Methods and means for engineering of non-ribosomal peptides

Abstract

The present invention relates to strategies for combining elements of non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) into functional enzymes to promote the production of peptides and polyketide of any sequence. The present invention is based on the use of conserved sequence motifs within the T domain (or T domain-like) gene of the NRPS / PKS, which is used according to the invention as fusion sites for the production of new (and artificial) NRPS / PKS enzymes that can produce any peptide or polyketones of interest. The present invention provides artificial NRPS / PKS of the invention, methods and means for generating them, and nucleic acid libraries encoding various modules useful for designing NRPS / PKS for any peptide sequence of interest.

Description

Field of the Invention

[0001] The present invention relates to strategies for combining elements of non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS) into functional enzymes to 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 NRPS / PKS, which are used according to the present invention as fusion sites for producing new (and artificial) NRPS / PKS enzymes that can produce any peptide or polyketide of interest. The present invention provides artificial NRPS / PKS of the present invention, methods and means for their production, and nucleic acid libraries encoding various modules that can be used to design NRPS / PKS for any peptide sequence of interest.

[0002] illustrate

[0003] The present invention relates to a system for producing and expressing chimeric nonribosomal peptide synthetases (NRPS), polyketide synthetases (PKS) or NRPS / PKS hybrid enzymes. NRPS, PKS or their hybrids are large multifunctional modular multidomain proteins or complexes that produce a variety of clinically relevant scaffolds. Their expression and modification are used for peptide production and product modification, respectively, and there are many difficulties. The present invention relates to a novel evolution-inspired strategy (EIS) for functional recombination, expression and production of artificial nonribosomal peptides (NRPs), polyketides (PKs) and their hybrids. The EIS method utilizes computer-detected and in vivo-validated splicing positions, which can both rationally modify target biosynthetic gene clusters (BGCs) and assemble new artificial / synthetic BGCs from scratch. The present invention discloses the specifically defined splicing positions, protein fragments and recombination strategies of this molecular synthetic biology assembly kit, as well as their code nucleic acids. Also disclosed are the cloning strategies of the nucleotide fragments of the present invention and their use in preparing functional NRPS / PKS enzymes to produce novel bioactive NRP / PK scaffold libraries.

[0004] Reprogramming biosynthetic assembly lines (NRPSs, PKSs, NRPS / PKS hybrids) has generated intense interest. This is not surprising, as the scaffolds of many antibiotics (penicillin 4), immunosuppressants (rapamycin 5), and anticancer therapeutics (bleomycin 6) in clinical use are generated via this pathway.

[0005] In essence, the NRPS7 and PKS8 modular megasynthetases generate highly functionalized biopolymers from a variety of monomers, called elongators. Hundreds of elongators have been reported9, typically derived from amino acids in the case of NRPSs10,11 and from malonate in the case of PKSs12. Due to their hierarchical and modular structure, they have been likened to assembly line processes. Multiple repeating modules of enzyme domains catalyze the incorporation of elongators into the growing chain along with any programmed additional chemical modifications, and then transfer the elongated chain to the next module. A typical minimal assembly line module consists of three “core” domains. First, a domain for selecting and activating elongators, either the adenylation (A) domain of NRPSs or the acyltransferase (AT) domain of PKSs. The activated substrate is then covalently attached to a pseudophosphopantetheine group of a small peptide carrier protein (PCP; NRPS), also known as the thiolation (T) domain or the acyl carrier protein (ACP; PKS) domain. Finally, condensation (C; NRPS) domains or ketosynthase (KS; PKS) enzymes link covalently bound substrates to growing peptide or polyketide chains. Despite their modular nature, which provides a direct relationship between enzyme domain sequence and product chemical structure13,14, past efforts to rationally reprogram them have met with limited success15.

[0006] Since 1995, the study of NRPSs has become a focus, when Marahiel et al. (WO200052152) demonstrated that NRPSs could be reprogrammed by exchanging the adenylation-thiolation dual domains (Marahiel et al., 1995). In the past 20 years, there have been many attempts 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 conferred by AA in the catalytic center (Conti et al. 1997, Stachelhaus et al. 1999). Using this code for conferring specificity (called the Stachelhaus code), it has been possible to predict and change the substrate specificity of the A domain in vitro (Khurana et al., 2010; Rausch et al., 2005; et al., 2011; Kries et al., 2014). The most obvious drawback of this attempt is that it is not applicable in vivo. A major reason for this drawback is that the C and TE domains are also selective, leading to substrate incompatibility (Belshaw et al., 1999; Traugel et al., 2000; Tseng et al., 2002).

[0007] Another attempt to change the known NRPS biosynthetic cluster (WO200130985, Marahiel et al.) is based on the exchange of single domains, double domains or entire modules, and the knowledge of the precisely defined boundaries (joints) between the various domains. With this invention, it is only possible to successfully change several NRPSs by introducing additional modules or deleting them. However, it is impossible to produce completely artificial NRPSs from artificial de novo combinations of modules or domains. This will produce new NRPSs that do not exist in nature, and will also produce new peptides. The problem with this exchange or combination is always the uncertainty of the compatibility between modules and / or domains. Due to the lack of a solution to the above-mentioned problems, very few peptide derivatives have been designed using this method, which illustrates the shortcomings of this method.

[0008] Another attempt to alter known NRPS biosynthetic clusters (WO2007014076, Walsh et al.) is based on mutagenesis of the so-called "assembly line", i.e., synthases. NRPS gene mutagenesis is not the subject of the present invention, although the method of the present invention can be combined with mutagenesis that changes the NRPS produced and causes changes in peptide synthesis. Such mutagenesis can help increase the diversity of NRPS libraries and the number of NRPS clones in the library.

[0009] It is therefore an object of the present invention to provide alternative methods for the design and expression of artificial NRPS / PKS that facilitate peptide and polyketide synthesis.

[0010] Brief description of the invention

[0011] In general, by way of brief description, the main aspects of the present invention can be described as follows:

[0012] In a first aspect, the present invention relates to a chimeric protein for producing a non-ribosomal peptide, comprising at least one chimeric thiolation domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)), which is composed of a first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment directly adjacent to a second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment comprises An amino acid sequence containing an N-terminal portion of an amino acid sequence derived from a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)), and wherein the second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment contains an amino acid sequence derived from the C-terminal portion of an amino acid sequence of a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)); wherein the chimeric thiol domain is a fully functional T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)); characterized in that:

[0013] The amino acid sequences of portions of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments are:

[0014] (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or

[0015] (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or

[0016] (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

[0017] In another first aspect, the present invention also relates to a method for producing a chimeric non-ribosomal peptide synthetase (NRPS) and / or polyketide (PKS) module, the method comprising:

[0018] (a) providing a first NRPS / PKS module sequence, which comprises at least a first T domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence);

[0019] (b) providing a second NRPS / PKS module sequence, which comprises at least a second T domain sequence;

[0020] (c) optionally, aligning the first T domain sequence with the second T domain sequence to identify at least one T domain fusion site;

[0021] (d) by cloning the first partial sequence and the second partial sequence directly adjacent to each other into a genetic construct to fuse them to obtain a chimeric modular sequence, wherein

[0022] (i) the first partial sequence is a nucleotide sequence encoding the amino acid sequence of the first NRPS / PKS module sequence directly located at the N-terminus of the identified T domain fusion site; and

[0023] (ii) the second partial sequence is a nucleotide sequence encoding the amino acid sequence of the second NRPS / PKS module sequence directly located at the C-terminus of the identified T domain fusion site;

[0024] Genetic constructs comprising chimeric NRPS and / or PKS modules are thereby generated.

[0025] In a second 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 at least two system units:

[0026] (a) a first system unit comprising at least the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment as described in any of the other aspects and embodiments of these aspects, and

[0027] (b) a second system unit comprising at least the second T domain fragment as described in any of the other aspects and embodiments of these aspects;

[0028] Each system unit may contain one or more additional NRPS / PKS domains, and a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS) or a chimeric NRPS / PKS hybrid may be obtained by fusing a first and a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl 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 peptidyl carrier (PCP)) of the second system unit.

