Methods of enhancing production of metabolites in bacteria

Abstract

The invention relates to genetically altered bacteria with autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) proteins or NLR-like proteins, and metabolites produced by such bacteria. The invention also relates to methods of producing such genetically altered bacteria.

Description

[0001] Methods of enhancing production of metabolites in bacteria

[0002] FIELD OF THE INVENTION

[0003] The invention relates to methods of producing metabolites in bacteria, the method comprising expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) in the bacteria. The invention also relates to bacteria expressing an autoactive NLR protein. The invention also relates to methods of identifying novel metabolites.

[0004] BACKGROUND OF THE INVENTION

[0005] Microbial secondary metabolites, also known as natural products, are a valuable source of numerous medical agents, including antimicrobial, immunosuppressive, anti-cancer and anti-inflammatory agents. One notable example is doxorubicin that belongs to a class of chemotherapy drug known as the anthracyclines that are extracted from Streptomyces peucetius bacterium. Doxorubicin slows or stops the growth of cancer cells by inhibiting the enzyme called topoisomerase 2.

[0006] However, there exists longstanding problems in harvesting microbial secondary metabolites, including obtaining sufficient yield levels, the need for specific media for the production of particular metabolites and limits of when in bacterial development these compounds are produced.

[0007] The genes involved in the production, transport, and regulation of secondary metabolites are often found adjacent to one another in microbial genomes, forming biosynthetic gene clusters (BGCs). Regulation of these gene clusters has been associated with bacterial developmental processes, sporulation, responses to antibiotics, and other signals and stressors. Many BGCs are silent under traditional laboratory conditions, making them difficult to functionally characterize, consequently, leaving a host of potentially important natural products undiscovered.

[0008] Given the vast current and potential applications of microbial secondary metabolites, there is a need to increase the yield and / or simplify production. In addition to this, given that many BGCs are silent under normal laboratory conditions there is a need to find ways of activating these gene clusters to facilitate the discovery of new secondary metabolites that could have important applications and be of economic value. The present invention addresses these needs.

[0009] SUMMARY OF THE INVENTION

[0010] We have identified that synthetically activating nucleotide binding oligomerisation domain-like receptors (NLR) and NLR-like proteins, such as AfsR and other AfsR-like proteins, triggers the expression of biosynthetic gene clusters or BGCs that are under NLR control. This is a novel strategy that not only boosts the yields of commercially and therapeutically valuable compounds produced by bacteria, such as Streptomyces, but secondly decouples the synthesis of these molecules from development- and nutrientdependent regulation. This in turn further simplifies not only the process, but in turn reduces the cost associated with producing commercially valuable metabolites in microorganisms.

[0011] Specifically, we have demonstrated that introducing autoactivating mutations into NLR and NLR-like proteins of microorganisms, and particularly in the nucleotide-binding domain or NOD, significantly increases production of these commercially valuable metabolites.

[0012] In one aspect of the invention there is provided a genetically altered bacteria, wherein said microorganism expresses an autoactive nucleotide-binding oligomerisation domain- like receptor (NLR) protein or NLR-like protein.

[0013] In another aspect of the invention, there is provided a method of producing a metabolite in bacteria, the method comprising expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein in the microorganism.

[0014] In another aspect of the invention, there is provided a method for identifying and / or selecting bacteria that will have increased secondary metabolism compared to a wildtype or control bacterium, the method comprising detecting in said bacteria at least one mutation in at least one NLR gene. In another aspect of the invention, there is provided a method of identifying one or more novel metabolites, the method comprising identifying a BGC and at least one associated NLR or NLR-like protein in a bacteria, expressing an autoactive variant of the BGC- associated NLR or NLR-like protein as defined herein and screening for the presence of one or more novel metabolites.

[0015] In another aspect of the invention, there is provided a metabolite obtained or obtainable by the method described herein.

[0016] The following embodiments apply to all aspects of the invention.

[0017] In one embodiment, the bacteria comprises at least one mutation in at least one nucleic acid sequence encoding a NLR or NLR-like protein. Preferably, the at least one mutation leads to autoactivation of the NLR or NLR-like protein.

[0018] In one embodiment, the NLR or NLR-like protein comprises at least one nucleotide oligomerisation domain (NOD), wherein the genetically altered microorganism has at least one mutation in the nucleotide oligomerisation domain. Preferably, the nucleotide oligomerisation domain comprises at least a hhGRExE motif, RNBS-B motif, GLPL motif, or MHD motif, and wherein the genetically altered bacteria has at least one mutation in at least one of the hhGRExE motif, RNBS-B motif, GLPL motif and MHD motif.

[0019] In one embodiment, the mutation is selected from at least one or any combination of a. a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149. More preferably, the substitution is a substitution from an R to a Q; and / or b. a mutation, preferably a substitution at position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23. More preferably, the substitution is a substitution from an I to a N or a substitution at position 1 of the GLPL motif, for example as defined in one of SEQ ID NO 150. More preferably, the substitution is a substitution from a G to a R; and / or c. a mutation, preferably a substitution at position 2 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151. More preferably, the substitution is a substitution from H to G; and / or d. a mutation, preferably a substitution at position 3 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151. More preferably, the substitution is a substitution from D to V.

[0020] In another embodiment, the one or more mutation is selected from one or more of the following, or any combinations thereof: a. a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and / or b. a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and / or c. a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; and / or d. a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V.

[0021] Preferably, the mutation is a substitution.

[0022] In one embodiment, the sequence of the NLR or NLR-like protein is selected from SEQ ID NO: 35 to 53 or a functional variant or homologue thereof. In another embodiment, the nucleic acid sequence that encodes a NLR or NLR-like protein is selected from SEQ ID NO: 54 to 72.

[0023] In another embodiment, the bacteria expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutated NLR or NLR-like protein. In one example, the nucleic acid sequence encodes a mutated NLR or NLR-like protein as defined in SEQ ID NO: 95 to 108, and wherein preferably the nucleic acid sequence is operably linked to a regulatory sequence.

[0024] In another embodiment, the bacteria expresses a nucleic acid construct comprising a nucleic acid sequence encoding a. a mutated NLR or NLR-like protein, preferably the mutated NLR or NLR- like protein of claims 4 to 10; or b. a NLR or NLR-like protein as defined in one of SEQ ID NO: 35 to 53 or a functional variant or homologue thereof; or c. an effector domain linked to a protein capable of oligomeristaion, preferably YFP, wherein preferably the nucleic acid sequenc encodes an effector domain as defined in one of SEQ ID NO: 127 to 145 or a functional variant or homologue thereof. In one embodiment, the method comprises culturing the bacteria and extracting the metabolite, wherein the metabolite is extracted at any developmental stage of the microorganism and / or following culture in any form of media, preferably a simple media. In another embodiment, the bacteria is cultured in solid or liquid media. In one embodiment, the method increases the level of metabolite production in the bacteria compared to the level of metabolite production in a wild-type or control bacteria. Preferably, the NLR or NLR-like protein is selected from AfsR, KY5 AfsR, AfsR-sl, AfsR- sv, AfsR-p, AfsR-g, AfsR-pr, AfsR-L, PteR, Orf2, AveR, NysRI, CdaR, GdmRI, bfmR, pimR, PolY, SanG, NemR, MycG1, AstG1, StaR, RebR, NeoR, SCO_0877, MilR, VemR, GdmRII, ttmRI, BafG, HbmRII, amphRI, snorA, fdmRI, pnxR2, sanR1, pokR1, llpRIV, gilS, OzmU, scnRI, MgsA, PlmR1, pnR2, pnR1, lmR1, FilR, LcmRIII, ChxA, SCAB1371, SgnR, ORF4, lct22, lct23, Orf15, Orf17, sky44, Orf(+2), AcoK, GutR, RslR3 , MalT, BpdS, PteF, CphR, PtnR, Asm18, orf18, SalRI, SalRII, AmphRII, FscRII, FscRIV, AbmH, TrdH, cvm7P, Kmy4, IdmG, OlmRI, Tmn5, CalR2, PolR, MlaH, BecH, cvm7, ttmRII, aas1, ttmRIII, MonH, SCO7173, PikD, NbmM, TmcN, fkbN, SlnR, IdnR1 , NysRIII, SamR0484, RapH, AmphRIII, FscRIII, Hgc1, Div8, Cuv20, OlmRII, Tei15, LipReg4, TvaN, TtnG, vioT, NysRII, AtmR, LooR, AmphRVI, RevU, AcuR1, AcuR2, AcuR3, SelRI, SelRII, SelRIII, SelRV, SelRVI, CppRI, CppRII, CppRIII, CppRV and ClaR and ClaR. More preferably, the NLR or NLR-like protein is AfsR. In one embodiment, the bacteria is a bacterium. In one embodiment, the bacteria is selected from the phyla Actinomycetota, Pseudomonadota, Bacillota or Myxococcota, most preferably Actinomycetota, and preferably is selected from the genus Streptomyces. In one embodiment, the bacteria is selected from S. coelicolor, S. peucetius, S. rapamycinus, S. nodosus, S. avermitilis, S. griseus, S. bingchenggensis, S. scabiei, S. cattleya, S. plantensis, S. roseosporus, S. ribosidificus, S. fradiae, S. kanamyceticus, S. niveus, S. orientalis, S. garyphalus, S. lincolnensis, S. pristinaespiralis, S. virginiae, S. vinaceus, S. capreolus, S. noursei, S. aureofaciens, S. fradiae, S. venezuelae, S. clavuligerus, S. griseus, S. rochei, S. hygroscopicus, S. acidiscabies, S. lomondensis, S. lividans and S. cacaoi.

[0025] Preferably, the metabolite is a secondary metabolite or specialised metabolite.

[0026] In one embodiment, the metabolite is selected from an antibiotic, antibiotic precursor, antibacterial metabolite, anti-cancer drug, pigment, growth hormone, immunosuppressant, agrochemical, antiviral or anti-fungal.

[0027] In one embodiment, the metabolite is selected from actinorhodin, pikromycin, doxorubicin, a-factor, pristinamycin i, pristinamycin ii, butenyl-spinosyn, filipin, thaxtomin a, lomofungin, avermectin, nystatin, calcium-dependent antibiotic, geldanamycin, ebafilomycin, pimaricin, natamycin, polyoxin, nikkomycin, nemadectin, mycotrienin (myc) members, ansatrienins (hydroxymycotrienin a and thiazinotrienomycin g), staurosporine, rebeccamycin, neomycin, milbemycin, venemycin, tetramycin, herbimycin pks, amphotericin, nogalamycin, fredericamycin, fd-594, a-74528, polyketomycin, lysolipin, gilvocarcin (v), oxazolomycin, natamycin, iso-migrastatin, migrastatin, dorrigocin, phospholactomycin, lactomycins a-c, lucensomycin, cycloheximide pyochelin, aranciamycin, lactonamycin, salinomycin, concanamycin a, skyllamycin a, himastatin, rishirilide(s), pentamycin, validamycin a, ansamitocin, ahba, candicidin, neoabyssomicin, tirandamycin, 5s clavam, kendomycin b, indanomycin, oligomycin, tetronomycin, ml- 449, be-14106, calcimycin (a23187), clavam, aminoansamycins, monensin pks, methymycin, neomethymycin, narbomycin, pikromycin, tautomycin, fk506, incednine, stambomycin, xcp, rapamycin, amphotericin b, hygrocins, divergolides, cuevaene a, teicoplanin, a-lipomycin, thioviridamide, tautomycetin, viomycin, at2433, loonamycin, reveromycin a, aculeximycin, selvamicin, npp, YM3163-A, avenolides, prodiginine, CDA, WS599B, holomycin, daunorubicin, daptomycin and cladoniamides.

[0028] In one embodiment, the metabolite may be doxorubicin. Alternatively, the metabolite is actinorhodin.

[0029] BRIEF DESCRIPTION OF THE FIGURES The invention is further described in the following non-limiting figures.

[0030] Figure 1. Streptomyces AfsR is an NLR: NLR signalling proteins are comprised of three characteristic domains; a sensor domain comprising of SSFRs, a central NOD (belonging to the NB-ARC / NACHT ATPase family) and an N-terminal effector domain responsible for propagating the signal downstream. This domain organization is most well-characterised in animal and plant NLRs but is conserved in prokaryote proteins such as AfsR in Streptomyces coelicolor. Domain organization represents annotations generated using InterProScan.

[0031] Figure 2. AfsR contains conserved activation-related residues: AfsR homologues share conserved activation-related residues, (a) Examples of characterised AfsR homologues shown to positively regulate the synthesis of various specialized metabolites (box), (b) Autoactivity-associated residues have previously been identified in the NOD of SINRC1 . Protein alignments of the SINRC1 NOD and that of Streptomyces AfsR homologues identify 4 of these residues that are conserved almost universally across the AfsR family. Black shading represents conserved residues which have previously been mutated to induce autoactivity in plant NLRs (12), grey shading represents a residue which in the wild-type allele is the amino acid previously introduced at that site in other NLRs to induce autoactivity. Arrows represent mutations taken forward in generating autoactive variants of S. coelicolor and S. peucetius AfsR. Sequence information for the listed AfsR homologous can be found in Table 4.

