阅读说明：本技术 用于非核糖体肽合成酶的组装和修饰的系统 (System for assembly and modification of non-ribosomal peptide synthetases ) 是由 凯南·波祖尤克 安娜贝尔·林克 海尔格·波特 于 2019-01-15 设计创作，主要内容包括：本发明涉及用于非核糖体肽合成酶(NRPS)的组装和修饰的系统。该系统使用了包含缩合亚结构域的新颖的定义明确的构建基块(单元)。该策略允许有效地组合被称为交换单元(XU<Sub>2.0</Sub>)的组装单元,而无需考虑其对后续NRPS腺苷酸化结构域的天然存在的特异性。本发明的系统允许容易地组装具有选择的任何氨基酸序列的NRPS,而没有由于天然存在的NRPS单元而引起的任何限制。该系统还允许使用创造性的XU<Sub>2.0</Sub>交换天然NRPS构建基块,从而产生经修饰的肽。本发明提供了所述系统、它们的单个交换单元、编码这些单元的核酸、及其方法和用途。(The present invention relates to a system for the assembly and modification of non-ribosomal peptide synthetases (NRPS). The system uses novel well-defined building blocks (units) comprising condensation subdomains. This strategy allows to efficiently combine units called switching units (XU) 2.0 ) Without regard to its naturally occurring specificity for a subsequent NRPS adenylation domain. The system of the invention allows easy assembly of NRPS with any amino acid sequence selected without any limitations due to naturally occurring NRPS units. The system also allowsPermitting use of the inventive XU 2.0 The native NRPS building blocks were exchanged, resulting in a modified peptide. The invention provides such systems, their individual exchange units, nucleic acids encoding these units, and methods and uses thereof.)

1. A system for the production of a non-ribosomal peptide synthetase (NRPS), wherein said system comprises at least one (preferably two) NRPS exchange units (XU)_2.0) Each of said exchange units being specific for a different or the same amino acid X used for assembling the NRPS, and wherein said XU_2.0Comprising at least one partially condensed (C) domain or partially condensed/epimerized (C/E) domain selected from: condensation domain receptor site subdomain (C) specific for a given amino acid X_Asub) A condensation/epimerization domain receptor site subdomain (C/E) specific for a given amino acid X_Asub) A condensation domain donor site subdomain (C) specific for a given amino acid X_Dsub) And a condensation/epimerization domain donor site subdomain (C/E) specific to a given amino acid X_Dsub）。

2. The system of claim 1, wherein at least one XU_2.0Comprising a C or C/E acceptor domain and a C or C/E donor domain separated by one or more NRPS domains other than the C or C/E domain.

3. The system of claim 1 or 2, wherein at least of the systemsFirst XU_2.0And a second XU_2.0Formed by said first XU when connected to each other_2.0And said second XU_2.0A part of a C or C/E domain.

4. The system of any one of claims 1 to 3, comprising at least two XUs_2.0Each of which has a different amino acid specificity X, preferably wherein each X is selected from specificity for any one or two, three, four or more natural or unnatural amino acid.

5. The system of any one of claims 1 to 7, further comprising an XU_2.0Terminating and/or initiating unit, wherein the XU_2.0The starter unit comprises as starter unit only the C or C/E domain donor subdomain having the domain structure C-A specifically for incorporation of acyl units (fatty acids and derivatives thereof)^X-T-C_Dsub ^XOr C-A^X-T-C/E_Dsub ^XAnd wherein the termination module comprises any one of: terminal condensed Domain (C)_term) An internal condensation (C) domain, an internal condensation and epimerization (C/E) bi-domain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (Re), oxidation (Ox) or Thioesterase (TE) domain.

6. The system according to any one of claims 1 to 7, further comprising an XU2.0 starting unit, preferably selected from the following formulae: c_Asub-A^X-T-C_Dsub ^X、C/E_Asub-A^X-T-C_Dsub ^X、C_Asub-A^X-T-C/E_Dsub ^XOr C/E_Asub-A^X-T-C/E_Dsub ^X。

7. The system of any one of claims 1 to 9,wherein at least two, preferably three, four or more XUs_2.0NRPS is provided when put into sequence.

8. The system of any one of claims 1 to 7, further comprising at least one XU having a modified domain_2.The modifying domain is for example E, MT or Ox or other modifying domain.

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

10. A library of nucleic acid molecules, wherein the library comprises at least two or more nucleic acid constructs, each encoding an NRPS crossover unit (XU) with amino acid specificity X_2.0) And wherein said XU_2.0Comprising at least one partially condensed (C) domain or partially condensed/epimerized (C/E) domain selected from: condensation domain receptor site subdomain with amino acid specificity X (C)_Asub) Condensation/epimerization domain receptor site subdomains (C/E) with amino acid specific X_Asub) A condensation domain donor site subdomain having an amino acid-specific X (C)_Dsub) And a condensation/epimerization domain donor site subdomain (C/E) with an amino acid specific X_Dsub）。

11. The library of claim 10, comprising a coding sequence encoding at least one XU_2.0Nucleic acid construct of a termination and/or initiation unit, wherein said XU_2.0The starter unit comprises as starter unit only the C or C/E domain donor subdomain with a specific for the incorporation of an acyl unit (lipid)Fatty acids and derivatives thereof) domain structure C-A^X-T-C_Dsub ^XOr C-A^X-T-C/E_Dsub ^XOr A^X-T-C_Dsub ^XOr A^X-T-C/E_Dsub ^XAnd wherein the termination module comprises any one of: terminal condensed Domain (C)_term) An internal condensation (C) domain, an internal condensation and epimerization (C/E) bi-domain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (Re), oxidation (Ox) or Thioesterase (TE) domain.

12. The library of claim 10 or 11, wherein each XU_2.0Encoded by a separate nucleic acid construct, preferably by a nucleic acid according to claim 9.

13. Library according to any one of claims 10 to 12, wherein the library comprises XUs of a system according to any one of claims 1 to 8_2.0。

14. A method for producing NRPS, the method comprising the steps of: assembling at least two NRPS switching units (XU)_2.0) Each of said exchange units having the same or different amino acid specificity X, and wherein said XU_2.0Comprising at least one partially condensed (C) domain or partially condensed/epimerized (C/E) domain selected from: condensation domain receptor site subdomain with amino acid specificity X (C)_Asub) Condensation/epimerization domain receptor site subdomains (C/E) with amino acid specific X_Asub) A condensation domain donor site subdomain having an amino acid-specific X (C)_Dsub) And a condensation/epimerization domain donor site subdomain (C/E) with an amino acid specific X_Dsub）。

15. A method for producing a non-ribosomal peptide having a specific sequence, the method comprising a rootThe method of assembling NRPS according to claim 14, wherein said NRPS is composed of XUs having specificity according to the non-ribosomal peptide to be produced_2.0The sequence of (a).

