Methods and means for engineering nonribosomal peptides

Abstract

The present invention relates to a strategy for combining elements of nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs) to function as enzymes that facilitate the production of peptides and polyketides of any sequence. The present invention is based on the use of conserved sequence motifs within the T domain (or T domain-like) genes of NRPSs / PKSs, which are used as fusion points to generate new (and artificial) NRPS / PKS enzymes that have the potential to produce any peptide or polyketide of interest. The present invention provides artificial NRPS / PKSs of the invention, methods and means for their generation, and nucleic acid libraries encoding various modules that can be used to design NRPS / PKSs for any peptide sequence of interest.

Description

[Technical Field]

[0001] The present invention relates to a strategy for combining elements of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) to function as enzymes that facilitate the production of peptides and polyketides of any sequence. The invention is based on the use of conserved sequence motifs within the T domain (or T domain-like) genes of NRPSs / PKSs, which are used as fusion points to generate new (and artificial) NRPS / PKS enzymes that have the potential to produce any peptide or polyketide of interest. The invention provides artificial NRPS / PKSs of the invention, methods and means for their generation, and nucleic acid libraries encoding various modules that can be used to design NRPS / PKSs for any peptide sequence of interest. [Background technology]

[0002] The present invention relates to a system for the generation and expression of chimeric nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs), or NRPS / PKS hybrid synthases. NRPSs, PKSs, or their hybrids are large, multifunctional, modular, multidomain proteins or complexes that generate a variety of clinically relevant scaffolds. Their expression and modification for peptide production and product engineering, respectively, present significant challenges. The present invention relates to a novel evolutionarily inspired strategy (EIS) for functionally recombining, expressing, and generating artificial nonribosomal peptides (NRPs), polyketides (PKs), and their hybrids. This EIS method leverages in silico-detected and in vivo-validated splicing sites to enable both the rational modification of targeted biosynthetic gene clusters (BGCs) and the de novo assembly of novel artificial / synthetic BGCs. The present invention discloses specifically defined splice sites, protein fragments, and recombination strategies for such molecular synthetic biology assembly kits, as well as the nucleic acids encoding them. Also disclosed are cloning strategies for the nucleotide fragments of the invention and their use in preparing functional NRPS / PKS enzymes and generating libraries of novel bioactive NRP / PK scaffolds.

[0003] Reprogramming biosynthetic assembly lines (NRPSs, PKSs, and NRPS / PKS hybrids) is of great interest, which is not surprising since the scaffolds for many clinically used antibiotic (penicillin, 1), immunosuppressant (rapamycin, 2), and anticancer (bleomycin, 3) therapeutic agents are generated by such pathways.

[0004] Essentially, NRPS (Non-Patent Document 4) and PKS (Non-Patent Document 5) modular megasynthases generate highly functionalized biopolymers from various monomers called extender units. Hundreds of extender units have been reported (Non-Patent Document 6), typically derived from amino acids in NRPSs (Non-Patent Document 7, Non-Patent Document 8) and malonates in PKSs (Non-Patent Document 9). Their hierarchical and modular structure resembles an assembly-line process. Multiple repeating modules of enzyme domains catalyze the incorporation of extender units into the growing chain, along with any programmed additional chemical modifications, before transferring the extended chain to the next module. A typical minimal assembly-line module consists of three "core" domains: first, a domain for selecting and activating the extender units: an adenylation (A) domain in NRPSs or an acyltransferase (AT) domain in PKSs. The activated substrate then covalently binds to the prosthetic phosphopantetheine group of a small peptidyl carrier protein (PCP; NRPS), also known as a thiolation (T) domain or acyl carrier protein (ACP; PKS) domain. Finally, a condensation (C; NRPS) domain or ketosynthase (KS; PKS) ligates the covalently bound substrate into the growing peptide or polyketide chain. Their modularity provides a direct relationship between the sequence of the enzyme domain and the chemical structure of the product (10, 11), but efforts to rationally reprogram them have met with limited success (12).

[0005] Since Marahiel et al. (Patent Document 1) demonstrated that NRPSs can be recombined through the exchange of adenylation-thiolation didomains in 1995, NRPS research has attracted attention (Marahiel et al. 1995). Over the past 20 years, numerous attempts have been made to reprogram NRPSs. Based on the crystal structure of the phenylalanine activation domain PheA (PDB-ID: 1AMU), Stachelhaus et al. were able to elucidate the specificity of AA addition to the catalytic center (Conti et al. 1997, Stachelhaus et al. 1999). Using this specificity-imparting code, known as the Stachelhaus code, it is possible to predict and modify the substrate specificity of the A domain in vitro (Khurana et al. 2010, Rausch et al. 2005, Rottig et al. 2011, Kries et al. 2014). The most obvious drawback of this approach is its inapplicability in vivo. One of the main reasons for this drawback is that the C and TE domains also have selectivity, resulting in substrate mismatch ( Belshaw et al. 1999 , Trauger et al. 2000 , Tseng et al. 2002 ).

[0006] Further attempts to alter known NRPS biosynthetic clusters (Patent Document 2, Marahiel et al.) have been based on the exchange of single domains, didomains, or entire modules, with knowledge of precisely defined boundaries (linkers) between individual domains. This work has only successfully altered a few NRPSs by introducing additional modules or deleting modules. However, it has not been possible to generate completely artificial NRPSs from the de novo combination of modules or domains. This results in the creation of new NRPSs not found in nature, as well as the generation of new peptides. The challenge of such exchanges and combinations has always been the uncertainty regarding the compatibility between modules and / or domains. The drawbacks stemming from the lack of a method to solve the above problems are illustrated by the fact that only a few peptide derivatives have been designed using this approach.

[0007] Another attempt to alter known NRPS biosynthetic clusters (Walsh et al., U.S. Patent No. 6,277,999) is based on so-called "assembly line" mutagenesis, i.e., synthase mutagenesis. While mutagenesis of NRPS genes is not the subject of this invention, the methods of this invention can be combined with mutagenesis to alter the NRPS produced and alter peptide synthesis. This mutagenesis may be useful for diversifying NRPS libraries and increasing the number of NRPS clones within the library. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] W0200052152 [Patent Document 2] W0200130985 [Patent Document 3] W02007014076 [Non-patent literature]

[0009] [Non-Patent Document 1] Fierro, F. et al. Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeatscontaining the penicillin gene cluster in Penicillium chrysogenum. Fungal GenetBiol 43, 618-629, doi:10.1016 / j.fgb.2006.03.001 (2006). [Non-patent document 2] Li, J., Kim, SG & Blenis, J. Rapamycin: one drug, many effects. Cell Metab 19, 373-379, doi:10.1016 / j.cmet.2014.01.001 (2014). [Non-licensed document 3] Murray, V., Chen, JK & Chung, LHThe Interaction of the Metallo-Glycopeptide Anti-Tumour Drug Bleomycin withDNA. Int J Mol Sci 19, doi:10.3390 / ijms19051372 (2018).

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Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

[0010] It is therefore an object of the present invention to provide an alternative approach to facilitate the design and expression of artificial NRPS / PKS for peptide and polyketide synthesis. [Means for solving the problem]

[0011] Generally and briefly, the main aspects of the present invention are as follows:

[0012] In a first aspect, the present invention relates to a chimeric protein for producing non-ribosomal peptides, the chimeric protein comprising at least one chimeric thiolation domain (T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP))) immediately flanked by a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment and a second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment is located at the N-terminal portion of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence. and the second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment comprises an amino acid sequence derived from the C-terminal portion of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence, wherein the chimeric thiolation domain is a fully functional T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) characterized in that the amino acid sequences of the portions of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments are as follows: (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different species that are heterologous to each other, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different NRPS / PKS genes of the same species, or (c) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene.

[0013] In an alternative first aspect, the present invention relates to a method for generating a chimeric non-ribosomal peptide synthetase (NRPS) and / or polyketide synthase (PKS) module, the method comprising: (a) providing a first NRPS / PKS module sequence comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequence; (b) providing a second NRPS / PKS module sequence comprising at least a second T domain sequence; (c) optionally aligning the first T domain sequence to the second T domain sequence to identify at least one T domain fusion point; (d) fusing the first subsequence to the second subsequence by cloning them immediately adjacent to each other into a genetic construct to obtain a chimeric module sequence, (i) the first subsequence is a nucleotide sequence encoding the amino acid sequence of a first NRPS / PKS module sequence located directly N-terminal to the identified T domain fusion point; and (ii) the second subsequence is a nucleotide sequence encoding the amino acid sequence of a second NRPS / PKS module sequence located directly C-terminal to the identified T domain fusion point; and these steps generate genetic constructs containing chimeric NRPS and / or PKS modules.

[0014] In a second aspect, the present invention provides a system comprising at least two system units: (a) a first system unit comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment as described in any one of the other aspects and embodiments of this aspect; and (b) a second system unit comprising at least a second T domain segment as described in any one of the other aspects and embodiments of this aspect; The present invention relates to a system for producing a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, comprising: Each system unit may contain one or more additional NRPS / PKS domains, and the chimeric nonribosomal peptide synthetase (NRPS), the chimeric polyketide synthase (PKS), or the chimeric NRPS / PKS hybrid is obtained by fusing a first system unit with a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the first system unit is directly fused to the amino acid sequence of the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the second system unit.

[0015] In a third aspect, the present invention relates to a method for producing a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, the method comprising the steps of: (a) providing a first NRPS or PKS gene sequence from a first microbial species; (b) providing a second NRPS or PKS gene sequence from a second microbial species; and (c) combining at least a portion of the first NRPS or PKS gene sequence with at least a portion of the second NRPS or PKS gene sequence to obtain a chimeric gene sequence by fusing the first and second sequences to each other such that at least one fusion point is located within the T domain, wherein the fusion product comprises a T domain consisting of the N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and the C-terminal sequence of the T domain of the second NRPS or PKS gene sequence; and (d) optionally, expressing the chimeric gene sequence to obtain a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0016] In a fourth aspect, the present invention relates to a method for combining or generating a combination of at least two NRPS / PKS modules from two different NRPS / PKS genes or from two separate locations within the same NRPS / PKS gene, the method comprising the steps of: (a) identifying a first target NRPS / PKS module to be directly linked N-terminally to a second target NRPS / PKS module; (b) identifying a second target NRPS / PKS module to be directly linked C-terminally to a second target NRPS / PKS module, the second target NRPS / PKS module being selected from modules located within an NRPS / PKS gene C-terminal to a third, non-target NRPS / PKS module; (c) combining the first target NRPS / PKS module with a second target NRPS / PKS module by directly linking the N-terminal portion of the T domain of the first NRPS / PKS module with the C-terminal portion of the T domain of a third, non-target NRPS / PKS module; Here, the combination of the N-terminal portion of the T domain of the first NRPS / PKS module and the C-terminal portion of the T domain of the third, non-target NRPS / PKS module constitutes a complete and functional T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)).

[0017] In a fifth aspect, the present invention relates to a combination product obtainable by the method of the fourth aspect.

[0018] In a sixth aspect, the present invention relates to a method for generating a nucleic acid construct encoding a system unit as described in other aspects and embodiments of this aspect, the method comprising the step of amplifying a sequence stretch of an NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, the hybridization site comprising a sequence encoding the amino acid consensus motif (T domain motif) set forth in SEQ ID NO: XY (FFxxGG(H / N / D)S, where x is any amino acid).