[0032] Figure 3. Introduction of putative autoactive mutations induces strong actinorhodin production: S. coelicolor strains encoding the putative autoactive versions of AfsR were streaked out onto solid TSA media and imaged every 24 hours. Top row indicates the combinations of amino acid substitutions in AfsR, M145 is the unmodified wildtype strain, pSS170 EV is the wildtype strain with an empty plasmid. Strains containing the autoactive residues began to display strong actinorhodin production after 48 hours, whilst the wild-type strains begin to turn purple after 72 hours. By 120 hours all strains exhibit a strong actinorhodin phenotype resulting in a dark coloration of the agar, although the phenotype of autoactive strains still appears stronger. Images are representative of biological duplicates. Figure 4. Autoactivity removes media-dependency and has a global effect on secondary metabolite production: Strains containing autoactive afsR alleles were streaked onto DNA media and imaged after 120 hours (a). Actinorhodin production was negligible in the wild-type Streptomyces coelicolor M145 and empty plasmid (EV)- containing strains even at this timepoint. However, the strain containing an additional wild-type allele exhibits some induction of actinorhodin production, which is still reduced compared to autoactive alleles. To quantify this, we grew the strains in the liquid equivalent media (DNB) and measured actinorhodin production via spectrophotometry (b). The production was stronger over the first 96 hours in an autoactive AfsR-expressing strain compared to the wild type, empty vector or wild type AfsR-containing strains. Individual datapoints for each of the 4 biological replicates are displayed along with the standard error. AfsR acts as a global regulator, regulating the production of red- pigmented prodiginines alongside synthesis of actinorhodin. (c) Introduction of an autoactive AfsR allele in a mutant background lacking actinorhodin production demonstrated an increase in prodiginine production, visible by an increase in red- pigment production and secretion. In particular, in R438Q / H603G each individual exconjugant is redder / pinker giving an overall stronger red / brown colouration compared to the wild type.

[0033] Figure 5. Autoactive residues are conserved across bacterial NLR-like proteins: In order to demonstrate the applicability of our approach to further NLR-like regulators, we generated protein sequence alignments. This analysis included additional regulators of primary and secondary metabolism also containing (a) the same domain architecture as AfsR, (b) lacking the long series of TPR repeats or (c) belonging to the domain architecture associated with the Large ATP-binding regulators of the LuxR family (LAL) family of regulators. All alignments were performed using MUSCLE and standard parameters, with black shading representing conserved residues previously mutated to induce autoactivity in other NLRs whilst grey shading represents that the residue in the wild-type allele is the amino acid previously introduced at that site in other NLRs to induce autoactivity.

[0034] Figure 6. Autoactive AfsR does not require an additional copy of wild-type AfsR for function in vivo. Analysis of actinorhodin production in an afsR deletion strain (DafsR) compared to the mutant complemented with either the wild-type afsR allele (DafsR +afsR) or an autoactive afsR allele (DafsR +afsR H603D / D604V). Actinorhodin production was assessed following the growth of the strains on (a) DNA for 5 days and (b) DNB for 7 days. Data presented in (b) shows the mean absorbance A640 per gram cell pellet + / - standard error obtained from five biological replicates for each strain.

[0035] Figure 7. Autoactive SpAfsR induces anthracycline production in S. peucetius. (a) S. peucetius strains containing the pKF351 empty vector (EV), an additional wild-type afsR allele (AfsR) or one of the three autoactive afsR variants were grown in ISP4 media. Anthracycline metabolite production was analysed at an absorbance of 495 nm and normalized to the corresponding wet pellet mass (in g). Shown are the mean absorbance + / - standard error from three biological replicates per strain, (b) Calculated fold-change of anthracycline production in the same strains based on absorbance measurements shown in (a).

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0038] The practice of the present invention will employ unless otherwise indicated, conventional techniques of microbiology, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.

[0039] As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences.

[0040] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

[0041] The aspects of the invention involve recombinant DNA technology.

[0042] For the purposes of the invention, “genetically altered bacteria”, “genetically modified bacteria” or “mutant bacteria” are bacteria that have been genetically altered compared to the naturally occurring wild type (WT) bacteria to express an autoactive NLR or NLR- like protein.

[0043] Nucleotide-binding oligomerisation domain-like receptors (NLRs) are a well-studied protein family with crucial roles in monitoring the cytoplasm for infection in both plants and animals. NLRs are defined by a tripartite domain organisation comprising an N- terminal effector domain, a central NTPase domain, also referred to as nucleotide binding and oligomerisation (NOD) domain and a C-terminal sensor domain that contains a series of superstructure forming repeats (SSFRs). The NOD module is either an NTPase of the NB-ARC (nucleotide-binding adaptor shared by APAF-1 , certain R gene products, and CED-4) or a NACHT (neuronal apoptosis inhibitory protein, MHC class II transcription activator, HET-E incompatibility locus protein from Podospora anserina, and telomerase-associated protein 1), whereas the SSFRs can be formed by domains such as leucine-rich repeats (LRRs), WD repeats, armadillo (ARM) repeats, ankyrin (ANK) repeats, or tetratricopeptide repeats (TPRs).

[0044] NLRs act as molecular switches, under basal conditions they exhibit an inactive resting state characterised by ADP binding to the NOD and autoinhibitory contacts within their structure. Upon recognition of a specific ligand via the SSFR domain, a conformational change occurs allowing a nucleotide exchange and binding of ATP which is accompanied with a relaxation of autoinhibitory contacts, resulting in an active state of the NLR. Active NLRs monomers can then oligomerise into multimeric protein complexes, also termed inflammasomes / resistosomes, via their NODs leading to downstream signalling through a diverse range of effector domains. In plants, NLRs recognise specific effector proteins secreted into the plant cell by pathogens The signalling induced upon activation of NLRs canonically results in localised programmed cell death to prevent the further spread of the infection. A similar mode-of-action is present in animal NLRs, although recognition is of more generic infection-associated molecular patterns with a resulting output of either localised cell death (pyroptosis) or transcriptional reprogramming.

[0045] NLR-like proteins are proteins that have a very similar domain organisation to canonical NLRs, for example, they may have a P-loop ATPase but not one that is predicted to be a STAND class ATPase. Alternatively, NLR-like proteins are NLR proteins that do not have the long series of superstructure-forming repeats that are found in traditional NLRs.

[0046] The ‘Antiviral STAND (signal transduction ATPases with numerous domains)’ (A vs) family of NLRs (containing variable effectors, a NACHT-like NOD and tetratri co peptide repeat SSFRs) are broadly distributed across prokaryotes and upon recognition of specific components of the infecting phage oligomerise into complexes reminiscent of the plant resistosome. Furthermore, a TIR (Toll / interleukin-1 receptor) effector domaincontaining NLR encoded in the P4 prophage of Klebsiella pneumoniae has been shown to provide broad antiphage immunity when expressed in E. coli. While this begins to establish an antiphage role, the role of NLR-like proteins as regulators of other processes, particularly specialised metabolism, has not been characterised. Specialised metabolism (also referred to as secondary metabolism) refers to metabolic processes resulting in molecules, which are advantageous but not essential for viability. Specialised metabolism represents a major source for molecules utilised in medicine and industry, with this exploitation of specialised metabolism particularly well-studied in the Streptomyces genus of actinobacteria. Streptomyces produce approximately 2 / 3 of clinical antibiotics along with compounds possessing a diverse range of anticancer, immunosuppressant and herbicidal biological activities.

[0047] As used herein an “NLR” or “NLR-like” protein may refer to a protein with at least one conserved domain selected from a sensor domain, an effector domain and a NOD. Preferably the protein has all three domains. In one embodiment, the sensor domain may comprise a nucleic acid sequence that encodes a sensor domain as defined in SEQ ID NO: 109 to 126, or a functional variant or homologue thereof.

[0048] In one embodiment, the effector domain may comprise a nucleic acid sequence that encodes an effector domain as defined in SEQ ID NO: 127 to 145, or a functional variant or homologue thereof.

[0049] In one embodiment, the NOD domain may comprise a nucleic acid sequence that encodes a NOD as defined in SEQ ID NO: 73 to 91 or 94 or a functional variant or homologue thereof.

[0050] The NOD domain may also comprise at least four highly conserved regions or domains. These are: the hhGRExE motif (two hydrophobic residues followed by glycine-arginine- glutamic acid then any amino acid then another glutamic acid), RNBS-B (Resistance Nucleotide Binding Site B), GLPL (glycine-leucine-proline-leucine) and the MHD (Methionine-Histidine-Aspartate motif). Accordingly, in one embodiment, the NLR or NLR-like protein may be defined as having at least one, at least two, at least three, but preferably all of four of the above domains.

[0051] In one embodiment, the hhGRExE motif may comprise one of the following amino acid sequences or a functional variant or homologue thereof: FTGRAAFV (SEQ ID NO: 27); FTGRSGFV (SEQ ID NO: 28), FTGRENLA (SEQ ID NO: 29), FTGRSSFV (SEQ ID NO: 30), FTGRVSFV (SEQ ID NO: 31), FTGRSAFV (SEQ ID NO: 32), FTGRTSFV (SEQ ID NO: 33) or FSGQQAEL (SEQ ID NO: 34), FTGRDEEL (SEQ ID NO: 146), FTGRDAFV (SEQ ID NO: 147), FTGRASFV (SEQ ID NO: 148).

[0052] In one embodiment, the RNBS-B motif may comprise one of the following amino acid sequences or a functional variant or homologue thereof: GCAALITSR (SEQ ID NO: 17), GCAALVTAR (SEQ ID NO: 18), GCAALVTSR (SEQ ID NO: 19), or TCGVIATGR (SEQ ID NO: 20), DCLAIVTSR (SEQ ID NO: 149).

[0053] In one embodiment, the GLPL motif may comprise one of the following amino acid sequences or a functional variant or homologue thereof: FLPLAIRIA (SEQ ID NO: 21), FLPLAIRVA (SEQ ID NO: 22) or RLPLALRIV (SEQ ID NO: 23), GLPLALRIT (SEQ ID NO: 150).

[0054] In one embodiment, the MHD motif may comprise one of the following amino acid sequences or a functional variant or homologue thereof: YHDLVRLY (SEQ ID NO: 24), FHDLIRAY (SEQ ID NO: 25) or FHDLVRLY (SEQ ID NO: 26), FHDLVRTY (SEQ ID NO: 151).

[0055] In Streptomyces and other bacteria, genes encoding the enzymes required for the biosynthesis of these specialised metabolites, along with corresponding transporters and regulators (including NLRs), are usually grouped together within so-called ‘biosynthetic gene clusters’ (BGCs). Sometimes, BGC expression can also be controlled by global regulators that are encoded outside of the BGC itself. Expression of many BGCs is often not optimised under standard laboratory conditions, resulting in either suboptimal production of known molecules or BGCs where none of the desired product is generated. These so-called “silent” BGCs represent a treasure trove for novel antibiotics that are urgently needed to treat infection caused by antimicrobial-resistant pathogens. In Streptomyces, the NLR AfsR functions as a global regulator of specialised metabolism. AfsR conforms to the NLR definition through the presence of a central NB-ARC NOD, a series of C-terminal tetratricopeptide repeats and an N-terminal bacterial transcriptional activator (BTAD) effector domain. As an NLR, the likely mechanism for AfsR involves recognition of an unidentified signal, resulting in its activation from an inactive state and subsequent DNA binding and transcriptional activation of downstream genes required for specialised metabolism through the activity of its N-terminal BTAD. In S. coelicolor, AfsR has been demonstrated to positively regulate the production of at least three canonical antibiotics made under standard laboratory conditions (including actinorhodin, prodigininesand the calcium-dependent antibiotic). However, despite its role as a central regulator in a model organism with a clear phenotype, how AfsR functions at the molecular level is still poorly understood.

[0056] Here we describe a novel experimental strategy with the potential to increase the production of commercially valuable compounds and to discover novel molecules with beneficial activities through the constitutive activation of prokaryote NLRs functioning as transcriptional regulators. This is of particular importance in the current age of increasing antimicrobial resistance and the need for novel experimental strategies to expand our repertoire of antibiotic chemistry. As an example, we demonstrate that the global regulator AfsR from S. coelicolor is an NLR (Fig. 1) and utilise this insight to generate a constitutively active version by introducing point mutations into its NOD domain (Fig. 2). Introducing these constitutively active versions of AfsR into wild type S. coelicolor significantly increased production of the antibiotic actinorhodin compared to the introduction of an additional wild-type copy of afsR (Fig. 3). Furthermore, constitutive activation of AfsR overcomes the otherwise media-dependent and developmentally controlled production of actinorhodin (Fig. 3, Fig. 4). Thus, constitutive activation of AfsR, and other AfsR-like regulators, represents a novel strategy for enhancing the synthesis of commercially important natural products produced through Streptomyces fermentation.