Technical Field

The present invention relates to a system for the assembly and modification of non-ribosomal peptide synthetase (NRPS). The system uses novel well-defined building blocks (units) comprising condensation subdomains. This strategy allows for efficient combining of units called eXchange units (XUs)_2.0) Without regard to its naturally occurring specificity for a subsequent NRPS adenylation domain. The system of the invention allows easy assembly of NRPS with any amino acid sequence selected without any limitations due to naturally occurring NRPS units. The system also allows the use of the inventive XU_2.0The native NRPS building blocks were exchanged, resulting in a modified peptide. The invention provides such systems, their individual exchange units, nucleic acids encoding these units, and methods and uses thereof.

Description of the invention

Non-ribosomal peptide synthetases (NRPS) and polyketide synthetases (PKS) are multifunctional enzyme complexes with modular structure (Marahiel 1997). Many natural products synthesized by these enzyme classes have medical and/or biotechnological significance because they possess medically relevant properties, including antimicrobial (e.g., teixobactin), antitumor (e.g., bleomycin), antifungal (fengycin) and immunosuppressive (cyclosporine) activity (Ling et al 2015; Ishizuka et al 1967; Loeffler et al 1986; Emmel et al 1989). Although peptide compounds produced by NRPS exhibit broad biological activity and broad structural diversity (e.g. non-proteinogenic amino acids, N-methylation, epimerization, heterocycles), a common synthetic approach, the so-called "multiple-carrier thiotemplatemehanisms", is shared.

The structure of NRPS is obligately modular. Modules are defined as catalytic units that incorporate a particular building block (e.g., amino acids) into the growing peptide chain (Marahiel 1997). The NRPS module can be subdivided into multiple domains, each responsible for a certain reaction step in peptide assembly. For example, a canonical extender module consists of three domains, called core domains:

-an adenylation (a) domain which selectively determines the substrate (usually an amino acid) and activates it to an aminoacyl-adenylate.

-Peptidyl Carrier Protein (PCP), also known as thiolated domain (T), which binds to the cofactor 4-phosphopantetheine, to which the activated Amino Acid (AA) is covalently bound by the formation of thioesters.

-the condensation (C) domain catalyzes the peptide bond formation between the aminoacyl or peptidyl residues located downstream and upstream.

The first (N-terminal) module (start module) of the NRPS module generally does not have a C-domain, while the last (C-terminal) module (stop module) generally comprises a Thioesterase (TE) domain (Marahiel et al, 1997). The TE domain is generally responsible for the release of linear (transfer to water molecules), cyclic or branched cyclic peptides (amide or ester bonds).

The NRPS may comprise the following domains: c (condensation), Cy (heterocycle), a (adenylation), T (thiolation) or PCP (peptidyl carrier protein), TE (thioesterase), E (epimerization), condensation/epimerization (C/E), MT (methyltransferase), Ox (oxidase) and Re (reductase) domains. NRPSs generally have the following structure: A-T- (C-A-T) n-C-A-T-TE, wherein A-T is an initiation module, C-A-T is an extension module, and C-A-T-TE is a termination module. In the individual modules, for example, the following changes may occur: replacement of C with Cy or C/E and insertion of E, MT, Ox or Re; TE is replaced by C or Re. A complete assembly line may have a start module, a stop module, and between zero and n-2 extension modules, where n is the number of monomers in the polymer product. Exceptions may exist to the rule; for example, an enterobacterin synthase, in which the TE domain acts as an oligomerizing enzyme, will therefore hook three of these dimer products together to form a hexameric peptide product, although it has only two modules.

NRPSs are generally modular, and a series of catalytic steps move from the amino terminus to the carboxy terminus of each polypeptide that makes up the NRPS. For example, NRPS producing tyrocidine (tyrocidine) consists of three genes producing three polypeptides. TycA comprises a start module; TycB contains three extension modules, while TycC contains six additional extension modules plus a termination module.

The PKS may comprise the following domains: KS (ketosynthase), AT (acyltransferase), T (thiolate), KR (ketoreductase), DH (dehydratase), ER (enoyl reductase), TE (thioesterase). PKSs generally have the following structure: AT-T- (KS-AT-T) n-TE. AT-T is the start module, KS-AT-T is the extension module, and TE is the stop module. The structure of PKS is very similar to that of NRPS. There are many examples of mixed PKS-NRPS systems (e.g., yersinin, epothilone, bleomycin) in which two types of assembly lines are put together to form a coherent unit. In each PKS module, either KR, KR and DH and ER or no other domains are found. These additional domains within the module determine the chemical functionality at the beta carbon (e.g., carbonyl, hydroxyl, alkene, or saturated carbon).

The strength of NRP and PK as potential drugs lies in their diversity and complex chemical structure. In general, it is the complexity of these natural products that makes them (or variants thereof) difficult to obtain synthetically. For NRP or PK, several examples of cost-effective synthetic routes have been developed with little success. In addition, various moieties on such molecules cannot be modified by organic synthesis or can only be produced in low yields using such techniques. The difficulty of synthesis and modification of natural products of NRP and PK highlights the need for alternative strategies to enhance the synthesis and creation of variants of these molecules.

Although NRPS has an obvious modular structure, it was practically difficult to exchange domains so that the resulting NRPS was effective before the inventors' previous invention (EP15002340) and the present invention. Substitution of one or more domains or modules for another generally results in low yields (e.g., a > 10-fold reduction) and the production of undesirable biosynthetic byproducts. These changes may be the result of disruption of protein-protein interactions, and may also be due to the substrate specificity of each of the C and TE domains. Thus, new methods of generating new NRPs and PKs are needed, and methods of increasing the yield of such NRPs and PKs are needed.

For more general information on NRPS and PKS, please see Cane et al (1998), Marahiel (1997), Sieber and Marahiel (2005), Smith and Tsai (2007) and Sussmuth and Mainz (2017).

After activation and covalent binding of the first AA by the A-T double domain initiator module, extension of the peptide proceeds by block condensation of building blocks which are then covalently linked to the T domain of the downstream (C-terminal) extension module (C-A-T) n (Sieber and Marahiel, 2005; or Sussmuth and Mainz, 2017). All extension reactions (formation of peptide and amide bonds) are mediated by a C domain about 450 AA long located between the upstream T and downstream a domains, and strictly unidirectional leads to downstream directed synthesis of NRPS products (Samel et al, 2007). The C domain catalyzes nucleophilic attack of the downstream T domain bound acceptor AA with its free alpha-amino group on the upstream T domain bound donor AA or the activated thioester of the peptide.

