2.03 originated from GarS was replaced by a XU.sub.2.03 from HCTA (FIG. 2).

[0095] Whereas NRPS-3 (FIG. 2) showed no detectable production of any peptide, NRPS-4 (FIG. 2) resulted in the production of 1 and 3 in 66 and 6% yield compared to the natural GxpS, as confirmed by MS/MS analysis and comparison of the retention times (FIG. 2,). In line with expectations from domain sequences, phylogenetics as well as structural idiosyncrasies of C/E- and C-domains (Rausch et al. 2007), it may be deduced from these results that C/E and C-domains cannot be combined with each other. Although NRPS-4 (FIG. 2) showed moderately reduced production titers, most likely due to the non-natural C.sub.Dsub-C.sub.Asub pseudo-dimer interface, the formal exchange of the non-specific XU.sub.2.01 from GxpS (Val/Leu) against the Val-specific XU.sub.2.01 from XtpS led to exclusive production of 1 and 3 without production of 2 and 4 (FIG. 2), indicating that the XU.sub.2.0 can also be used for increasing product specificity and reducing side products (WO 2017/020983).

[0096] Additional GameXPeptide derivatives were generated (FIG. 2a, NRPS-5) by combining building blocks according to the definition of XU and XU.sub.2.0 (WO 2017/020983). Three fragments (1: C1-A1-T1-C/E2 of BicA; 2: A2-T2-C3-A3-T3-C/E4-A4-T4-C/E.sub.Dsub5 of GxpS; 3: C/E.sub.Asub5-A5-T5-C.sub.term of BicA) from two NRPSs (BicA: Xenorhabdus budapestensis DSM 16342; GxpS: Photorhabdus luminescens TT01) were used as construction material (Bode et al. 2012, Fuchs et al. 2012). The expected two Arg containing cyclic pentapeptides 6 and 7 were produced in yields of 2.25 and 0.17 mg/L and were structurally confirmed by chemical synthesis. Both peptides only differ in Leu or Phe at position three from the relaxed substrate specificity of XU.sub.2.03 from GxpS. Despite a drop of production rate in comparison to the WT NRPS, the inventors successfully demonstrated that the recently published XU (WO 2017/020983) as well as the novel XU.sub.2.0 reliably broaden the possibilities of successfully reprogramming NRPS and to heterologously synthesize tailor-made peptides.

[0097] To show the general applicability of the novel XU.sub.2.0 building block an artificial NRPS was designed de novo from building blocks of Gram-positive origin (NRPS for the production of bacitracin (Konz et al. 1998) from Bacillus licheniformis ATCC 10716 and surfactin (Cosmina et al. 1993) from Bacillus subtilis MR 168), since all aforementioned recombined NRPS are of Gram-negative origin. The expected pentapeptide (8) containing the bacitracin NRPS derived thiazoline ring was produced in yields of 21.09 mg/L (FIG. 2b), showing the universal nature of the XU.sub.2.0.

Example 4: Amending the Starter Unit

[0098] Up to date there is no publication describing the successful exchange of a starter unit against an internal module NRPS-fragment. However, possible identified problems which would need to be solved for example are: (I) as starter-A-domains in general comprise some kind of upstream sequence of variable length with unknown function and structure, it is difficult to define an appropriate artificial leader sequence, and (II) necessary interactions at the C-A interface may be important for adenylation activity and A-domain stability, like indicated by recently published studies (Li et al. 2016, Meyer et al. 2016). Therefore, the first step in order to approach the concrete problem three recombinant GxpS constructs (NRPS-7-9) with internal domains as starting units were created (FIG. 3):

[0099] NRPS-7: as all starter A-domains have at least a preceding C-A linker sequence A1-T1-C.sub.Dsub2 of GxpS was exchanged against C2A3-linker-A3-T3-C.sub.Dsub4 of XtpS;

[0100] NRPS-8: as there are several examples of NRPS carrying parts of a C-domain (e.g. BicA) in front of the starter A-domain A1-T1-C.sub.Dsub2 of GxpS was exchanged against C.sub.Asub-A3-T3-C.sub.Dsub4 of Xtps;

[0101] NRPS-9: as there are biosynthetic templates available, exhibiting catalytically inactive starter C-domains (e.g. AmbS), A1-T1-C.sub.Dsub2 of GxpS was altered to C3-A3-T3-C.sub.Dsub4 of XtpS.

[0102] Whereas NRPS-7 (FIG. 3) did not show production of the desired peptides, NRPS-8 synthesized 1-3 in yields of 0.35 and 0.44 mg/L, and NRPS-9 produced 1 in yields of 0.31 and 0.34 mg/L, as confirmed by MS/MS analysis and comparison of the retention times (FIG. 3). Obtained results revealed that internal A-domains can be used as starter domains, if the upstream C.sub.Asub or C-domain is kept in front of the A-domainindicating the importance of a functional C-A interface for A-domain activity. Yet, from drop in production titers, further questions arise that must be answered by future work. One reason for decreased synthesis rates might be for example the observed codon usage and the lowered GC-content at the beginning of WT NRPS encoding genes, which can have a major impact on transcriptional and/or translational efficiency in conjunction with protein folding issues.