[0057] Moreover, given the high sequence conservation of AfsR homologs throughout the Streptomyces species, equivalent mutations to introduce autoactive residues in these homologues can be used to trigger the expression of a wider range of biosynthetic gene clusters. One very exciting example to test this idea is AfsR from S. peucetius, which positively regulates the production of the anticancer drug doxorubicin. Furthermore, we show that when this approach for identifying autoactivity-associated residues can be applied to a broader range of NLR-like regulators outside of the AfsR clade as there is broad conservation of such residues (Fig. 5). For example, the manipulation of NLR-like regulators involved in the regulation of primary metabolism opens up additional possibilities for rewiring the production of desired molecules through increasing the production of necessary building blocks. Furthermore, there is a broad array of metabolic regulators, which have an altered NLR-like architecture and carry a P-loop ATPase instead of a NB-ARC module in their NOD, such as the MalT clade of regulators (Fig. 5c). Applying our described approach to these regulators still identifies conserved residues, meaning this pipeline can be applied to activate bacterial regulators containing NODs outside of the canonical NB-ARC class.

[0058] Taken together, our data confirms that the genetic engineering of autoactive mutations in NLRs predicted to transcriptionally activate specialised metabolism represents a novel strategy for increasing yields of bacterial-derived bioactive molecules.

[0059] Accordingly, in a first aspect of the invention there is provided a genetically altered bacterium, expressing an autoactive nucleotide binding domain oligomerisation domain- like receptor (NLR) protein or NLR-like protein. For completeness, NLR proteins are also referred to in the literature as a nucleotide-binding domain and leucine-rich repeatcontaining protein. Such terms may be used interchangeably.

[0060] By “autoactive” or “autoavtivation” is meant that the bacteria expresses a NLR or NLR- like protein that is active independent of any ligand - that is, the NLR or NLR-like protein does not require an agonist or (lack of) an antagonist for activity. Such a bacteria may be referred to as expressing a constitutively or a substantially constituvely active NLR or NLR-like protein. Alternatively, the bacteria may be referred to as having a gain of function mutation in a NLR or NLR-like gene. By “active” may mean that the NLR or NLR- like protein is able to activate downstream signalling, that for example, may lead to the expression of BGCs, and in particular, expression of a desired metabolite. The autoactive mutation may also enable ATP binding which is required for NLR oligomerisation and downstream signalling.

[0061] In one embodiment the NLR or NLR-like protein is selected from, AfsR, KY5 AfsR AfsR- sl, AfsR-sv, AfsR-p, AfsR-g, AfsR-pr, AfsR-L, PteR, Orf2, AveR, NysRI, CdaR, GdmRI, bfmR, pimR, PolY, SanG, NemR, MycG1 , AstG1 , StaR, RebR, NeoR, SCO_0877, MilR, VemR, GdmRII, ttmRI BafG, HbmRII, amphRI, snorA, fdmRI, pnxR2, sanR1 , pokR1 , IIpRIV, gilS, OzmU, scnRI, MgsA, PlmR1 , pnR2, pnR1 , lmR1 , FilR, LcmRIII, ChxA, SCAB1371 , SgnR, ORF4, Ict22, Ict23, Orf15, Orf17, sky44, Orf(+2), AcoK, GutR, RslR3 , MalT, BpdS, PteF, CphR, PtnR, Asm18, orf18, SalRI, SalRII, AmphRII, FscRII, FscRIV, AbmH, TrdH, cvm7P, Kmy4, IdmG, OlmRI, Tmn5, CalR2, PoIR, MlaH, BecH, cvm7, ttmRII, aas1 , ttmRIII, MonH, SCO7173, PikD, NbmM, TmcN, fkbN, SlnR, IdnRI , NysRIII, SamR0484, RapH, AmphRIII, FscRIII, Hgc1 , Div8, Cuv20, OlmRII, Tei15, LipReg4, TvaN, TtnG, vioT, NysRII, AtmR, LooR, AmphRVI, RevU, AcuR1 , AcuR2, AcuR3, SelRI, SelRII, SelRI II, SelRV, SeIRVI, CppRI, CppRII, CppRIII, CppRV and ClaR and ClaR.

[0062] In one embodiment the NLR or NLR-like protein is selected from AfsR (for example as described in BAA14186.1 , AGV55404.1 EFG08453.1 , or AEY89983.1), KY5 AfsR (SEQ ID NO: 92 and 93), AfsR-sl (for example as described in EFD67634.1), AfsR-sv (for example as described in ABR08660.1), AfsR-p (for example as described in CAH 10136.1), AfsR-g (for example as described in BAA83790.1) , AfsR-pr (for example as described in EDY62716.1) , AfsR-L (for example as described in WP_190816242.1), PteR (for example as described in BAC68120.2) , Orf2 (for example as described in BAO31545.1), AveR (for example as described in BAA84600.1) , NysRI for example as described in AAF71778.1), CdaR (for example as described in AAD18045.1), GdmRI (for example as described in AA006930.1) , bfmR (for example as described in BAJ33109.1), pimR (for example as described in CAE51066.1) , PolY (for example as described in ABX24502.1) , SanG (for example as described in AAV31783.1), NemR (for example as described in BAF85834.1) , MycG1 (for example as described in AFG19405.1), AstG1 (for example as described in AHH25581.1), StaR (for example as described in EFG04608.1 or AYA20393.1), RebR (for example as described in AAN01213.1), NeoR (for example as described in BAD95834.1), SCO_0877 (for example as described in CAB62668.1), MilR (for example as described in ADI03855.1), VemR (for example as described in CCA53779.1) , ttmRI (for example as described in AFW98290.1), BafG (for example as described in ADC79626.1), HbmRII (for example as described in AAY28234.1), amphRI (for example as described in AAV37059.1), snorA (for example as described in CAA12016.1), fdmRI (for example as described in AAQ08934.1), pnxR2 (for example as described in BAJ52674.1), sanR1 (for example as described in ADG86334.1), pokR1 (for example as described in ACN64819.1), IIpRIV (for example as described in CAM34372), gilS (for example as described in AAP69594.1), Ozmll (for example as described in ABS90482.1), scnRI (for example as described in ADX66458), MgsA (for example as described in ACY01386.1) , PlmR1 (for example as described in AAQ84162.1), pnR2 (for example as described in AFJ05085.1), pnR1 (for example as described in AFJ05084.1), lmR1 (for example as described in BBC48406.1), FilR (for example as described in AKX77827.1), LcmRIII (for example as described in QSE03598.1), ChxA (for example as described in AFO59862.1), SCAB1371 (for example as described in CBG67361.1), SgnR (for example as described in AQT01397.1), ORF4 (for example as described in ABL09952.1) Ict22 (for example as described in ABX71105.1), Ict23 (for example as described in ABX71106.1) Orf15 (for example as described in AEZ53968.1), Orf17 (for example as described in AAZ94408.1), sky44 (for example as described in AEA30287.1) Orf(+2) (for example as described in CBZ42156.1), AcoK (for example as described in VGL85338.1) GutR (for example as described in AAA20676.1), RslR3 (for example as described in AHL46726.1), MalT (for example as described in AAA83888.1), BpdS (for example as described in AAB52543.1), PteF ( for example as described in BAB69312.1), CphR (for example as described in AAN08756.1), MalT ( for example as described in CAL14010.1 or AEV84884.1 or ABR79175.1), PtnR (for example as described in TGZ15159.1), Asm18 (for example as described in AAM54096.1), orf18 (for example as described in ABH05068.1), SalRI (for example as described in ABG02267.1), SalRII (for example as described in ABG02266.1), AmphRII (for example as described in AAV37060.1), FscRII (for example as described in AAQ82552.1), FscRIV (for example as described in AAQ82554.1 , AbmH (for example as described in AVI57436.1), TrdH (for example as described in ADY38540.1), cvm7P (for example as described in EFG04565.1), Kmy4 (for example as described in UHY14120.1), IdmG (for example as described in ACN69983.1), OlmRI (for example as described in BAB69189.1), Tmn5 (for example as described in BAE93719.1), CalR2 (for example as described in AAM94777.1), PoIR (for example as described in ABX24503.1), PoIR (for example as described in AFP55322.1), MlaH (for example as described in ACO94482.1), BecH (for example as described in ACO94455.1), CalR2 (for example as described in AEH42478.1), cvm7 (for example as described in ABK96928.1), ttmRII (for example as described in AFW98288.1) aas1 (for example as described in AZM68330.1), ttmRIII (for example as described in AFW98289.1), MonH (for example as described in AAO65802.1 , SCO7173 (for example as described in CAD55526.1), PikD (for example as described in AAC68887.1) NbmM (for example as described in AAM88362.1), TmcN (for example as described in ABI94385.1 , fkbN (for example as described in AFV94638.1), SlnR (for example as described in AEZ53964.1) IdnRI (for example as described in BAP34706.1), NysRIII (for example as described in AAF71780.1), SamR0484 (for example as described in AKZ60150.1), RapH (for example as described in CAA60471.1), AmphRIII (for example as described in AAV37061.1), FscRIII (for example as described in AAQ82553.1 ) StaR (for example as described in AYA20393.1), Hgc1 (for example as described in AFV30245.1) Div8 (for example as described in AHG95688.1), Cuv20 ((for example as described in AGO98703.1), OlmRII (for example as described in BAC70612.1), Tei15 (for example as described in CAG15028.1), LipReg4 (for example as described in ABB05095.1), TvaN (for example as described in BAN83929.1), TtnG (for example as described in ABV91292.1), vioT (for example as described in AAP92511.1), NysRII (for example as described in AAF71779.1), AtmR (for example as described in ABC02804.1), LooR(for example as described in QJU69506.1), AmphRVI (for example as described in AAV48835.1), Revll (for example as described in BAK64651.1), AcuR1 (for example as described in AHH99950.1 , AcuR2 (for example as described in AHH99949.1), AcuR3 (for example as described in AHH99948.1), SelRI (for example as described in ALE82596.1), SelRII (for example as described in ALE82597.1), SelRIII (for example as described in ALE82598.1), SelRV (for example as described in ALE82603.1), SeIRVI (for example as described in ALE82604.1), CppRI (for example as described in ABV83232.1), CppRII (for example as described in ABV83233.1), CppRIII (for example as described in ABV83234.1), CppRV (for example as described in ABV83238.1) and ClaR (for example as described in AEO12719.1).

[0063] Preferably the NLR or NLR-like protein is AfsR. More preferably the NLR or NLR-like protein is AfsR and the bacterium belongs to the genus Streptomyces.

[0064] In one embodiment, there is provided a genetically altered bacterium, expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein, wherein said bacterium comprises at least one mutation in at least one nucleic acid sequence encoding a NLR or NLR-like protein.

[0065] In one embodiment, the mutation is any mutation in a NLR or NLR-like gene that leads to autoactivation of the NLR or NLR-like protein.

[0066] In plants and animals, in the absence of an activation stimulus, NLRs exist in their monomeric ADP-bound form, which represents the resting 'off state. Upon recognition of an effector via the C-terminal NLR sensor domain, ADP is exchanged for ATP. This, in turn, induces an intramolecular conformational shift, which switches the NLR from the autoinhibited (‘off’) to the active 'on' state. This structural rearrangement results in protein oligomerisation via the NOD domain, which for some NLRs resembles a wheel-like structure called inflammasomes (animals) or resistosomes (plants). Oligomerisation in turn leads to the concomitant activation of the effector domain, which in turn mediates an inflammatory or cell death response - or as described herein for the first time, in the production of metabolites.

[0067] Mutation of NLR / NLR-like proteins, such as for example the mutations described herein, result in a stimulus-independent NLR activation through altering the intramolecular peptide contacts that promote ATP binding. The residues mutated are either predicted to affect directly or indirectly nucleotide-binding.

[0068] Accordingly, in one embodiment, the mutation is any mutation that increases or promotes (directly or indirectly) the binding of ATP via the NOD domain in NLRs or NLR-like protein. By an “increase” may be meant at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the amount of ATP bound to wild-type or control NLR or NLR- like protein (i.e. that does not carry a mutation of the invention). The amount of ATP bound can be measured by any technique in the art, for example using a malachite green phosphate detection kit.

[0069] In an alternative embodiment, the mutation is any mutation that alters the ratio of ATP: ADP bound to the (population of) NLR or NLR-like proteins such that more ATP is bound than ADP in a population of NLR or NLR-like proteins. As an example, the ratio of ATP: ADP may be altered (at the population level (of proteins) rather than the individual NLR protein level) to more than 1 :10 or at least 2:10 or at least 3:10, or at least 4:10 or at least 5:10). The ratio of ATP to ADP bound can be measured by any technique in the art, for example using an NTPase activity, such as the malachite green phosphate assay.