The biochemical characterization of the C domain reveals insights into its catalytic action and substrate specificity. Through deletion experiments Stachelhaus and colleagues (1998) found that the C domain is essential for the formation of peptide bonds. Furthermore, sequence alignment of several C domains reveals a highly conserved HHXXDG sequence motif (the so-called "His motif") which is also present in the acyltransferase (e.g., chloramphenicol acetyl transferase), NRPS E, and Cy domains (De Crecy-Lagard et al, 1995). Mutation of the second His residue in the conserved motif abolished activity in the condensation assay (Sieber and Marahiel, 2005).

The structure comprising the NRPS C domain has been determined by X-ray crystallography: independent C-domain (Keting et al, 2002), C-T double domain (Samel et al, 2007) and C-A-T-TE termination module (Tanovic et al, 2008). The C domain has a pseudo-dimeric configuration, with both the N and C terminal subdomains having a folded core in the CoA-dependent acyltransferase superfamily (Blouff et al, 2013). The active site is at the bottom of the "canyon" formed by the two subdomains and is covered by a "latch-up" spanning from the C subdomain to the N subdomain. The catalytic center comprising the HHXXXDG (where X represents any residue) motif has two binding sites: one for electrophilic donor substrates (acyl groups of growing chains) and one for nucleophilic acceptor substrates (activated amino acids) (Rausch et al, 2007).

Although little is known about the catalysis of the reaction C domain, biochemical characterization of the different C domains of Brevibacterium casein peptide synthetase (Belshaw et al, 1999; Clugston et al, 2003; Samel et al, 2007) reveals insights into their substrate specificity. All C domain characterizations were performed in vitro and the same method was used to investigate the acceptance of the internal C domain for the substrate. The upstream and/or downstream T-domains are chemoenzymatically initiated (synthetic peptidyl-Ppan arm transfer) with acceptor substrates by using permissive PPTase Sfp (Belshaw et al, 1999; Samel et al, 2007). In summary, this approach indicates that the receptor sites of the C domain exhibit strong stereoselectivity and significant side chain selectivity (Rausch et al, 2007). The selectivity for a particular side chain appears to be less pronounced at the donor site, which exhibits strong stereoselectivity. The C domain following the E domain exhibits specificity for the configuration of the C-terminal residue bound at the donor site, since the preceding E domain does not specifically catalyze the epimerization from L to D, but rather provides a mixture of configurations. The C domain immediately downstream of the E domain appears to be D-specific for upstream donors and L-specific for downstream acceptors, thus catalyzing the condensation reaction between D and L residues (Clugston et al, 2003).

The C domain can be subdivided into functional and phylogenetic subtypes (Rausch et al, 2007). Within the extension module is a "standard" C domain, for example:^LC_La domain that catalyzes the formation of a peptide bond between two L-AA; and^DC_La domain that links the L amino acid to a growing peptide that ends with the D amino acid (Rausch et al,2007) the initial C domain acylating the first amino acid with a carboxylic acid (typically β -hydroxy fatty acid), while the heterocyclic (CY) domain catalyzes the formation of a peptide bond and subsequent cyclization of cysteine, serine or threonine residues (Rausch et al, 2007).

The most common method of multi-enzyme activation is through the TE domain, which belongs to the α/β -hydrolase superfamily (lipases, proteases and esterases) (Du and Lu, 2009). These enzymes are about 280 amino acids long and are fused to most of the C-terminal T domain of the termination module (Sieber and Marahiel, 2005; Kohli et al, 2001). In the final step of peptide assembly, the T domain-peptidyl thioester is subjected to nucleophilic attack by the active site serine of the TE domain to form a peptide-O-TE intermediate (Kohli et al, 2001). Deacylation of the intermediate involves hydrolysis (attacking the exogenous nucleophile) to release the linear peptide or, in the case of cyclic products, reaction of an intramolecular nucleophile (N-, O-, or C-nucleophile). Hydrolytic release is observed for peptides such as vancomycin, whose peptide backbone is constrained by further post-synthetic oxidative crosslinking reactions. The circularized TE domain provides diversity and complexity because in the cyclization reaction, various groups can be nucleophiles: n-terminal amino groups (head-to-tail cyclization; e.g., tyrocidin A and S), side chain nucleophiles (branched cyclic molecules; e.g., bacitracin A and daptomycin), and the beta-hydroxy group of beta-hydroxy fatty acids (e.g., surfactin) (Kohli et al, 2001).

Bruner et al (2002) solved the first TE crystal structure (SrfTE) of the surfactant biosynthetic cluster. In general, the NRPS TE domain is monomeric and consists of an α/β hydrolase fold, with a catalytic triad ((Ser/Cys) - (His) - (Asp/Glu/Ser)) used for substrate binding and catalysis by a covalently bound peptide, a thioesterase intermediate. In addition, the TE domain was found to exist in two different conformations, an on state and an off state. The difference between the two states is limited to a region of 40 amino acid residues that covers most of the active site of the enzyme, called the lid region.

Unlike many other catalytic domains involved in non-ribosomal peptide biosynthesis, the TE domain is highly diverse and therefore no model exists to predict TE loading or release selectivity (Horsman et al, 2015). Phylogenetic analysis of TE sequences shows that they do not cluster according to the type of unloading chemistry they catalyze.

The TE domain operates by a two-step mechanism, loading followed by release (Horsman et al, 2015). The active site Ser side chain alcohol is activated by a conserved His-Asp dimer, thereby increasing its nucleophilicity. The substrate to which the T domain binds is close to Ser activated by the 4' Ppant cofactor. The cap region is assumed to be open to accommodate the presence of the thioester substrate. The deprotonated and conserved active site Ser attacks the substrate thioester and stabilizes the formed charged tetrahedral intermediate in an oxyanion pore by hydrogen bonding of the two primary amide groups. This intermediate can be resolved by loss of the 4' Ppant thiolate to yield the acyl-TE intermediate. The second step (unloading) involves the release of the acyl group. This step begins with the proximity of an intramolecular or intermolecular nucleophile. Townsend and colleagues (2010, 2014) believe that the active site histidine ion is deprotonated by the leaving thiolate and is therefore able to activate the entering nucleophile (Korman et al, 2010; Gaudelli and Townsend, 2014). The nucleophile is added to the carbonyl group of the acyl-TE intermediate and the tetrahedral intermediate is again stabilized by oxygen anion pores. Eventually, the serinol alkylate is released as a consistent proton, and the product leaves the active site.