[0029] 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:

[0030] (a) providing a first NRPS or PKS gene sequence derived from a first microbial species,

[0031] (b) providing a second NRPS or PKS gene sequence derived from a second microbial species, and

[0032] (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 two sequences to each other, such that at least one fusion site is located within the T domain, and wherein the fusion product comprises a T domain consisting of an N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and a C-terminal sequence of the T domain of the second NRPS or PKS gene sequence, and

[0033] (d) Optionally, a chimeric gene sequence is expressed to obtain a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0034] In a fourth aspect, the present invention relates to a method for combining or generating a combination of at least two NRPS / PKS modules derived from two different NRPS / PKS genes or from two separate locations within the same NRPS / PKS gene, the method comprising the following steps:

[0035] (a) identifying a first target NRPS / PKS module that will directly bind to a second target NRPS / PKS module at the N-terminus;

[0036] (b) identifying a second target NRPS / PKS module that will directly bind to the second target NRPS / PKS module at the C-terminus, and wherein the second target NRPS / PKS module is selected from a module located at the C-terminus of a third non-target NRPS / PKS module within the NRPS / PKS gene;

[0037] (C) combining the first targeted NRPS / PKS module with the second targeted NRPS / PKS module by directly attaching the N-terminal portion of the T domain of the first NRPS / PKS module to the C-terminal portion of the T domain of a third non-target NRPS / PKS module;

[0038] 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 functional T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)).

[0039] In a fifth aspect, the present invention relates to a combination product obtained by the method of the fourth aspect.

[0040] In the sixth aspect, the present invention relates to a method for producing a nucleic acid construct, which encodes the system unit described in other aspects and embodiments of this aspect, and the method includes the step of amplifying a sequence fragment of the NRPS / PKS gene using a degenerate primer pair, wherein the degenerate primer pair hybridizes with at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene under stringent conditions, wherein the hybridization site contains a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO:XY:FFxxGG(H / N / D)S, wherein x is any amino acid.

[0041] In the seventh aspect, the present invention relates to a method for producing a library of nucleic acid constructs, each nucleic acid construct encoding a system unit as described in other aspects and embodiments of this aspect, wherein the method comprises the step of amplifying a first sequence fragment of a NRPS / PKS gene using a degenerate primer pair, wherein the degenerate primer pair hybridizes under stringent conditions with at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, wherein the hybridization site comprises a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO:XY:FFxxGG(H / N / D)S; and wherein the method comprises a second amplification of at least a second sequence fragment of the NRPS / PKS gene using a degenerate primer pair derived from the same or one or more different NRPS / PKS genes.

[0042] In an eighth aspect, the present invention relates to a genetic construct system for expressing a chimeric NRPS / PKS, wherein the system comprises at least one genetic construct comprising a chimeric NRPS and / or PKS module produced by the method of the preceding aspects. DETAILED DESCRIPTION OF THE INVENTION

[0044] In the following, the elements of the present invention will be described. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce more embodiments. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments explicitly described. This specification should be understood to support and include embodiments that combine two or more explicitly described embodiments, or embodiments that combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. In addition, any arrangement and combination of all described elements in this application should be considered to be disclosed by the description of this application, unless the context otherwise indicates.

[0045] definition

[0046] The term "partial domain" or "partial C or C / E domain" or similar expressions shall refer to a nucleic acid sequence encoding an incomplete (non-full-length) NRPS C or C / E domain or its protein sequence. The term thus describes a C or C / E domain sequence that does not contain the donor and acceptor sites of the NRPS C or C / E domain.

[0047] An "assembly" refers to a group of domains. Multiple components include a NRPS. One or more polypeptides may comprise a module. The combination of modules may catalyze a series of reactions to form a larger molecule. In one example, a module may comprise a C (condensation) domain, an A (adenylation) domain, and a peptidyl carrier protein domain.

[0048] For more structural information on A domains, C domains, dual domains, domain-domain interfaces and complete modules, see Conti et al. (1997), Sundlov et al. (2013), Samuel et al. (2007), Tanovic et al. (2008), Strieker and Marahiel (2010), Mitchell et al. (2012) and Tan et al. (2015). Are these references from another patent? The list could be updated, e.g. to match Drake et al., Izore et al., Reimer et al., NRPS Review: Süssmuth & Mainz.

[0049] "Starting module" refers to the N-terminal module that can provide the first monomer to another module (for example, extension or termination module). In some cases, other modules are not the second, but any module (such as the case of Nocardicin (Nocardicin) NRPS) of the C-terminal follow-up: in the case of NRPS, the starting module comprises, for example, A (adenylation) domain and PCP (peptidyl carrier protein) or T (sulfhydration) domain. The starting module can also comprise initial C domain and / or E (epimerization) domain. In the case of PKS, possible starting modules comprise AT (acetyltransferase) domain and acyl carrier protein (ACP) domain. The starting module is preferably located at the amino terminal of the polypeptide of the first module of the assembly line, and each assembly line preferably comprises a starting module.

[0050] "Extension module" refers to a module that adds a monomer to another monomer or polymer. The extension module may include C (condensation), Cy (heterocyclization), E, ​​C / E, MT (methyltransferase), A-MT (adenylation and methylation binding domain), Ox (oxidase) or Re (reductase) domain; A domain; or T domain. The extension domain may further include additional E, Re, DH (dehydration), MT, NMet (N-methylation), AMT (transaminase) or Cy domains. In addition, the extension module may be PKS-derived, comprising various domains (ketosynthase (KS) domain, acyltransferase (AT) domain, ketoreductase (KR) domain, dehydratase (DH) domain, enoylreductase (ER) domain, acyl carrier protein (ACP) domain or sulfhydration (T) domain) connecting amino acid building blocks and carboxylic acid building blocks.

[0051] "Termination module" refers to a module that releases a molecule (e.g., NRP, PK, or a combination thereof) from an assembly line. The molecule can be released by, for example, hydrolysis or cyclization. The termination module may include a TE (thioesterase), a C term Or Re domain. The termination module is preferably located at the carboxyl terminus of the NRPS or PKS polypeptide. The termination module may further comprise additional enzymatic activity (e.g., oligomerase activity).

[0052] "Domain" refers to a polypeptide sequence or a fragment of a larger polypeptide sequence that has one or more specific enzymatic activities (i.e., a C / E domain has C and E functions in one domain) or another conserved function (i.e., a tethering function as an ACP or T domain). Thus, a single polypeptide can contain multiple domains. Multiple domains can form a module. Examples of domains include C (condensation), Cy (heterocyclization), A (adenylation), T (sulfhydration), 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).

[0053] "Non-ribosomally synthesized peptide", "non-ribosomal peptide" or "NRP" refers to any polypeptide that is not produced by the ribosome. NRPs can be linear, cyclized or branched and contain acyl chains? --> lipopeptides?, aldehydes?, amines?, proteinogenic, natural or unnatural amino acids or any combination thereof. NRPs include peptides produced in an assembly line-like manner (= modular features of the enzyme system, allowing step-wise addition of building blocks to form the final product).

[0054] "Polyketone" refers to a compound containing multiple keto→keto? units.

[0055] "Non-ribosomal peptide synthetase" or "non-ribosomal peptide synthetase" or "NRPS" refers to a polypeptide or a series of interacting polypeptides that produce non-ribosomal peptides and are therefore able to catalyze peptide bond formation in the absence of ribosomal components.

[0056] "Polyketide synthase" (PKS) refers to a polypeptide or a series of polypeptides that produce polyketides. "Altered amount" refers to the amount of change by increasing or decreasing. The increase or decrease can be 3%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

[0057] "Non-ribosomal peptide synthetase / polyketide synthase hybrid" or "hybrid of a non-ribosomal peptide synthetase and a polyketide synthase" or "NRPS / PKS hybrid" or "hybrid of an NRPS and PKS" or "hybrid of a PKS and an NRPS" refers to an enzyme system comprising any domain or module derived from a non-ribosomal peptide synthetase and a polyketide synthase, thereby producing the respective hybrid natural product.

[0058] "Altered structure" refers to any change in chemical bonding (eg, covalent or non-covalent bonding) compared to a reference structure.

[0059] "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 compared to the naturally occurring sequence. A mutation can be, but is not limited to, an insertion, a deletion, a frameshift mutation, or a missense mutation. The term also describes a protein encoded by a mutant nucleic acid sequence.

[0060] "Variant" refers to a polypeptide or polynucleotide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity with a reference sequence. Sequence identity is usually measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by applying a substitution / scoring matrix (e.g., PAM, Blosum, GONET, JTT) to assign homology to various substitutions, deletions and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where probability scores between e-3 and e-150 indicate closely related sequences (Altschul et al., 1990).