[0070] In a further alternative embodiment, the mutation is any mutation that increases the oligomerisation of the NLR or NLR-like protein. By an “increase” may be meant an increase in oligomerisation of least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the level of oligomerisation in a wild-type or control NLR or NLR-like protein (i.e. that does not carry a mutation of the invention). The amount of NLR / NLR-like oligomerisation can be measured by any technique in the art, for example the oligomerisation of a NLR or NLR-like protein can be measured by, BN-PAGE or Mass Photometry.

[0071] In one embodiment the genetically altered bacterium described herein is characterised by an increase in the production of at least one metabolite, compared to a wild-type or control bacterium. Preferably the metabolite is an antibiotic, antibiotic precursor, antibacterial metabolite, anti-cancer drug, pigment, growth hormone, immunosuppressant, agrochemical, antiviral or anti-fungal. In one embodiment the at least one metabolite is selected from actinorhodin, pikromycin, doxorubicin, a-factor, pristinamycin i, pristamycin ii, butenyl-spinosyn, filipin, thaxtomin a, lomofungin, avermectin, nystatin, calcium-dependent antibiotic, geldanamycin, bafilomycin, pimaricin, natamycin, polyoxin, nikkomycin, nemadectin, mycotrienin (myc) members, ansatrienins (hydroxymycotrienin a and thiazinotrienomycin g), staurosporine, rebeccamycin, neomycin, milbemycin, venemycin, tetramycin, herbimycin pks, amphotericin, nogalamycin, fredericamycin, fd-594, a-74528, polyketomycin, lysolipin, gilvocarcin (v), oxazolomycin, natamycin, iso-migrastatin, migrastatin, dorrigocin, phospholactomycin, lactomycins a-c, lucensomycin, cycloheximide pyochelin, aranciamycin, lactonamycin, salinomycin, concanamycin a, skyllamycin a, himastatin, rishirilide(s), pentamycin, validamycin a, ansamitocin, ahba, candicidin, neoabyssomicin, tirandamycin, 5s clavams, kendomycin b, indanomycin, oligomycin, tetronomycin.ml- 449, be-14106, calcimycin (a23187), clavam, aminoansamycins, monensin pks, methymycin, neomethymycin, narbomycin, pikromycin, tautomycin, fk506, incednine, stambomycin, xcp, rapamycin, amphotericin b, hygrocins, divergolides, cuevaene a, teicoplanin, a-lipomycin, thioviridamide, tautomycetin, viomycin, ecis, at2433, loonamycin, reveromycin a, aculeximycin, selvamicin, npp, and cladoniamides. More preferably, the at least one metabolite is selected from actinorhodin, calcium-dependent antibiotic, prodiginine, undecylprodigiosin, amphotericin, rapamycin, FK506, avermectins, watasemycin, pristinamycin, pristinamycin II, thaxtomin A, WS599B, pikromycin, holomycin, doxorubicin, daunorubicin, A-factor validomycin A, YM3163-A, avenolides, prodiginine, CDA, holomycin, daunorubicin, daptomycin and lomofungin.

[0072] As used herein, an increase may refer to an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 or 95% or more compared to the amount of metabolite produced by a wild-type or control bacteria. Alternatively, the increase may at least a one-fold two-fold, three-fold, four-fold or fivefold increase or more compared the amount of metabolite produced by a wild-type or control bacteria.

[0073] In one embodiment, the amino acid sequence of the NLR or NLR-like protein is selected from SEQ ID NO: 37 to 55, 94 (or a sequence recited in table 1), or a functional variant or homologue thereof. In a further embodiment, the nucleic acid sequence of the NLR or NLR-like protein is selected from SEQ ID NO: 56 to 74, or 95 or a functional variant or homologue thereof.

[0074] As described above, and shown in Figure 1 , NLR and NLR-like proteins generally comprise three domains; a sensor domain, a central nucleotide-binding oligomerization domain or NOD domain and an effector domain. In one embodiment the one or more mutations are in the sensor domain of the NLR or NLR-like protein. The sensor domain may comprise a nucleic acid sequence that encodes a sensor domain as defined in SEQ ID NO: 109 to 126, or a functional variant or homologue thereof.

[0075] In one embodiment the one or more mutations are in the effector domain of the NLR or NLR-like protein. The effector domain may comprise a nucleic acid sequence that encodes an effector domain as defined in SEQ ID NO: 127 to 145, or a functional variant or homologue thereof.

[0076] In a preferred embodiment the one or more mutations are in the nucleotide-binding oligomerization domain (NOD) of the NLR or NLR-like protein. The NOD domain may comprise a nucleic acid sequence that encodes a NOD as defined in SEQ ID NO: 73 to 91 or 94, or a functional variant or homologue thereof.

[0077] Within the NOD of NLR and NLR-like proteins there are typically at least four highly conserved regions or domains. These are: the hhGRExE motif (two hydrophobic residues followed by glycine-arginine-glutamic acid then any amino acid then another glutamic acid), RNBS-B (Resistance Nucleotide Binding Site B), GLPL (glycine-leucine- proline-leucine) and the MHD (Methionine-Histidine-Aspartate motif). Accordingly, in one embodiment, the genetically altered bacterium comprises at least one mutation in at least one nucleic acid sequence encoding at least one of the hhGRExE, RNBS-B, GLPL and the MHD motif. In one embodiment, the underlying nucleic acid sequence encodes a hhGRExE motif as defined in one of the following amino acid sequences or a functional variant or homologue thereof:

[0078] FTGRAAFV (SEQ ID NO: 27); FTGRSGFV (SEQ ID NO: 28), FTGRENLA (SEQ ID NO: 29), FTGRSSFV (SEQ ID NO: 30), FTGRVSFV (SEQ ID NO: 31), FTGRSAFV (SEQ ID NO: 32), FTGRTSFV (SEQ ID NO: 33) or FSGQQAEL (SEQ ID NO: 34). FTGRDEEL (SEQ ID NO: 146), FTGRDAFV (SEQ ID NO: 147), FTGRASFV (SEQ ID NO: 148).

[0079] In one embodiment, the nucleic acid sequence encodes a RNBS-B motif as defined in one of the following sequences or a functional variant or homologue thereof: GCAALITSR (SEQ ID NO: 17), GCAALVTAR (SEQ ID NO: 18), GCAALVTSR (SEQ ID NO: 19), or TCGVIATGR (SEQ ID NO: 20), DCLAIVTSR (SEQ ID NO: 149).

[0080] In one embodiment, the nucleic acid sequence encodes a GLPL motif as defined in one of the following sequences or a functional variant or homologue thereof:

[0081] , FLPLAIRIA (SEQ ID NO: 21), FLPLAIRVA (SEQ ID NO: 22) or RLPLALRIV (SEQ ID NO: 23), GLPLALRIT (SEQ ID NO: 150).

[0082] In one embodiment, the nucleic acid sequence encodes a MHD motif as defined in one of the following sequences or a functional variant or homologue thereof.

[0083] YHDLVRLY (SEQ ID NO: 24), FHDLIRAY (SEQ ID NO: 25) or FHDLVRLY (SEQ ID NO: 26), FHDLVRTY (SEQ ID NO: 151).

[0084] The at least one mutation that is introduced into at least one nucleic acid sequence encoding at least one NLR or NLR-like protein can be selected from the following mutation types:

[0085] 1. a "missense mutation", which is a change in the nucleic acid sequence that results in the substitution of one amino acid for another amino acid;

[0086] 2. an "insertion mutation" of one or more nucleotides or one or more amino acids, due to one or more codons having been added in the coding sequence of the nucleic acid;

[0087] 3. a "deletion mutation" of one or more nucleotides or of one or more amino acids, due to one or more codons having been deleted in the coding sequence of the nucleic acid;

[0088] 4. a "frameshift mutation", resulting in the nucleic acid sequence being translated in a different frame downstream of the mutation. A frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides; wherein any one or more of the above mutations leads to autoactivation of the NLR or NLR-like protein. Preferably, the mutation is at least one point mutation. A point mutation is a mutation in which one single nucleotide base is added, deleted or changed. Most preferably, the mutation is a substitution of one or more amino acids.

[0089] In one embodiment, the mutation is selected from one or more of the following mutations, and any combination thereof: a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149. More preferably, the substitution is a substitution from an R to a Q; and / or a mutation, preferably a substitution at position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23. More preferably, the substitution is a substitution from an I to a N; and / or a mutation, preferably a substitution at position 1 of the GLPL motif, for example as defined in one of SEQ ID NOs 150. More preferably, the substitution is a substitution from G to a R; and / or a mutation, preferably a substitution at position 2 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151 . More preferably, the substitution is a substitution from H to G; and / or a mutation, preferably a substitution at position 3 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151 . More preferably, the substitution is a substitution from D to V.

[0090] Examples of these substitutions are shown in Figure 2.

[0091] In another embodiment, the one or more mutation is selected from one or more of the following, and any combinations thereof: a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and / or a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and / or a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; and / or a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V.

[0092] In a further embodiment, the genetically altered bacterium comprises at least two mutations. Preferably, the at least two mutations may be selected from: a mutation, preferably a substitution at position 2 and 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution at position 2 is a H to G, and the substitution at position 3 is a D to V; or a mutation, preferably a substitution at position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23, wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 1 of the GLPL motif, for example as defined in one of SEQ ID NOs 150, wherein preferably the substitution is a G to R, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution is a D to V; or a mutation, preferably a substitution position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23, wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution at position 2 is a H to G; or a mutation, preferably a substitution position 1 of the GLPL motif, for example as defined in one of SEQ ID NO 150, wherein preferably the substitution is a G to a R, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution at position 2 is a H to G; or a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149, wherein preferably, the substitution is a substitution from an R to a Q, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution is a H to G; or a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149, wherein preferably, the substitution is a substitution from an R to a Q, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution at position 3 is a D to V; or a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149, wherein preferably, the substitution is a substitution from an R to a Q, and a mutation, preferably a substitution position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23, wherein preferably the substitution is an I to N. a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149, wherein preferably, the substitution is a substitution from an R to a Q, and a mutation, preferably a substitution position 1 of the GLPL motif, for example as defined in one of SEQ ID NOs 150, wherein preferably the substitution is an G to R. Examples of these substitutions are shown in Figure 2.

[0093] In another embodiment, the one or more mutation is selected from one or more of the following: a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; or a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; or a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N.

[0094] In a further embodiment, the genetically altered bacterium comprises at least three mutations. Preferably, the at least three mutations may be selected from: a mutation, preferably a substitution at position 9 of the RNBS-B motif , for example as defined in one of SEQ ID NOs 17 to 20 or 149, wherein preferably the mutation is an R to a Q, and a mutation, preferably a substitution at position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 3 of the MH D motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 9 of the RNBS-B motif , for example as defined in one of SEQ ID NOs 17 to 20 or 149, wherein preferably the mutation is an R to a Q, and a mutation, preferably a substitution at position 1 of the GLPL motif, for example as defined in one of SEQ ID NOs 150 wherein preferably the substitution is a G to R, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs 17 to 20 or 149 wherein preferably the mutation is an R to a Q, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is a H to G, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an H to G, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an D to V; or a mutation, preferably a substitution at position 1 of the GLPL motif, for example as defined in one of SEQ ID NOs 150 wherein preferably the substitution is a G to R, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an H to G, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an D to V.

[0095] Examples of these substitutions are shown in Figure 2.

[0096] In another embodiment, at least three mutations are selected from the following: a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N, and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G, and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V; or a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G, and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V.

[0097] In a further embodiment, the genetically altered bacterium comprises at least four mutations. Preferably, the at least four mutations may be a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs 17 to 20 or 149 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an H to G.

[0098] In a further embodiment, the genetically altered bacterium comprises at least four mutations. Preferably, the at least four mutations may be a mutation, preferably a substitution at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs 17 to 20 or 149 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 1 of the GLPL motif, for example as defined in one of SEQ ID NOs 150 wherein preferably the substitution is G to R, and a mutation, preferably a substitution at position 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 2 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 wherein preferably the substitution is an H to G.

[0099] In a further embodiment, the genetically altered bacterium comprises at least four mutations. Preferably, the at least four mutations may be a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G.

[0100] Examples of these substitutions are shown in Figure 2.

[0101] In another embodiment, at least four mutations are a mutation, preferably a substitution at position 438 of SEQ ID NO: 44 ora corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and a mutation, preferably a substitution at position 494 of SEQ ID NO: 44 ora corresponding position in a homologous sequence, wherein preferably the substitution is a I to N, and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 ora corresponding position in a homologous sequence, wherein preferably the substitution is a D to V; and a mutation, preferably a substitution at position 603 of SEQ ID NO: 44 ora corresponding position in a homologous sequence, wherein preferably the substitution is a H to G.