Trauger (2000) and Tseng (2002) obtained major insights into the specificity of TE substrates. By using synthetic SNAC peptides (N-acetylcysteine), they were able to show that the TE domain was selective for stereochemistry as well as the side chain of the N-terminal AA residue. They also revealed that AA next to the AA forming the peptidyl-O-TE (C-terminal AA) is important for peptide hydrolysis and cyclization, while all other AAs seem to be unimportant in the produced peptide. Furthermore, Kohli et al (2001) revealed that the TE domain excised from Brevibacterium casein NRPS accepts various SNAC-peptides as substrates for cyclization, varying in length and composition.

The obvious feature of most fungal NRPS is the substitution of the TE domain by either the terminal C, Re or T domain (Haynes et al, 2011). In addition to nad (p) H-dependent Re domains, the C domain may also be involved in peptide release (Kopp and Marahiel, 2007). Although most bacterial NRPSs use TE domains for circularization, fungal NRPSs, as well as some NRPSs from bacteria (including the genera Photorhabdus and Xenorhabdus), employ this complementation strategy (Gao et al, 2012; Reimer et al, 2013).

In macrocyclic fungal NRPSs (e.g. cyclosporin a, staurosporine aureus A, apicidin and ferrichrome a), each respective NRPS catalyzes peptide release by a terminal condensation (Cterm) domain (Gao et al, 2012). In the NRPS paradigm, the C domain is canonical sorted to catalyze the growing peptidyl-S-T from module n using the active site histidine as the general base_nWith activated aminoacyl-S-T_n+1Peptide bond formation therebetween. Thus, it is surprising that Cterm domains are able to perform equivalent head-to-tail chaining of TE domains. This reaction relies on the serine residue of the HHxxxDxxS motif being highly conserved in the active site of nucleophilic catalysis, the nucleophile being an intramolecular amino group rather than the next AA (Kopp and Marahiel, 2007). Gao et al (2012) found that Cterm cyclization activity requires the presence of a T domain. Furthermore, by constructing recombinant T-Cterm bi-domains, they were able to demonstrate that the non-homologous T-domains do not interact with downstream Cterm domains. Thus, the protein-protein interaction between Cterm and the upstream T domain appears to be specific and may rely on the C domain to recognize all of the unique T domain sequence elements. However, although the terminal C domain is cited as controlling the cyclization reaction of NRPS-based intermediates, there is no experimental evidence of its proposed catalytic activity (Haynes et al, 2011).

In addition to the Cterm domain that catalyzes the release of the peptide by cyclization, there is a Cterm domain that catalyzes the formation of an amide bond between the peptide to which the linear T domain is bound and an amine in the environment (Reimer et al, 2013; Fuchs et al, 2012; Gao et al, 2012; Cai et al, 2017). An example is the non-ribosomal rhabdopeptide biosynthetic cluster from Xenorhabdus nematophila (Xenorhabdus nematophila). Here, the Cterm domain may be involved in the condensation of biogenic amines (e.g.phenylethylamine from the decarboxylation of phenylalanine) with peptide intermediates during release (Reimer et al, 2013; Fuchs et al, 2012).

Since 1995, Marahiel et al (WO200052152) were able to show that it is possible to recombine NRPS by exchanging adenylation-thiolation double domains, and NRPS research has been the focus (Marahiel et al, 1995). Over the last two decades, many attempts have been made to re-encode NRPS. Based on the crystal structure of the phenylalanine-activating domain PheA (PDB-ID:1AMU), Stachelhaus et al were able to elucidate AA conferring specificity at the catalytic center (Conti et al, 1997; Stachelhaus et al, 1999). Using this specificity-conferring code (denoted as Stachelhaus code), the substrate specificity of the A domain can be predicted and altered in vitro (Khurana et al, 2010; Rausch et al, 2005;

etc., 2011; kries et al, 2014). The most obvious disadvantage of this attempt is that it is not applicable in vivo. One major reason for this deficiency is that the C and TE domains are also selective, resulting in substrate incompatibility (Belshaw et al, 1999; Truger et al, 2000; Tseng et al, 2002).

Another attempt to alter the known NRPS biosynthetic cluster (WO200130985, Marahiel et al) is based on the knowledge of the exchange of single domains, double domains or whole modules and precisely defined boundaries (linkers) between the individual domains. With this invention, it is only possible to successfully change some NRPSs by introducing additional modules or deleting them. However, it is impossible to produce a completely artificial NRPS from the manual de novo combination module. This will result in new NRPSs, which are not found in nature, which will also produce new peptides. The problem of such exchanges or combinations is always an uncertainty as to the compatibility of the modules and/or domains with each other. The disadvantage due to the lack of a solution to the above problem is illustrated by the fact that few artificial peptides have been designed by this method.

Another attempt to alter the known NRPS biosynthetic cluster (WO2007014076, Walsh, etc.) is based on mutagenesis of so-called "assembly lines" (i.e. synthetases). Mutagenesis of the NRPS gene is not the subject of the present invention, although the methods of the invention may be combined with mutagenesis which will alter the NRPS produced and cause altered peptide synthesis. Such mutagenesis can be used to increase the diversity of the NRPS library and the number of NRPS clones in the library.

Since the a domain is the initial gate-keeping enzyme, the production of modified peptide products requires substitution or modification of the a domain specifying the target residue in the native peptide. Researchers have adopted three general strategies to achieve this goal: (I) (ii) replacement of a or paired a-T domains, thereby activating a replacement substrate; (II) a targeted alteration of the substrate binding pocket of the a domain; (III) substitution of domain units C-A or C-A-T as inseparable pairs. These strategies were complemented by recombinant studies that attempted to redesign the NRPS by T, T-C-A, communication domain and A-T-C exchange. However, apart from the latter and recently issued strategy (Bozhhuuyuk et al, 2017), a concept known as a crossover unit (XU), scientists have failed to introduce well-defined, reproducible and validated guidelines for engineering modified NRPS (WO 2017/020983).

The XU concept provides three simple rules for designing, cloning and producing NRPs with the desired AA composition, structure and length: (I) use of A-T-C or A-T-C/E as XU, (II) fusion of XU in the CA linker at the conserved WNATE sequence, and (III) must consider the specificity of the downstream C domain. With XU, a naturally occurring NRPS assembly line was reconstructed; novel peptide derivatives and novel artificial NP-like peptides were generated. The drawback of the XU concept is that the natural downstream C domain specificity must be observed, which obviously limits its applicability, and the C domain specificity must be met at both the donor and acceptor sites. This disadvantage can be accepted if there are a large number of XUs with different downstream C domains. Due to these limitations, at least two XUs must also be exchanged to generate a new peptide derivative having an AA position different from the primary sequence of the wild-type (WT) peptide. However, a more flexible system is highly desirable.