[0061] In a first aspect, the present invention relates to a chimeric protein for producing a non-ribosomal peptide, comprising at least one chimeric thiolation domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP))) consisting of a first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment directly adjacent to a second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment The fragment comprises an amino acid sequence derived from the N-terminal portion of the amino acid sequence of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)), and wherein the second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment comprises an amino acid sequence derived from the C-terminal portion of the amino acid sequence of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP); wherein the chimeric thiol domain is a fully functional T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)); characterized in that:

[0062] The amino acid sequences of the plurality of T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments are:

[0063] (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or

[0064] (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or

[0065] (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

[0066] In another first aspect, the present invention also relates to a method for producing a chimeric non-ribosomal peptide synthetase (NRPS)- and / or polyketide (PKS) module, the method comprising:

[0067] (a) providing a first NRPS / PKS module sequence, which comprises at least a first T domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence);

[0068] (b) providing a second NRPS / PKS module sequence, which comprises at least a second T domain sequence;

[0069] (c) optionally, aligning the first T domain sequence with the second T domain sequence to identify at least one T domain fusion site;

[0070] (d) by cloning the first partial sequence and the second partial sequence directly adjacent to each other into a genetic construct to fuse them to obtain a chimeric module sequence, wherein

[0071] (i) the first partial sequence is a nucleotide sequence encoding the amino acid sequence of the first NRPS / PKS module sequence directly located at the N-terminus of the identified T domain fusion site; and

[0072] (ii) the second partial sequence is a nucleotide sequence encoding the amino acid sequence of a second NRPS / PKS module sequence located directly at the C-terminus of the identified T domain fusion site;

[0073] Genetic constructs comprising chimeric NRPS and / or PKS modules are thereby generated.

[0074] The present invention is based on the amino acid sequences of the NRPS A-TC tridomains of more than 225 different species, including the amino acid sequences of Photorhabdus and Xenorhabdus species, as well as the evolutionary analysis of the amino acid sequences of representative NRPSs of the genera Firmicutes, Actinomycetes, Actinomycetes and other Proteobacteria, and surprisingly found highly conserved sites within the T domain. The analysis of the present invention identifies the conserved FFxxGGxS motif in the T domain, which forms the basis for the production of the chimeric protein of the present invention. This sequence motif in the NRPS sequence in the T domain is surprisingly effective as a fusion site for combining NRPS sequences that are not naturally combined in a single gene (so this combination is chimeric / artificial).

[0075] A preferred embodiment of the present invention is that the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment is directly adjacent to and connected to the N-terminus of the second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment.

[0076] 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.

[0077] According to the present invention, it may be further preferred that the chimeric protein of the present invention further comprises one or more known NRPS and / or PKS domains, in particular one or more adenylation (A) domains, one or more condensation (C) domains, one or more epimerization (E) domains, one or more reductase (R) domains, one or more C / E domains, one or more oxidation (Ox) domains, one or more reduction (Red) domains, one or more heterocyclization (Cy) domains, one or more initiation C domains, one or more methylation (Mt) domains, one or more A-Mt domains, one or more A-Ox domains, one or more communication (Com) domains, one or more mitochondrial (MIT ... any or any combination of a plurality of formylation (F) domains, one or more X (X) domains and / or a terminal thioesterase (TE) domain or one or more terminal C domains; and / or a PKS domain, one or more acyltransferase (AT) domains, one or more ketosynthase (KS) domains, one or more dehydratase (DH) domains, one or more ketoreductase (KR) domains, one or more enoylreductase (ER) domains, one or more methyltransferase (Mt) O- or C domains, one or more docking (DD) domains, one or more thioesterase (TE) domains, one or more PLP-dependent cysteine ​​lyase (SH) domains, acyl carrier protein (ACP). NRPS, fungi and bacteria or related domains and domain structures are well known to those skilled in the art.

[0078] In another embodiment, the chimeric T domain is preferably composed of an N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence and a C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence.

[0079] According to the present invention, a chimeric protein is preferred, wherein the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence and the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence are fused together at a fusion site, and wherein the fusion site is located such as Figure 3 Fusion sites 1 to 7 are shown at + / - 5 amino acids around them, with fusion sites 3 and 4 being preferred.

[0080] Another embodiment of the present invention is directed to a chimeric protein, wherein the fusion site is located directly before or after any amino acid position of the conserved T domain motif -FFxxGG(H / N / D)S-, wherein x is any amino acid.

[0081] In another embodiment of part (a) of the first aspect, the protein further comprises at least one NRPS / PKS domain located at the N-terminus of the chimeric T domain and derived from a first microbial species, and at least one NRPS / PKS domain located at the C-terminus of the chimeric T domain and derived from a second microbial species, with the proviso that the first and the second microbial species are different.

[0082] Another preferred embodiment is that the chimeric protein comprises a NRPS / PKS domain sequence capable of catalyzing the synthesis of peptides and / or hybrid peptide-polyketide.

[0083] Preferred embodiments, such as in the case of the first alternative aspect, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are not identical, preferably wherein the first T domain sequence and the second T domain sequence are not identical.

[0084] Another preferred embodiment, for example in the case of the first alternative aspect, is 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 directly adjacent to each other.

[0085] In another preferred embodiment, for example in the case of the first alternative aspect, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are derived from two different NRPS / PKS genes; preferably two different bacterial and / or fungal species.

[0086] In the case of, for example, the first alternative aspect, preferably, the T domain comprises a T domain linker sequence and a T domain protein domain sequence.

[0087] In the case of, for example, the first alternative aspect, preferably, the T domain fusion site is selected to be located at one of the following positions with reference to the aligned sequence of the T domain structure from N-terminus to C-terminus: T domain linker domain, Helix-1, Loop-1, Helix-2, Loop-2, Helix-3, Helix-4, wherein:

[0088] (a) within the T domain linker domain or at the N-terminus,

[0089] (b) within the Loop-1 domain;

[0090] (c) In Helix-2;

[0091] (d) At the C-terminus of Helix-4.

[0092] In a first alternative aspect, the method further comprises expressing the chimeric NRPS and / or PKS module from the genetic construct.

[0093] In a second 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 at least two system units:

[0094] (a) a first system unit, which comprises at least the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment as described in any one of the other aspects and embodiments of this aspect, and

[0095] (b) a second system unit, which comprises at least the second T domain fragment as described in any one of the other aspects and embodiments of this aspect;

[0096] Each system unit may contain one or more additional NRPS / PKS domains, and a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS) or a chimeric NRPS / PKS hybrid may be obtained by fusing a first and a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl 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 peptidyl carrier (PCP)) of the second system unit.

[0097] In a preferred embodiment, the sequence of the first system unit and the second system unit comprises the amino acid sequence of multiple portions of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments, which is:

[0098] (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or

[0099] (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or

[0100] (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

[0101] 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:

[0102] (a) providing a first NRPS or PKS gene sequence from a first microbial species,

[0103] (b) providing a second NRPS or PKS gene sequence from a second microbial species, and

[0104] (C) combining at least a portion of a first NRPS or PKS gene sequence with at least a portion of a second NRPS or PKS gene sequence to obtain a chimeric gene sequence by fusing the two sequences to each other, such that at least one fusion site is located within the T domain, and wherein the fusion product comprises a T domain consisting of an N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and a C-terminal sequence of the T domain of the second NRPS or PKS gene sequence, and

[0105] (d) Optionally, a chimeric gene sequence is expressed to obtain a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0106] 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 following steps:

[0107] (a) identifying a first target NRPS / PKS module that directly binds to a second target NRPS / PKS module at its N-terminus;

[0108] (b) identifying a second target NRPS / PKS module that directly binds to the second target NRPS / PKS module at its C-terminus, and wherein the second target NRPS / PKS module is selected from a module located at the C-terminus of a third non-target NRPS / PKS module within the NRPS / PKS gene;

[0109] (C) combining a first targeted NRPS / PKS module with a second targeted NRPS / PKS module by directly attaching the N-terminal portion of the T domain of the first NRPS / PKS module to the C-terminal portion of the T domain of a third non-target NRPS / PKS module;

[0110] 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 functional T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)).

[0111] In a preferred embodiment of the invention, the first and third NRPS / PKS modules each comprise at least a thiolation domain.

[0112] Also preferably, said combination may involve one or more additional NRPS / PKS modules and / or domains at the N-terminus of said first target NRPS / PKS module.

[0113] Alternatively or additionally, the combination may involve one or more additional NRPS / PKS modules and / or domains at the C-terminus of the second target NRPS / PKS module.

[0114] A preferred embodiment also relates to the first target NRPS / PKS module and the third non-target NRPS / PKS module being different.

[0115] In a fifth aspect, the invention relates to a combination product obtained by the process of the fourth aspect.