[0019] In a seventh aspect, the present invention relates to a method for generating a library of nucleic acid constructs each encoding a system unit as described in other aspects and in embodiments of this aspect, the method comprising the step of amplifying a first sequence stretch of the NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, the hybridization site comprising a sequence encoding the amino acid consensus motif (T domain motif) set forth in SEQ ID NO: XY(FFxxGG(H / N / D)S), and the method comprising a second amplification of at least a second sequence stretch of the NRPS / PKS gene using a degenerate primer pair derived from the same or one or more different NRPS / PKS genes.

[0020] In an eighth aspect, the present invention relates to a system of genetic constructs for expression of a chimeric NRPS / PKS, the system comprising at least one genetic construct comprising a chimeric NRPS and / or PKS module produced by the method of the preceding aspect. [Brief explanation of the drawings]

[0021] [Figure 1] Phosphopantetheinylation of the apo-T (PCP) domain to the holoenzyme by Sfp. Top: Crystal structure of Sfp and its substrates, coenzyme A and Mg2+ (PDB-ID: 1QR0)37. Center: The phosphopantetheine moiety of coenzyme A (red) is covalently attached by Sfp to a conserved serine residue in the T (PCP) domain. Bottom: Crystal structure of the T (PCP) domain solved by Weber et al. (PDB-ID: 1DNY)38. [Figure 2]Overview of the applied in silico workflow. To gain a deeper understanding of NRPS evolution and detect evolutionary hotspots of recombination, NRPSs from the genera Photohabdus and Xenorhabdus were analyzed in silico. First, fine-grained phylogenetic reconstruction (a) was performed using the software DualBrothers24, along with tree topology analysis (b). The obtained results suggest that different parts of the T domain exhibit different topologies, indicating different evolutionary origins. Next, the recombination detection program RDP425 was applied to detect potential evolutionary hotspots (c). RDP4 suggested that homologous recombination often occurs within the A domain or immediately before the T domain, and near the invariant serine. Further nucleotide sequence analysis revealed the presence of an AT skew in the conserved region immediately (5') of the invariant serine, which is theoretically ideal for promoting homologous recombination. Further phylogenetic reconstruction (d) of the T domain, divided into N- and C-terminal subunits by an invariant serine, shows distinct clustering relative to each other, suggesting that during evolution, homologous recombination may become more common within than before the T domain. Taking a, b, c, and d together, leads to the evolutionary hypothesis shown in (e). [Figure 3]Recombination point screening to detect functional splice positions within the T domain. To analyze whether functional recombination of NRPS building blocks can be achieved by targeting the T domain, the Xenoamicin72-producing NRPS Cstart-A1-Tp1 from X. stockiae was fused to pT3-C / E4-A4-T4-C / E5-A5-T5-TE from the GameXPetptide-producing NRPS GxpS. Splice positions 1, 3, and 4 demonstrated the production of the expected lipopeptide. Top: Schematic of the resulting chimeric NRPS (left) and the chemical structure of the lipopeptide (right). Middle: Ribbon representation of the crystallized T domain (left) of EntF (PDB: 4ZXJ). Tested fusion points (1-7) are highlighted in black. The fusion point resulting in a catalytically active NRPS is marked with a red arrow. An EIC with m / z [M+H]+ = 444.3 generated in E. coli DH10B::mtaA is shown on the right. Numbers represent individual recombinant NRPSs (1-7). Bottom: Sequence alignment of the targeted T domains. Functional recombination sites are highlighted in red. Center: Color coding of the NRPS components used is shown. The following symbols are used for domain assignment: A: adenylation domain, large circle; T: thiolation domain, rectangle; C: condensation domain, triangle; C / E: double condensation / epimerization domain, diamond; TE: thioesterase domain, small circle. [Figure 4]Proof of concept - fusion site 3. NRPS-1 to NRPS-3 were generated to test whether the evolutionary-inspired novel fusion site 3 could be applied to NRPS reprogramming. Top: Schematic of recombinant NRPS-1 to NRPS-3 and related products. Middle: Color coding of the NRPS components used is shown. The following symbols are used for domain assignment: A: adenylation domain, large circle; T: thiolation domain, rectangle; C: condensation domain, triangle; C / E: double condensation / epimerization domain, diamond; TE: thioesterase domain, small circle; R: reductase domain, square; and the synthetic zipper (SZ) pair used, SZ17:18. Bottom: Structures of peptides 1 to 12 generated from NRPS-1 to NRPS-3 expressed in E. coli DH10B::mtaA. [Figure 5] De novo design of chimeric NRPSs to produce lipopeptide aldehydes. NRPS-4 through NRPS-8 were created to determine whether the evolutionarily inspired novel fusion site 3 could be applied to de novo designed NRPSs from scratch. Top: Schematic of recombinant NRPS-4 through NRPS-8 and related products. Middle: Color coding of the NRPS components used is indicated. See legend to Figure 4 for domain assignments. Bottom: Structures of peptides 13 through 17 generated from NRPS-4 through NRPS-8 expressed in E. coli DH10B::mtaA. [Figure 6] Conversion of NRPS into an artificial NRPS / PKS hybrid synthetase (part 1). Top: Schematic of recombinant NRPS-PKS-9 and related products. Middle: Color coding of the NRPS and PKS components used is shown. For domain assignments, see legend to Figure 4. Additional symbols: KS, DH, and KR domains, small circles. Bottom: Structures of peptides 18–23 generated from NRPS-PKS-9 expressed in E. coli DH10B::mtaA. [Figure 7]Conversion of NRPSs into artificial NRPS / PKS hybrid synthetases (part 2). Top: Schematic of the recombinant PKS-NRPS-10. Middle: Color coding of the NRPS and PKS components used is shown. See legends to Figures 4 and 6 for domain assignments. Bottom: Structures of 24 generated from PKS-NRPS-10 expressed in E. coli DH10B::mtaA. [Figure 8] Sequence alignment of selected T domains. To validate and compare fusion points 1 and 4, a series of recombinant NRPSs were generated (see Figures 9-11). Highlighted by arrows are the recombination sites applied to generate NRPS-11 through NRPS-24. [Figure 9] Creating chimeric NRPSs by recombining components unrelated in GC content and codon usage (part 1). A series of recombinant NRPSs were created to validate and compare the applicability of fusion points 1 and 4. Fusion point 1: NRPS-11 and NRPS-13. Fusion point 4: NRPS-12 and NRPS-14. Top: Schematic of unpublished WT-NRPS and recombinant NRPS-11 to NRPS-14. Middle: Color coding of the NRPS components used is indicated. For domain assignments, see legend to Figure 4. Bottom: Structures of 25 to 27 generated from NRPS-11 to NRPS-14 expressed in E. coli DH10B::mtaA. [Figure 10] Creating chimeric NRPSs by recombining components unrelated by GC content and codon usage (Part 2). A series of recombinant NRPSs were created to validate and compare the applicability of fusion points 1 and 4. Fusion point 1: NRPS-15 and NRPS-17. Fusion point 4: NRPS-16 and NRPS-18. Top: Schematic of recombinant NRPS-15 through NRPS-19. Middle: Color coding of the NRPS components used is indicated. For domain assignments, see the legend to Figure 4. Bottom: Structures of 28 and 29 generated from NRPS-15 through NRPS-18 expressed in E. coli DH10B::mtaA. [Figure 11]Creating chimeric NRPSs by recombining components unrelated by GC content and codon usage (Part 3). A series of recombinant NRPSs were created to validate and compare the applicability of fusion points 1 and 4. Fusion point 1: NRPS-19 and NRPS-21. Fusion point 4: NRPS-20 and NRPS-22. Top: Schematic of recombinant NRPS-19 to NRPS-22. Middle: Color coding of the NRPS components used is indicated. For domain assignments, see legend to Figure 4. Bottom: 30 structures generated from NRPS-19 to NRPS-22 expressed in E. coli DH10B::mtaA. [Figure 12] Schematic diagram of the T-domain degenerate primer library. Steps A to G are described in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0022] The elements of the present invention are described below. While these elements are described with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description herein should be understood to support and encompass embodiments combining two or more of the explicitly described embodiments, or combining one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, all permutations and combinations of elements described herein should be considered disclosed by the description herein, unless the context indicates otherwise.

[0023] definition

[0024] The terms "partial domain" or "partial C or C / E domain," or similar terms, refer to a nucleic acid sequence encoding an incomplete (non-full-length) NRPS C or C / E domain or its protein sequence. Thus, the terms describe the majority of the sequence of an NRPS C or C / E domain that does not include both the donor and acceptor sites of the NRPS C or C / E domain.

[0025] "Assembly" refers to a set of domains. Multiple assemblies comprise NRPSs. One or more polypeptides may comprise modules. The combination of modules catalyzes a series of reactions to form larger molecules. In one example, a module can comprise a C (condensation) domain, an A (adenylation) domain, and a peptidyl carrier protein domain.

[0026] For details regarding the structure of A domains, C domains, didomains, interdomain interfaces, and complete modules, see Conti et al. (1997), Sundlov et al. (2013), Samel et al. (2007), Tanovic et al. (2008), Strieker and Marahiel (2010), Mitchell et al. (2012), and Tan et al. (2015), Drake et al., Izore et al., Reimer et al., NRPS Review: Sussmuth & Mainz.

[0027] "Initiator module" refers to an N-terminal module that can provide the first monomer to another module (e.g., an elongator module or a terminator module). In some cases, the other module is not the second but rather one of the modules following at the C-terminus (e.g., in the case of nocardicin NRPS). In the case of NRPSs, an initiator module may include, for example, an A (adenylation) domain and a PCP (peptidyl carrier protein) or T (thiolation) domain. An initiator module may also include the first C domain and / or an E (epimerization) domain. In the case of PKSs, possible initiator modules include an AT (acetyltransferase) domain and an acyl carrier protein (ACP) domain. The initiator module is preferably located at the amino terminus of the polypeptide of the first module in the assembly line, and each assembly line preferably includes one initiator module.

[0028] "Extender module" refers to a module that adds a monomer to another monomer or polymer. An extender module can contain a C (condensation) domain, a Cy (heterocyclization) domain, an E domain, a C / E domain, an MT (methyltransferase) domain, an A-MT (adenylation and methylation domain combination) domain, an Ox (oxidase) domain, or a Re (reductase) domain, an A domain, or a T domain. An extender domain can further contain an additional E domain, a Re domain, a DH (dehydration) domain, an MT domain, an NMet (N-methylation) domain, an AMT (aminotransferase) domain, or a Cy domain. Additionally, extender modules may be derived from PKSs and contain the respective domains that link the amino acid and carboxylic acid components (ketosynthase (KS), acyltransferase (AT), ketoreductase (KR), dehydratase (DH), enoylductase (ER), acyl carrier protein (ACP), or thiolation (T)).

[0029] "Termination module" refers to a module that releases a molecule (e.g., an NRP, a PK, or a combination thereof) from the assembly line. The molecule may be released, for example, by hydrolysis or cyclization. The termination module may include a TE (thioesterase) domain, a Cterm domain, or a Re domain. The termination module is preferably located at the carboxy terminus of the NRPS or PKS polypeptide. The termination module may further include additional enzymatic activities (e.g., oligomerase activity).