In order to be suitable for a wide range of applications, the disadvantages of the XU concept have to be reduced. It is therefore an object of the present invention to establish a more convenient method of circumventing the specificity of the C domain. This approach would greatly reduce the number of NRPS building blocks necessary to produce or alter a particular peptide and would enable the creation of artificial natural product libraries with hundreds to thousands of entities for bioactive screening.

In a first aspect, the above mentioned problem is solved by a system for the production of a non-ribosomal peptide synthetase (NRPS), wherein the system comprises at least one (preferably two) NRPS exchange units (XU)_2.0) Each specific for a different or the same amino acid X used to assemble the NRPS, and wherein XU_2.0Comprising at least one partial condensation (C) domain or partial condensation/epimerization (C/E) domain selected from the group consisting of a condensation domain receptor site subdomain (C) specific for a given amino acid X_Asub) A condensation/epimerization domain receptor site subdomain (C/E) specific for a given amino acid X_Asub) A condensation domain donor site subdomain (C) specific for a given amino acid X_Dsub) And a condensation/epimerization domain donor site subdomain (C/E) specific to a given amino acid X_Dsub)。

In the context of the present invention, the name "X" refers to the amino acid specificity of any NRPS module or exchange unit of the invention (e.g. a partial or complete C or C/E domain, or an adenylated a domain). The specificity of such a domain or module may include specificity for one or more species of amino acids. For example, certain domains (e.g., a domains) are specific not only for one amino acid species, but also for two, three, four, or five or more different amino acid species. For example, the A domain is specific for a variety of amino acids that are also accepted by the downstream C or C/E domains, resulting in the production of several different peptides. Thus, the invention also includes such domains specific for a variety of amino acid species.

The system of the invention preferably comprises an XU_2.0Wherein XU_2.0Comprising at least one C or C/E domain consisting only of a partial sequence of said C or C/E domainPreferably consisting of only donor or acceptor sites of said C or C/E domain. In this respect, the XU of the present invention_2.0May comprise a partial C or C/E domain and optionally additionally a complete C or C/E domain comprising C or C/E donor and acceptor sites. Accordingly, the XU of the present invention_2.0Preferably characterized by the presence of at least one partial C or C/E domain. In some cases, a system according to the invention may be preferred, wherein the at least one XU_2.0Does not include fully assembled C or C/E domain. However, the system of the present invention may preferably comprise more than one XU_2.0And thus may include both XU's in which only part of the C or C/E domain is present_2.0Including XU comprising an intact C or C/E domain_2.0And, further, optionally, may comprise additional NRPS switching units that do not comprise part of the C or C/E domain. Thus, in some embodiments, the system of the present invention includes at least one XU_2.0In some embodiments, in combination with: (i) other XU or XUs_2.0And/or (ii) other prior art switching units, and/or (iii) other naturally occurring NRPS sequences. In addition to this, the system of the invention may also comprise a switching unit for or originating from the PKS.

For the present invention, the following definitions should be used:

the term "partial domain/partial domain" or "partial C or C/E domain" or similar expression refers to a nucleic acid sequence or protein sequence thereof that encodes an incomplete (not full-length) NRPS C or C/E domain. Thus, the term describes a C or C/E domain sequence that does not simultaneously contain donor and acceptor sites of the NRPS C or C/E domain.

"Module" refers to a group of domains. The plurality of components includes NRPSs. One or more of the polypeptides may comprise a module. The combination of modules can catalyze a series of reactions to form larger molecules. In one example, the module can comprise a C (condensation) domain, an a (adenylation) domain, and a peptidyl carrier protein domain.

For more structural information about the a-domain, C-domain, bi-domain, domain-domain interface and complete module, see Conti et al (1997), Sundlov et al (2013), Samel et al (2007), tannovic et al (2008), Strieker and Marahiel (2010), Mitchell et al (2012) and Tan et al (2015).

An "initiating module" refers to an N-terminal module that is capable of providing a first monomer to another module (e.g., an extension or termination module). In some cases, the other module is not the second module, but is a subsequent C-terminal module (e.g. nocardiin NRPS): in the case of NRPS, the starter module comprises, for example, an a (adenylation) domain and a PCP (peptidyl carrier protein) or T (thiolation) domain. The starter module may also comprise a starter C domain and/or an E (epimerization) domain. For PKS, possible initiation modules include an AT (acetyltransferase) domain and an Acyl Carrier Protein (ACP) domain. The starter module is preferably at the amino terminus of the polypeptide of the first module of the assembly line, and each assembly line preferably comprises one starter module.

"extension module" refers to a module in which a monomer is added to another monomer or polymer. The extension module may comprise a C (condensation), Cy (heterocyclic), E, C/E, MT (methyltransferase), a-MT (combined adenylation and methylation domain), Ox (oxidase), or Re (reductase) domain; a domain; or a T domain. The extension domain may further comprise additional E, Re, DH (anhydro), MT, NMet (N-methylation), AMT (aminotransferase), or Cy domains. In addition, the extension module may be of PKS origin, comprising individual domains (ketosynthase (KS), Acyltransferase (AT), Ketoreductase (KR), Dehydratase (DH), Enoyl Reductase (ER), thiolation (T)) linking the amino acid building block with the carboxylic acid building block.

"stop module" refers to a module that releases a molecule (e.g., NRP, PK, or a combination thereof) from an assembly line. The molecule may be released by, for example, hydrolysis or cyclization. The termination module may comprise TE (thioesterase), C_termOr a Re domain. The termination module is preferably at the carboxy terminus of the polypeptide of the NRPS or PKS. The termination module may further comprise additional enzymatic activity (e.g., an oligomerisation activity).

"Domain" refers to a polypeptide sequence or fragment of a larger polypeptide sequence that has one or more specific enzymatic activities (i.e., the 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 may comprise multiple domains. Multiple domains may form a module. Examples of domains include C (condensation), Cy (heterocyclic), A (adenylation), T (thiolation), TE (thioesterase), E (epimerization), C/E (condensation/epimerization), MT (methyltransferase), Ox (oxidase), Re (reductase), KS (ketosynthase), AT (acyltransferase), KR (ketoreductase), DH (dehydratase), and ER (enoyl reductase).

"non-ribosomally synthesized peptide," "non-ribosomal peptide," or "NRP" refers to any polypeptide that is not produced by ribosomes. NRPs may be linear, cyclic, or branched, and comprise proteinogenic, natural or unnatural amino acids, or any combination thereof. NRPs include peptides produced in an assembly line-like manner (a modular feature of the enzyme system, allowing stepwise addition of building blocks to form the final product).