Example 5: The Creation of Functionalized Peptides

[0103] Besides simply creating NRP derivatives, one useful application of NRPS reprogramming is the incorporation of AAs that contain alkyne or azide groups into peptides, allowing reactions like Cu(I)-catalyzed or strain-promoted Huisgen cyclizationalso known as click reactions (Sletten & Bertozzi 2009; Kolb & Sharpless 2003). Yet, although NPRS and A domains have been examined exhaustively for several years, no general method for the simple functionalization of NRPs has emerged.

[0104] A broad range of AAs are accepted by the A3 domain of GxpS resulting in a large diversity of GameXPeptides (Bode et al. 2012, Nollmann et al. 2014). Moreover, by using a -.sup.18O.sub.4-ATP pyrophosphate exchange assay for adenylation activity (Phelan et al. 2009, Kronenwerth et al. 2014) and adding substituted AAs to growing E. coli cultures expressing GxpS, the respective A3-domain was identified as being able to activate (in vitro) and incorporate (in vivo 10) several ortho- (o), meta- (m) and para- (p) substituted phenylalanine derivatives, including 4-azido-L-phenylalanine (p-N.sub.3-Phe) and O-propargyl-L-tyrosine (Y-Tyr).

[0105] In order to create functionalized NRPs the Val specific XU.sub.2.03 of the xenotetrapeptide (Kegler et al. 2014) (9) producing NRPS (XtpS) from X. nematophila HGB081 was exchanged against XU.sub.2.03 of GxpS, resulting in the production of six new xenotetrapeptide derivatives (10-15) in yields of 0.17-106 mg/L (FIG. 4). After adding p-N.sub.3-Phe and Y-Tyr to growing E. coli cultures expressing recombined XtpS (NRPS-10), six functionalized peptides (16-21) have been identified in yields of 5-228 mg/L. Compounds 17, 8 and 19 were structurally confirmed by chemical synthesis. Due to the relaxed substrate specificity of the introduced A3-domain, compounds 9-21 differ at position three. Moreover, although the A4-domain of XtpS shows an exclusive specificity for Val in the WT NRPS, peptides 11, 13, 16 and 21 produced by NRPS-10 additionally differ in Val or Leu at position four. The observed specificity shift might be due to the hybrid C/E4-domain upstream of A4 from NRPS-10. Leu is the original substrate downstream of the introduced XU.sub.2.03 of GxpS (FIG. 1b) in its natural context, indicating that the overall structure of C-domain's along with resulting transformed C-A interface interactions might have some kind of influence to A-domain's substrate specificity. Recently, similar but in vitro observed effects were reported regarding A-domains from sulfazecin (Li et al. 2016) and microcystin (Meyer et al. 2016). This effect could also be used to increase the specificity of A-domains to prevent the formation of side products. Further investigations will shed light on this remarkable and yet unreported effect.

Example 6: The Biotechnological Creation of Natural Product Like Peptide Libraries

[0106] To address the issue of biologically relevant chemical space the modern drug-discovery approach applies screening libraries based on NPs (Harvey et al. 2015). NP collections exhibit a wide range of pharmacophores, a broad range of stereochemistry and have the property of metabolite-likeness providing a high degree of bioavailability. Yet, the NP discovery process is as expansive as time consuming (Lefevre et al. 2008). Consequently, for bioactivity screenings the random recombination of certain NRPS fragments would be a powerful means to create focused artificial NP-like libraries. The definition of XU.sub.2.0 building blocks, including the targeted and automated reprograming of C-domain specificities, brought this goal within grasp.

[0107] For an initial approach, GxpS was chosen for the generation of a focused peptide library created via a one-shot yeast based TAR cloning approach (Schimming et al. 2014, Gietz et al. 2007). Here, the third position of the peptide (D-Phe) was randomized (FIG. 5) using six unique XU.sub.2.0 building blocks from six NRPS (KolS (Bode et al. 2015), AmbS.sub.mir (Schimming et al. 2014), Pax (Fuchs et al. 2011), AmbS.sub.ind, XllS; for detail see FIG. 8), resulting in the production of 1 and four new GameXPeptide derivatives (22-25) in yields of 3-92 mg/L that were structurally confirmed by chemical synthesis.

[0108] For the generation of a second and structurally more diverse library, positions 1 (D-Val) and 3 (D-Phe) of GxpS were selected in parallel for randomization (FIG. 6). From the experimental setup theoretically 48 different cyclic and linear peptides could have been expected. Screening of 50 E. coli clones resulted in the identification of 18 unique cyclic and linear peptides (1, 4, 11, 13, 24, 26-36) differing in length and AA composition. Since only 7 out of 18 identified peptides belong to the expected set of peptides, homologues recombination in yeast based reprogramming of NRPS also allows the production of unexpected peptides due to unexpected homologues recombination events resulting in an additional layer of peptide diversification, as recorded in previous experiments (WO 2017/020983).