[0102] In a preferred embodiment, the genetically altered bacterium comprises at least two mutations, wherein the mutation is a substitution at position 2 and 3 of the MHD motif, for example as defined in one of SEQ ID NOs 24 to 26 or 151 , wherein preferably the substitution at position 2 is a H to G, and the substitution at position 3 is a D to V. Alternatively, the genetically altered bacterium comprises at least two mutations, wherein the mutation is preferably a substitution at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; and a mutation, preferably a substitution at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V.

[0103] In a preferred embodiment the genetically altered bacterium is S. coelicolor or S. peucetius.

[0104] In another example, as shown in Figure 7, when the bacteria is S. peucetius the mutation is selected from one of the mutations described below. In an embodiment, the genetically altered bacterium comprises at least one, two, three or at least four mutations. Preferably, the at least four mutations may be any mutation selected from the following: a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G. In a more preferred embodiment the genetically altered bacterium comprises all four of these mutations.

[0105] In an embodiment, when the genetically altered bacteriuim is S. peucetius said genetical Illy altered bacteria has a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V.

[0106] In an embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G.

[0107] In an embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q.

[0108] In an embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N.

[0109] In another embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G.

[0110] In another embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q.

[0111] In another embodiment, when the genetically altered bacteriuim is S. peucetius said genetical Illy altered bacteria has a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N.

[0112] In another embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G, and a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q.

[0113] In another embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N.

[0114] In another embodiment, when the genetically altered bacteriuim is S. peucetius said geneticalllly altered bacteria has a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N.

[0115] In an embodiment, the genetically altered bacterium comprises at least three mutations. Preferably, selected from the following: a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G. In a more preferred embodiment the genetically altered bacterium comprises all of these mutations. In an embodiment, the genetically altered bacterium comprises at least three mutations. Preferably, selected from the following: a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V, and a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G. In a more preferred embodiment the genetically altered bacterium comprises all of these mutations.

[0116] In an embodiment, the genetically altered bacterium comprises at least three mutations. Preferably, selected from the following: a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 594 SEQ ID NOs 39 wherein preferably the substitution is an H to G. In a more preferred embodiment the genetically altered bacterium comprises all of these mutations.

[0117] In an embodiment, the genetically altered bacterium comprises at least three mutations. Preferably, selected from the following: a mutation, preferably a substitution at position 429 of SEQ ID NO 39 wherein preferably the substitution is an R to Q, and a mutation, preferably a substitution at position 485 of SEQ ID NO 39 wherein preferably the substitution is an I to N, and a mutation, preferably a substitution at position 595 of SEQ ID NO: 39 wherein preferably the substitution is an D to V. In a more preferred embodiment the genetically altered bacterium comprises all of these mutations.

[0118] By “corresponding position in a homologous sequence” is meant an equivalent position in a similar protein or nucleic acid sequence, due to common evolutionary origin or structural conservation.

[0119] Homologous positions or as used herein “corresponding positions in homologous sequences” can thus be determined by performing sequence alignments once the homologous sequence has been identified. For example, homologues can be identified using a BLAST search of the bacterial genome of interest using the S. coelicolor AfsR sequence as a query (i.e. the sequence provided in SEQ ID NO: 47). As shown in Figure 3, all single AfsR mutants, D604V, H603G, I494N and R438Q produced higher levels of actinorhodin at an earlier time-point compared to the wild-type (M145), exemplified by the dark purple colour on all plates. Particularly high levels of actinorhodin were observed in the D604V and R438Q mutants. Figure 3 also shows that the time actinorhodin was produced was earlier in double, triple, and quadruple mutants compared to the single mutants and the wild-type (M145), with a darker pigment also being observed, which also indicates a higher overall level compared to both single mutants and the wild-type (M145).

[0120] In a preferred embodiment, the NLR or NLR-like protein is AfsR and the genetically altered bacterium comprises at least one mutation, as described above, in at least one nucleic acid encoding AsfR. The nucleic acid may encode an AsfR protein as defined in SEQ ID NO: 35 to 44 or 47 or a functional variant or homologue thereof. More preferably, the AsfR protein may comprise or consist of an amino acid sequence as defined in SEQ ID NO: 35 to 44 or 47 or a functional variant or homologue thereof.

[0121] In another aspect of the invention there is provided a genetically altered bacterium, wherein the bacterium comprises at least one mutation, wherein the mutation is the addition of one or more additional copy of a NLR or NLR-like gene. For example, one or more additional copy of a NLR or NLR-like gene as defined in one of SEQ ID NOs 54 to 72.

[0122] In another embodiment, there is provided a genetically altered bacterium, wherein the bacterium comprises at least one mutation in at least one nucleic acid sequence encoding at least one NLR or NLR-like protein, wherein the NLR or NLR-like nucleic acid comprises or consists of a. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID NOs: 35 to 53; or b. a nucleic acid sequence as defined in one of SEQ ID NOs: 54 to 72 or c. a nucleic acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (b); or d. a nucleic acid sequence encoding a NLR or NLR-like polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to the nucleic acid sequence of any of (a) to (c);

[0123] In another aspect of the invention, a transgenic construct expressing a (mutated) autoactive NLR or NLR-like protein may be introduced into the genetically altered bacterium to produce a genetically altered bacterium expressing an autoactive NLR or NLR-like protein. Accordingly, in a further aspect, there is provided a genetically altered bacterium that comprises and / or expresses at least one nucleic acid construct, wherein the nucleic acid construct comprises a nucleic acid sequence encoding an NLR or NLR- like protein, for example as defined in SEQ ID NO: 35 to 53, or an auto active NLR or NLR-like protein. For example, the autoactive protein may be selected from one of the mutated NLR or NLR-like proteins described herein.

[0124] In one embodiment a nucleic acid construct expressing a mutated NLR or NLR-like nucleic acid sequence (that is mutated to be autoactive by, for example, introducing at least one of the mutations described above) can be produced using PCR mutagenesis. In this example, the desired mutation is introduced into a nucleic acid construct or plasmid using primers (with overlapping sequences and the desired mutation at the 3’ end - for example, those described in Table 3) in a PCR protocol that amplifies individual fragments or the entire NLR or NLR-like nucleic acid sequence the plasmid template, which are then assembled using Gibson Assembly. The parent template is removed using a methylation-dependent endonuclease (i.e. Dpnl), and bacteria are transformed with the assembled plasmid-PCR product. Plasmids are then isolated from the resulting colonies, and screened for the correct size. Finally, the positive clones are sequenced to confirm the desired modification and the absence of additional modifications.

[0125] In a further embodiment, the nucleic acid construct comprises and / or expresses a NLR or NLR-like protein as defined in any one of SEQ ID NOs: 95 to 108 or functional variants thereof.

[0126] In a further embodiment, the NLR or NLR-like protein and / or the bacteria and / or the one or more metabolite is selected from Table 1.

[0127] Table 1 - NLRs and NLR-like proteins Preferably, the nucleic acid sequences encoding the NLR or NLR-like proteins are operably linked to a regulatory sequence. Examples of suitable regulatory sequences include promoters, such as a constitutive or strong promoter. Alternatively, the promoter is the natural promoter. The nucleic acid constructs may also be introduced into the bacterium by conjugation or transformation, as described in further detail below.

[0128] In another aspect of the invention, it is possible to express an autoactive NLR or NLR- like protein by expressing just one domain - preferably the effector domain - in the bacterium. An example of an effector domain is shown in SEQ ID NO: 127 to 145 or a functional variant thereof. Preferably, the effector domain of the NLR or NLR-like protein may be fused to another protein, preferably a protein that self-oligomerizes such as a fluorescent protein, such as YFP. The effector domain as described above may be expressed from a nucleic acid construct. Accordingly, in a further aspect of the invention, there is provided a genetically altered bacterium that comprises and / or expresses a nucleic acid construct comprising a nucleic acid sequence encoding a NLR or NLR-like effector domain, as described above for example, where said effector domain may be further fused to YFP that self-oligomerises.

[0129] In another aspect of the invention, there is provided a method of producing at least one metabolite in bacteria, the method comprising expressing an autoactive nucleotide- binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein in the microorganism.

[0130] In another aspect of the invention, there is provided a method of increasing the production of a metabolite in a bacterium, the method comprising expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein in the microorganism. Preferably, said increase is relative to a control or wild-type bacteria.

[0131] Levels of a given metabolite in a bacteria can be measured by any standard technique in the art, for example, but not limited to liquid chromatography-mass spectrometry (LC- MS).

[0132] As shown in Figure 4B, the AfsR H603G / D604V double mutant has a ~3-fold increase in actinorhodin production compared to the M145 wild-type strain after 96 hours. Accordingly, in one embodiment, said increase is at least one-fold, two-fold, three-fold, four-fold, five-fold or more compared to a wild-type or control microorganism. In another embodiment, an increase may refer to an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 or 95% or more compared to the level in a wild-type or control microorganism. The increases observed are preferably independent of the nutrient conditions, and / or media, and / or the stage development of the bacteria.

[0133] In another aspect of the invention there is a method of reducing the time taken for a given bacteria to produce at least one target metabolite compared to a wild-type or control bacteria, wherein the method comprises expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein in the microorganism. The metabolite may be produced immediately, after 12 hours, after 24 hours, after 48 hours, after 72 hours or after 96 hours of bacterial culture. Alternatively, the metabolite may be produced after 1 day, 2 days, 3 days, 3 days, 4 days, 5 days, 6 days or 7 days of bacterial culture. Alternatively, the metabolite may be produced 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7, weeks, 8 weeks, 9 weeks or 10 weeks of bacterial culture. The time taken to produce the metabolite will vary depend on the bacteria and the metabolite. Preferably, the metabolite is produced at any time in the bacteria’s development cycle, most preferably the metabolite is also produced on any medium that the bacteria is grown on.

[0134] The method may comprise either mutating at least one NLR or NLR-like protein, for example by introducing one or more of the mutations described above, introducing and expressing a nucleic acid construct comprising an autoactive NLR or NLR-like protein or an effector domain, optionally further fused to a second protein that self-oligomerizes as described above.

[0135] In one embodiment, the metabolite is a secondary metabolite. A secondary metabolite may also be referred to as a specialised metabolite. Alternatively, the metabolite is a primary metabolite (i.e. those metabolites that are essential for growth of the bacterium).

[0136] The metabolite may be an antibiotic, antibiotic precursor, antibacterial, anti-cancer drug, pigment, growth hormone, antiviral, agrochemical, immunosuppresant or anti-fungal agent.

[0137] In one embodiment the metabolite may be selected from actinorhodin, pikromycin, doxorubicin, a-factor, pristinamycin i, pristinamycin ii, butenyl-spinosyn, filipin, thaxtomin a, lomofungin, avermectin, nystatin, calcium-dependent antibiotic, geldanamycin, ebafilomycin, pimaricin, natamycin, polyoxin, nikkomycin, nemadectin, mycotrienin (myc) members, ansatrienins (hydroxymycotrienin a and thiazinotrienomycin g), staurosporine, rebeccamycin, neomycin, milbemycin, venemycin, tetramycin, herbimycin pks, amphotericin, nogalamycin, fredericamycin, fd-594, a-74528, polyketomycin, lysolipin, gilvocarcin (v), oxazolomycin, natamycin, iso-migrastatin, migrastatin, dorrigocin, phospholactomycin, lactomycins a-c, lucensomycin, cycloheximide pyochelin, aranciamycin, lactonamycin, salinomycin, concanamycin a, skyllamycin a, himastatin, rishirilide(s), pentamycin, validamycin a, ansamitocin, ahba, candicidin, neoabyssomicin, tirandamycin, 5s clavam, kendomycin b, indanomycin, oligomycin, tetronomycin, ml- 449, be-14106, calcimycin (a23187), clavam, aminoansamycins, monensin pks, methymycin, neomethymycin, narbomycin, pikromycin, tautomycin, fk506, incednine, stambomycin, xcp, rapamycin, amphotericin b, hygrocins, divergolides, cuevaene a, teicoplanin, a-lipomycin, thioviridamide, tautomycetin, viomycin, at2433, loonamycin, reveromycin a, aculeximycin, selvamicin, npp, YM3163-A, avenolides, prodiginine, CDA, WS599B, holomycin, daunorubicin, daptomycin and cladoniamides.

[0138] More preferably the metabolite is selected from actinorhodin, calcium-dependent antibiotic, prodiginine, undecylprodigiosin, amphotericin, rapamycin, FK506, avermectins, watasemycin, pristinamycin, pristinamycin II, thaxtomin A, WS599B, pikromycin, holomycin, doxorubicin, daunorubicin, A-factor and validomycin A.

[0139] The genetically altered bacterium described herein may produce one or more of the above-described metabolites.