[0116] In the sixth aspect, the present invention relates to a method for producing a nucleic acid construct, which encodes the system unit described in other aspects and embodiments of this aspect, and the method includes the step of amplifying a sequence fragment of the NRPS / PKS gene using a degenerate primer pair, wherein the degenerate primer pair hybridizes with at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene under stringent conditions, wherein the hybridization site contains a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO:XY:FFxxGG(H / N / D)S, wherein x is any amino acid.

[0117] In the seventh aspect, the present invention relates to a method for producing a library of nucleic acid constructs, each nucleic acid construct encoding a system unit as described in other aspects and embodiments of this aspect, wherein the method comprises the step of amplifying a first sequence fragment of a NRPS / PKS gene using a degenerate primer pair, wherein the degenerate primer pair hybridizes under stringent conditions with at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, wherein the hybridization site comprises a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO:XY:FFxxGG(H / N / D)S; and wherein the method comprises amplifying at least a second sequence fragment of the NRPS / PKS gene for a second time using a degenerate primer pair from the same or one or more different NRPS / PKS genes.

[0118] In an eighth aspect, the present invention relates to a genetic construct system for expressing a chimeric NRPS / PKS, wherein the system comprises at least one genetic construct comprising a chimeric NRPS and / or PKS module produced by the method of the preceding aspects.

[0119] Specifically, some aspects described in the following embodiments and projects are preferred:

[0120] Item 1. A chimeric protein for producing a non-ribosomal peptide, comprising at least one chimeric thiol domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP))) consisting of a first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment directly adjacent to a second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment comprises The invention relates to an amino acid sequence of the N-terminal portion of the sequence of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)), and wherein the second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment comprises an amino acid sequence from the C-terminal portion of the amino acid sequence of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)); wherein the chimeric thiol domain is a fully functionalized T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)); characterized in that:

[0121] The amino acid sequences of portions of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments are:

[0122] (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or

[0123] (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or

[0124] (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

[0125] Item 2. A chimeric protein of Item 1, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment is directly adjacent to and connected to the N-terminus of the second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment.

[0126] Item 3. The chimeric protein of Item 1 or Item 2, wherein the chimeric protein is a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid.

[0127] Item 4. The chimeric protein of any one of Items 1 to 3, further comprising one or more known NRPS and / or PKS domains, in particular one or more adenylation (A) domains, one or more condensation (C) domains, one or more isomerization (E) domains, one or more reductase (R) domains, one or more C / E domains, one or more oxidation (Ox) domains, one or more reduction (Red) domains, one or more heterocyclization (Cy) domains, one or more initiation C domains, one or more methylation (Mt) domains, one or more A-Mt domains, one or more A-Ox domains, one or more communication (Com) domains, one or more multiple formylation (F) domains, X (X) domains and / or one or more terminal thioesterase (TE) domains or terminal C domains; and / or any or any combination of PKS domains, one or more acyltransferase (AT) domains, one or more ketosynthase (KS) domains, one or more dehydratase (DH) domains, one or more ketoreductase (KR) domains, one or more enoylreductase (ER) domains, one or more methyltransferase (Mt) O- or C domains, one or more docking (DD) domains, one or more thioesterase (TE) domains, one or more PLP-dependent cysteine ​​lyase (SH) domains, acyl carrier protein (ACP).

[0128] Item 5. A chimeric protein as described in any one of Items 1 to 4, wherein the chimeric T domain is composed of the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) and the amino acid sequence of the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)).

[0129] Item 6. The chimeric protein of any one of Items 1 to 5, wherein the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence and the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence are fused together at a fusion site, and wherein the fusion site is located at Figure 3 Fusion sites 1 to 7 are shown + / - 5 amino acids around them, with fusion sites 3 and 4 being preferred.

[0130] Item 7. The chimeric protein of Item 6, wherein the fusion site is located directly before or after any amino acid position of the conserved T domain motif -FFxxGG(H / N / D)S-, wherein x is any amino acid.

[0131] Item 8. The chimeric protein of any one of Items 1 to 7, wherein in Item 1(a), the protein further comprises at least one NRPS / PKS domain located at the N-terminus of the chimeric T domain and derived from a first microbial species, and at least one NRPS / PKS domain located at the C-terminus of the chimeric T domain and derived from a second microbial species, provided that the first and the second microbial species are different.

[0132] Item 9. The chimeric protein of any one of Items 1 to 8, wherein the chimeric protein comprises a NRPS / PKS domain sequence capable of catalyzing peptide and / or hybrid peptide-polyketide synthesis.

[0133] Item 10. A system for producing a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, comprising at least two system units:

[0134] (a) a first system unit, which comprises at least the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment described in any one of items 1 to 9, and

[0135] (b) a second system unit, comprising at least the second T domain fragment described in any one of items 1 to 9;

[0136] Each system unit may contain one or more additional NRPS / PKS domains, and a chimeric non-ribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS) or a chimeric NRPS / PKS hybrid may be obtained by fusing a first and a second system unit such that the amino acid sequence of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl 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 peptidyl carrier (PCP)).

[0137] Item 11. The system of Item 10, wherein the sequences of the first system unit and the second system unit comprise the amino acid sequences of multiple portions of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments, which are:

[0138] (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or

[0139] (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or

[0140] (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

[0141] Item 12. A method for producing a chimeric nonribosomal peptide synthetase (NRPS), a chimeric polyketide synthase (PKS), or a chimeric NRPS / PKS hybrid, the method comprising the steps of:

[0142] (a) providing a first NRPS or PKS gene sequence from a first microbial species,

[0143] (b) providing a second NRPS or PKS gene sequence from a second microbial species,

[0144] (C) combining at least a portion of a first NRPS or PKS gene sequence with at least a portion of a second NRPS or PKS gene sequence, by fusing the two sequences to each other to obtain a chimeric gene sequence, such that at least one fusion site is located within the T domain; and wherein the fusion product comprises a T domain consisting of an N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and a C-terminal sequence of the T domain of the second NRPS or PKS gene sequence, and

[0145] (d) Optionally, chimeric gene sequences are expressed to obtain chimeric nonribosomal peptide synthetases (NRPSs), chimeric polyketide synthetases (PKSs), or chimeric NRPS / PKS hybrids.

[0146] 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:

[0147] (a) identifying a first target NRPS / PKS module that will directly bind to a second target NRPS / PKS module at its N-terminus;

[0148] (b) identifying a second target NRPS / PKS module that will directly bind to the second target NRPS / PKS module at the C-terminus, and wherein the second target NRPS / PKS module is selected from a module located at the C-terminus of a third non-target NRPS / PKS module within the NRPS / PKS gene;

[0149] (C) combining a first targeted NRPS / PKS module with a second targeted NRPS / PKS module by directly attaching the N-terminal portion of the T domain of the first NRPS / PKS module to the C-terminal portion of the T domain of a third non-target NRPS / PKS module;

[0150] 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 functional T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)).

[0151] Item 14. A method according to Item 13, wherein the first and third NRPS / PKS modules each comprise at least a thiol domain.

[0152] Item 15. A method according to Item 13 or 14, wherein the combination may involve one or more additional NRPS / PKS modules and / or domains at the N-terminus of the first target NRPS / PKS module.

[0153] Item 16. The method of any one of Items 13 to 15, wherein the combination may involve one or more additional NRPS / PKS modules and / or domains at the C-terminus of the second target NRPS / PKS module.

[0154] Item 17. A method according to any one of Items 13 to 15, wherein the first target NRPS / PKS module and the third non-target NRPS / PKS module are not the same.

[0155] Item 18. A combination product obtained by the method of any one of Items 13 to 17.

[0156] Item 19. A method for producing a nucleic acid construct encoding the system unit described in Item 10, the method comprising the step of amplifying a sequence fragment of the NRPS / PKS gene using a degenerate primer pair, which hybridizes with at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene under stringent conditions, wherein the hybridization site contains a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ IDNO:XY:FFxxGG(H / N / D)S, wherein x is any amino acid.

[0157] Item 20. A method for producing a library of nucleic acid constructs, each of which encodes the system unit of Item 10, wherein the method comprises the step of amplifying a first sequence fragment of an NRPS / PKS gene using a degenerate primer pair, the degenerate primer pair hybridizing under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, wherein the hybridization site comprises a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO: XY: FFxxGG(H / N / D)S; and

[0158] Wherein the method comprises amplifying at least a second sequence segment of the NRPS / PKS gene a second time using a degenerate primer pair from the same or one or more different NRPS / PKS genes.