[0109] Randomizing adjacent positions via a one shot yeast based TAR cloning approach assumes a standardized nucleotide sequence (40-80 base pairs) for homologues recombination. (Schimming et al. 2014, Gietz et al. 2007). By exploring the T-C didomain crystal structure of TycC5-6 (PDB-ID: 2JGP), the helix 5 (I253-F265) next to the C-domain's pseudo-dimer linker was identified as an ideal target for homologues recombination. Following, an artificial 5 helix was designed to randomize position 2 (L-Leu) and 3 (D-Phe) of GxpS, being an integral part of all resulting recombinant C3 domainsconnecting XU.sub.2.02 and 3. The applied as helix was defined as the consensus sequence of all involved XU.sub.2.0 building blocks (FIG. 7a). Screening of 25 E. coli clones revealed the synthesis of 7 cyclic and linear GameXPeptides (1, 23, 38-42), showing the general applicability of redesigning 5 with respect to randomly reprogramming biosynthetic templates (FIG. 7b).

[0110] Very recently the concept of XU was published, enabling the guided reprogramming of NRPS for the first time (WO 2017/020983). Nevertheless, the inventors attempted to develop a more user-friendly and straightforward way to achieve the de novo design of NRPS from scratch. The novel XU.sub.2.0 building block presented here brings crucial advantages compared with all other up to now published strategies (Win et al. 2015, Calcott & Ackerley 2014). In comparison to the state-of-the-art XU concept, the XU.sub.2.0 enables reprogramming of NRPSs by solely altering one unit since the XU.sub.2.0 automatically modulates C-domain specificities. Moreover, much less building blocks are required to introduce any changes into the appropriate biosynthetic template. For example, to create any peptide based on the 20 proteinogenic AAs only 80 XU.sub.2.0 building blocks are necessaryonly four of each: C.sub.Dsub-A-T-C.sub.Asub, C.sub.Dsub-A-T-C/E.sub.Asub, C/E.sub.Dsub-A-T-C/E.sub.Asub, and C/E.sub.Dsub-A-T-C.sub.Asub. In contrast, 800 XU building blocks would be necessary to generate the same spectrum of peptides. Consequently, the introduction of the XU.sub.2.0 enormously simplifies and broadens the possibilities of biotechnological applications with regard to optimize bioactive agents via NRPS engineering.

[0111] In summary the inventors demonstrated how C-domain specificities can be avoided (FIG. 1) and how this knowledge can be applied to flexibly reprogram NRPSs and to functionalize NRPs by incorporating XU.sub.2.0 building blocks accepting non-natural AAs like p-N.sub.3-Phe and Y-Tyr (FIG. 4,). As azides and alkynes readily undergo bioorthogonal click reactions, p-N.sub.3-Phe and Y-Tyr incorporating NRPs can be used for further derivatization (Sletten & Bertozzi 2009, Kolb & Sharpless 2003). Consequently, NRPSs able to incorporate clickable AAs into the produced peptides provide a powerful means of isolating, labelling, and modifying biologically active peptides (Kries et al. 2015).

[0112] The true strength of the XU.sub.2.0's flexibility, however, lay in the ability to generate random NP-like peptide libraries for subsequent bioactivity screenings. The simple randomization of two building blocks from GxpS and subsequent screening of only 50 E. coli clones led to the identification of 19 novel peptides. The possible automation of screening in line with assays for bioactivity, e.g. via intelligent droplet based microfluidics, opens up entirely new opportunities of identifying novel lead compounds in the future. Especially in the area of anti-infective research underlying results might be an issue very important in practice, namely to fight upcoming antimicrobial resistances.

Materials and Methods

Cultivation of Strains

[0113] 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 5 g/L NaCl). Solid media contained 1.5% (w/v) agar. S. cerevisiae strain CEN.PK 2-1C 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 1.5% (w/v) agar. Kanamycin (50 g/ml) and G418 (200 g/ml) were used as selection markers. E. coli was cultivated at 37 C. all other strains were cultivated at 30 C.