[0030] By "domain" is meant a polypeptide sequence or fragment of a larger polypeptide sequence that has one or more specific enzymatic activities (i.e., a C / E domain has the functions of C and E in one domain) or another conserved function (i.e., as a binding function for an ACP domain or a T domain). Thus, a single polypeptide may contain multiple domains. Multiple domains may form a module. Examples of domains include C (condensation), Cy (heterocyclization), A (adenylation), T (thiolation), TE (thioesterase), E (epimerization), C / E (condensation / epimerization), MT (methyltransferase), Ox (oxidase), Re (reductase), KS (ketosynthase), AT (acyltransferase), KR (ketoreductase), DH (dehydratase), and ER (enoylreductase).

[0031] "Non-ribosomally synthesized peptide," "non-ribosomal peptide," or "NRP" refers to any polypeptide that is not produced by a ribosome. NRPs may be linear, cyclic, or branched, and may contain acyl chains, lipopeptides, aldehydes, amines, and may contain proteinogenic, natural, or unnatural amino acids, or any combination thereof. NRPs include peptides produced in an assembly-line-like manner (i.e., the modular nature of enzymatic systems allows for the stepwise addition of building blocks to form the final product).

[0032] "Polyketide" means a compound containing multiple ketone units (ketides).

[0033] "Nonribosomal peptide synthetase" or "nonribosomal peptide synthase" or "NRPS" refers to a polypeptide or series of interacting polypeptides that produces a nonribosomal peptide, thereby catalyzing peptide bond formation in the absence of ribosomal components.

[0034] "Polyketide synthase" (PKS) means a polypeptide or series of polypeptides that produce a polyketide. "Altering the amount" means altering by increasing or decreasing the amount. The increase or decrease can be 3%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0035] "Non-ribosomal peptide synthetase / polyketide synthase hybrid" or "non-ribosomal peptide synthetase and polyketide synthase hybrid" or "NRPS / PKS hybrid" or "NRPS and PKS hybrid" or "PKS and NRPS hybrid" means an enzyme system that includes any domains or modules from a non-ribosomal peptide synthetase and a polyketide synthase to produce the respective hybrid natural product.

[0036] By "altering the structure" is meant changing the chemical bonds (eg, covalent or non-covalent bonds) compared to a reference structure.

[0037] "Mutation" refers to a change in a nucleic acid sequence such that the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid change from the naturally occurring sequence. The mutation may be, but is not limited to, an insertion mutation, a deletion mutation, a frameshift mutation, or a missense mutation. The term also refers to the protein encoded by the mutated nucleic acid sequence.

[0038] "Variant" refers to a polypeptide or polynucleotide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to a reference sequence. Sequence identity is typically measured using sequence analysis software (e.g., sequence analysis software packages from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705, USA; programs: BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX). This type of software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications (substitution / scoring matrices: e.g., PAM, Blosum, GONET, JTT). Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. As an exemplary approach to determining the degree of identity, the BLAST program can be used, with probability scores between e-3 and e-150 indicating closely related sequences (Altschul et al., 1990).

[0039] In a first aspect, the present invention relates to a chimeric protein for producing nonribosomal peptides, comprising at least one chimeric thiolation domain (T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP))) directly flanked by a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment and a second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment is derived from the N-terminal portion of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence. and the second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment comprises an amino acid sequence derived from the C-terminal portion of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence, wherein the chimeric thiolation domain is a fully functional T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) characterized in that the amino acid sequences of the portions of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments are as follows: (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different species that are heterologous to each other, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different NRPS / PKS genes of the same species, or (c) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene.

[0040] In an alternative first aspect, the present invention relates to a method for generating a chimeric non-ribosomal peptide synthetase (NRPS) and / or polyketide synthase (PKS) module, the method comprising: (a) providing a first NRPS / PKS module sequence comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequence; (b) providing a second NRPS / PKS module sequence comprising at least a second T domain sequence; (c) optionally aligning the first T domain sequence to the second T domain sequence to identify at least one T domain fusion point; (d) fusing the first subsequence to the second subsequence by cloning them immediately adjacent to each other into a genetic construct to obtain a chimeric module sequence, (i) the first subsequence is a nucleotide sequence encoding the amino acid sequence of a first NRPS / PKS module sequence located directly N-terminal to the identified T domain fusion point; and (ii) the second subsequence is a nucleotide sequence encoding the amino acid sequence of a second NRPS / PKS module sequence located directly C-terminal to the identified T domain fusion point; and these steps generate genetic constructs containing chimeric NRPS and / or PKS modules.

[0041] The present invention is based on the surprising exploitation of a highly conserved site within the T domain, based on an evolutionary analysis of over 225 aligned amino acid sequences of NRPS ATC3 domains from various species, including Photorhabdus and Xenorhabdus species, as well as representative NRPSs from Firmicutes, actinomycetes, cyanobacteria, and other proteobacteria. This analysis identified a conserved FFxxGGxS motif within the T domain, which forms the basis for generating the chimeric proteins of the present invention. This sequence motif within the NRPS sequence of the T domain is surprisingly effective as a fusion point for combining NRPS sequences that are not naturally combined within a single gene (thus, such combinations are chimeric and artificial).

[0042] In a preferred embodiment of the invention, a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment is directly adjacent to and connected to the N-terminus of a second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment.

[0043] In another preferred embodiment, the chimeric protein is a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0044] According to the present invention, the chimeric protein of the present invention may further comprise one or more known NRPS and / or PKS domains, in particular an adenylation (A) domain, a condensation (C) domain, an epimerization (E) domain, a reductase (R) domain, a C / E domain, an oxidase (Ox) domain, a reduction (Red) domain, a heterocyclization (Cy) domain, a starter C domain, a methylation (Mt) domain, an A-Mt domain, an A-Ox domain, a communication (Com) domain, a formylation (F) domain, an X (X) domain, and / or a terminal thioesterase (TE) domain or a terminal C domain; and / or a PKS domain. It may be preferable to include any one or any combination of acyltransferase (AT) domains, ketosynthase (KS) domains, dehydratase (DH) domains, ketoreductase (KR) domains, enoylreductase (ER) domains, methyltransferase (Mt) O- or C-domains, docking (DD) domains, thioesterase (TE) domains, PLP-dependent cysteine lyase (SH) domains, and acyl carrier proteins (ACPs). NRPSs, fungi, and bacteria, or related domains and domain structures, are well known to those skilled in the art.

[0045] In another embodiment, the chimeric T domain preferably consists of an N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence and a C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence.

[0046] The chimeric protein of the present invention is preferably one in which the amino acid sequence of an N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) and the amino acid sequence of a C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) are fused together at a fusion site, and the fusion site is preferably located + / - 5 amino acids around fusion sites 1 to 7, preferably around fusion sites 3 and 4, as shown in Figure 3 of the present application.

[0047] A further embodiment of the invention relates to chimeric proteins in which the fusion site is immediately before or after any amino acid position of the conserved T domain motif (FFxxGG(H / N / D)S, where x is any amino acid).

[0048] In a further embodiment of section (a) of the first aspect, the protein further comprises at least one NRPS / PKS domain N-terminal to the chimeric T domain derived from a first microbial species and at least one NRPS / PKS domain C-terminal to the chimeric T domain derived from a second microbial species, wherein the first microbial species and the second microbial species are not identical.

[0049] In another preferred embodiment, the chimeric protein comprises a sequence of an NRPS / PKS domain capable of catalyzing the synthesis of a peptide and / or hybrid peptide-polyketide.

[0050] In a preferred embodiment, for example in the context of the first alternative aspect, the first and second NRPS / PKS module sequences are not identical, and preferably the first and second T domain sequences are not identical.

[0051] In another preferred embodiment, for example in the context of the first alternative aspect, the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are derived from the same NRPS / PKS gene but are not immediately adjacent to each other.

[0052] In yet another preferred embodiment, for example in the context of the first alternative aspect, the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are derived from two different NRPS / PKS genes, preferably from two different bacterial and / or fungal species.

[0053] For example, in the context of the first alternative aspect, preferably the T domain comprises both a T domain linker sequence and a T domain protein domain sequence.

[0054] For example, in the context of the first alternative embodiment, the T domain fusion point may preferably be selected to be located at one of the following positions in the aligned sequence, based on the T domain structure including from N- to C-terminus: T domain linker domain, helix 1, loop 1, helix 2, loop 2, helix 3, helix 4: (a) within the T-domain linker domain or at the N-terminus of the T-domain linker domain; (b) in the loop 1 domain; (c) in helix 2; (d) C-terminus of helix 4.

[0055] In an alternative first embodiment, the method further comprises expressing the chimeric NRPS and / or PKS module from a genetic construct.

[0056] In a second aspect, the present invention relates to a system for generating a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, comprising: At least two system units: (a) a first system unit comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment as described in any one of the other aspects and embodiments of this aspect; and (b) a second system unit comprising at least a second T domain segment as described in any one of the other aspects and embodiments of this aspect; A system comprising: Each system unit may contain one or more additional NRPS / PKS domains, and the chimeric nonribosomal peptide synthetase (NRPS), the chimeric polyketide synthase (PKS), or the chimeric NRPS / PKS hybrid is obtained by fusing a first system unit with a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the first system unit is directly fused to the amino acid sequence of the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the second system unit.

[0057] In a preferred embodiment, the sequences of the first system unit and the second system unit comprise amino acid sequences of portions of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments, these are, (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different species that are heterologous to each other, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different NRPS / PKS genes of the same species, or (c) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene; is.

[0058] In a third aspect, the present invention relates to a method for producing a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, the method comprising the steps of: (a) providing a first NRPS or PKS gene sequence from a first microbial species; (b) providing a second NRPS or PKS gene sequence from a second microbial species; and (c) combining at least a portion of the first NRPS or PKS gene sequence with at least a portion of the second NRPS or PKS gene sequence to obtain a chimeric gene sequence by fusing the first and second sequences to each other such that at least one fusion point is located within the T domain, wherein the fusion product comprises a T domain consisting of the N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and the C-terminal sequence of the T domain of the second NRPS or PKS gene sequence; and (d) optionally, expressing the chimeric gene sequence to obtain a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0059] In a fourth aspect, the present invention relates to a method for combining or generating a combination of at least two NRPS / PKS modules from two different NRPS / PKS genes or from two separate locations within the same NRPS / PKS gene, the method comprising the steps of: (a) identifying a first target NRPS / PKS module to be directly linked N-terminally to a second target NRPS / PKS module; (b) identifying a second target NRPS / PKS module to be directly linked C-terminally to a second target NRPS / PKS module, the second target NRPS / PKS module being selected from modules located within an NRPS / PKS gene C-terminal to a third, non-target NRPS / PKS module; (c) combining the first target NRPS / PKS module with a second target NRPS / PKS module by directly linking the N-terminal portion of the T domain of the first NRPS / PKS module with the C-terminal portion of the T domain of a third, non-target NRPS / PKS module; Here, the combination of the N-terminal portion of the T domain of the first NRPS / PKS module and the C-terminal portion of the T domain of the third, non-target NRPS / PKS module constitutes a complete and functional T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)).

[0060] In a preferred embodiment of the invention, the first and third NRPS / PKS modules each comprise at least one thiolation domain.

[0061] Also preferably, the binding may involve one or more additional NRPS / PKS modules and / or domains at the N-terminus of the first target NRPS / PKS module.

[0062] Alternatively or additionally, the binding may involve one or more further NRPS / PKS modules and / or a domain at the C-terminus of the second target NRPS / PKS module.