[0159] Item 21. A method for producing a chimeric non-ribosomal peptide synthetase (NRPS) and / or polyketide (PKS) module, the method comprising:

[0160] (a) providing a first NRPS / PKS module sequence, which comprises at least a first T domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence);

[0161] (b) providing a second NRPS / PKS module sequence, which comprises at least a second T domain sequence;

[0162] (c) optionally, aligning the first T domain sequence with the second T domain sequence to identify at least one T domain fusion site;

[0163] (d) fusing the first partial sequence and the second partial sequence directly adjacent to each other into a genetic construct to obtain a chimeric modular sequence, wherein

[0164] (i) the first partial sequence is a nucleotide sequence encoding the amino acid sequence of the first NRPS / PKS module sequence directly located at the N-terminus of the identified T domain fusion site; and

[0165] (ii) the second partial sequence is a nucleotide sequence encoding the amino acid sequence of a second NRPS / PKS module sequence located directly at the C-terminus of the identified T domain fusion site;

[0166] Genetic constructs comprising chimeric NRPS and / or PKS modules are thereby generated.

[0167] Item 22. The method of Item 21, wherein the first NRPS / PKS module sequence and the second NRPS / PKS module sequence are not identical, preferably, wherein the first T domain sequence and the second T domain sequence are not identical.

[0168] Item 23. The method of Item 21 or Item 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 directly adjacent to each other.

[0169] Item 24. The method of Item 21 or Item 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 RPS / PKS genes of two different bacterial and / or fungal species.

[0170] Item 25. The method of any one of Items 21 to 24, wherein the T domain comprises a T domain linker sequence and a T domain protein domain sequence.

[0171] Item 26. The method of any one of Items 21 to 25, wherein the T domain fusion site is selected to be located at one of the following positions of the aligned sequence of the T domain structure from the N-terminus to the C-terminus: T domain linker domain, Helix-1, Loop-1, Helix-2, Loop-2, Helix-3, Helix-4, wherein:

[0172] (a) within the T domain linker domain or at the N-terminus,

[0173] (b) within the Loop-1 domain;

[0174] (c) In Helix-2;

[0175] (d) At the C-terminus of Helix-4.

[0176] Item 27. The method of any one of Items 21 to 26, further comprising expressing a chimeric NRPS and / or PKS module from a genetic construct.

[0177] Item 28. A genetic construct system for expressing a chimeric NRPS / PKS, wherein the system comprises at least one genetic construct comprising a chimeric NRPS and / or PKS module produced by any one of Items 21 to 27.

[0178] As used herein, the terms "the present invention," "according to the present invention," "according to the present invention" and the like refer to all aspects and embodiments of the present invention described and / or claimed herein.

[0179] As used herein, the term "comprising" should be understood to include "including" and "consisting of", both of which are specific and therefore according to the embodiments disclosed separately of the present invention. "And / or" as used herein is considered to be a specific disclosure of each of two specific features or components, whether or not the other is included. For example, "A and / or B" is considered to be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein. In the context of the present invention, the terms "about" and "approximately" represent an interval of accuracy that will be understood by those skilled in the art, still ensuring the technical effect of the feature in question. The term generally indicates a deviation of ±20%, ±15%, ±10%, such as ±5% from the indicated value. As will be understood by those of ordinary skill in the art, the specific such deviation of the numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have greater such deviations than artificial or engineered technical effects. It will be understood by those of ordinary skill in the art that the specific such deviation of the numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have greater such deviations than artificial or engineered technical effects. When an indefinite or definite article is used when referring to a singular noun, for example "a", "an" or "the", this includes a plural of that noun unless otherwise stated.

[0180] It will be appreciated that applying the teachings of the present invention to a particular problem or situation, as well as incorporating variations or additional features of the present invention (such as further aspects and embodiments) therein, will be within the capabilities of one of ordinary skill in the art based on the teachings contained herein.

[0181] Unless the context dictates otherwise, the descriptions and definitions of the features described above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0182] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0183] BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

[0184] These figures show:

[0185] Figure 1 :Sfp phosphorylates the apo-T(PCP) domain into the full enzyme. Top: Sfp and its substrates coenzyme A and Mg 2+(PDB-ID:1QR0) 37 Crystal structure of. Middle: The phosphopantetheine moiety of coenzyme A (red) is covalently attached to a conserved serine residue in the T(PCP) domain via Sfp. Bottom: Crystal structure of the T(PCP) domain solved by Weber et al. (PDB-ID: 1DNY) 38 .

[0186] Figure 2 : Overview of the in silico workflow. To better understand NRPS evolution and to detect evolutionary hotspots of recombinant NRPSs from Photo- and Xenorhabdus sp., in silico analyses were performed. Initially, the software DualBrothers 24 Fine-grained phylogenetic reconstruction analysis (a) and tree topology analysis (b) were performed. The results showed that different parts of the T domain showed different topological structures, indicating different evolutionary origins. Next, the recombination detection program RDP4 was applied 25 to detect potential evolutionary hotspots (c). RDP4 often showed that homologous recombination occurred within the A domain or just before the T domain and close to the invariant serine. Further nucleotide sequence analysis revealed the presence of an AT offset in the conserved region just before the invariant serine (5'), which is theoretically very suitable for promoting homologous recombination. Further phylogenetic reconstruction (d) showed that the clustering of the T domain split into N- and C-terminal subunits at the invariant serine was different from each other, indicating that during evolution, homologous recombination may be more common within the T domain than in front of it. Combining a, b, c, and d, we came up with the evolutionary hypothesis described in (e).

[0187] Figure 3 : Recombination site screening probes functional splice sites within the T domain. To analyze whether the T domain can target functional recombinant NRPS building blocks, the Xenoamicin NRPS-producing protein from X. stockiae was 72Cstart-A1-Tp1 was fused to pT3-C / E4-A4-T4-C / E5-A5-T5-TE of GameXPetptide, which produces the NRPS GxpS. Splice positions 1, 3 and 4 show the production of the expected lipopeptide. Top: Schematic diagram of the resulting chimeric NRPS (left) and lipopeptide chemical structure (right). Middle: Ribbon diagram of the crystallized T domain of EntF (PDB: 4ZXJ) (left), with the tested fusion sites (1-7) highlighted in black. The fusion sites that produce catalytically active NRPSs are marked with red arrows. The right side shows the EIC of m / z [M+H] + = 444.3 produced in Escherichia coli DH10B (E. coli DH10B)::mtaA. The numbers refer to individual recombinant NRPSs (1-7). Bottom: Sequence alignment of the target T domain. Functional recombination sites are highlighted in red. Middle panel: Schematic representation of the color code used for NRPS building blocks. For domain assignment, the following symbols are used: A, adenylation domain, large circle; T, thiolation domain, rectangle; C, condensation domain, triangle; C / E, dual condensation / epimerization domain, diamond; TE, thioesterase domain, small circle.

[0188] Figure 4 : Proof of concept – fusion site 3. To verify whether the new evolution-inspired fusion site 3 could be used to reprogram NRPSs, NRPS-1 to -3 were created generically. Top: Schematic representation of recombinant NRPS-1 to -3 and the associated products. Middle: Schematic representation of the color code for the NRPS building blocks used. For domain assignments, the following symbols are used: A, adenylation domain, large circle; T, thiolation domain, rectangle; C, condensation domain, triangle; C / E, dual condensation / epimerization domain, diamond; TE, thioesterase domain, small circle, R, reductase domain, square, and the synthetic zipper (SZ) pair used, SZ17:18. Bottom: Structure of peptides 1-12 produced by NRPS-1 to -3 expressed in E. coli DH10B::mtaA.

[0189] Figure 5 : Shown is the de novo design of chimeric NRPSs that produce lipopeptide-aldehydes. To verify whether the novel evolution-inspired fusion site 3 could be used for de novo design, NRPS-4 to -8 were created. Top: Schematic representation of recombinant NRPS-4–8 and related products. Middle: Schematic representation of the color code used for NRPS building blocks. For domain assignments, see Figure 4 Bottom: Structures of peptides 13-17 produced by NRPS-4 to -8 expressed in E. coli DH10B::mtaA.

[0190] Figure 6: Conversion of NRPSs into artificial NRPS / PKS hybrid synthetases (Part 1). Top: Schematic representation of recombinant NRPS-PKS-9 and related products. Middle: Schematic representation of the color code used for NRPS and PKS building blocks. For domain assignments, see Figure 4 ; Other symbols: KS, DH and KR domains, small circles. Bottom: Structure of 18-23 produced by NRPS-PKS-9 expressed in E. coli DH10B::mtaA.