"polyketide" refers to a compound comprising a plurality of keto units.

"non-ribosomal peptide synthetase" or "non-ribosomal peptide synthase" or "NRPS" refers to a polypeptide or series of interacting polypeptides that produces non-ribosomal peptides, which are thus capable of catalyzing peptide bond formation in the absence of ribosomal components.

"polyketide synthase" (PKS) refers to a polypeptide or series of polypeptides that produce polyketides. "changing amount" means changing the amount by increasing or decreasing. The increase or decrease may be 3%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more.

By "non-ribosomal peptide synthetase/polyketide synthase hybrid" or "hybrid of non-ribosomal peptide synthetase and polyketide synthase" or "NRPS/PKS hybrid" or "hybrid of NRPS and PKS" or "hybrid of PKS and NRPS" is meant an enzyme system comprising any domain or module from the non-ribosomal peptide synthetase and the polyketide synthase, resulting in a respective hybrid natural product.

By "altering the structure" is meant any change in chemical (e.g., covalent or non-covalent) bonds as compared to a reference structure.

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

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

In some embodiments, a system according to the present invention is preferred, wherein at least one XU_2.0Comprising a C or C/E acceptor domain and a C or C/E donor domain separated by one or more NRPS domains other than the C or C/E domain. In this embodiment, each individual unit consists of one C or C/E acceptor domain and one C or C/E donor domain separated by a non-C or non-C/E domain (preferably by an adenylated A domain). In other embodiments, the system is superiorOptionally, wherein each unit within the system comprises (i) only one C or C/E acceptor domain or only one C or C/E donor domain; or (ii) comprises only a C or C/E acceptor domain and only one C or C/E donor domain, wherein the two domains are spatially separated by one or more other NRPS domains.

Preferably, the XU of the present invention_2.0At least comprises the following structure: z^XY^XOr Y^XZ^x(ii) a Wherein Z is a partial C or C/E domain, preferably C (or C/E)_AsubOr C (or C/E)_Dsub ^XAnd wherein Y is any one or more of the same or different (or both) NRPS/PKS domains or modules having similar or the same specificity X, wherein X represents the amino acid specificity of a domain or module for one or more amino acid species. In addition, XU_2.0Other modules or domains may be included at the N or C terminus.

In other preferred alternative or additional embodiments of the present invention, at least one XU_2.0Having a structure according to any one of the following formulas:

a.C_Asub ^X-A^X-T-C_Dsub ^X,

b.C/E_Asub ^X-A^X-T-C/E_Dsub ^X,

c.C_Asub ^X-A^X-T-C/E_Dsub ^Xor is or

d.C/E_Asub ^X-A^X-T-C_Dsub ^X,

Other elements included in the system of the present invention may be selected from one or more of the following formulas:

e.C^X-A^X-T-C_Dsub ^X

f.C/E^X-A^X-T-C_Dsub ^X

g.C^X-A^X-T-C/E_Dsub ^X

h.C/E^X-A^X-T-C/E_Dsub ^X

i.C_Asub ^X-A^X-T-C^X

j.C/E_Asub ^X-A^X-T-C/E^X

k.C_Asub ^X-A^X-T-C/E^X

l.C/E_Asub ^X-A^X-T-C^X

m.C_start-A^X-T-C_Dsub ^X,

n.A^X-T-C_Dsub ^X,

o.C_start-A^X-T-C/E_Dsub ^X

p.A^X-T-C/E_Dsub ^X,

q.C_Asub ^X-A^X-T-TE

r.C/E_Asub ^X-A^X-T-TE

s.C_Asub ^X-A^X-T-C_term

t.C/E_Asub ^X-A^X-T-C_term

u.C_Asub ^X-Y^X-C_Dsub ^X

v.C/E_Asub ^X-Y^X-C/E_Dsub ^X

w.C_Asub ^X-Y^X-C/E_Dsub ^X

x.C/E_Asub ^X-Y^X-C_Dsub ^X

y.C^X-Y^X-C_Dsub ^X

z.C/E^X-Y^X-C_Dsub ^X

aa.C^X-Y^X-C/E_Dsub ^X

bb.C/E^X-Y^X-C/E_Dsub ^X

cc.C_Asub ^X-Y^X-C^X

dd.C/E_Asub ^X-Y^X-C/E^X

ee.C_Asub ^X-Y^X-C/E^X

ff.C/E_Asub ^X-Y^X-C^X

gg.C_start-Y^X-C_Dsub ^X,

hh.C_start-Y^X-C/E_Dsub ^X

ii.Y^X-C_Dsub ^X

jj.Y^X-C/E_Dsub ^X

kk.C_Asub ^X-Y^x-TE

ll.C/E_Asub ^X-Y^x-TE

mm.C_Asub ^X-Y^x-C_term

nn.C/E_Asub ^X-Y^x-C_term

wherein

C_Asub ^XOr C/E_Asub ^XIs C with amino acid specificity X_AsubOr C/E_AsubAnd X is specific for one or more amino acid species (e.g., for amino acids X1-Xn),

Y^Xrepresents one or a consecutive series of any NRPS or PKS domain or module (e.g.A, T, E, C, MT, A-MT, Cy) with amino acid specificity X,

A^xis an adenylation domain with an amino acid specificity X,

c or C/E is C or C/E with an amino acid specificity X,

C_Dsub ^Xor C/E_Dsub ^XIs C with amino acid specificity X_DsubOr C/E_DsubAnd is and

TE is a thioesterase domain, C_termAre terminal condensation domains, both of which are involved in NRPS regeneration.

In some embodiments of the invention, the system of the inventionXU_2.0Domains with promiscuous amino acid specificity may be included.

In some embodiments of the invention, the XU of the system of the invention_2.0Domains with different amino acid specificities may be included. Thus, the amino acid specificity of a part of the C or C/E subdomain may be different from that of another and/or A domain of the same unit. However, it is generally preferred that the XU of the present invention_2.0Comprising domains and modules having the same or at least overlapping amino acid specificity X. Of course, this does not exclude, on the contrary, that, for the purposes of the present invention, the system comprises a plurality of XUs with different amino acid specificities X_2.0Thereby assembling non-ribosomal peptides having different amino acid residues.

Abbreviations for the domains have been defined above. For the description of the invention, the marking_DsubShall refer to the C or C/E donor subdomain, and_Asubreference should be made to the C or C/E receptor subdomain. X shall denote the amino acid specificity of the corresponding domain.

In this context, it is noted that X may be selected from any naturally or non-naturally occurring amino acid. Also within the system, each unit in the system may have a different given amino acid X, or the same given amino acid X as one or more other exchange units in the system.