[0063] In a further preferred embodiment, the first target NRPS / PKS module and the third non-target NRPS / PKS module are not identical.

[0064] In a fifth aspect, the present invention relates to a combination product obtainable by the method of the fourth aspect.

[0065] In a sixth aspect, the present invention relates to a method for generating a nucleic acid construct encoding a system unit described in other aspects and embodiments of this aspect, the method comprising the step of amplifying a sequence stretch of an NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, the hybridization site comprising a sequence encoding the amino acid consensus motif (T domain motif) set forth in SEQ ID NO: XY (FFxxGG(H / N / D)S, where x is any amino acid).

[0066] In a seventh aspect, the present invention relates to a method for generating a library of nucleic acid constructs each encoding a system unit as described in other aspects and in embodiments of this aspect, the method comprising the step of amplifying a first sequence stretch of the NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, the hybridization site comprising a sequence encoding the amino acid consensus motif (T domain motif) set forth in SEQ ID NO: XY(FFxxGG(H / N / D)S), and the method comprising a second amplification of at least a second sequence stretch of the NRPS / PKS gene using a degenerate primer pair derived from the same or one or more different NRPS / PKS genes.

[0067] NRPS / PKS. In an eighth aspect, the present invention relates to a system of genetic constructs for the expression of chimeric NRPS / PKSs, the system comprising at least one genetic construct comprising a chimeric NRPS and / or PKS module produced by the method of the previous aspect.

[0068] In particular, the particular aspects described are preferred in the following embodiments and items:

[0069] Item 1. A chimeric protein for producing nonribosomal peptides, comprising at least one chimeric thiolation domain (T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP))) consisting of a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment immediately adjacent to a second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment, the first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment comprises an amino acid sequence derived from the N-terminal portion of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence, and the second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment comprises an amino acid sequence derived from the C-terminal portion of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence, and the chimeric thiolation domain is a fully functional T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)), wherein the amino acid sequences of the portions of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments are as follows: (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different species that are heterologous to each other, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different NRPS / PKS genes of the same species, or (c) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene; A chimeric protein characterized by:

[0070] Item 2. The chimeric protein of Item 1, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment is directly adjacent to and connected to the N-terminus of the second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment.

[0071] Item 3. The chimeric protein according to item 1 or 2, wherein the chimeric protein is a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0072] Item 4. One or more known NRPS and / or PKS domains, in particular an adenylation (A) domain, a condensation (C) domain, an epimerization (E) domain, a reductase (R) domain, a C / E domain, an oxidase (Ox) domain, a reduction (Red) domain, a heterocyclization (Cy) domain, a starter C domain, a methylation (Mt) domain, an A-Mt domain, an A-Ox domain, a communication (Com) domain, a formylation (F) domain, an X (X) domain, and / or a terminal thioesterase (TE) domain or a terminal C domain; and / or a PKS domain 4. The chimeric protein according to any one of Items 1 to 3, further comprising an acyltransferase (AT) domain, a ketosynthase (KS) domain, a dehydratase (DH) domain, a ketoreductase (KR) domain, an enoylreductase (ER) domain, a methyltransferase (Mt) O- or C-domain, a docking (DD) domain, a thioesterase (TE) domain, a PLP-dependent cysteine lyase (SH) domain, or an acyl carrier protein (ACP).

[0073] Item 5. The chimeric protein according to any one of Items 1 to 4, wherein the chimeric T domain consists of an N-terminal T domain (a PKS acyl carrier protein (ACP) or an NRPS petidyl carrier (PCP)) amino acid sequence and a C-terminal T domain (a PKS acyl carrier protein (ACP) or an NRPS petidyl carrier (PCP)) amino acid sequence.

[0074] Item 6. The chimeric protein according to any one of Items 1 to 5, wherein the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) and the amino acid sequence of the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) are fused together at a fusion site, and the fusion site is located around + / - 5 amino acids of fusion sites 1 to 7 shown in Figure 3, preferably fusion sites 3 and 4.

[0075] Item 7. The chimeric protein according to Item 6, wherein the fusion site is located immediately before or after any amino acid position of the conserved T domain motif (FFxxGG(H / N / D)S, where x is any amino acid).

[0076] Item 8. The chimeric protein according to any one of Items 1 to 7, wherein in (a) of Item 1, the protein further comprises at least one NRPS / PKS domain on the N-terminal side of the chimeric T domain derived from a first microbial species and at least one NRPS / PKS domain on the C-terminal side of the chimeric T domain derived from a second microbial species, wherein the first microbial species and the second microbial species are not identical.

[0077] Item 9. The chimeric protein according to any one of Items 1 to 8, wherein the chimeric protein comprises a sequence of an NRPS / PKS domain capable of catalyzing the synthesis of a peptide and / or a hybrid peptide-polyketide.

[0078] Item 10. At least two system units: (a) a first system unit comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment according to any one of items 1 to 9; and (b) a second system unit including at least a second T domain segment according to any one of items 1 to 9; 1. A system for producing a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, comprising: Each system unit may contain one or more additional NRPS / PKS domains, and the chimeric nonribosomal peptide synthetase (NRPS), the chimeric polyketide synthase (PKS), or the chimeric NRPS / PKS hybrid is obtained by fusing a first system unit with a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the first system unit is directly fused to the amino acid sequence of the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the second system unit.

[0079] Item 11. The sequences of the first system unit and the second system unit comprise amino acid sequences of portions of the T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments, these are, (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different species that are heterologous to each other, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences from different NRPS / PKS genes of the same species, or (c) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene; Item 11. The system according to item 10,

[0080] Item 12. A method for producing a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, comprising: The method comprises the steps of: (a) providing a first NRPS or PKS gene sequence from a first microbial species; (b) providing a second NRPS or PKS gene sequence from a second microbial species; and (c) combining at least a portion of the first NRPS or PKS gene sequence with at least a portion of the second NRPS or PKS gene sequence to obtain a chimeric gene sequence by fusing the first and second sequences to each other such that at least one fusion point is located within the T domain, wherein the fusion product comprises a T domain consisting of the N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and the C-terminal sequence of the T domain of the second NRPS or PKS gene sequence; and (d) optionally, expressing the chimeric gene sequence to obtain a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid; A method comprising:

[0081] Item 13. A method for combining or generating a combination of at least two NRPS / PKS modules from two different NRPS / PKS genes or from two separate locations within the same NRPS / PKS gene, the method comprising the steps of: (a) identifying a first target NRPS / PKS module to be directly linked N-terminally to a second target NRPS / PKS module; (b) identifying a second target NRPS / PKS module to be directly linked C-terminally to a second target NRPS / PKS module, the second target NRPS / PKS module being selected from modules located within an NRPS / PKS gene C-terminally to a third, non-target NRPS / PKS module; (c) combining the first target NRPS / PKS module with a second target NRPS / PKS module by directly linking the N-terminal portion of the T domain of the first NRPS / PKS module with the C-terminal portion of the T domain of a third, non-target NRPS / PKS module; and wherein the combination of the N-terminal portion of the T domain of the first NRPS / PKS module and the C-terminal portion of the T domain of the third non-target NRPS / PKS module constitutes a complete and functional T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)).

[0082] Item 14. The method of Item 13, wherein the first and third NRPS / PKS modules each contain at least one thiolation domain.

[0083] Item 15. The method of items 13 or 14, wherein binding may involve one or more additional NRPS / PKS modules and / or domains N-terminal to the first target NRPS / PKS module.

[0084] Item 16. The method of any one of Items 13 to 15, wherein binding may involve one or more additional NRPS / PKS modules and / or a domain C-terminal to the second target NRPS / PKS module.

[0085] Item 17. The method of any one of Items 13 to 15, wherein the first target NRPS / PKS module and the third non-target NRPS / PKS module are not identical.

[0086] Item 18. A combination product obtained by the method according to any one of Items 13 to 17.

[0087] Item 19. A method for generating a nucleic acid construct encoding the system unit according to Item 10, comprising: The method comprises amplifying a sequence stretch of an NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, The hybridization site comprises a sequence encoding the amino acid consensus motif (T domain motif) set forth in SEQ ID NO: XY (FFxxGG(H / N / D)S, where x is any amino acid).

[0088] Item 20. A method for generating a library of nucleic acid constructs encoding the system units according to Item 10, comprising: The method comprises amplifying a first sequence stretch of an NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, The hybridization site comprises a sequence encoding the amino acid consensus motif (T domain motif) set forth in SEQ ID NO: XY (FFxxGG(H / N / D)S), and a second amplification of at least a second sequence stretch of the NRPS / PKS gene using a degenerate primer pair derived from the same or one or more different NRPS / PKS genes.

[0089] Item 21. A method for producing chimeric nonribosomal peptide synthetase (NRPS) and / or polyketide synthase (PKS) modules, comprising: The method comprises: (a) providing a first NRPS / PKS module sequence comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequence; (b) providing a second NRPS / PKS module sequence comprising at least a second T domain sequence; (c) optionally aligning the first T domain sequence to the second T domain sequence to identify at least one T domain fusion point; (d) fusing the first subsequence to the second subsequence by cloning them immediately adjacent to each other into a genetic construct to obtain a chimeric module sequence, (i) the first subsequence is a nucleotide sequence encoding the amino acid sequence of a first NRPS / PKS module sequence located directly N-terminal to the identified T domain fusion point; and (ii) the second subsequence is a nucleotide sequence encoding the amino acid sequence of a second NRPS / PKS module sequence located directly C-terminal to the identified T domain fusion point; and and a method wherein these steps produce a genetic construct comprising a chimeric NRPS and / or PKS module.

[0090] Item 22. The method of Item 21, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are not identical, and preferably, the first T domain sequence and the second T domain sequence are not identical.

[0091] Item 23. The method of Items 21 or 22, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are derived from the same NRPS / PKS gene but are not immediately adjacent to each other.

[0092] Item 24. The method of Item 21 or 22, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are derived from two different NRPS / PKS genes, preferably from two different bacterial and / or fungal species.

[0093] Item 25. The method of any one of Items 21 to 24, wherein the T domain comprises both a T domain linker sequence and a T domain protein domain sequence.

[0094] Item 26. The T domain fusion point is located at the following position in the aligned sequence based on the T domain structure including the N-terminus to C-terminus: T domain linker domain, helix 1, loop 1, helix 2, loop 2, helix 3, and helix 4: (a) within the T-domain linker domain or at the N-terminus of the T-domain linker domain; (b) in the loop 1 domain; (c) in helix 2; (d) C-terminus of helix 4; 26. The method according to any one of items 21 to 25, wherein the amino acid sequence is selected to be located at any one of the positions.

[0095] Item 27. The method of any one of items 21 to 26, further comprising expressing the chimeric NRPS and / or PKS module from the genetic construct.

[0096] Item 28. A system of genetic constructs for expression of a chimeric NRPS / PKS, the system comprising at least one genetic construct containing a chimeric NRPS and / or PKS module produced by the method of any one of Items 21 to 27.