[0191] Figure 7 : Turning NRPSs into artificial NRPS / PKS hybrid synthetases (Part 2). Top: Schematic representation of the recombinant PKS-NRPS-10. Middle: Illustration of the color code used for NRPS and PKS building blocks. For domain assignments, see Figure 4 and Figure 6 Bottom: Structure of 24 produced by PKS-NRPS-10 expressed in E. coli DH10B::mtaA.

[0192] Figure 8 : Sequence alignment of selected T domains. To validate and compare fusion sites 1 and 4, a series of recombinant NRPSs were created (see Figure 9-11 ). Highlighted by arrows are the recombination sites used to generate NRPS-11 to -24.

[0193] Fig. 9 : Recombining non-related building blocks for GC content and codon usage to create chimeric NRPSs (Part 1). To validate and compare the suitability of fusion sites 1 and 4, a series of recombinant NRPSs were created; fusion site 1: NRPS-11 and -13; fusion site 4: NRPS-12 and -14. Top: Schematic representation of an unpublished WT NRPS and recombinant NRPSs-11 to -14. Middle: Illustration of the color code used for NRPS building blocks. For domain assignments, see Figure 4 Bottom: Structures of 25-27 produced by NRPS-11 to -14 expressed in E. coli DH10B::mtaA.

[0194] Fig.10 : Recombining non-related building blocks for GC content and codon usage to create chimeric NRPSs (Part 2). To validate and compare the suitability of fusion sites 1 and 4, a series of recombinant NRPSs were created; fusion site 1: NRPS-15 and -17; fusion site 4: NRPS-16 and -18. Top: Schematic representation of recombinant NRPSs-15 to -19. Middle: Illustration of the color code used for NRPS building blocks. For domain assignments, see Figure 4Bottom: Structures of 28 and 29 produced by NRPS-15 to -18 expressed in E. coli DH10B::mtaA.

[0195] Fig.11 : Recombination of unrelated building blocks of varying GC content and codon usage to generate chimeric NRPSs (Part 3). To validate and compare the suitability of fusion sites 1 and 4, a series of recombinant NRPSs were created; fusion site 1: NRPS-19 and -21; fusion site 4: NRPS-20 and -22. Top: Schematic representation of recombinant NRPSs-19 to -22. Middle: Illustration of the color code used for NRPS building blocks. For domain assignments, see Figure 4 Bottom: Structure of 30 produced by NRPS-19 to -22 expressed in E. coli DH10B::mtaA.

[0196] Fig.12 : Schematic diagram of the T domain degenerate primer library. Steps A to G are described in the example. Example

[0197] Certain aspects and embodiments of the present invention will now be described by way of example and with reference to the description, drawings and tables described herein. These examples of the methods, uses and other aspects of the present invention are representative only and should not be considered to limit the scope of the present invention to only these representative examples.

[0198] Background: Thiolation domain (T domain)

[0199] The T domain is the only NRPS domain without autonomous catalytic activity and is responsible for transporting substrates and elongation intermediates to the catalytic center. 26 The approximately 100AA long protein belongs to the acyl carrier protein (ACP-)-like superfamily and is located at the C-terminus of the A domain and the N-terminus of the C domain ( Figure 5 B).

[0200] The A domain catalyzes two half reactions27. In the second half reaction, the activated (adenylated) substrate is transferred to the terminal cysteamine thiol group of the 4'-phosphopantetheine (4'Ppan) cofactor covalently attached to the T domain. The T domain and its 4'-phosphopantetheine cofactor represent a holoprotein that is derived from apolipoprotein by post-translational modification28. Approximately 100% of the coenzyme A (CoA)-derived phosphopantetheine group with a terminal thiol is present. The long covalent group is linked via a phosphodiester to a strictly conserved serine side chain, which is surrounded by a homology pattern of AA residues of folded apolipoprotein 29-33. Transfer of 4'Ppan to apolipoprotein is accomplished by the action of NRPS-specific 4'-phosphopantetheinyltransferases (PPTases), which are usually encoded as part of NRPS biosynthetic gene clusters ( Figure 1 ) (e.g., Sfp of Bacillus subtilis) 34-37.

[0201] Weber and colleagues used NMR spectroscopy to characterize the T domain of the third module of the Bacillus brevis tyrosine synthase ( Figure 1 ) solved this first T domain structure38. Weber et al. revealed that the T domain is a twisted four-helix bundle with an extended loop between the first two helices and revealed that the invariant serine residue, the site of cofactor binding, is located at the interface between this loop and the second helix. In addition, no binding pocket was found within the T domain, consistent with the lack of substrate selectivity. In addition, the cofactor does not interact with the protein but is accommodated in the solvent. In terms of functionality and structural folding, the T domain is similar to the acyl carrier proteins (ACPs) from fatty acid synthases and PKS26. However, the sequence homology between them is only present in the immediate vicinity of the invariant serine residues39-41. The most obvious difference between them is the overall charge of the proteins. The ACP surface has mainly acidic side chains, while the T domain is less polar. This finding corresponds to the charge of the related 4'-phosphopantetheine transferases (AcpS and Sfp-like phosphopantetheine transferases (PPtases)). Furthermore, crystal structures of carrier proteins—as excised domains, as stand-alone individual proteins, and upon recognition by PPtase, such as Sfp from Bacillus subtilis and AcpS from Streptomyces coelicolor or Streptococcus pneumoniae—reveal that Sfp has a V-shaped interface for 4′Ppan cofactor modification by the T domain, whereas AcpS has three catalytic sites for post-translational modifications28,38,39,42–50.

[0202] The AT dual domain is defined as the initiation module because two proteins are required to activate and covalently bind the first substrate for subsequent peptide assembly26. During non-ribosomal synthesis, the T domain transfers reaction intermediates between different catalytic centers and adopts alternative conformations in the process42. The crystal structure of the termination module of the surfactant protein AC (SrfA-C) solved by Tanovic et al. (2008) Fig.12) made a significant contribution to the understanding of the different domain arrangements during the catalytic cycle51. In this structure, the PCP domain is positioned to interact with the condensation domain. However, the attachment site of the 4'PPan arm of the T domain is located 60 and 100 m from the active sites of the A and TE domains, respectively. This suggests that a large conformational rearrangement is required to deliver the pantetheine thiol group to the other domains. The T domain must adopt at least three functionally relevant and distinct orientations. The first conformation allows the transfer of the aminoacyl group to the thiol of 4'Ppan via binding to the A domain. The remaining two orientations within the extension module have the T domain approaching the catalytic center of the preceding C domain in its acceptor site and the catalytic center of the succeeding C domain in its donor site.

[0203] Due to evolutionary recombination analysis ( Figure 2 ) does not naturally lead to a specific splice site, but rather to a region (sequence stretch) that may promote homologous recombination, and fusion site screening was initially performed ( Figure 3 )—to verify whether chimeric NRPSs could be generated by targeting the T domain and to identify fusion sites that produced optimal peptides. Briefly, this screen identified three functional fusion sites ( Figure 3 ).

[0204] In particular, fusion sites 3 and 4 are located within a region (I-III) that has some very interesting features:

[0205] (I) First, they are located in the immediate vicinity of the invariant serine residue that serves as the 4'Ppan attachment site ( Figure 1 ), which is surrounded by a conserved pattern of AA residues (FFxxGG(H / D)S) and is homologous to regions of the PKS ACP domain.

[0206] (II) Secondly, at the nucleotide level, another striking feature can be observed. A region spanning about 50 nucleotides is not only AT-rich and conserved in different unrelated bacterial species, but also in GC-rich organisms such as actinomycetes, but also has unequal frequencies of the four DNA bases on the two strands of the respective DNA molecule. This mathematically unequal distribution, known as AT bias, 52, 53, not only represents a statistical deviation, but more importantly, it indicates genetic and codon stability that seems to resist mutation - that is, transition mutations are statistically significantly underestimated, so that AT bias dominates and point mutations are unlikely to change the AA encoded at that site.

[0207] (III) Third, when performing phylogenetic reconstruction, the two T domain parts located upstream (N-terminus) and downstream (C-terminus) of the above-mentioned conserved AA sequence ( Figure 2d) do show distinct clustering. 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 the adjacent C domains. When phylogenetic reconstruction ( Figure 2 a) and tree topology analysis ( Figure 2 b) This behavior is also observed at 24, suggesting that this region represents a potential evolutionary recombination site that has not yet been noticed.

[0208] In summary (I-III), these computer observations ( Figure 2 ) and fusion site screening from in vivo ( Figure 3 ) led us to hypothesize that the TCA unit (fusion site 1), the part-TCAT part (pT-CA-Tp) unit (fusion sites 3 & 4), and their combinations ((1,3), (1,4), (3,4), (4,3), (4,1), (3,1)) could serve as ideal starting points for evolution-inspired megasynthase engineering ( Figure 2 e).