Expression and Cultivation of His-Tagged Proteins

[0114] For overproduction and purification of the 72 Da His-tagged A domain GxpS_A3 5 ml of an overnight culture in LB medium of E. coli BL21 (DE3) cells harboring the corresponding expression plasmid and the TaKaRa chaperone-plasmid pTf16 (TAKARA BIO INC.) were used to inoculate 500 ml of autoinduction medium (464 ml LB medium, 500 l 1 M MgSo.sub.4, 10 ml 505052, 25 ml 20NPS) containing 20 g/mL chloramphenicol, 50 g/mL kanamycin and 0.5 mg/ml L-arabinose (Nishihara et al. 2000). The cells were grown at 37 C. up to an OD.sub.600 of 0.6. Following the cultures were cultivated for additional 48 h at 18 C. The cells were pelleted (10 min, 4,000 rpm, 4 C.) and stored overnight at 20 C. For protein purification the cells were reuspended in binding buffer (500 mM NaCl, 20 mM imidazol, 50 mM HEPES, 10% (w/v) glycerol, pH 8.0). For cell lysis benzonase (Fermentas, 500 U), protease inhibitor (Complete EDTA-free, Roche), 0.1% Triton-X and lysozym (0.5 mg/ml, 20,000 U/mg, Roth) were added and the cells were incubated rotating for 30 min. After this the cells were placed on ice and lysed by sonication. Subsequently, the lysed cells were centrifuged (25,000 rpm, 45 min, 4 C.). The yielded supernatant was passed through a 0.2 m filter and loaded with a flow rate of 0.5 ml/min on a 1 ml HisTrap HP column (GE Healthcare) equilibrated with binding buffer. Unbound protein was washed off with 10 ml binding buffer. Impurities were washed off with 5 ml 8% elution buffer (500 mM NaCl, 500 mM imidazol, 50 mM HEPES, 10% (w/v) glycerol, pH 8.0). The purified protein of interest was eluted with 39% elution buffer. Following, the purified protein containing fraction was concentrated (Centriprep Centrifugal Filters UltacelYM50, Merck Millipore) and the buffer was exchanged to 20 mM Tris-HCl (pH 7.5) using a PD-10 column (Sephadex G-25 M, GE Healthcare).

Cloning of GxpS_A3

[0115] The adenylation domain GxpS_A3 was cloned from Photorhabdus luminescens TTO1 genomic nomic DNA by PCR using the pCOLA_Gib_A3 Insert forward and reverse oligonucleotides shown in Tab. 1. The plasmid backbone of pCOLADUET-1 (Merck/Millipore) was amplified using the DUET_Gib forward and reverse oligonucleotides shown in Tab 1. The 1,900 bp PCR product was cloned via Gibson Assembly Cloning Kit (NEB) according to the manufacturers' instructions into pCOLADUET-1.

-.SUP.18.O.SUB.4.-ATP Pyrophosphat Exchange Assay

[0116] The -.sup.18O.sub.3-ATP Pyrophosphat Exchange Assay was performed as published previously (Kronenwerth et al. 2014, Phelan et al. 2009). After an incubation period of 90 min at 24 C. the reactions were stopped by the addition of 6 l 9-aminoacridine in acetone (10 mg/ml) for MALDI-Orbitrap-MS analysis.

MALDI-Orbitrap-MS

[0117] Samples were prepared for MALDI-analysis as a 1:1 dilution in 9-aminoacridine in acetone (10 mg/ml) and spotted onto a polished stainless steel target and air-dried. MALDI-Orbitrap-MS analyses were performed with a MALDI LTQ Orbitrap XL (Thermo Fisher Scientific, Inc., Waltham, Mass.) equipped with a nitrogen laser at 337 nm. The following instrument parameters were used: laser energy, 27 J; automatic gain control, on; auto spectrum filter, off; resolution, 30,000; plate motion, survey CPS. Mass spectra were obtained in negative ion mode over a range of 500 to 540 m/z. The mass spectra for ATP-PP.sub.i exchange analysis were acquired by averaging 50 consecutive laser shots. Spectral analysis was conducted using Qual Browser (version 2.0.7; Thermo Fisher Scientific, Inc., Waltham, Mass.).

Cloning of Biosynthetic Gene Clusters

[0118] 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 Eurofins Genomics (Tab. 1). Fragments with homology arms (40-80 bp) were amplified in a two-step PCR program For PCR Phusion High-Fidelity DNA polymerase (Thermo Scientific), Q5 High-Fidelity DNA polymerase (New England BioLabs) and Velocity DNA polymerase (Bioline) were used. Polymerases were used according to the manufacturers' instructions. DNA purification was performed from 1% TAE agarose gel using Invisorb Spin DNA Extraction Kit (STRATEC Biomedical AG). Plasmid isolation from E. coli was done by alkaline lysis.

Overlap Extnesion PCR-Yeast Homologous Recombination (ExRec)

[0119] Transformation of yeast cells was done according to the protocols from Gietz and Schiestl (Gietz et al. 2007). 100-2,000 ng of each fragment was used for transformation. Constructed plasmids were isolated from yeast transformants and transformed in E. coli DH10B::mtaA by electroporation. Successfully transformed plasmids were isolated from E. coli transformants and verified by restriction digest.