[0097] As used herein, the terms "of the invention," "according to the invention," "in accordance with the invention," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0098] As used herein, the term "comprises" is interpreted as encompassing both "comprises" and "consisting of," both meanings being specifically intended and, therefore, separately disclosed embodiments in accordance with the present invention. As used herein, "and / or" is interpreted as specifically disclosing each of the two specified features or components, with or without the other. For example, "A and / or B" is deemed to specifically disclose (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein. In the context of the present invention, the terms "about" and "approximately" indicate a range of accuracy within which a person skilled in the art would understand that the technical effect of the feature in question is still guaranteed. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g., ±5% from the indicated numerical value. As will be understood by those skilled in the art, the specific deviation regarding the numerical value of a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects are generally more likely to have a deviation than artificial or engineered technical effects. As will be understood by those skilled in the art, the specific deviation regarding the numerical value of a given technical effect depends on the nature of the technical effect. For example, natural or biological technological effects are generally subject to greater deviations than man-made or engineered technological effects. Where an indefinite or definite article is used when referring to a singular noun (e.g., "a", "an", "the"), the plural of that noun is also included unless otherwise specified.

[0099] It will be understood that adapting the teachings of the present invention to a particular problem or environment, and incorporating variations or additional features of the present invention (such as further aspects and embodiments), will be within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.

[0100] Unless the context requires otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0101] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety. [Example]

[0102] Certain aspects and embodiments of the present invention are described, by way of example, with reference to the description, figures, and tables set forth herein. Such examples of methods, uses, and other aspects of the invention are merely representative and should not be construed as limiting the scope of the invention to only such representative examples.

[0103] Background: Thiolated domain (T domain)

[0104] The T domain is the only NRPS domain that does not have autonomous catalytic activity and is responsible for transporting substrates and elongation intermediates to the catalytic center. 26 The approximately 100 AA long protein belongs to the acyl carrier protein (ACP)-like superfamily and is located C-terminal to the A domain and N-terminal to the C domain (Figure 5B).

[0105] The A domain catalyzes two half-reactions 27 In the second half-reaction, the activated (adenylated) substrate is transferred to the terminal cysteamine thiol group of the 4'Ppan cofactor covalently bound to the T domain. The T domain, together with its 4'Ppan cofactor, represents a holoprotein derived from the apocarrier protein by post-translational modification. 28 A phosphopantetheinyl, approximately 20 Å long, prosthetic group derived from coenzyme A (CoA) with a terminal thiol is covalently linked via a phosphodiester to a strictly conserved serine side chain surrounded by a homologous pattern of AA residues in the folded apocarrier protein. 29-33 The transfer of 4'Ppan to the apoprotein is achieved by the action of NRPS-specific 4'-phosphopantetheinyltransferases (PPTases), which are often encoded as part of NRPS biosynthetic gene clusters (e.g., Sfp in B. subtilis) (Figure 1). 34-37 .

[0106] The first T domain structure was elucidated by Weber et al. using NMR spectroscopy of the T domain of the third module of B. brevis tyrocidine synthetase (Figure 1). 38 Weber et al. showed that the T domain is a distorted four-helix bundle with an extended loop between the first two helices, and that the invariant serine residue that binds the cofactor is located at the interface between this loop and the second helix. Furthermore, no binding pocket is found within the T domain, consistent with a lack of substrate selectivity. Furthermore, the prosthetic group does not interact with the protein and is accommodated in solvent. In terms of functionality and structural fold, the T domain is similar to the acyl carrier proteins (ACPs) of fatty acid synthases and PKSs. 26 However, the sequence homology between them exists only in the immediate vicinity of the invariant serine residue. 39-41 The most obvious difference between them is the overall charge of the proteins. The surface of ACPs has predominantly acidic side chains, while the T domain is less polar. This finding corresponds to the charge of related 4'Ppan transferases (PPtases such as AcpS and Sfp). Furthermore, crystal structures of the carrier proteins (as excised domains, as independent single proteins, and upon recognition by PPtases such as Sfp from B. subtilis and AcpS from Streptomyces coelicolor or Streptococcus pneumoniae) reveal that Sfp has one V-shaped interface for 4'Ppan cofactor modification in the T domain, whereas AcpS has three catalytic sites for post-translational modification. 28,38,39,42-50 .

[0107] The AT didomain is defined as the initiation module because both proteins are required to activate and covalently bind the first substrate for subsequent peptide assembly. 26 During nonribosomal synthesis, T domains transfer reactive intermediates between different catalytic centers, adopting alternative conformations in the process.42 The crystal structure of the surfactin AC (Srf AC) termination module (Figure 12) solved by Tanovic et al. (2008) has made a major contribution to our understanding of the various domain arrangements during the catalytic cycle. 51 In this structure, the PCP domain was positioned to interact with the condensation domain. However, the binding sites of the 4'Ppan arm of the T domain were 60 and 45 A from the active sites of the A and TE domains, respectively, suggesting that a large conformational rearrangement is required to deliver pantethethiol to the other domains. The T domain must adopt at least three functionally relevant, distinct orientations. In the first conformation, binding to the A domain allows aminoacyl transfer to the thiol group of the 4'Ppan. The remaining two orientations within the extender module are those in which the T domain approaches the catalytic center of the preceding C domain in the acceptor site and the next C domain in the donor site.

[0108] While evolutionary recombination analysis (Figure 2) naturally does not reveal specific splicing sites, there are regions (sequence stretches) that may promote homologous recombination. Therefore, a fusion point screen was first performed (Figure 3) to confirm whether the T domain could be targeted to generate chimeric NRPSs and to identify fusion sites that would result in optimal peptide production. Briefly, this screen identified three functional fusion sites (Figure 3).

[0109] In particular, fusion sites 3 and 4 are located within a region with very interesting features (I)-(III).

[0110] (I) First, they are located in close proximity to an invariant serine residue that serves as the 4′Ppan binding site (Fig. 1 ) and are surrounded by a conserved pattern of AA residues (FFxxGG(H / D)S), homologous to a region of PKS ACP domains.

[0111] (II) Second, at the nucleotide level, a further remarkable feature is observed: a region spanning about 50 nucleotides is not only AT-rich and conserved among various unrelated bacterial species, even within GC-rich organisms such as Streptomyces, but also shows unequal frequencies of four DNA bases on both single strands of each DNA molecule. This mathematically unequal distribution is called AT skew. 52,53 This is called a "genomic skew" and not only represents a statistical bias, but more importantly, a genetic and codon stability that appears to be resistant to mutations: translocations are significantly underrepresented statistically, leading to a predominance of AT skew, and point mutations are unlikely to alter the AA encoded at this site.

[0112] (III) Third, both T-domain portions located upstream (N-terminus) and downstream (C-terminus) of the aforementioned conserved AA stretch (Fig. 2d) show different clustering when performing phylogenetic reconstruction. The N-terminal portions of the T-domains cluster like their respective upstream A-domains, whereas the C-terminal portions of the T-domains cluster like their neighboring C-domains. This behavior is also observed when performing phylogenetic reconstruction (Fig. 2a) and tree topology analysis (Fig. 2b). 24 , indicating that this region represents a potential evolutionary recombination site that has not yet been noticed.

[0113] Taken together, these in silico observations (Fig. 2) and the results of in vivo fusion point screening (Fig. 3) lead us to hypothesize that the TCA unit (fusion point 1), the partial TCAT / partial (pT-CA-Tp) unit (fusion points 3 and 4), and their combinations ((1,3), (1,4), (3,4), (4,3), (4,1), and (3,1)) may be ideal starting points for evolutionarily inspired megasynth(et)ase engineering (Fig. 2e).

[0114] An example is given as follows:

[0115] Example 1: Proof of Concept

[0116] This example demonstrates that the NRPS extension (NRPS-1) and starter units (NRPS-2 and -3) are functionally interchangeable via fusion point 3 (Fig. 3).

[0117] Prior to this work, we identified the GameX Peptide (Gxp) AE-producing synthetase (GxpS). 3,54 We created an S-type mutant of this artificial / synthetic two-component NRPS (Figure 4) to confirm that fusion point 3 could indeed be applied to the creation of new Gxp derivatives. Targeting the GxpS domains T3 and T4 enabled us to functionally exchange the leucine-specifying component pT3-C4-A4-Tp4 of GxpS with the arginine-specifying component pT3-C4-A4-Tp4 of the bicornucoplast-producing NRPS (BicA) from X. budapestensis (Figure 4). HPLC-MS / MS analysis of methanolic culture extracts detected five arginine-containing peptides (1-5, Figure 4). Peptides 1 and 2 are the result of the unpaired activity of NRPS-1 subunit 2, while peptides 3-5 represent the predicted full-length Gxp derivatives. Due to the loose substrate specificity of A1 (Val, Leu) and A3 (Phe, Leu), peptides 3-5 differ at positions 1 and 3. Furthermore, NRPS-2 and NRPS-3 are homologous to odilorhabdin from X. nematophila HGB081, respectively. 55 and xenoamicin III from X. stockiae 56 Synthetic NRPSs were created by substituting GxpS A3-Tp3 of subunit 2 of S-type GxpS for two different start components (Cstart-A-Tp) (Figure 4). Both NRPSs exhibited biosynthetic activity, synthesizing lipopeptides 6–12. NRPS-3 produced only the predicted lipopeptides (10–12), whereas NRPS-2 also synthesized two C-terminally truncated versions of lipopeptide 6.

[0118] Example 2: De novo design of a chimeric NRPS that produces lipopeptide aldehydes

[0119] In this example (NRPS-4 to NRPS-8), fusion point 3 was utilized to exchange the termination unit and insert a reductase (R) domain at the end. 57,58 We demonstrate how to create a synthetic NRPS with the R domain (Figure 5). The R domain not only releases the synthesized peptide and regenerates the NRPS machinery, but also introduces a functional / reactive group (warhead) into the synthesized peptide by catalyzing the NAD(P)H-dependent two-electron reduction of a thioester to an aldehyde, which can then be further reduced to an alcohol. 59 Peptide aldehydes are often associated with protease inhibitors, for example, in the reversible binding of ferutamide B to the active site threonine of the Mycobacterium tuberculosis proteasome. 60 .

[0120] However, inspired by the potent proteasome inhibitor ferutamide B, we combined components of up to five different NRPSs from four different Photorhabdus and Xenorhabdus strains (Figure 5). The resulting chimeric assembly line enzymes, NRPS-4 through NRPS-8, were produced in E. coli DH10B::mtaA, and culture extracts were analyzed by HPLC-MS / MS. All of the NRPSs produced demonstrated catalytic activity to produce the desired lipopeptide aldehydes 13 through 19.

[0121] When tested against the yeast proteasome, compounds 15 and 17 indeed showed the expected inhibitory activity (low pM range).

[0122] Example 3: Conversion of an NRPS into an artificial NRPS / PKS hybrid synthetase

[0123] NRPS-PKS-9 (Fig. 6) and PKS-NRPS-10 (Fig. 7) were generated to examine whether NRPS and PKS components could be artificially fused using identified fusion sites 3 and 1, respectively, to generate catalytically active NRPS / PKS hybrid synthetases.

[0124] Therefore, we again utilized the S-type GxpS3 model system to convert an NRPS into an artificial NRPS-PKS hybrid synthetase (Figure 6), which produces glidobactin. 61 This was achieved by removing the GxpS termination unit (pT4-C / E5-A5-TE) from pT4 and replacing it with a PKS termination unit (pT3-KS-AT-DH-KR-ACP-TE). Briefly, the resulting chimeric NRPS-PKS-9 synthesized six distinct peptides (20–25). Peptides 20–23 lacked the desired terminal ketide group and were synthesized by the sole activity of the NRPS moiety, whereas peptides 24 and 25 were the desired full-length hybrid NRPS-PKS products.