[0140] In another aspect of the invention, there is also provided a method of making at least one metabolite in a bacterium, wherein the method comprises expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein, as described herein, in the bacteria. Preferably the method comprises culturing the bacteria and extracting the metabolite, wherein the metabolite is extracted at any developmental stage of the microorganism. Preferably the bacterium is cultured in standard media (e.g. not specialised production media). In other words, production of the metabolite is media-independent.

[0141] As used herein by “media-independent” is meant that the bacterium / bacteria can be cultured on any type of media to produce the metabolite. Typically for metabolite production a production media is required, which is more complex (i.e. could be a complex media). Often a production media may contain about 10% w / v of solids. By media is independent may therefore mean either a liquid or solid culture, that is either complex or simple. Preferably, a simple media will be used to reduce production costs.

[0142] As described above, the method may comprise either mutating at least one NLR or NLR- like protein, for example by introducing one or more of the mutations described above, introducing and expressing a nucleic acid construct comprising an autoactive NLR or NLR-like protein or an effector domain, optionally further fused to a second protein that self-oligomerizes as described above.

[0143] In one embodiment, the bacteria are genetically altered by using conjugation. Such methods are described by “Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. 2000. Practical Streptomyces Genetics. John Innes Foundation.”

[0144] Conjugation is the process by which one bacterium transfers genetic material to another through direct contact. During conjugation, one bacterium serves as the donor of the genetic material, and the other serves as the recipient. This process has been exploited for use in genetic engineering as described below.

[0145] Conjugation can be used to transfer genetic material such as a plasmid into bacteria so that a gene of interest can be, knocked-in knocked-out, complemented, or over expressed. Preferably, in the context of the present invention conjugation is used to integrate an autoactive NLR or NLR-like protein into a bacterial genome, preferably into a Streptomyces genome. In one embodiment, mutagenesis is used to introduce mutations in a plasmid-borne gene which is then moved into the host Streptomyces strain via conjugation. This plasmid integrates into the Streptomyces genomes via single- homologous recombination at a conserved phage attachment site. Consequently, the resulting strain will have a wild-type NLR and an autoactive NLR copy in the genome.

[0146] Intergeneric transfer of plasmids carrying a nucleic acid sequence of interest, such as an autoactive NLR or NLR-like protein interest preferably uses the E. coli strain ET12567 [pUZ8002],

[0147] Non-replicating plasmids can be used in conjugation methods that integrate into the genome, preferably the plasmid integrates site specifically at the φC31 or φBT1 phage attachment sites, or via insert-directed homologous recombination.

[0148] Alternatively, self-replicating plasmids can be used that will not integrate into the genome but need to be maintained using a suitable selecting agent. Resulting exconjugants are selected on agar plates using a suitable selecting agent (e.g. antibiotic) so that only bacteria can grow that have integrated or received the plasmid containing the nucleic acid sequence of interest (e.g. autoactive NLR).

[0149] Following nucleic acid transfer, putatively conjugated bacteria can be evaluated using techniques known in the art for example, using Southern blot analysis, for the presence of one or more of the above-described mutations.

[0150] In another embodiment, the mutation is introduced using mutagenesis or targeted genome editing. That is, in one embodiment, the invention relates to a method and bacterium that has been generated by genetic engineering methods as described, and does not encompass naturally occurring bacteria.

[0151] Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.

[0152] A preferred genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in US 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defence against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I- III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

[0153] One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted.

[0154] Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms.

[0155] The single guide RNA (sgRNA) is the second component of the CRISPR / Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20bp. Using techniques known in the art it is possible to design sgRNA molecules that target an NLR or NLR-like protein, and in particular, the NOD as described herein. In one embodiment, the sgRNA molecules target a sequence selected from SEQ ID No: 54 to 72 or a variant thereof as defined herein.

[0156] Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.

[0157] In a preferred embodiment of any aspect of the invention described herein, sgRNA can be used with a modified Cas9 protein, such as nickase Cas9 or nCas9 or a “dead” Cas9 (dCas9) fused to a “Base Editor” - such as an enzyme, for example a deaminase such as cytidine deaminase, or TadA (tRNA adenosine deaminase) or ADAR or APOBEC. These enzymes are able to substitute one base for another. As a result, no DNA is deleted, but a single substitution is made. Alternatively, the method may use sgRNA together with a template or donor DNA construct, to introduce a targeted substitution, and in particular one of the substitutions described herein. In this embodiment, the introduction of a template DNA strand, following a sgRNA-mediated snip in the doublestranded DNA, can be used to produce a specific targeted mutation in the gene using homology directed repair. As a further alternative, prime editing can be used to introduce the specific mutation. Here a catalytically impaired Cas9 endonuclease is fused to an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) that is both specific to the target site and encodes the desired edit.

[0158] In an alternative embodiment, the nuclease used may be Cpf1 or MAD7.

[0159] Alternatively, the mutation is introduced using suicide plasmids or recombineering, for example using Lambda Red (Arroyo-Olarte RD, Bravo Rodriguez R, Morales-Rios E. Genome Editing in Bacteria: CRISPR-Cas and Beyond. Microorganisms. 2021 Apr 15;9(4):844).

[0160] Accordingly, in one embodiment, the method comprises a. selecting a bacteria; b. transfecting said bacteria with at least one sgRNA as described above; c. allowing said bacteria to reproduce through binary fission; d. selecting one or more bacteria obtained according to paragraph (c) that have one or more mutations in at least one NLR or NLR-like protein, and preferably, in the NOD of said NLR or NLR-like protein.

[0161] Accordingly, any of the genome editing constructs or any of the nucleic acid constructs described herein, may be introduced into said microorganism through either conjugation or transformation. These terms referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. The exogenous polynucleotide may or may not be be integrated into the host genome. Preferably, the exogenous polynucleotides are integrated at an ectopic locus.

[0162] Transformation methods include the use of electroporation, chemicals that increase free DNA uptake and heat-shock techniques. To select transformed bacteria, the bacteria obtained in the transformation are grown on agar plates using a suitable selection agent so that only the transformed microorganisms can grow. Alternatively, the transformed bacteria are screened for the presence of a selectable marker such as antibiotic resistance, or a fluorescent protein gene.

[0163] Following DNA or nucleic acid transfer, putatively transformed bacteria may also be evaluated, for instance using Southern analysis, for the presence of one or more of the above-described mutations.

[0164] In a further embodiment of any of the methods described herein, the method may further comprise at least one or more of the steps of assessing the phenotype of the genetically altered bacteria, measuring at least one of an increase in at least one secondary metabolite production. In other words, the method may involve the step of screening the bacteria for the desired phenotype.

[0165] Bacteria obtained or obtainable by the methods described above which carry the above described nucleic acid constructs or carry a functional mutation in an NLR or NLR-like protein are also within the scope of the invention.

[0166] We have also identified a further application of the current NLR engineering approach as a discovery tool for novel natural products. Most bacteria, and in particular Streptomyces, many BGCs are silent under standard laboratory conditions, and therefore there is a huge untapped reservoir of exploitable compounds. We can therefore identify NLR-like regulators encoded within silent BGCs using tools such as antiSMASH (Blin K, Shaw S, Kloosterman AM, Charlop-Powers Z, van Wezel GP, Medema MH, Weber T. antiSMASH 6.0: improving cluster detection and comparison capabilities. Nucleic Acids Res. 2021 Jul 2;49) and then generate a constitutively active variant of these regulators to induce production of the uncharacterised compound. This could represent a more-targeted complement to existing strategies for activating silent BGCs such as the heterologous expression or refactoring within the native host.

[0167] By a biosynthetic gene cluster or BGC is meant a physically clustered group of two or more genes in a particular genome that together encode a biosynthetic pathway for the production of a specialized metabolite (including its chemical variants). Biosynthetic gene clusters (BGCs) are responsible for the production of various secondary metabolites. BGCs can be identified using techniques known in the art such as genome mining with tools such as antiSMASH. A BGC may comprise or be associated with a known NLR or NLR-like protein. Using the methods of the present invention we can mutate the endogenous NLR or NLR-like (associated with the target BGC) to. From this we can determine whether a new metabolite is produced. The presence of a new metabolite can be determined using standard techniques in the art, for example, untargeted metabolomics NMR or LC-MS.

[0168] Accordingly, in a further aspect of the invention, there is provided a method of identifying one or more novel metabolites, the method comprising identifying a BGC and at least one associated NLR or NLR-like protein in a bacteria, expressing an autoactive variant of the BGC-associated NLR or NLR-like protein (for example by expressing an additional autoactive copy of the NLR or NLR-like protein, and screening for the presence of one or more novel metabolites).

[0169] In one embodiment, the method may comprise identifying one or more biosynthetic gene clusters within the genome and then analysing said cluster for one or more sequences that could be defined as an NLR (e.g. by screening for structures characteristic of NLR or NLR-like sequences, such as sequences with a P-loop-loop ATPase and / or a series of superstructure-forming repeats). The identified BGC may be characterised as a “silent BGC”. A “silent BGC” may be defined as one where no known product or metabolite is associated with it and / or the BGC has not been previously characterised.

[0170] A novel metabolite(s) obtained by the above method are also within the scope of the invention, as are genetically altered bacteria expressing the autoactive NLR or NLR-like protein.

[0171] The term “variant” or “functional variant” as used throughout with reference to any of the sequences described herein refers to a variant gene sequence or part of the gene sequence (such as a fragment) which retains the biological function of the full non-variant sequence (e.g. is either autoactive (for example, the NLR or NLR-like protein has an increased ratio of ATP:ADP bound or an increased rate of oligomerisation) or the variant produces or increases the production of the target metabolite). A functional variant also comprises a variant of the gene of interest, which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in nonconserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence that results in the production of a different amino acid at a given site that does not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N- terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0172] As used in any aspect of the invention described throughout a “variant” or a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %,

[0173] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

[0174] 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

[0175] 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

[0176] 97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence.

[0177] Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When the percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

[0178] The skilled person would understand that suitable homologues and the homologous positions in these sequences can be identified by sequence comparisons (e.g. BLAST, alignments) and identifications of conserved domains. Phylogenetic tree analysis using nucleotide or amino acid sequences can be used to establish orthology to an NLR gene. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example by assaying for changes in metabolite production), RNA-seq (transcriptomic changes) or (as some metabolites may be pigmented) assaying for a colour change in the growth medium (similar to actinorhodin, as described in the Examples). Homologous positions or as used herein “corresponding positions in homologous sequences” can thus be determined by performing sequence alignments once the homologous sequence has been identified. For example, homologues can be identified using a BLAST search of the bacterial genome of interest - for example., using the S. coelicolor AfsR sequence as a query, or using one of the sequences defined in SEQ ID Nos: 35 to 53 or 54 to 72.

[0179] Suitable homologues can be identified by sequence comparisons and identifications of conserved domains. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when expressed.

[0180] The term homologue, as used herein, also designates an NLR gene orthologue from other bacterial species. Suitable homologues can be identified by sequence comparisons and identifications of conserved domains as described above. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example by leading to the production of secondary metabolites.

[0181] A homologue may also have, in increasing order of preference, at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%,

[0182] 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%,

[0183] 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,

[0184] 98%, or at least 99% overall sequence identity to the amino acid sequences referenced herein or to the nucleic acid sequences referenced herein. Functional variants of NLR gene homologues as defined above are also within the scope of the invention.

[0185] Thus, the nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other microorganisms, for example bacteria. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Topology of the sequences, structural homology and the characteristic domain architecture can also be considered when identifying and isolating homologues. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen microbe. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

[0186] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0187] In a further embodiment, a variant as used herein can comprise a nucleic acid sequence encoding an NLR polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to a nucleic acid sequence as defined herein.

[0188] Preferably, a bacterium according to the present invention belongs to the phyla Actinomycetota, Pseudomonadota, Bacillota or Myxococcota, most preferably Actinomycetota.

[0189] Preferably, said bacteria belongs to the order Streptomycetales, Pseudonocardiales, Enterobacterales, Bacilliales, Corynebacteriales, Micrococcales, Micromonosporales, Myxococcales, Mycobacteriales, Pseudomonadales, Actinomycetales or Streptosporangiales.