In other embodiments, each individual XU_2.0Preferably do not contain functionally assembled condensation (C) domains or condensation/epimerization (C/E) domains. Using the system of the present invention, only two separate XUs_2.0Will result in functional C or C/E domain.

In other embodiments, the system preferably includes at least two XUs_2.0When connected to each other, they form a first XU_2.0A partial C or C/E domain and a second XU_2.0A part of a C or C/E domain. Preferably, the first and second XUs_2.0Are different in kind, in other words, are preferably donor or acceptor domains.

Yet another aspect of the present inventionA preferred embodiment provides a system comprising at least two XUs_2.0Each of which is specific for a different amino acid X, preferably wherein each X is selected from any natural or unnatural amino acid.

According to the invention, the amino acid X is selected from a proteinogenic amino acid, a non-proteinogenic amino acid, a D amino acid or an L amino acid, or a non-standard amino acid, or a combination thereof.

Additionally, the present invention provides in some embodiments a system further comprising an XU_2.0Terminating and/or initiating unit, wherein the XU_2.0The starter unit comprises only the C or C/E domain donor subdomain (domain structure C-A)^X-T-C_Dsub ^XOr C-A^X-T-C/E_Dsub ^XSpecifically for incorporation of acyl units (fatty acids and derivatives thereof)) as starter units, and wherein the termination module comprises any one of the following: terminal condensed Domain (C)_term) An internal condensation (C) domain, an internal condensation and epimerization (C/E) bi-domain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (Re), oxidation (Ox) or Thioesterase (TE) domain.

For example, in certain embodiments, the system preferably includes an XU having any one of the following formulas_2.0A starting unit:

C_Asub-A^X-T-C_Dsub ^X,

C/E_Asub-A^X-T-C_Dsub ^X,

C_Asub-A^X-T-C/E_Dsub ^X,

C/E_Asub-A^X-T-C/E_Dsub ^Xor is or

C_start-A^X-T-C_Dsub ^X,

A^X-T-C_Dsub ^X,

C_start-A^X-T-C/E_Dsub ^XOr is or

A^X-T-C/E_Dsub ^X,

Wherein except C_AsubOr C/E_AsubOutside the domain, a complete C may be present_starOr C or no C domain of any type.

The system of the invention preferably has at least two, preferably three, four, or more XUs_2.0Which when put into sequence provides NRPS. The number of units is not subject to any limitation and will depend on the expected complexity of the system or the peptide to be produced. A system may include at least 2, 5, 10, 20, 30, 40, 50, 100, 500, or more units. And these units may have the same or different amino acid specificities X.

Any two XUs of the present invention_2.0Can be assembled in the C or C/E domain donor and acceptor site between the ring region. The loop region is the region of the C or C/E domain that connects the two halves of the C or C/E domain's pseudo-dimeric structure (Keting et al, 2002; Samel et al, 2007; Tanovic et al, 2008; Blouff et al, 2013). Preferably, the loop region is between amino acids 261 and 271-named according to the crystal structure of the TycC 5-6T-C double domain (PDB-ID:2JGP) -for the C or C/E domain.

In some other embodiments, the systems of the invention may include at least one XU having a modified domain_2.Such as E, MT or Ox or other modifying domain.

In some embodiments, the system of the invention may be one in which each XU_2.0A system encoded by a nucleic acid sequence. Thus, the system is a system of nucleic acid constructs. In other embodiments, the system is a system of amino acid or protein sequences, such as NRPS.

For each amino acid X, it is further preferred that the following XU comprises the formula_2.0The system of each of: c_Asub ^X-A^X-T-C_Dsub ^X、C/E_Asub ^X-A^X-T-C/E_Dsub ^X、C_Asub ^X-A^X-T-C/E_Dsub ^X、C/E_Asub ^X-A^X-T-C_Dsub ^X。

The system according to the invention may comprise said XU for two or more amino acids X_2.0Preferably for a plurality of amino acids, preferably wherein for each natural amino acid the system comprises one of the following: c_Asub ^X-A^X-T-C_Dsub ^X、C/E_Asub ^X-A^X-T-C/E_Dsub ^X、C_Asub ^X-A^X-T-C/E_Dsub ^XAnd C/E_Asub ^X-A^X-T-C_Dsub ^X。

In another aspect of the present invention, there is provided a method for producing a peptide, the method comprising the step of expressing or assembling an NRPS assembled using the system according to the present invention.

In another aspect of the invention, there is provided a library of nucleic acid molecules, wherein the library comprises at least two or more nucleic acid constructs, each nucleic acid construct encoding an XU_2.0And each has the same or different amino acid specificity, and wherein the XU_2.0Comprising at least one partially condensed (C) domain or partially condensed/epimerized (C/E) domain selected from: condensation domain receptor site subdomain with amino acid specificity X (C)_Asub) Condensation/epimerization domain receptor site subdomains (C/E) with amino acid specific X_Asub) A condensation domain donor site subdomain having an amino acid-specific X (C)_Dsub) And a condensation/epimerization domain donor site subdomain (C/E) with an amino acid specific X_Dsub). In certain embodiments, the XU_2.0May not contain fully assembled C or C/E domains.

The libraries of the invention may comprise a library encoding at least one XU_2.0Nucleic acid construct of a termination and/or initiation unit, wherein said XU_2.0The starter unit comprises only the C or C/E domain donor subdomain (domain structure C-A)^X-T-C_Dsub ^XOr C-A^X-T-C/E_Dsub ^XSpecially adapted for incorporation of acyl units (fatty acids and their esters)Derivative) as a starting unit, and wherein the termination module comprises any one of: terminal condensed Domain (C)_term) An internal condensation (C) domain, an internal condensation and epimerization (C/E) bi-domain, a cyclization (Cy) domain, an epimerization (E) domain, a reduction (Re), oxidation (Ox) or Thioesterase (TE) domain.

Libraries of the invention may be preferred, wherein each XU_2.0Encoded by a separate nucleic acid construct. Preferably, the library comprises XU of the system described herein before_2.0。

A method for producing NRPS, the method comprising the steps of: assembling at least two NRPS exchange units (XU) each specific for a different or the same amino acid X_2.0) And wherein XU_2.0Comprising at least one partially condensed (C) domain or partially condensed/epimerized (C/E) domain selected from: condensation domain receptor site subdomain (C) specific for a given amino acid X_Asub) A condensation/epimerization domain receptor site subdomain (C/E) specific for a given amino acid X_Asub) A condensation domain donor site subdomain (C) specific for a given amino acid X_Dsub) And a condensation/epimerization domain donor site subdomain (C/E) specific to a given amino acid X_Dsub) (ii) a Wherein the XU_2.0Does not contain fully assembled C or C/E domain. Preferably, the NRPS is assembled from the nucleic acid constructs of the library of the invention and expresses said NRPS.