Heterologous Expression of NRPS Templates and LC-MS Analysis

[0120] Constructed plasmids were transformed into E. coli DH10B::mtaA. Strains were grown overnight in LB medium containing 50 g/mL kanamycin. 100 l of an overnight culture were used for inoculation of 10 ml cultures, containing 0.02 mg/ml L-arabinose and 2% (v/v) XAD-16. 50 g/mL kanamycin were used as selection markers. After incubation for 72 h at 22 C., respectively, 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 1 mL methanol and a 1:10 dilution was used for LC-MS analysis as described previously (Fuchs et al. 2013 & 2014). All measurements were carried out by using an Ultimate 3000 LC system (Dionex) coupled to an AmaZonX (Bruker) electron spray ionization mass spectrometer. High-resolution mass spectra were obtained on a Dionex Ultimate 3000 RSLC Coupled to a Bruker micro-TOF-Q II equipped with an ESI Source set to positive ionization mode. The software DataAnalysis 4.3 (Bruker) was used to evaluate the measurements.

Homology-Modelling

[0121] The homology-modelling was performed as described previously {Fuchs:2013cv}. For homology modelling, the 1.85 crystal structure of PCP-C bidomain TycC 5-6 from tyrocidine syntethase (TycC) of Brevibacillus brevis (PDB-ID: 2JGP) were used {Samel:2007eh}. The sequence identity of GxpS_C3 in comparison to TycC 5-6 is 34.8%, respectively. The final models have a root-mean-square deviation (RMSD) of 1.4 respectively, in comparison to the template structures.

10. Peptide Quantification

[0122] All peptides were quantified using a calibration curve of synthetic 1 (for quantification of 1-4), 5 (for quantification of 5, 34, 35, 37, 38 and 39), 9 (for quantification of 9, 14 and 15), 10 (for quantification of 10, 11, 12, 13, 26, 27, 28, 29, 30, 31 and 36), 17 (for quantification of 16 and 17), 18, 19 (for quantification of 19, 20 and 21), 22, 23, 24 (for quantification of 24 and 39), 25 (for quantification of 25 and 33), and cyclo[RLflL] (for quantification of 32 and 40) using HPLC/MS measurements as described above. Triplicates of all experiments were measured.