[0190] Even more preferably, said bacteria belong to the genus Streptomyces. Preferably, said bacteria is selected from Streptomyces coelicolor, Streptomyces lividans, Streptomyces venezuelae, Streptomyces peucetius, Streptomyces griseus, Streptomyces pristinaespiralis, Saccharopolyspora pogona, Streptomyces avermitilis, Streptomyces acidiscabies, Streptomyces lomondensis, Streptomyces noursei, Streptomyces hygroscopicus, Kitasatospora setae, Streptomyces natalensis, Streptomyces cacaoi subsp. Asoensis, Streptomyces ansochromogenes, Streptomyces cyaneogriseus subsp. Noncyanogenus, Streptomyces flaveolus, Streptomyces sp. XZQH13, Streptomyces clavuligerus, Lentzea aerocolonigenes (Lechevalieria aerocolonigenes) (Saccharothrix aerocolonigenes), Streptomyces fradiae,

[0191] Streptomyces bingchanggensis, Streptomyces ahygroscopicus, Streptomyces lohii, Streptomyces nodosus, Streptomyces nogalater, Streptomyces sp. TA-0256, Streptomyces sp. SANK 61196, Streptomyces diastatochromogenes, Streptomyces tendae, Streptomyces griseoflavus, Streptomyces al bus, Streptomyces chattanoogensis, Streptomyces platensis, Streptomyces sp. HK803, Streptomyces sp. ACT232, Streptomyces filipinensis, Streptomyces cyanogenus, Streptomyces sp. YIM 56141, Streptomyces scabies, Streptomyces gilvosporeus, Streptomyces echinatus, Streptomyces rishiriensis, Streptomyces neyagawaensis, Streptomyces sp. Acta 2897, Streptomyces himastatinicus, Bacillus subtilis, Streptomyces bottropensis, Escherichia coli, Rhodococcus sp., Pseudarthrobacter chlorophenolicus, Yersinia enterocolitica, Actinoplanes sp. SE50 / 110, Streptomyces sp. S816, Actinosynnema pretiosum subsp., Auranticum, Kitasatospora putterlickiae, Streptomyces sp. FR-008, Streptomyces koyangensis, Streptomyces sp. SCSIO1666, Verrucosispora sp., Streptomyces antibioticus, Streptomyces sp. NRRL 11266, Micromonospora echinospora, Streptomyces aureochromogenes, Streptomyces sp. MP39-85, Streptomyces sp. DSM 21069, Streptomyces chartreusis, Streptomyces ahygroscopicus, Streptomyces sp. S35, Streptomyces virginiae, Streptomyces coelicolor A3(2), Streptomyces narbonensis, Streptomyces sp. CK4412, Streptomyces tsukubaensis, Streptomyces sp. ML694-90F3, Streptomyces ambofaciens, Streptomyces rapamycinicus , Streptomyces nodosus , Streptomyces sp. LZ35, Streptomyces sp. W112, Streptomyces avermitilis, Actinoplanes teichomyceticus, Kitasatospora aureofaciens, Streptomyces olivoviridis, Streptomyces griseochromogenes , Streptomyces vinaceus, Actinomadura melliaura, Nocardiopsis flavescens, Streptomyces sp. SN-593, Kutzneria albida, Pseudonocardia sp. HH 130629- 09, Pseudonocardia autotrophica, Streptomyces rochei, Streptomyces hygroscopicus, Streptomyces acidiscabies, Streptomyces lomondensis, Streptomyces avermitilis, Streptomyces lividans or Streptomyces uncialis.

[0192] More preferably said bacteria is selected from S.coelicolor, S.peucetius, S.rapamycinus, S.nodosus, S. avermitilis, S.griseus, S.bingchenggensis, S.scabiei, S.cattleya, S.plantensis, S.roseosporus, S.ribosidificus, S.fradiae, S.kanamyceticus, S. niveus, S.orientalis, S.garyphalus, S.lincolnensis, S.pristinaespiralis, S.virginiae, S.vinaceus, S.capreolus, S.noursei, S.aureofaciens, S.fradiae, S.venezuelae, S.clavuligerus, S.griseus and S.cacaoi.

[0193] The invention also extends to products obtained or obtainable from a genetically altered bacterium of the invention. A product obtained or obtainable from the invention may be an antibiotic, antiviral compound, agrochemical, antibiotic precursor, antibacterial, anticancer drug, pigment, growth hormone or anti-fungal. Preferably, a product derived by from the invention may be Actinorhodin, Pikromycin, Doxorubicin, A-factor, Pristinamycin I, butenyl-spinosyn, Filipin, Thaxtomin A, Lomofungin, Avermectin, Nystatin, Calcium-dependent antibiotic, Geldanamycin, Bafilomycin, Pimaricin, natamycin, polyoxin, Nikkomycin, Nemadectin, mycotrienin (MYC) members, ansatrienins (hydroxymycotrienin A and thiazinotrienomycin G), Staurosporine, Rebeccamycin, Neomycin, Milbemycin, Venemycin, Tetramycin, Herbimycin PKS, Amphotericin, Nogalamycin, Fredericamycin, FD-594, A-74528, Polyketomycin, Lysolipin, Gilvocarcin (V), Oxazolomycin, Natamycin, iso-Migrastatin, Migrastatin, Dorrigocin, Phospholactomycin, Lactomycins A-C, lucensomycin, cycloheximide pyochelin, Aranciamycin, Lactonamycin, Salinomycin, Concanamycin A, Skyllamycin A, Himastatin , Rishirilide(s), Pentamycin, Validamycin A, Ansamitocin, AHBA, candicidin, neoabyssomicin, tirandamycin, 5S clavam, Kendomycin B, Indanomycin, Oligomycin, Tetronomycin, ML-449, BE-14106, Calcimycin (A23187), Clavam, aminoansamycins, Monensin PKS, Methymycin, neomethymycin, narbomycin, pikromycin, Tautomycin, FK506, incednine, Stambomycin, Xcp, Rapamycin, Amphotericin B, Hygrocins, Divergolides, Cuevaene A, Teicoplanin, a-lipomycin, Thioviridamide, Tautomycetin, Viomycin, AT2433, Loonamycin, Reveromycin A, Aculeximycin, Selvamicin, NPP, or cladoniamides.

[0194] More preferably, a product derived by from the invention may be actinorhodin, calciumdependent antibiotic, prodiginine, undecylprodigiosin, amphotericin, rapamycin, FK506, avermectins, watasemycin, pristinamycin, pristinamycin II, thaxtomin A, WS599B, pikromycin, holomycin, doxorubicin, daunorubicin, A-factor, or validomycin A.

[0195] A control bacterium as used herein according to all of the aspects of the invention is a bacterium, which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control bacteria do not have one or more of the above-described mutations. In one embodiment, the control bacterium is a wild type bacterium. The control bacterium is typically of the same bacterial species, preferably having the same genetic background as the modified bacteria.

[0196] While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0197] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0198] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0199] The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0200] The invention is now described in the following non-limiting examples.

[0201] EXAMPLES

[0202] Example 1 - Streptomyces AfsR is an NLR with a number of autoactivity- associated residues

[0203] The NLR AfsR is highly-conserved across Streptomyces species, which have been shown to produce a range of commercially valuable bioactive molecules (Fig. 2a). This conservation, and the role of AfsR from S. coelicolor (ScAfsR) in regulating the production of the blue-pigmented antibiotic actinorhodin, make it an ideal proof-of- concept for identifying likely autoactive mutations. Protein sequence alignments of Streptomyces AfsR NOD sequences identify conserved residues between them that we predict are associated with autoactivity (Fig. 2c). This includes an arginine in the RNBS- B region, an isoleucine adjacent to the GLPL motif and both the histidine and aspartic acid of the MHD motif, with the only exception being the isoleucine near the GLPL motif, which is replaced with leucine in Streptomyces pristinaespiralis AfsR. Intriguingly, in S. pristinaespiralis AfsR and Streptomyces rochei SRO_3163 an arginine is located in place of this glycine in the GLPL motif (Fig. 2c). Autoactivity of the plant NLR NRC1 by substituting this glycine in the GLPL motif for an arginine, opening up the possibility that there has been selection at this site to increase activity in S. pristinaespiralis AfsR.

[0204] Example 2 - Introduction of point mutants induces autoactivity in ScAfsR

[0205] Given the high conservation of these four autoactivity-associated residues across the AfsR clade of NLRs, we set out to confirm whether mutating them represents a viable strategy for inducing autoactivity. Sc-afsR was cloned under its native promoter, both with and without putative autoactivity-inducing mutations, and introduced into the genome of wild-type S. coelicolor in trans. The resulting strains were streaked onto TSA agar and imaged over the course of 5 days. These plates demonstrate that introduction of wild type afsR results in a slight increase of the purple coloration indicative of increased actinorhodin (Fig. 3). However, this increase pales in comparison to the level of actinorhodin production of S. coelicolor strains expressing ‘autoactive’ alleles of afsR (Fig. 3). Of these, the R438Q and D604V mutations appear to be causal for inducing autoactivity. Aside from general overproduction, we also hypothesised that autoactive AfsR should remove the developmental and media-dependent regulation of actinorhodin production. Actinorhodin production is tightly coordinated with cellular development and is switched on during the transition of growth from vegetative to aerial mycelium. Previous RNA-seq data suggest that afsR is however constitutively expressed through growth. This suggests that prior to this developmental transition, AfsR is held in an inactive state, which can be overcome by autoactive mutations. This was supported by earlier actinorhodin production being visible when grown on TSA. Most of the strains harbouring the putative autoactivity-inducing mutations already demonstrate a strong purple colouration after 48 hours whilst the unmodified wild type strain M145, a strain harbouring the empty vector or wild type AfsR only begin to develop the purple colouration after 72 hours (Fig. 3). The media dependency of actinorhodin production, and the abolishment of this media-dependent phenotype in S. coelicolor strains producing autoactive AfsR can be seen when the strains are instead streaked onto media where actinorhodin production is usually negligible even after 120 hours of growth (Fig. 4a). We further quantified actinorhodin production in liquid DNB culture using spectrophotometry. Production of actinorhodin in an autoactive AfsR strain (containing the H603G and D604V substitutions) was significantly greater compared to the wild type carrying an empty vector or an additional wild-type copy of afsR (Fig. 4b). The production of actinorhodin on a standard media rather than a specific production media could be extremely useful when applied to trigger the production of molecules, which are ordinarily only synthesised on complex, expensive production media.

[0206] AfsR also positively regulates the production of the red-pigmented prodiginine natural product, although this colouration is normally masked by actinorhodin. To confirm that autoactive ScAfsR can trigger the production of other specialised metabolites, we also conjugated an autoactive afsR allele containing the R438Q and H603G mutations into an S. coelicolor mutant (M511) in which the actinorhodin cluster-specific regulator Actll- ORF4 is deleted and hence actinorhodin production is absent. In this genetic background, introduction of an autoactive afsR allele resulted in enhanced red pigmentation, confirming that autoactive ScAfsR has a wider effect on the activation of BGCs (Fig. 4c).

[0207] Example 3 - Autoactive AfsR boosts yields of actinorhodin in the absence of the native wild-type allele

[0208] In our initial proof-of-concept experiments, autoactive alleles of afsR were introduced in trans into the wild-type strain S. coelicolor M 145. This resulted in strains that contain both afsR at the native locus, and an additional copy of autoactive afsR integrated at the ΦBT1 locus. Whilst these data demonstrate that our autoactive approach does not require a clean knockout of the NLR of interest, we wanted to confirm that autoactive AfsR is not dependent on a wild-type copy of AfsR for functionality. Therefore, we introduced the empty vector, wild-type afsR or autoactive afsR (H603G / D604V) constructs into an existing AafsR mutant strain (M513) at the ΦBT1 locus. We then repeated the visual screen and spectrophotometry assay to assess actinorhodin production in the AafsR genetic background (Fig. 6).

[0209] Overall, the level of actinorhodin production in the AafsR background was consistent with the results obtained using the wild-type background (Fig. 6). We observed minimal actinorhodin production in the AafsR background strain or the strain carrying the wildtype afsR allele (Fig. 6a). In line with our previous results (Fig. 3 and 4), expression of an autoactive afsR allele (H603G / D604V) resulted in a striking actinorhodin overproduction phenotype. These results were further supported by quantifying actinorhodin production in cultures of the same strains grown in YEME-TSB media using spectrophotometric analysis (Fig. 6b). Together, our findings confirm that autoactive AfsR can function independently of the native wild-type protein.

[0210] Example 4 - Applying autoactivation of AfsR homologues to increase the production of anthracyclines in S. peucetius

[0211] To further explore how autoactive NLRs can be utilised to overproduce commerciallyrelevant molecules (Fig. 2a), we chose the AfsR homologue from S. peucetius (SpAfsR), which has been characterised as a positive regulator of doxorubicin biosynthesis. Doxorubicin (brand name Adriamycin) belongs to the anthracycline group of chemotherapeutic agents, still produced semi-synthetically via fermentation of its natural producer, S. peucetius. Thus, reliance upon S. peucetius for its doxorubicin production offers a clear opportunity to apply our approach to an industrially relevant process through generation of autoactive SpAfsR.