[0125] PKS-NRPS-10 is a further example demonstrating how an NRPS can be converted into an NRPS-PKS hybrid (Figure 7). In contrast to NRPS-PKS-9, here, fusion point 1 incorporates the Cstart-A1 initiation unit of an unpublished cyclic lipopeptide-producing NRPS (locus tag XINNV2_12405) from X. innexii DSM16336, which is expressed in Myxococcus xanthus DK1622. 63 Myxochromid of origin 62 The resulting assembly line synthesized the desired PK-NRP hybrid product 26, even though both starter units were derived from unrelated organisms with very different GC contents (Xenorhabdus sp.: ∼44%, Myxococcus sp.: ∼70%).

[0126] Example 4: Creation of chimeric NRPSs by recombining unrelated components that differ in GC content and codon usage

[0127] NRPS-PKS-9 and PKS-NRPS-10 not only demonstrate for the first time that naturally occurring NRPS proteins can be functionally converted into NRPS-PKS hybrids, but also show that PKS-NRPS-10 can combine unrelated components with significantly different GC content with each other.

[0128] To further investigate and characterize the novel fusion points defined by the ELS method, we generated chimeric proteins NRPS-11 to NRPS-24 (Figures 9 to 11). The purpose was to clone the unpublished WT-NRPS-1 (X. innexii DSM16336, locus tag XINNV2_12405, Figure 9) into Pseudomonas sp. MYb11, respectively. 70 Massetolide A from Serratia sp. SCBI, S. marcescens DSM 12481, X. bovienii SS-2004, X. sp. KK7.4, X. doucetiae DSM 17909, X. sp. PB61.4, and B. subtilis 168 64 , Serrawettin W2 65 , Serrawettin W1 66 , Xefoampeptide 67 , unpublished NRPS (locus tag XEKKV2_12060), protegomycin 56 , Chaiyaphumin 68 , and Plipastatin 69 Furthermore, each reprogrammed NRPS was designed and generated with two variants, applying both fusion sites 1 and 4. All resulting reprogrammed NRPSs exhibited biocatalytic activity with both variants (fusion sites 1 and 4) and produced the desired cyclic lipopeptides (27-33).

[0129] Conclusion:

[0130] We were able to confirm that all of the evolutionary ideas and in silico-detected fusion points (1, 3, 4; Figure 3) could be applied in vivo to engineer and de novo generate synthetic NRPSs and NRPS-PKS hybrids. Interestingly, all reprogrammed NRPSs and NRPS-PKS hybrids exhibited good to very good production titers, comparable to or even exceeding WT production levels.

[0131] Overall, splicing positions 1, 3, and 4 can all be used to generate functionally novel biosynthetic pathways. The major strength of the EIS method described above appears to be its versatility, suggesting that recombination within the T domain is not only an effective method for reprogramming NRPSs in the laboratory for synthetic biology purposes, but that nature may actually use the T domain as a recombination point to insert and delete components from existing BGCs or to recombine two BGCs by intragenomic homologous recombination. However, all state-of-the-art methods 1-3,71 Compared to previous work, we were able to demonstrate for the first time that building blocks can be functionally produced in E. coli to synthesize novel peptides and peptide-polyketide hybrids, regardless of their origin, preferred codon usage, or GC content of the source strain. Finally, compared to the total number of characterized extender units in all NRPSs and PKS9s, each bacterial species encodes BGCs using only a very limited range of extender units. Thus, the EIS method can expand the chemically accessible space beyond the limits set by nature.

[0132] Example 5: Generation of a library encoding modules

[0133] The library construction strategy is shown in Figure 3. T domains from diverse organisms, such as Xenorhabdus, Pseudomonas, and Burkholderia, are not highly conserved across species. However, when the T domain and the subsequent C or CE domain encoded by a single organism are compiled and aligned (Figure 3A), a conserved sequence spanning 10–12 amino acids can be observed. The compiled T domain and the subsequent E domain from the genus Xenorhabdus show distinct conserved sequence patches with an even higher degree of sequence identity. By analyzing the DNA sequence underlying this conserved amino acid patch, we can design degenerate primers (Figure 3B) that can potentially target and amplify 70–90% of the T domains in the more than 50 T domain-containing organisms for which we have evaluated this method (e.g., Pseudomonas, Xenorhabdus, Burkholderia, Mycetohabitans, etc.).

[0134] Using a degenerate primer set in a PCR reaction using genomic DNA (gDNA) as a template, a library of DNA fragments encoding modules can be obtained. This library is then ligated into an entry vector. Ligation is performed using a dephosphorylated vector and 5'-phosphorylated PCR product. The entry vector encodes the homology arms required for the Gibson cloning step into an NRPS (cloning based on a stretch of homologous DNA region) in the following steps (see Figure 3E and Figure 3F). The first PCR module library can be ligated into any entry vector flanked by any homology arms. To clone the module library into an NRPS context, a second PCR must be performed. The primer set for this PCR binds to the homology arms and overlaps the insert by 3–5 base pairs. Due to the overlap between the primer pair and the ligated insert, only "correctly" ligated inserts are amplified (see Figure 3E). The Gibson cloning step (Figure 3F) inserts the module library into the selected NRPS biosynthetic site. The resulting clones can be used for production culture in E. coli or other suitable prokaryotic organisms. The resulting nonribosomal peptides incorporate a variety of amino acids at the insert site of the modular library (Figure 3G). In this example, it is the third amino acid. It is important to note that the module inserted at the third position defines the stereochemistry of the amino acid at the second position, thereby increasing the number of peptides that can be generated.