TABLE-US-00001 TABLE1 Oligonucleotidesusedinthiswork. Oligo- Plas- nucleo- SEQ mid tide Sequence(5.fwdarw.3) Template ID pCO- pCOLA CATCACCATCATCACCACCCTCAACAACCTGTCACGGC P. 1 LA_gx _Gib_A lumine pS_A3 3Insert ne- FW scens TT01 pCOLA CAGCCTAGGTTAATTAAGCTGTTAAGTCAGATCAATCAGCGGCAAC 2 _Gib_A 3Insert RV DUET_ CAGCTTAATTAACCTAGGCTG pCO- 3 Gib_F LADU W ET-1 DUET_ GTGGTGATGATGGTGATG 4 Gib_RV pFF1 KB-RT- TCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCG P. 5 NRPS_ 6 CCTCCGCTTCACAATTC lumine 1 ne- scens TT01 KB-RT- TGGAATGCGACCGAAGAACC 6 7 KB-RT- CACATACCTGAGTAGGATACGGTTCTTCGGTCGCATTCCAAGTTTTC X. 7 8 AGCAACAACTGGC buda- pes- tensis DSM 16342 KB-RT- TGTTTTGCCTGCATCGGAACGCACGTTGTTGCTGGAAACGTGGAAT 8 9 ACAACGGAAACTGC KB-RT- CGTTTCCAGCAACAACG P. 9 10 lumine ne- scens TT01 KB-RT- TTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCATGAAAG 10 11 ATAGCATGGCTAAAAAGG pFF1_ KB-RT- TCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCG P. 11 NRPS_ 6 CCTCCGCTTCACAATTC lumine 2 ne- scens TT01 KB-RT- TGGAATGCGACCGAAGAACC 12 7 KB-RT- AAGCCATTGACGCTGCCAG X. 13 15 buda- pes- tensis DSM 16342 KB-RT- TGTTTTGCCTGCATCGGAACGCACGTTGTTGCTGGAAACGTGGAAT 14 9 ACAACGGAAACTGC KB-RT- CGTTTCCAGCAACAACG P. 15 10 lumine ne- scens TT01 KB-RT- TTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCATGAAAG 16 11 ATAGCATGGCTAAAAAGG KB-RT- CACATACCTGAGTAGGATACGGTTCTTCGGTCGCATTCCAATTTTCC P. 17 13 AGTAATAACTCCCGCTC lumine ne- scens TT01 KB-RT- TCAATATCCTGATTATGCGGTCTGGCAGCGTCAATGGCTTTCAGGTG 18 14 AAGGAGTACAGGC pFF1 AL- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT X. 19 NRPS GxpS- GAAAGATAGCATGGCTAAAAAGG nema- 3 2-1 tophila ATCC 19061 AL- CCCAATCAACATATCGGTAAAAAAGCGAGTATGTTCCATCTGGCTCA 20 GxpS- CCCCCTGGTGGGCC 2-2 AL- CCCGTACCTTTCCGTAATCTGGTCGCTCAGGCCCACCAGGGGGTGA X. 21 GxpS- GCCAGATGGAACATACTCG mira- 2-3 niensis DSM 17902 AL- CGTCCGACGCCAATAATCACTCTGTGCCTGTACTCCTTCACCTGAAA 22 GxpS- ACCACTGGCGTTGCC 2-4 AL- TCAGGTGAAGGAGTACAGGCAC P. 23 GxpS- lumine 2-5 ne- scens TT01 AL- GACACCCTGCCGAGCC 24 GxpS- 2-6 AL- CCGGTCCCGTTCCGCCATTTAGTGGCACAGGCTCGGCAGGGTGTC X. 25 GxpS- CAAGGCGCTGTTCTCACTG bovi- 2-7 enii SS200 4 AL- CTCAGCCAACATTTCAGTAAAGAAACGGGTATGTTCAGCCTGACTCA 26 GxpS- CGCTCAGGGTCTGGG 2-8 AL- AGTCAGGCTGAACATACCCG P- 27 GxpS- lumine 2-9 ne- scens TT01 AL- TTTGCTCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCC 28 GXpS- AGCGCCTCCGCTTCAC 2-10 pFF1_ AL- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT X. 29 NRPS_ GxpS- GAAAGATAGCATGGCTAAAAAGG nema- 4 2-1 tophila ATCC 19061 AL- CCCAATCAACATATCGGTAAAAAAGCGAGTATGTTCCATCTGGCTCA 30 GXpS- CCCCCTGGTGGGCC 2-2 AL- CCCGTACCTTTCCGTAATCTGGTCGCTCAGGCCCACCAGGGGGTGA X. 31 GXpS- GCCAGATGGAACATACTCG mira- 2-3 niensis DSM 17902 AL- CGTCCGACGCCAATAATCACTCTGTGCCTGTACTCCTTCACCTGAAA 32 GXpS- ACCACTGGCGTTGCC 2-4 AL- TCAGGTGAAGGAGTACAGGCAC P. 33 GxpS- lumine 2-5 ne- scens TT01 AL- GACACCCTGCCGAGCC 34 GxpS- 2-6 AL- GCCGGTCCCGTTCCGCCATTTAGTGGCACAGGCTCGGCAGGGTGT X. 35 GxpS- CAGTCAGGAAGCCCACACC mira- 2-11 niensis DSM 17902 AL- CTCAGCCAACATTTCAGTAAAGAAACGGGTATGTTCAGCCTGACTCA 36 GXpS- CCCCCAACCGAACC 2-12 AL- AGTCAGGCTGAACATACCCG P. 37 GxpS- lumine 2-9 ne- scens TT01 AL- TTTGCTCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCC 38 GXpS- AGCGCCTCCGCTTCAC 2-10 pFF1_ ML020 AGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGT pFF1_ 39 NRPS_ TTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCATGAAAG NRPS 5 ATAACATTGCTACAGTG _0 FF_305 CCAATAATCACTCTGTGCCTG 40 FF_306 GAACTGGTTGCACTTTACGC 41 FF_307 CATCCCTAACCGGGACTG 42 FF_308 CATACCTTGACGGACAGGGCGGCAACCTGCCAGCGCCGGCACCCT X. 43 TCCGCAATCTGGTAGCGCAGTCCCGGTTAGGGATGAGTCAGGCAG buda- CCCATACC Pes- tensis DSM 16342 FF_309 AACAACACCGGTAAACAGTTCTTCACCTTTGCTCATGAACTCGCCAG 44 AACCAGCAGCGGAGCCAGCGGATCCGGCGCGCCCTATTGCTCTGC TGATATCAGAA pFF1_ ML_P1 GACCAGACAGAACATCACCG pFF1_ 45 NRPS_ gxpS_ 6 WT ML_P2 GGCCCAATCCTATACGCC 46 ML_P3 CTTACCAAGCGCCACAAGG 47 ML_P4 AGAATCGGAACAACACCGGTAAACAGTTCTTCACCTTTGCTCATGAA 48 CTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCGCCTCCGC TTCA ML_P5 