[0209] The following examples show:

[0210] Example 1: Proof of Concept

[0211] This example is used to demonstrate that the NRPS elongation (NRPS-1) and initiation units (NRPS-2 & 3) can be functionally interchanged via fusion site 3 ( Figure 3 ).

[0212] Prior to the present work, an S-type variant of GameXPeptide (Gxp)AE was created that produced the synthetase (GxpS) 3, 54. This artificial / synthetic two-component NRPS ( Figure 4 ) to verify that fusion site 3 can indeed be used to generate new Gxp derivatives. By targeting GxpS domains T3 and T4, it was possible to functionally exchange the leucine of the building block pT3-C4-A4-Tp4 specifying GxpS with the arginine of the building block pT3-C4-A4-Tp4 specifying bicanthrin production of the NRPS (BicA) from X. budapestensis ( Figure 4 ). HPLCMS / MS analysis of methanolic culture extracts resulted in the detection of five arginine-bearing peptides (1-5; Figure 4). Among them, peptides 1 and 2 are the result of the unpaired activity of NRPS-1 subunit 2, while peptides 3-5 represent the expected full-length Gxp derivatives. Due to the loose substrate specificity of A1 (Val, Leu) and A3 (Phe, Leu) 3-5, there are differences at positions 1 and 3. In addition, NRPS-2 and -3 were created by replacing GxpS A3-Tp3 of the second subunit of S-type GxpS with two different starting building blocks (CstarTA-Tp) from odilorhabdin55 and xenoamicin III56 of synthetic NRPSs from Xenorhabdus nematophila HGB081 and Xenorhabdus stockiae, respectively. Figure 4 Both NRPSs showed biosynthetic activity and synthesized lipopeptides 6–12. NRPS-3 produced only the expected lipopeptide (10–12), whereas NRPS-2 also synthesized two C-terminally truncated versions of lipopeptide 6.

[0213] Example 2: De novo design of chimeric NRPS to produce lipopeptide-aldehyde compounds

[0214] The present examples (NRPS-4 to 8) are intended to highlight how fusion site 3 can be used to exchange the termination unit to generate synthetic NRPSs with a terminal reductase (R) domain 57, 58 ( Figure 5 The R domain not only releases the synthesized peptide and thus 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 the thioester to acetaldehyde, which can be further reduced to ethanol 59. Peptide aldehydes are often associated with protease inhibitors, for example, by fellutamide B60, which reversibly binds to the threonine in the active site of the M. tuberculosis proteasome.

[0215] However, inspired by the potent proteasome inhibitor fellutamide B, building blocks of up to five different NRPSs from four different Photorhabdus and Xenorhabdus strains were added ( Figure 5 The resulting chimeric assembly line enzymes NRPS-4 to 8 were produced in E. coli DH10B::mtaA and culture extracts were analyzed by HPLCMS / MS. All produced NRPSs showed catalytic activity to produce the desired lipopeptide aldehydes 13-19.

[0216] When tested on the yeast proteasome, compounds 15 and 17 indeed showed the expected inhibitory activity (low μM range).

[0217] Example 3: Conversion of NRPS into artificial NRPS / PKS hybrid synthetases

[0218] Create NRPS-PKS-9( Figure 6 ) and PKS-NRPS-10( Figure 7 ) to find out whether the identified fusion sites 3 and 1 could also be used to artificially fuse NRPS and PKS building blocks, respectively, to generate catalytically active NRPS / PKS hybrid synthetases.

[0219] Therefore, we again used our S-type GxpS3-type model system to convert NRPSs into artificial NRPS-PKS hybrid synthetases ( Figure 6 This was achieved by removing the GxpS terminator unit (pT4-C / E5-A5-TE) and replacing it with the PKS terminator unit from Glidobactin that produced the hybrid synthetase 61 (pT3-KS-ATDH-KR-ACP-TE). In brief, the resulting chimeric NRPS-PKS-9 synthesized six different peptides (20-25). While peptides 20-23 did not carry the desired terminal keto group and were synthesized by the sole activity of the NRPS portion, peptides 24 and 25 were the desired full-length hybrid NRPS-PKS products.

[0220] PKS-NRPS-10 is another example that highlights how an NRPS can become a NRPS-PKS hybrid ( Figure 7 ). In contrast to NRPS-PKS-9, fusion site 1 was used here to exchange the Cstart-A1 starting unit of an undisclosed cyclic lipopeptide (site tag XINNV2_12405) producing NRPS from X. innexii DSM16336 for KS1-AT1-DH1-ER1-KR1-KR1-T1-T1-C2-A2 of Myxochromid62 producing the synthase (MchAB) from Myxococcus xanthus DK162263. Although both starting units were derived from unrelated organisms with very different GC contents (Xenorhabdus sp.: ~44%; Myxococcus sp.: ~70%), the resulting assembly line synthesized the desired PK-NRP hybrid product 26.

[0221] Example 4: Recombination of unrelated building blocks of varying GC content and codon usage to generate chimeric NRPSs

[0222] Not only did NRPS-PKS-9 and PKS-NRPS-10 demonstrate for the first time that naturally occurring NRPS proteins could be functionally converted into NRPS-PKS hybrids, but PKS-NRPS-10 also showed that unrelated building blocks with vastly different GC contents could bind to each other.

[0223] To further investigate and characterize the novel fusion sites defined by the EIS approach, a chimeric protein NRPS11-24 ( Figure 9-11 ). The goal was to transform the unpublished WTNRPS-1 (X. innexii DSM16336, locus tag XINNV2_12405; Fig. 9 ) was combined with starting building blocks of Massetolide A64, Serrawettin W265, Serrawettin W166, Xefoampeptide67, an undisclosed NRPS (locus tag XEKKV2_12060), Protegomycin56, Chaiyaphumin68 and Plipastatin69 to produce synthetases from Pseudomonas sp MYb1170, Serratia sp SCBI, Serratia marcescens DSM 12481, X. bovienii SS-2004, X. sp. KK7.4, X. doucetiae DSM 17909, X. sp PB61.4 and Bacillus subtilis 168, respectively. Furthermore, each reprogrammed NRPS was designed and produced in two variants, using fusion sites 1 and 4. All the resulting reprogrammed NRPSs showed biocatalytic activity in both variants (fusion sites 1 and 4), producing the desired cyclolipopeptides (27-33).

[0224] in conclusion

[0225] The in vivo suitability of all evolution-inspired and in silico detected fusion sites could be confirmed ( 1 , 3 , and 4 ; Figure 3 ), to modify and de novo generate synthetic NRPSs and NRPS-PKS hybrids. Interestingly, all reprogrammed NRPSs and NRPS-PKS hybrids showed good to very good production titers comparable to or even exceeding wild-type production levels.

[0226] In general, all splice positions 1, 3 and 4 can be used to functionally generate novel biosynthetic pathways. The great advantage of the EIS approach described above seems to be its versatility, suggesting that recombination within the T domain is not only an efficient method for reprogramming NRPSs for synthetic biology purposes in the laboratory, but that nature may actually use the T domain as a recombination site to insert and delete building blocks from existing BGCs or to reconstitute two BGCs by intragenomic homologous recombination. However, in contrast to all state-of-the-art approaches 1-3, 71, it was possible to show for the first time ever that building blocks functionally independent of their origin, preferred codon usage and GC content of their source strain can be generated in E. coli to synthesize novel peptides and peptide-polyketide hybrids. Last but not least, each bacterial species encodes BGCs with only a very limited range of stretch units, compared to the total number of characteristic stretch units for all NRPSs and PKSs 9. Thus, the EIS approach allows us to expand the chemically available space beyond the limits set by nature.

[0227] Example 5: Module code library generation

[0228] Library construction strategy Figure 3 As shown in Figure 2, the T domains of various organisms such as Xenorhabdus, Pseudomonas, and Burkholderia are not highly conserved between species. However, when the T domain encoded by one organism is followed by a C or CE domain and is edited and aligned ( Figure 3 A) A conserved sequence spanning 10-12 amino acids can be observed. The compiled T domain followed by the E domain of the genera Xenorhabdus showed a different conserved sequence patch with an even higher degree of sequence identity. Analysis of the underlying DNA sequence of this conserved amino acid patch allowed the design of degenerate primers ( Figure 3 B), a degenerate primer set that can potentially target and amplify 70-90% of the T domains in organisms containing more than 50 T domains (e.g., Pseudomonas, Xenorhabdus, Burkholderia, Mycetohabitans), was evaluated.