[0135] References

[0136] References include: 1. Bozhuyuk, KAJ et al. De novo design and engineering of non-ribosomalpeptide synthetases. Nat Chem 10, 275-281 , doi:10.1038 / nchem.2890 (2018). 2 Bozhuyuk, K. A. J. et al. Modification and de novo design of non-ribosomalpeptide synthetases using specific assembly points within condensation domains.Nat Chem II , 653-661 , doi:10.1038 / s41557-019-0276-z (2019). 3 Bozhuyuk, K., Watzel, J., Abbood, N. & Bode, H. B. Synthetic Zippers asan Enabling Tool for Engineering of Non-Ribosomal Peptide Synthetases. AngewChem Int Ed Engl, doi:10.1002 / anie.202102859 (2021). 4 Fierro, F. et al. Transcriptional and bioinformatic analysis of the 56.8 kbDNA region amplified in tandem repeats containing the penicillin gene clusterin Penicillium chrysogenum. Fungal Genet Biol 43, 618-629,doi:10.1016 / j.fgb.2006.03.001 (2006). 5 Li, J., Kim, S. G. & Blenis, J. Rapamycin: one drug, many effects. CellMetab 19, 373-379, doi:10.1016 / j.cmet.2014.01.001 (2014). 6 Murray, V., Chen, J. K. & Chung, L. H. The Interaction of theMetallo-Glycopeptide Anti-Tumour Drug Bleomycin with DNA. Int J Mol Sci 19, doi:10.3390 / ijms19051372(2018). 7 Sussmuth, R. D. & Mainz, A. Nonribosomal Peptide Synthesis-Principles andProspects. Angew Chem Int Ed Engl 56, 3770-3821 , doi:10.1002 / anie.201609079 (2017). 8 Nivina, A., Yuet, K. P, Hsu, J. & Khosla, C. Evolution and Diversity ofAssembly- Line Polyketide Synthases. Chem Rev 119, 12524-12547,doi:10.1021 / acs.chemrev.9b00525 (2019). 9 Flissi, A. et al. Norine: update of the nonribosomal peptide resource.Nucleic Acids Res 48, D465-D469, doi:10.1093 / nar / gkz1000 (2020). 10 Caboche, S., Leclere, V., Pupin, M., Kucherov, G. & Jacques, P.Diversity of monomers in nonribosomal peptides: towards the prediction oforigin and biological activity. J Bacteriol 192, 5143-5150,doi:10.1128 / JB.00315-10 (2010). 11 Walsh, C. T., O'Brien, R. V. & Khosla, C. Nonproteinogenic amino acidbuilding blocks for nonribosomal peptide and hybrid polyketide scaffolds. AngewChem Int Ed Engl 52, 7098-7124, doi:10.1002 / anie.201208344 (2013). 12 Ray, L. & Moore, B. S. Recent advances in the biosynthesis of unusualpolyketide synthase substrates. Nat Prod Rep 33, 150-161,doi:10.1039 / c5np00112a (2016). 13 Blin, K. et al. antiSMASH 4.0-improvements in chemistry prediction and genecluster boundary identification. NucleicAcids Res 45, W36-W41, doi:10.1093 / nar / gkx319(2017). 14 Skinnider, M. A., Merwin, N. J., Johnston, C. W & Magarvey, N. A. PRISM3: expanded prediction of natural product chemical structures from microbialgenomes. NucleicAcids Res 45, W49-W54, doi:10.1093 / nar / gkx320 (2017). 15 Bozhuyuk, K. A., Micklefield, J. & Wilkinson, B. Engineering enzymaticassembly lines to produce new antibiotics. Curr Opin Microbiol 51, 88-96,doi:10.1016 / j.mib.2019.10.007 (2019). 16 Dutta, S. et al. Structure of a modular polyketide synthase. Nature 510,512-517, doi:10.1038 / nature13423 (2014). 17 Whicher, J. R. et al. Structural rearrangements of a polyketide synthasemodule during its catalytic cycle. Nature 510, 560-564, doi:10.1038 / nature13409(2014). 18 Drake, E. J. et al. Structures of two distinct conformations ofholo-non-ribosomal peptide synthetases. Nature 529, 235-238,doi:10.1038 / nature16163 (2016). 19 Miller, B. R., Drake, E. J., Shi, C., Aldrich, C. C. & Gulick, A. M.Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-likeProteins Support a Highly Dynamic Domain Architecture. J Biol Chem 291,22559-22571, doi: 10.1074 / jbc.M 116.746297 (2016). 20 Jenke-Kodama, H. & Dittmann, E. Evolution of metabolic diversity:insights from microbial polyketide synthases. Phytochemistry 70, 1858-1866,doi:10.1016 / j.phytochem.2009.05.021 (2009). 21 Zhang, L. et al. Characterization of Giant Modular PKSs Provides Insightinto Genetic Mechanism for Structural Diversification of AminopolyolPolyketides. Angew Chem Int Ed Engl 56, 1740-1745, doi:10.1002 / anie.201611371(2017). 22 Wlodek, A. et al. Diversity oriented biosynthesis via accelerated evolutionof modular gene clusters. Nat Commun 8, 1206, doi: 10.1038 / s41467-017-01344-3(2017). 23 Baunach, M., Chowdhury, S., Stallforth, P. & Dittmann, E. The Landscapeof Recombination Events That Create Nonribosomal Peptide Diversity. Mol BiolEvol 38, 2116-2130, doi:10.1093 / molbev / msab015 (2021). 24 Minin, V. N., Dorman, K. S., Fang, F. & Suchard, M. A. Dual multiplechange-point model leads to more accurate recombination detection.Bioinformatics 21, 3034-3042, doi:10.1093 / bioinformatics / bti459 (2005). 25 Martin, D. P, Murrell, B., Golden, M., Khoosal, A. & Muhire, B. RDP4:Detection and analysis of recombination patterns in virus genomes. Virus Evol 1,vev003, doi:10.1093 / ve / vev003 (2015). 26 Sieber, S. A. & Marahiel, M. A. Molecular mechanisms underlyingnonribosomal peptide synthesis: approaches to new antibiotics. Chem Rev 105,715-738, doi:10.1021 / cr0301191 (2005). 27 Beer, R. et al. Creating functional engineered variants of the single-modulenonribosomal peptide synthetase IndC by T domain exchange. Mol Biosyst 10,1709-1718, doi:10.1039 / c3mb70594c (2014). 28 Koglin, A. & Walsh, C. T. Structural insights into nonribosomal peptideenzymatic assembly lines. Nat Prod Rep 26, 987-1000, doi:10.1039 / b904543k(2009). 29 Zhou, Z., Lai, J. R. & Walsh, C. T. Directed evolution of aryl carrierproteins in the enterobactin synthetase. Proc Natl Acad Sci U SA 104,11621-11626, doi: 10.1073 / pnas.0705122104 (2007) . 30 Finking, R. & Marahiel, M. A. Biosynthesis of nonribosomal peptides!Annu Rev Microbiol 58, 453-488, doi:10.1146 / annurev.micro.58.030603.123615(2004). 31 Lai, J. R., Koglin, A. & Walsh, C. T. Carrier protein structure andrecognition in polyketide and nonribosomal peptide biosynthesis. Biochemistry45, 14869-14879, doi:10.1021 / bi061979p (2006). 32 Mofid, M. R., Finking, R., Essen, L. O. & Marahiel, M. A.Structure-based mutational analysis of the 4'-phosphopantetheinyl transferasesSfp from Bacillus subtilis: carrier protein recognition and reaction mechanism.Biochemistry 43, 4128-4136, doi:10.1021 / bi036013h (2004). 33 Mofid, M. R., Finking, R. & Marahiel, M. A. Recognition of hybridpeptidyl carrier proteins / acyl carrier proteins in nonribosomal peptidesynthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp. JBiol Chem 277, 17023-17031, doi: 10.1074 / jbc. M200120200 (2002). 34 Lambalot, R. H. et al. A new enzyme superfamily - the phosphopantetheinyltransferases. Chem Biol 3, 923-936, doi:10.1016 / s1074-5521(96)90181-7 (1996). 35 Walsh, C. T., Gehring, A. M., Weinreb, P. H., Quadri, L. E. & Flugel, R.S. Post-translational modification of polyketide and nonribosomal peptidesynthases. Curr Opin Chem Biol 1 , 309-315, doi:10.1016 / s1367-5931(97)80067-1 (1997). 36 Quadri, L. E. et al. Characterization of Sfp, a Bacillus subtilisphosphopantetheinyl transferase for peptidyl carrier protein domains in peptidesynthetases. Biochemistry 37, 1585-1595, doi: 10.1021 / bi9719861 (1998). 37 Reuter, K., Mofid, M. R., Marahiel, M. A. & Ficner, R. Crystal structureof the surfactin synthetase-activating enzyme sfp: a prototype of the4'-phosphopantetheinyl transferase superfamily. EMBO J 18, 6823-6831 , doi:10.1093 / emboj / 18.23.6823 (1999). 38 Weber, T., Baumgartner, R., Renner, C., Marahiel, M. A. & Holak, T. A.Solution structure of PCP, a prototype for the peptidyl carrier domains ofmodular peptide synthetases. Structure 8, 407-418, doi:10.1016 / s0969-2126(00)00120-9 (2000). 39 Holak, T. A., Kearsley, S. K., Kim, Y. & Prestegard, J. H.Three-dimensional structure of acyl carrier protein determined by NMRpseudoenergy and distance geometry calculations. Biochemistry 27, 6135-6142,doi:10.1021 / bi00416a046 (1988). 40 Parris, K. D. et al. Crystal structures of substrate binding to Bacillussubtilis holo-(acyl carrier protein) synthase reveal a novel trimericarrangement of molecules resulting in three active sites. Structure 8, 883-895,doi:10.1016 / s0969-2126(00)00178-7 (2000). 41 Crump, M. P. et al. Solution structure of the actinorhodin polyketidesynthase acyl carrier protein from Streptomyces coelicolor A3(2). Biochemistry36, 6000-6008, doi:10.1021 / bi970006+ (1997). 42 Koglin, A. et al. Conformational switches modulate protein interactions inpeptide antibiotic synthetases. Science 312, 273-276,doi:10.1126 / science.1122928 (2006). 43 Kim, Y, Ohlrogge, J. B. & Prestegard, J. H. Motional effects on NMRstructural data. Comparison of spinach and Escherichia coli acyl carrierproteins. Biochem Pharmacol 40, 7-13, doi: 10.1016 / 0006-2952(90)90171 -g(1990). 44 Zornetzer, G. A., Fox, B. G. & Markley, J. L. Solution structures ofspinach acyl carrier protein with decanoate and stearate. Biochemistry 45,5217-5227, doi:10.1021 / bi052062d (2006). 45 Alekseyev, V. Y, Liu, C. W, Cane, D. E., Puglisi, J. D. & Khosla, C.Solution structure and proposed domain domain recognition interface of an acylcarrier protein domain from a modular polyketide synthase. Protein Sci 16,2093-2107, doi:10.1110 / ps.073011407 (2007). 46 Weber, T. et al. antiSMASH 3.0-a comprehensive resource for the genomemining of biosynthetic gene clusters. Nucleic Acids Res 43, W237-243,doi:10.1093 / nar / gkv437 (2015). 47 Findlow, S. C., Winsor, C., Simpson, T. J., Crosby, J. & Crump, M. P.Solution structure and dynamics of oxytetracycline polyketide synthase acylcarrier protein from Streptomyces rimosus. Biochemistry 42, 8423-8433, doi:10.1021 / bi0342259 (2003). 48 Johnson, M. A., Peti, W, Herrmann, T., Wilson, I. A. & Wuthrich, K.Solution structure of Asl1650, an acyl carrier protein from Anabaena sp. PCC7120 with a variant phosphopantetheinylation-site sequence. Protein Sci 15, 1030-1041, doi:10.1110 / ps.051964606 (2006). 49 Sharma, A. K., Sharma, S. K., Surolia, A., Surolia, N. & Sarma, S. P. Solutionstructures of conformationally equilibrium forms of holo-acyl carrier protein(PfACP) from Plasmodium falciparum provides insight into the mechanism ofactivation of ACPs. Biochemistry 45, 6904-6916, doi:10.1021 / bi060368u (2006). 50 Kim, Y. & Prestegard, J. H. A dynamic model for the structure of acylcarrier protein in solution. Biochemistry 28, 8792-8797,doi:10.1021 / bi00448a017 (1989). 51 Tanovic, A., Samel, S. A., Essen, L. O. & Marahiel, M. A. Crystalstructure of the termination module of a nonribosomal peptide synthetase.Science 321, 659-663, doi: 10.1126 / science.1159850 (2008). 52 Grigoriev, A. Analyzing genomes with cumulative skew diagrams. Nucleic AcidsRes 26, 2286-2290, doi: 10.1093 / nar / 26.10.2286 (1998). 53 Lobry, J. R. Asymmetric substitution patterns in the two DNA strands ofbacteria. Mol Biol Evol 13, 660-665, doi:10.1093 / oxfordjournals.molbev.a025626(1996). 54 Nollmann, F. I. et al. Insect-specific production of new GameXPeptides inphotorhabdus luminescens TTO1, widespread natural products in entomopathogenicbacteria. Chembiochem 16, 205-208, doi:10.1002 / cbic.201402603 (2015). 55 Pantel, L. et al. Odilorhabdins, Antibacterial Agents that Cause Miscodingby Binding at a New Ribosomal Site. Mol Cell 70, 83-94 e87, doi:10.1016 / j.molcel.2018.03.001(2018). 56 Behsaz, B. et al. Integrating genomics and metabolomics for scalablenon-ribosomal peptide discovery. Nat Commun 12, 3225,doi:10.1038 / s41467-021-23502-4 (2021). 57 Du, L. & Lou, L. PKS and NRPS release mechanisms. Nat Prod Rep 27,255-278, doi:10.1039 / b912037h (2010). 58 Tietze, A., Shi, Y. N., Kronenwerth, M. & Bode, H. B. NonribosomalPeptides Produced by Minimal and Engineered Synthetases with Terminal ReductaseDomains. Chembiochem 21 , 2750-2754, doi:10.1002 / cbic.202000176 (2020). 59 Chhabra, A. et al. Nonprocessive [2 + 2]e- off-loading reductase domainsfrom mycobacterial nonribosomal peptide synthetases. Proc Natl Acad Sci II S A109, 5681- 5686, doi: 10.1073 / pnas.1118680109 (2012). 60 Lin, G., Li, D., Chidawanyika, T., Nathan, C. & Li, H. Fellutamide B isa potent inhibitor of the Mycobacterium tuberculosis proteasome. Arch BiochemBiophys 501, 214-220, doi:10.1016 / j.abb.2010.06.009 (2010). 61 Oka, M. et al. Chemical modification of the antitumor antibioticglidobactin. J Antibiot (Tokyo) 41 , 1812-1822, doi:10.7164 / antibiotics.41.1812(1988). 62 Wenzel, S. C. et al. Structure and biosynthesis of myxochromides S1-3 inStigmatella aurantiaca: evidence for an iterative bacterial type I polyketidesynthase and for module skipping in nonribosomal peptide biosynthesis.Chembiochem 6, 375-385, doi:10.1002 / cbic.200400282 (2005). 63 Krug, D. et al. Discovering the hidden secondary metabolome of Myxococcusxanthus: a study of intraspecific diversity. Appl Environ Microbiol 74, 3058-3068, doi: 10.1128 / AEM.02863-07 (2008). 64 de Bruijn, I., de Kock, M. J., de Waard, P, van Beek, T. A. &Raaijmakers, J. M. Massetolide A biosynthesis in Pseudomonas fluorescens. JBacteriol 190, 2777-2789, doi:10.1128 / JB.01563-07 (2008). 65 Petersen, L. M., LaCourse, K., Schoner, T. A., Bode, H. & Tisa, L. S.Inactivation of the Major Hemolysin Gene Influences Expression of theNonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp.Strain SCBI. J Bacteriol 199, doi:10.1128 / JB.00333-17 (2017). 66 Li, H., Tanikawa, T., Sato, Y, Nakagawa, Y. & Matsuyama, T. Serratiamarcescens gene required for surfactant serrawettin W1 production encodesputative aminolipid synthetase belonging to nonribosomal peptide synthetasefamily. Microbiol Immunol 49, 303-310, doi: 10.1111 / j.1348-0421,2005.tb03734.x (2005). 67 Kegler, C. & Bode, H. B. Artificial Splitting of a Non-Ribosomal PeptideSynthetase by Inserting Natural Docking Domains. Angew Chem Int Ed Engl 59,13463-13467, doi:10.1002 / anie.201915989 (2020). 68 Grundmann, F. et al. Antiparasitic chaiyaphumines from entomopathogenicXenorhabdus sp. PB61.4. J Nat Prod 77, 779-783, doi:10.1021 / np4007525 (2014). 69 Batool, M., Khalid, M. H., Hassan, M. N. & Fauzia Yusuf, H. Homologymodeling of an antifungal metabolite plipastatin synthase from the Bacillussubtilis 168. Bioinformation 7, 384-387, doi: 10.6026 / 97320630007384 (2011). 70 Kissoyan, KAB et al. Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. CurrBiol 29, 1030-1037 e1035, doi:10.1016 / j.cub.2019.01.050 (2019). 71 Calcott, MJ, Owen, JG & Ackerley, DF Efficient rational modification of non-ribosomal peptides by adenylation domain substitution. NatCommun 11, 4554, doi: 10.1038 / S41467-020- 18365-0 (2020). 72 Zhou, Q. et al. Structure and biosynthesis of xenoamicins from entomopathogenic Xenorhabdus. Chemistry 19, 16772-16779, doi:10.1002 / chem.201302481 (2013).