TCGGTCAGCCCAAACGGTAATGTCGGTTCATCCACTTCTGCCAACAT X. 49 GTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCGACACCCTGCCGA nema- GCC tophila HGB0 81 ML_P6. ATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACC 50 1 CGTTTTTTTGGGCTAACAGGAGGAATTCCATGCGGGCAATGGTGAA CC pFF1_ ML_Pl GACCAGACAGAACATCACCG pFF1_ 51 NRPS_ gxpS_ 7 WT ML_P2 GGCCCAATCCTATACGCC 52 ML_P3 CTTACCAAGCGCCACAAGG 53 ML_P4 AGAATCGGAACAACACCGGTAAACAGTTCTTCACCTTTGCTCATGAA 54 CTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCGCCTCCGC TTCA ML_P5 TCGGTCAGCCCAAACGGTAATGTCGGTTCATCCACTTCTGCCAACAT X. 55 GTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCGACACCCTGCCGA nema- GCC tophila HGB0 81 ML_P6. ATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACC 56 3 CGTTTTTTTGGGCTAACAGGAGGAATTCCATGTCTGATGAAGGCGT GC pFF1 ML_P1 GACCAGACAGAACATCACCG pFF1_ 57 NRPS_ gxpS_ 8 WT ML_P2 GGCCCAATCCTATACGCC 58 ML_P3 CTTACCAAGCGCCACAAGG 59 ML_P4 AGAATCGGAACAACACCGGTAAACAGTTCTTCACCTTTGCTCATGAA 60 CTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCGCCTCCGC TTCA ML_P5 TCGGTCAGCCCAAACGGTAATGTCGGTTCATCCACTTCTGCCAACAT X. 61 GTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCGACACCCTGCCGA nema- GCC tophila HGB0 81 ML_P6. ATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACC 62 2 CGTTTTTTTGGGCTAACAGGAGGAATTCCATGGCATTTACCGAAAAG ATCTGCG pFF1_ ML_P7 CGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCA X. 63 NRPS_ TACCCGTTTTTTTGGGCTAACAGGAGGAATTCCATGAAAGATAGCAT nema- 9 GGCTAAAAAGGG tophila HGB0 81 ML_P8 CTATCGGCAATTCAAGTAACACCGGTGCATCTGCCAACGTCCGACG 64 CCAATAATCACTCTGTGCCTGTACTCCTTCACCTGAAAATACCTGCC GCTGCC ML_P9 GCTTGTCTGAATCAACAACCTGATCCGCTGCCGCCATTGACCATTCA 65 ATATCCTGATTATGCTGCCTGGCAGCGGCAGGTATTTTCAGGTGAA GGAGTACAGGC ML_P1 GCACTTCCGACAATCCAAATGACAACGTTGGCTCATCTACCTCAGCC 66 0 AACATATCGGTAAAGAAACGGGTATGTTCTGCCTGACTGACACCCT GCCGAGCC ML_P1 GGCTTGCCTCTTGGGGCAAATGGATAGCCTGCCTGCGCCGGTCCC X. 67 1 GTTCCGCCATTTAGTGGCACAGGCTCGGCAGGGTGTCAGTCAGGC nema- AGAACATACCCG tophila HGB0 81 ML_P1 CCAGAATCGGAACAACACCGGTAAACAGTTCTTCACCTTTGCTCATG 68 2 AACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCGCCTCC ACTTCG pFF1_li KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC X. 69 bra- AmbF-1 GGTTGAACGCTTACAATCC mira- ry_1 niensis DSM 17902 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTG 70 AmbF-2 ATACCCAGCCGGGCTTG KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC X. 71 AmbW- ATCGGAACGGGTACAAATTC indica 1 DSM 17382 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTTA 72 AmbW- CCCCCATCCGTGCCTG 2 KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC P. 73 Thr1 GGCAGCACAGATACAGTCTC lumine ne- scens TT01 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCA 74 Thr2 CGCCCAACCGGACC KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC X. 75 Arg1 TGCTGATCGTATTCAGGTGCAG buda- pes- tensis DSM 16342 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCA 76 Arg2 CGCCCAACCGGACC KB- GTCTAAATCAACAGCCAGATCCGTTGTTGCCATTGACCATTCAATAT X. 77 Ser1 CCTGATTATGCGGCTTGGCAGCGGCAGGTATTTCAGGGTGACCGCC sze- TGAC ntirmai iDSM 16338 KB- TCCGCCAACCCAAATAGCAGCGTCGGTTCATCCACCTCAGCCAACA 78 Ser2 TGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCACACTCAGGATT TGAGCGATAAAG KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTCA X. 79 Lys1 GGGTGAGGTACTGGAAAAGC nema- tophila HGB0 81 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTTA 80 Lys2 CACTGCGGGTTTGGGC AL- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT pFF1_ 81 GxpS-1 GAAAGATAGCATGGCTAAAAAGG gxpS_ WT AL- AAATACCTGCCGCTGCC 82 GxpS-2 AL- AGTCAGGCAGAACATACTCGCTTCTTTAC 83 GxpS-3 AL- TTTGCTCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCC 84 GxpS-4 AGCGCCTCCGCTTCAC pFF1_li KB- TTCTGCCAACATGTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCTT X. 85 bra- xeyS-C CCCCCAACCAGGACTG indica ry2 DSM 17382 KB- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT 86 xeyS-N GAAAGATAACATGGCTACAACG KB- TTCTGCCAACATGTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCCA X. 87 BicA-C TCCCCAACCAGGACTG buda- pes- tensis DSM 16342 