Further, there is provided a method for producing a non-ribosomal peptide having a specific sequence, the method comprising assembling an NRPS according to the method for producing an NRPS of the present invention, wherein the NRPS is composed of XU having a specificity according to the peptide to be produced_2.0The sequence of (a).

Then, a further aspect of the invention provides a biological cell comprising a nucleic acid construct as described before in the context of the library of the invention. Thus, as an aspect, the present invention may also provide a library of biological cells (strains), wherein each biological cell (strain) comprises the nucleic acid construct of the library.

The non-ribosomal peptides of the invention can be linear or cyclic peptides. When the peptide is cyclic, the NRPS preferably comprises a cyclization domain (i.e., Thioesterase (TE), reductase (Red), terminal condensation (Cterm), or C/E domain) in the termination block. The non-ribosomal peptides produced according to the description of the invention are preferably non-naturally occurring non-ribosomal peptides.

Thus, another aspect of the present invention relates to a method for modifying a provided NRPS coding sequence, the method comprising the steps of: providing an NRPS coding sequence, preferably a full-length NRPS coding sequence, e.g., a wild-type or naturally occurring NRPS coding sequence; and XU as defined by the invention_2.0Introducing said NRPS coding sequence, preferably by said XU_2.0The encoded domains replace and/or complement the respective domains of the provided NRPS. The substitution preferably modifies the sequence or structure of the peptide product produced by the NRPS. XU of the invention_2.0May be used to introduce additional amino acid(s), remove amino acid(s), substitute amino acid(s), and/or alter peptide structure (cyclic or linear peptides). The XU of the invention in the process is preferably carried out by_2.0Introduction of (2): will code XU_2.0XU of the donor or acceptor site of part of the C or C/E domain of_2.0The fragments are fused to the respective ends of the donor or acceptor site of the provided NRPS coding sequence, thereby obtaining the introduced XU_2.0And chimeric C or C/E domains of NRPS provided.

The invention will now be further described in the following examples with reference to the figures and sequences, without however being limited thereto. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety. In the drawings:

FIG. 1: modulation of C domain substrate specificity. (a) The C domain excised from the T-C double domain TycC5-6 (PDB-ID:2JGP) of Brevibacillus brevis casein peptide synthetase (TycC) of Brevibacillus brevis (Brevibacillus brevis) is shown as N-terminal (yellow) and C-terminal (blue) in a band diagram (top). In the frame: c_Dsub–C_AsubEnlargement of the joint, bar representationContributing linker AA, red label fusion site. Bottom: c from Photorhabdus and Xenorhabdus_Dsub–C_AsubSequence logo (logo) of linker sequence. (b) Schematic representation of WT GxpS, recombinant NRPS-1 and NRPS-2, and the corresponding peptide yields obtained from three replicates. For peptide nomenclature, the standard one-letter AA code and lower case letters of D-AA are used. (c) Schematic representation of BicA with modules and switching units (XU and XU) highlighted_2.0). All A domains are assigned specificities. For domain assignment, the following notation is used: a (big circle), T (rectangle), C (triangle), C/E (diamond), TE (C end small circle).

FIG. 2: de novo design of recombinant NRPS for peptide production. (a) The resulting recombinant Gxps (NRPS-3-5) and the corresponding number of GameXPeptide derivatives 1, 3, 6 and 7 determined in three replicates. (b) Recombinant NRPS-6 was synthesized 8. The building blocks are gram positive. Bottom: the color code of the NRPS used to construct the base block (see fig. 8 for details). For the assignment of domain symbols, see FIG. 1.

FIG. 3: NRPS initiates swapping of cells. Schematic representation of recombinant GxpS (NRPS-7-9) and the corresponding peptide yields obtained from three replicates. For peptide nomenclature, the standard one-letter AA code and lower case letters of D-AA are used. For the assignment of domain symbols, see FIG. 1. Bottom: the color code of the NRPS used to construct the base block (see fig. 8 for details).

FIG. 4: creating functionalized heterogeneous tetrapeptide derivatives. Schematic representation of WT XtpS, recombinant NRPS-10 and the corresponding peptide yields obtained from three replicates. For peptide nomenclature, the standard one-letter AA code and lower case letters of D-AA are used. For the assignment of domain symbols, see FIG. 1. Bottom: the color code of the NRPS used to construct the base block (see fig. 8 for details).

FIG. 5: targeted randomization of GxpS at position 3. Schematic representation of all possible recombinant NRPSs (upper left) and the corresponding a domain specificities (lower left). The detected peptides (solid line) and corresponding peptide yields (upper right) obtained from three replicates. The dashed line represents an unidentified GxpS derivative. For peptide nomenclature, the standard one-letter AA code and lower case letters of D-AA are used. For the assignment of domain symbols, see FIG. 1. Bottom: the color code of the NRPS used to construct the base block (see fig. 8 for details).

FIG. 6: a randomized library of positions 1 and 3 of GxpS was created. Schematic representation of all possible recombinant NRPSs (upper left) and the corresponding a domain specificities (lower left). The detected peptides and corresponding peptide yields obtained from three replicates (right). For peptide nomenclature, the standard one-letter AA code and lower case letters of D-AA are used. For the assignment of domain symbols, see FIG. 1. Bottom: the color code of the NRPS used to construct the base block (see fig. 8 for details).

FIG. 7: randomization of adjacent positions. (a) The crystal structure of TycC6(PDB-ID:2JGP) is subdivided into an N-terminal subdomain (grey) and a C-terminal subdomain (light red). The subdomain linker is highlighted in red and the target region for homologous recombination in yeast (I253-F265) is highlighted in green (39 nucleotides). The consensus sequence used to generate library 3 is shown in the lower right. (b) Schematic representation of all possible recombinant NRPSs (upper left) and the corresponding a domain specificities (lower left). The detected peptides and corresponding peptide yields obtained from three replicates (right). For peptide nomenclature, the standard one-letter AA code and lower case letters of D-AA are used. For the assignment of domain symbols, see FIG. 1. Bottom: the color code of the NRPS used to construct the base block (see fig. 8 for details).

FIG. 8: schematic of all NRPSs used in this work. GxpS, BicA, XtpS, HCTA, PaxB, KolS, AmbS from X.miraniensis have been previously described_mirAnd AmbS from x_ind. For GarS producing garganturanin, see Genbank accession number PRJNA 224116. For xenolinicin-like synthetases, see Genbank accession number PRJNA 328553. For XeyS producing xindeyrin, see Genbank accession number PRJNA 328572.

Examples