[0229] Using degenerate primer sets in PCR reactions with genomic DNA (gDNA) as template, a modular library of encoding DNA fragments can be obtained. This library is then ligated into an entry vector. The dephosphorylated vector is used to ligate with the 5'-phosphorylated PCR product. The entry vector encodes at its ligation site the homology arms necessary for the Gibson cloning step (cloning of homologous DNA region fragments) that will enter the NRPS in the subsequent step (see Figure 3 E and 3F). The initial PCR module library can be ligated into any entry vector on either side of any chosen homology arms. In order to clone the module library into the NRPS context, a second round of PCR must be performed. The primer sets for this PCR bind to the homology arms and overlap the insert by 3-5 base pairs. The overlap of the primer pairs with the ligated insert ensures unique amplification of the "correctly" ligated insert (see Figure 3 E). Gibson cloning steps ( Figure 3 F) Inserting the module library into the selected NRPS biosynthesis. The resulting clones can be used in production culture of E. coli or any other suitable prokaryotic organism. The generated non-ribosomal peptides are doped with various amino acids ( Figure 3 G). In the example given, it is the third amino acid. It is noteworthy that the module inserted at position 3 defines the stereochemistry of the amino acid at position 2, thereby expanding the number of possible peptides that can be generated.

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Claims

1. A chimeric protein for producing a non-ribosomal peptide, comprising at least one chimeric thiolation domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP))), wherein the chimeric thiolation domain is composed of a first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment directly adjacent to a second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment, wherein the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment comprises An amino acid sequence containing an N-terminal portion derived from an amino acid sequence of a T domain (PKS acyl carrier protein (ACP) or an NRPS peptidyl carrier (PCP)), and wherein the second T domain (PKS acyl carrier protein (ACP) or an NRPS peptidyl carrier (PCP)) fragment contains an amino acid sequence derived from the C-terminal portion of an amino acid sequence of a T domain (PKS acyl carrier protein (ACP) or an NRPS peptidyl carrier (PCP)); wherein the chimeric thiol domain is a fully functional T domain (PKS acyl carrier protein (ACP) or an NRPS peptidyl carrier (PCP)); characterized in that: The amino acid sequences of portions of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments are: (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

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

3. The chimeric protein of claim 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.

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

5. The chimeric protein according to any one of claims 1 to 4, wherein the chimeric T domain consists of the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence and the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence.

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

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

8. The chimeric protein of any one of claims 1-7, wherein in claim 1(a), the protein further comprises at least one NRPS / PKS domain located at the N-terminus of the chimeric T domain and derived from a first microbial species, and at least one NRPS / PKS domain located at the C-terminus of the chimeric T domain and derived from a second microbial species, with the proviso that the first and second microbial species are different.

9. The chimeric protein of any one of claims 1-8, wherein the chimeric protein comprises a NRPS / PKS domain sequence capable of catalyzing peptide and / or hybrid peptide-polyketide synthesis.

10. A system for producing a chimeric nonribosomal 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, which comprises at least the first T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragment of any one of claims 1 to 9, and (b) a second system unit, which comprises at least the second T domain fragment according to any one of claims 1 to 9; Each system unit may contain one or more additional NRPS / PKS domains, and wherein the chimeric non-ribosomal peptide synthetase (NRPS), the chimeric polyketide synthase (PKS) or the chimeric NRPS / PKS hybrid can be obtained by fusing the first and the second system units, so that the N-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence of the first system unit is directly fused to the C-terminal T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequence of the second system unit.

11. The system according to claim 10, wherein the sequences of the first system unit and the second system unit comprise the amino acid sequences of portions of the T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) amino acid sequences of the first and second T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) fragments, which are: (a) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from different species that are heterologous to each other; or (b) a T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence from a different NRPS / PKS gene from the same species; or (c) T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequences derived from two different T domains (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) of the same NRPS / PKS gene.

12. A method for producing a chimeric nonribosomal 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 derived from a first microbial species, (b) providing a second NRPS or PKS gene sequence derived from a second microbial species, (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 two sequences to each other, such that at least one fusion site is located within the T domain, and wherein the fusion product comprises a T domain consisting of an N-terminal sequence of the T domain of the first NRPS or PKS gene sequence and a 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 the chimeric non-ribosomal peptide synthetase (NRPS), chimeric polyketide synthase (PKS) or chimeric NRPS / PKS hybrid.

13. A method for combining or generating a combination of at least two NRPS / PKS modules derived 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 that will directly bind to a second target NRPS / PKS module at its N-terminus; (b) identifying a second target NRPS / PKS module that will bind directly to the second target NRPS / PKS module at the C-terminus, and wherein the second target NRPS / PKS module is selected from a module located at the C-terminus of a third non-target NRPS / PKS module within the NRPS / PKS gene; (C) combining the first targeted NRPS / PKS module with the second targeted NRPS / PKS module by directly attaching 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; 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 functional T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)).

14. The method of claim 13, wherein the first and the third NRPS / PKS modules each comprise at least one thiolation domain.

15. The method of claim 13 or 14, wherein the combination may involve one or more additional NRPS / PKS modules and / or domains at the N-terminus of the first target NRPS / PKS module.

16. The method according to any one of claims 13 to 15, wherein the combination may involve one or more additional NRPS / PKS modules and / or domains at the C-terminus of the second target NRPS / PKS module.

17. The method according to any one of claims 13 to 15, wherein the first targeted NRPS / PKS module and the third non-targeted NRPS / PKS module are not the same.

18. A combination product obtained or obtainable by a method as claimed in any one of claims 13 to 17.

19. A method for producing a nucleic acid construct encoding the system unit of claim 10, the method comprising the step of amplifying a sequence fragment of a NRPS / PKS gene using a degenerate primer pair, wherein the degenerate primer pair hybridizes under stringent conditions to at least 10 nucleic acids of a hybridization site within the NRPS / PKS gene, wherein the hybridization site comprises a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO:XY:FFxxGG(H / N / D)S, wherein x is any amino acid.

20. A method for generating a library of nucleic acid constructs, each encoding a system unit according to claim 10, wherein the method comprises the step of amplifying a first sequence segment of a 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, wherein the hybridization site comprises a sequence encoding an amino acid consensus motif (T domain motif) according to SEQ ID NO: XY: FFxxGG(H / N / D)S; and The method comprises amplifying at least a second sequence fragment of the NRPS / PKS gene for a second time using the degenerate primer pair derived from the same or one or more different NRPS / PKS genes.

21. A method for producing a chimeric non-ribosomal peptide synthetase (NRPS) and / or polyketide (PKS) module, the method comprising: (a) providing a first NRPS / PKS module sequence, which comprises at least a first T domain (T domain (PKS acyl carrier protein (ACP) or NRPS peptidyl carrier (PCP)) sequence); (b) providing a second NRPS / PKS module sequence, which comprises at least a second T domain sequence; (c) optionally, aligning the first T domain sequence with the second T domain sequence to identify at least one T domain fusion site; (d) by cloning the first partial sequence and the second partial sequence directly adjacent to each other into a genetic construct to fuse them to obtain a chimeric module sequence, wherein i. the first partial sequence is a nucleotide sequence encoding the amino acid sequence of the first NRPS / PKS module sequence directly located at the N-terminus of the identified T domain fusion site; and ii. the second partial sequence is a nucleotide sequence encoding the amino acid sequence of the second NRPS / PKS module sequence directly located at the C-terminus of the identified T domain fusion site; Genetic constructs comprising chimeric NRPS and / or PKS modules are thereby generated.

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

23. The method of claim 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 directly adjacent to each other.

24. The method of claim 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.

25. The method of any one of claims 21-24, wherein the T domain comprises a T domain linker sequence and a T domain protein domain sequence.

26. The method of any one of claims 21-25, wherein the T domain fusion site is selected to be located at one of the following positions with reference to the aligned sequence of the T domain structure from N-terminus to C-terminus: T domain linker domain, Helix-1, Loop-1, Helix-2, Loop-2, Helix-3, Helix-4, wherein: (a) within the T domain linker domain or at the N-terminus, (b) within the Loop-1 domain; (c) within the Helix-2; (d) at the C-terminus of Helix-4.

27. The method of any one of claims 21-26, further comprising expressing the chimeric NRPS and / or PKS module from the genetic construct.

28. A genetic construct system for expressing a chimeric NRPS / PKS, wherein the system comprises at least one genetic construct comprising a chimeric NRPS and / or PKS module produced according to any one of claims 21-27.

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