KB- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT 88 BicA-N GAAAGATAACATTGCTACAGTGG KB- TTCTGCCAACATGTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCAA X. 89 17902- CGCCCAGCCGGGCTTGAGC mira- C niensis DSM 17902 KB- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT 90 17902- GAAAAATGATAAGGTGATGACTCTGC N KB- TTCTGCCAACATGTCGGTAAAGAATCGGTGATGTTCTGTCTGGTCCA X. 91 2022-C CCCCCTGGTGGGCC nema- tophila HGB0 81 KB- ACTGTTTCTCCATACCCGTTTTTTTGGGCTAACAGGAGGAATTCCAT 92 2022-N GAAAGATAGCATGGCTAAAAAGG KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTCA X. 93 XLSer1 GGGTGACCGCCTGAC sze- ntirmai iDSM 16338 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCA 94 XLSer2 CACTCAGGATTTGAGCGATAAAG KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC X. 95 AmbF-1 GGTTGAACGCTTACAATCC mira- niensis DSM 17902 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTG 96 AmbF-2 ATACCCAGCCGGGCTTG KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC X. 97 AmbW- indica 1 ATCGGAACGGGTACAAATTC DSM 17382 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTTA 98 AmbW- CCCCCATCCGTGCCTG 2 KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC P. 99 Thr1 GGCAGCACAGATACAGTCTC lumine ne- scens TT01 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCA 100 Th2 CGCCCAACCGGACC KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTC X. 101 Arg1 TGCTGATCGTATTCAGGTGCAG buda- pes- tensis DSM 16342 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCA 102 Arg2 CGCCCAACCGGACC KB- ACCATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTCA X. 103 Lys1 GGGTGAGGTACTGGAAAAGC nema- tophila HGB0 81 KB- CTCAGCCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTTA 104 Lys2 CACTGCGGGTTTGGGC AL- AGTCAGGCAGAACATACTCGCTTCTTTAC pFF1_ 105 GxpS gxpS_ P3 WT AL- TTTGCTCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCC 106 GxpS- AGCGCCTCCGCTTCAC P4 KB- GACCAGACAGAACATCACCG 107 Lib3-1 KB- AAATACCTGCCGCTGCC 108 Lib3-2 pFF1_li KB-XL- CCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCACACTC X. 109 bra- X3RV AGGATTTGAGCG sze- ry3 ntirmai iDSM 16338 KB-XL- ATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTCAGG 110 X3FW GTGACCGCCTGACC KB-XI- CCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCATACCT X. 111 ambX3 AGACGTGCCTGTGC indica RV DSM 17382 KB-XI- ATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTCGGC 112 ambX3 AGAACGGATACAAATTC FW KB-Kol CCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCACGCC P. 113 X3-RV CAACCGGACC lumine ne- scens TT01 KB-Kol ATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTCGGC 114 X3- AGCACAGATACAGTC FW KB-Kol AAATACCTGCCGCTGCCAAGCCGCATAATCAGGATATTGAATCGTCA P. 115 X2RV ACGGTAGCAACGG lumine ne- scens TT01 KB-Kol ACCTTTCCGCAATCTGGTGGCTCAGGCTCGGCAGGGGGTTAGTCAG 116 X2FW GCTGAGCATACCCG KB- CCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTCACGCC X. 117 BicAX3 CAACCGGACC buda- RV pes- tensis DSM 16342 KB- ATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTCTGC 118 BicAX3 TGATCGTATTCAGGTGC FW KB-Bb TCATGAACTCGCCAGAACCAGCAGCGGAGCCAGCGGATCCCAGCG pFF1_ 119 2RV CCTCCGCTTCACAATTC gxpS_ WT KB-Bb AGTCAGGCAGAACATACTCGC 120 2FW KB-Bb AACCCCCTGCCGAGCC pFF1_ 121 1RV gxpS_ WT KB-Bb AACCCCCTGCCGAGCC 122 1FW KB- AAATACCTGCCGCTGCCAAGCCGCATAATCAGGATATTGAATTGCCA X. 123 ambX2 ATGGTGGCAAGGG indica RV DSM 17382 KB- ACCTTTCCGCAATCTGGTGGCTCAGGCTCGGCAGGGGGTTAGCCA 124 ambX2 GACAGAGCACACCCG FW KB- CCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTGATACCC X. 125 17902 AGCCGGGCTTGTGC mira- X3RV niensis DSM 17902 KB- ATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTCGGT 126 17902 TGAACGCTTACAATCC X3FW KB- CCAACATGTCAGTAAAGAAGCGAGTATGTTCTGCCTGACTGACACC X. 127 2022 CTGCCGAGCC nema- X3RV tophila HGB0 81 KB- ATTCAATATCCTGATTATGCGGCTTGGCAGCGGCAGGTATTTTCTGA 128 2022 TGAAGGCGTGCAGG X3FW KB- AAATACCTGCCGCTGCCAAGCCGCATAATCAGGATATTGAATGGTC X. 129 2022 AATGGCGGCAGCGG nema- X2RV tophila HGB0 81 KB- ACCTTTCCGCAATCTGGTGGCTCAGGCTCGGCAGGGGGTTAGTCAG 130 2022 GAAGCGTACACGCG X2FW

LITERATURE

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