[0137] Drawing Terminology Figure 1 Coenzyme A Thiolation domain Figure 2 Area of recombination A and A-subdomains move independently TCA-domains or T / 2-CAT / 2 move together Figure 3 Helix-1 Loop-1 Helix-2 Loop-2 Helix-3 Helix-4 Figure 4 Products domain Synthetic zipper Peptide Figure 5 Products Figure 6 Products Ketosynthase Acyltransferase Dehydratase Ketoreductase Figure 7 Products Figure 8 T-domain linker T-domain Consensus Figure 9 Products unpublished Protegomycin Chaiyaphumin Chaiyaphumin Figure 10 Products unpublished locus tag Figure 11 Products unpublished Figure 12 T-domain Alignment Conserved Sequence Patch Degenerate primer design Degenerate primer PCR using gDNA template Creating module coding library R can be L- or D- amino acid, proteinogenic or non-proteinogenic amino acid Peptide product with diverse function atposition 3 and L- and D-stereochemistry of amino acid 2 Ligation PCR product Entry vector Homology arms of choice Second round PCR wrong orientation correct orientation e.g.

Claims

1. A chimeric protein for generating a non-ribosomal peptide, comprising at least one chimeric thiolated domain (T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP))) consisting of a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment directly adjacent to a second T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment, The first T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment includes an amino acid sequence derived from the N-terminal portion of the T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence, and the second T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment includes an amino acid sequence derived from the C-terminal portion of the T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence, and the chimeric thiolated domain is a fully functional T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)), The amino acid sequences of the T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) portion of the first and second T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments are as follows: (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from different species that are heterogeneous, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from different NRPS / PKS genes of the same species, (c) T domain sequences (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene, A chimeric protein characterized by the following:

2. The chimeric protein according to claim 1, wherein the first T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment is directly adjacent to and connected to the N-terminus of the second T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment.

3. The chimeric protein according to claim 1 or 2, wherein the chimeric protein is a chimeric non-ribosomal peptide synthase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

4. One or more known NRPS and / or PKS domains, particularly adenylation (A) domains, condensation (C) domains, epimerization (E) domains, reductase (R) domains, C / E domains, oxidase (Ox) domains, reduction (Red) domains, heterocyclization (Cy) domains, starter C (C) domains, methylation (Mt) domains, A-Mt domains, A-Ox domains, communication (Com) domains, formylation (F) domains, X (X) domains, and / or terminal thioesterase (TE) domains or terminal C domains; and / or PKS domains. The chimeric protein according to claim 1, further comprising an acyltransferase (AT) domain, a ketosynthase (KS) domain, a dehydratase (DH) domain, a ketoreductase (KR) domain, an enoyl reductase (ER) domain, a methyltransferase (Mt) O- or C- domain, a docking (DD) domain, a thioesterase (TE) domain, a PLP-dependent cysteine ​​lyase (SH) domain, and an acyl carrier protein (ACP).

5. The chimeric protein according to claim 1, wherein the chimeric T domain consists of an N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence and a C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequence.

6. The chimeric protein according to claim 1, wherein the amino acid sequence of an N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) and the amino acid sequence of a C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) are fused together at a fusion site, and the fusion site is located around the + / - 5 amino acids of fusion sites 1 to 7 shown in Figure 3, or is fusion sites 3 and 4.

7. The chimeric protein according to claim 6, wherein the fusion site is located immediately before or after any amino acid position of a conserved T domain motif (FFxxGG(H / N / D)S, where x is any amino acid).

8. The chimeric protein according to claim 1(a), wherein the protein further comprises at least one NRPS / PKS domain at the N-terminal end of a chimeric T domain derived from a first microbial species and at least one NRPS / PKS domain at the C-terminal end of a chimeric T domain derived from a second microbial species, wherein the first microbial species and the second microbial species are not the same.

9. The chimeric protein according to claim 1, comprising a sequence of NRPS / PKS domains capable of catalyzing the synthesis of peptides and / or hybrid peptide-polyketides.

10. At least two system units: (a) A first system unit comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segment as described in claim 1, and (b) A second system unit comprising at least the second T-domain segment described in claim 1, A system for generating chimeric non-ribosomal peptide synthase (NRPS), chimeric polyketide synthase (PKS), or chimeric NRPS / PKS hybrids, including Each system unit may comprise one or more further NRPS / PKS domains, and the chimeric non-ribosomal peptide synthetase (NRPS), the chimeric polyketide synthase (PKS), or the chimeric NRPS / PKS hybrid is obtained by fusing a first system unit with a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the first system unit is directly fused with the amino acid sequence of the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the second system unit.

11. The sequences of the first and second system units include the amino acid sequences of the portions of the T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) amino acid sequences of the first and second T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) segments, these are, (a) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from different species that are heterogeneous, or (b) T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) sequences derived from different NRPS / PKS genes of the same species, (c) T domain sequences (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)) of the same NRPS / PKS gene, The system according to claim 10.

12. A method for generating chimeric non-ribosomal peptide synthase (NRPS), chimeric polyketide synthase (PKS), or chimeric NRPS / PKS hybrids, The method involves the following steps: (a) A step of providing a first NRPS or PKS gene sequence derived from a first microbial species, (b) A step of providing a second NRPS or PKS gene sequence derived from a second microbial species, and (c) A step of combining at least a portion of a first NRPS or PKS gene sequence and at least a portion of a second NRPS or PKS gene sequence in order to obtain a chimeric gene sequence by fusing the first and second sequences together such that at least one fusion site is located within a T domain, wherein the fusion product includes a T domain consisting of the N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and the C-terminal sequence of the T domain of the second NRPS or PKS gene sequence, and (d) A step of expressing a chimeric gene sequence in order to obtain a chimeric non-ribosomal peptide synthase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid. Methods that include...

13. A method for combining or generating a combination of at least two NRPS / PKS modules from two different NRPS / PKS genes, or from two different locations within the same NRPS / PKS gene, the method comprising the following steps: (a) A step of identifying a first target NRPS / PKS module to be directly bound to the N-terminus of a second target NRPS / PKS module, (b) A step of identifying a second target NRPS / PKS module to be directly bound to the C-terminal side of a second target NRPS / PKS module, wherein the second target NRPS / PKS module is selected from modules located within the C-terminal NRPS / PKS gene relative to a third non-target NRPS / PKS module. (c) A step of combining the first target NRPS / PKS module with the second target NRPS / PKS module by directly binding the N-terminal portion of the T-domain of the first NRPS / PKS module to the C-terminal portion of the T-domain of the third non-target NRPS / PKS module. It includes, Herein, the combination of the N-terminal portion of the T-domain of the first NRPS / PKS module and the C-terminal portion of the T-domain of the third non-targeted NRPS / PKS module constitutes a complete and functional T-domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP)), in this method.

14. The method according to claim 13, wherein the first and third NRPS / PKS modules each include at least one thiolated domain.

15. The method according to claim 13 or 14, wherein the binding may involve one or more further NRPS / PKS modules and / or a domain located on the N-terminal side of a first target NRPS / PKS module.

16. The method according to claim 13 or 14, wherein the binding may involve one or more further NRPS / PKS modules and / or a domain located on the C-terminal side of a second target NRPS / PKS module.

17. The method according to claim 13 or 14, wherein the first targeted NRPS / PKS module and the third non-targeted NRPS / PKS module are not identical.

18. A combination product obtained or obtainable by the method of claim 13 or 14.

19. A method for generating a nucleic acid construct that encodes the system unit described in claim 10, The method includes a step of amplifying the sequence stretch of the NRPS / PKS gene using a degenerate primer pair that hybridizes under stringent conditions to at least 10 nucleic acids of the hybridization site within the NRPS / PKS gene, A method wherein the hybridization site includes a sequence encoding an amino acid consensus motif (T domain motif) represented by sequence number XY (FFxxGG(H / N / D)S, where x is any amino acid).

20. A method for generating a library of nucleic acid constructs that encode the system unit described in claim 10, The method includes the step of amplifying a first sequence stretch of the NRPS / PKS gene using a pair of degenerate primers that hybridize under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, The hybridization site includes a sequence encoding the amino acid consensus motif (T domain motif) shown in sequence number XY (FFxxGG(H / N / D)S), And, The method comprises a second amplification of at least a second sequence stretch of an NRPS / PKS gene using a pair of degenerate primers derived from the same or one or more different NRPS / PKS genes.

21. A method for producing chimeric non-ribosomal peptide synthase (NRPS) and / or polyketide synthase (PKS) modules, This method is (a) Providing a first NRPS / PKS module sequence comprising at least a first T domain (PKS acyl carrier protein (ACP) or NRPS petidyl carrier (PCP) sequence), (b) A step of providing a second NRPS / PKS module sequence comprising at least a second T domain sequence, (c) Aligning a first T-domain sequence with a second T-domain sequence in order to identify at least one T-domain fusion site, (d) A step of fusing a first partial sequence with a second partial sequence within a gene construct by cloning them so that they are directly adjacent to each other, in order to obtain a chimeric module sequence, (i) The first subsequence is a nucleotide sequence encoding the amino acid sequence of a first NRPS / PKS module sequence located directly on the N-terminal side of the identified T-domain fusion site, and (ii) The second subsequence is a nucleotide sequence that encodes the amino acid sequence of a second NRPS / PKS module sequence located directly on the C-terminal side of the identified T-domain fusion site, It includes, Furthermore, a method comprising these steps for generating a gene construct containing chimeric NRPS and / or PKS modules.

22. The method according to claim 21, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are not identical, and the first T-domain sequence and the second T-domain sequence are not identical.

23. The method according to claim 21 or 22, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence originate from the same NRPS / PKS gene but are not directly adjacent to each other.

24. The method according to claim 21 or 22, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are derived from two different bacterial and / or fungal species, which are derived from two different NRPS / PKS genes.

25. The method according to claim 21, wherein the T domain includes both a T domain linker sequence and a T domain protein domain sequence.

26. The T-domain fusion point is located at the following positions in the aligned sequence relative to the T-domain structure, which includes the N-terminus to the C-terminus: T-domain linker domain, helix 1, loop 1, helix 2, loop 2, helix 3, and helix 4: (a) Within the T-domain linker domain or at the N-terminus of the T-domain linker domain, (b) Within the Loop 1 domain, (c) Inside Helix 2, (d) C-terminus of helix 4, The method according to claim 21, wherein the position is selected to be located at any one of the following positions.

27. The method according to claim 21, further comprising expressing a chimeric NRPS and / or PKS module from a gene construct.

28. A system of gene constructs for the expression of chimeric NRPS / PKS, the system comprising at least one gene construct containing a chimeric NRPS and / or PKS module produced by the method of claim 21.

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