[0212] Our initial AfsR protein sequence alignments and structural modelling (data not shown) revealed that the autoactivity-associated residues that we targeted in S. coelicolor AfsR are conserved in S. peucetius AfsR (Fig. 2b). We therefore generated three corresponding SpAfsR mutant variants, SpAfsR*-1 (D595V), SpAfsR*-2 (H594G / D595V) and SpAfsR*-3 (R429Q / I485N / H594G / D595V) which represent combinations of point mutations shown to be efficacious at inducing autoactivity in ScAfsR (Fig. 3, 4 and 6). The Sp-afsR mutant alleles were integrated into the S. peucetius genome at the ΦC31 locus and expressed from the native promoter. Anthracycline production by these strains becomes easily visible by a red-brown colouration of the ISP4 culture medium and was further quantified spectrophotometrically at 495 nm (Fig. 7a). Our spectrophotometric analysis showed that introduction of an additional wild-type allele into S. peucetius increased anthracycline production by approximately 5-fold relative to the empty vector control. In contrast, we observed a striking increase in anthracycline production in cells expressing any of the three autoactive afsR alleles. On average, introducing the autoactive alleles increased the absorbance by 27.8-fold compared to cells carrying the empty vector (Fig. 7b). Thus, constitutive activation of AfsR, and other AfsR-like regulators, represents a novel strategy for enhancing the synthesis of commercially important natural products produced through Streptomyces fermentation. Collectively, our results show that the expression of autoactive AfsR homologues with the aim to increase the production of antibiotics is not limited to S. coelicolor but can also be applied to improve the synthesis of medically important molecules such as the anticancer drug doxorubicin.

[0213] Example 5 - Constitutive activation could be utilised to increase the production of diverse metabolites and uncover novel chemical diversity

[0214] In addition, we noticed that outside of the AfsR clade there is a wide array of prokaryote NLR-like proteins with characterised biological roles, which are also amenable to this approach. The AfsR domain organisation, and NB-ARC domain specifically, is also present in other regulators of both primary and specialised metabolism, such as GutR which regulates expression of glucitol dehydrogenase (GutB) in Bacillus subtilis and PolY which regulates Streptomyces cacaoi subsp. Asoensis polyoxin production. In both of these examples, amino acid alignments confirm conservation of multiple residues we mutated in ScAfsR (Fig. 5a). We also identified several examples of NLR-like proteins involved in transcriptional regulation with an altered NLR domain organisation, but which share several of the residues associated with autoactivity. For example, AfsR-L from Saccharopolyspora pogona which regulates production of butenyl-spinosin in Saccharopolyspora pogona but lacks the extended series of C-terminal tetratri coptide repeats (Fig. 5b) or the so-called “large ATPase-like” (LAL) regulators (Fig. 5c). This group of putative NLRs include regulators of primary metabolism such as Escherichia coli MalT and regulators of secondary metabolism such as those involved in the production of commercially-valuable compounds rapamycin (RapH in Streptomyces rapamycinus) and amphotericin (AmphRI / AmphRII / AmphRIII / AmphRVI in Streptomyces nodosus). LAL regulators have a predicted NOD-SSFR-effector organisation, and whilst they lack the complete set of four autoactivity-associated residues manipulated in ScAfsR there remains conserved autoactivity-associated residues (Fig. 5c), further demonstrating the wider applicability of our approach.

[0215] Example 6 - Materials and Methods

[0216] Strain and culture conditions:

[0217] Strains and oligonucleotides utilised in this work are described in Table 2 and Table 3 respectively. All plasmid propagation was performed using Escherichia coli K-12 strain TOP10, selected on solid Difco Nutrient Agar (DNA) containing 50 pg / ml hygromycin B or 50 pg / mL apramycin and propagated in liquid LB media also containing 25 pg / ml hygromycin B. S. coelicolor was routinely maintained on solid SFM media. S. peucetius was routinely maintained on solid SFM medium or liquid tryptic soy broth (TSB) medium

[0218] Generation of autoactive ScAfsR strains:

[0219] For cloning of Sc-afsR SCO4426), its native promoter was defined as the 300 bp upstream of a transcriptional start site (TSS) present at the SCO4426 gene in previously generated S. coelicolor TSS data. This promoter and the coding sequence of afsR was then amplified from the St6F11 cosmid (https: / / strepdb.streptomyces.org.uk) using Q5 polymerase with the resulting fragment inserted into pl J10770 cut with Ndel / Hindll I using Gibson assembly. Point mutations were introduced using Gibson assembly by including the chosen changes at the end of forward and reverse primers for the amplification of afsR fragments, with resulting fragments assembled into the plJ 10770 using the same method. After propagation in E. coli TOP10, resulting colonies were screened via colony PCR using the GoTaq master mix with the plasmid isolated from positive colonies using the Qiagen miniprep kit following manufacturer’s instructions. Plasmid sequences were confirmed through Sanger sequencing using the Eurofins Mix2Seq kit. Electrocompete nt E. coli ET12567 / pUZ8002 was transformed with the sequence-confirmed plasmids for conjugation into S. coe / / co / or M145 / M511 / M513 using established protocols.

[0220] Identifying autoactivity-conferring residues:

[0221] Protein sequences of NLRs were annotated using InterProScan v5.60-92.0 and the NOD sequence extracted by removing: 1) sequence upstream of the beginning of the P-loop NTPase domain (IPR027417) and 2) sequence downstream of the start of the SSFR Prokaryote NLRs of interest; in our proof-of-concept S. coelicolor AfsR (ScAfsR) (SCO4426, UniProt ID: P25941). The resulting NOD sequence of SINRC1 and prokaryote NLRs were then aligned using EMBL-EBI MUSCLE (htps: / / www.ebi.ac.uk / Tools / msa / muscle / ) under standard parameters. Residues associated with autoactivity were then annotated on the alignment, such residues conserved in the prokaryote NOD can be mutated using a substitution strategy.

[0222] This approach was followed with minor modifications for identifying conserved autoactivity-associated residues in NLRs with alternative domain organisations. In NLRs with a central SSFR domain flanked by an N-terminal NOD and C-terminal effector, the NOD sequence was extracted by removing all sequence downstream from the start of the annotated SSFR domain. Occasionally, such NLRs have homologues which lack an annotated SSFR domain despite sequence homology and the appearance of repeats in AlphaFold structural predictions. In this case, the NOD sequence was estimated by generating a structural prediction of the putative NLR using the ColabFold notebook, with the predicted aligned error (PAE) of the highest-scoring model utilised to define putative domain boundaries. The NOD sequence was then extracted by removing all sequence downstream of the boundary between the P-loop containing domain and the subsequent putative SSFR domain and fed into the above approach.

[0223] NLR phylogenetic analysis:

[0224] NLR P-loop ATPase domains (IPR027417) were extracted using InterProScan v5.60-

[0225] 92.0, and aligned using EMBL-EBI MUSCLE under the standard parameters. Alignments were trimmed using clipKIT in gappy mode. Phylogenies were generated based on these trimmed alignments using the IQTREE web server. The correct substitution model was selected using ModelFinder and all trees contained 10,000 ultrafast bootstraps. Resulting trees were visualised in iTOL using the spreadsheet editor tool for annotations.

[0226] Assaying actinorhodin production:

[0227] Actinorhodin production was visually screened by streaking strains out onto TSA and DNA media and imaging every 24 hours. TSA is a commonly used growth media to assay actinorhodin production. Total levels of actinorhodin produced by S. coelicolor strains expressing wild-type or autoactive AfsR were quantified from cells grown in liquid culture as previously described by Calvelo et al (2021). A starter culture of each strain was grown for approximately 72 hours in 30 ml tryptic soy broth (TSB); production cultures were then set up by adding 500 pl of normalised seed culture to 30 ml of Difco nutrient broth (DNB) production media. In the case of strains created in the M513 background, 30 ml production cultures consisting of 50% yeast extract-malt extract medium (YEME) and 50% TSB were instead inoculated with 106spores. Samples were then taken every 24 hours, 800 μl of culture was combined with 200 μl 5M KOH and vortexed to mix. Samples were then centrifuged at 5000 x g at room temperature for 5 minutes and 200 μl of the supernatant loaded into a 96-well plate to measure the absorbance at 640 nm using a SPECTROstar platereader (BMG labtech). Samples were then centrifuged at 13,000 rpm for 20 minutes and the supernatant aspirated before being weighed, the absorbance was then normalised against this wet pellet mass. Quantification was performed on four biological replicates. All data analysis and visualisation was then performed using R in RStudio and the tidyverse suite of packages.

[0228] Assaying anthracycline production:

[0229] Anthracycline production was also quantified in S. peucetius using spectrophotometry. TSB seed cultures were inoculated using mycelial stocks, and grown until confluence. The optical density at 600 nm of these cultures were normalised and equal volumes of each culture were used to inoculate International Streptomyces Project 4 (ISP4) medium. Cultures were incubated at 30°C and 250 rpm shaking, with 2 ml samples taken at indicated timepoints. Samples were pelleted and the supernatant was aspirated off. The pellet mass was weighed before being resuspended in 500 μl 100% methanol and shaken at 1400 rpm on a ThermoMixer (Eppendorf) for 10 minutes. Samples were then spun down at 13,000 x g for 10 minutes, and 200 pl of the supernatant was loaded into a 96-well plate. The absorbance was measured at 495 nm using a SPECTROstar platereader (BMG labtech) before being normalised against wet pellet mass. Quantification was performed on at least three biological replicates, with all data analysed and visualised using R as described above.

[0230] Table 2. Strains used.

[0231]

[0232] Table 3. Oligonucleotides used

[0233]

[0234]

[0235] Table 4. Sequences used in AfsR phylogenetic tree (Fig. 2b)

[0236] SEQUENCE LISTING

[0237]

[0238] Transgenic constructs hhGRExe domain : FTGRVSFV

[0239] RNBS-B domain: GCAALITSR

[0240] GLPL domain: FLPLAIRIA

[0241] MHD domain: YHDLVRLY

[0242] Yellow highlights are mutated amino acids.

[0243] NLR Sensor Domains

Claims

CLAIMS:

1. A genetically altered bacteria, wherein said bacteria expresses an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR- like protein.

2. The genetically altered bacteria of claim 1 , wherein the NLR or NLR-like protein is selected from AfsR, KY5 AfsR AfsR-sl, AfsR-sv, AfsR-p, AfsR-g, AfsR-pr, AfsR- L, PteR, Orf2, AveR, NysRI, CdaR, GdmRI, bfmR, pimR, PolY, SanG, NemR, MycG1 , AstG1 , StaR, RebR, NeoR, SCO_0877, MilR, VemR, GdmRII, ttmRI, BafG, HbmRII, amphRI, snorA, fdmRI, pnxR2, sanR1 , pokR1 , IIpRIV, gilS, OzmU, scnRI, MgsA, PlmR1 , pnR2, pnR1 , lmR1 , FilR, LcmRIII, ChxA, SCAB1371 , SgnR, ORF4, Ict22, Ict23, Orf15, Orf17, sky44, Orf(+2), AcoK, GutR, RslR3 , MalT, BpdS, PteF, CphR, PtnR, Asm18, orf18, SalRI, SalRII, AmphRII, FscRII, FscRIV, AbmH, TrdH, cvm7P, Kmy4, IdmG, OlmRI, Tmn5, CalR2, PoIR, MlaH, BecH, cvm7, ttmRII, aas1 , ttmRIII, MonH, SCO7173, PikD, NbmM, TmcN, fkbN, SlnR, IdnRI , NysRIII, SamR0484, RapH, AmphRIII, FscRIII, Hgc1 , Div8, Cuv20, OlmRII, Tei15, LipReg4, TvaN, TtnG, vioT, NysRII, AtmR, LooR, AmphRVI, RevU, AcuR1 , AcuR2, AcuR3, SelRI, SelRII, SelRIII, SelRV, SeIRVI, CppRI, CppRII, CppRIII, CppRV and ClaR.

3. The genetically altered bacteria of claim 2, wherein the NLR or NLR-like protein is AfsR.

4. The genetically altered bacteria of any preceding claim, wherein said bacteria comprises at least one mutation in at least one nucleic acid sequence encoding a NLR or NLR-like protein.

5. The genetically altered bacteria of claim 4, wherein the at least one mutation leads to autoactivation of the NLR or NLR-like protein.

6. The genetically altered bacteria of any preceding claim, wherein the NLR or NLR- like protein comprises at least one nucleotide oligomerisation domain (NOD), and wherein the genetically altered microorganism has at least one mutation in the nucleotide oligomerisation domain.

7. The genetically altered microorganism of claim 6, wherein the nucleotide oligomerisation domain comprises at least a hhGRExE motif, RNBS-B motif, GLPL motif, or MHD motif, and wherein the genetically altered bacteria has at least one mutation in at least one of the hhGRExE motif, RNBS-B motif, GLPL motif and MHD motif.

8. The genetically altered bacteria of any of claims 4 to 7, wherein the mutation is selected from at least one or any combination of a. a mutation, preferably a substitution, at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149, wherein preferably, the substitution is a substitution from an R to a Q; and / or b. a mutation, preferably a substitution, at position 1 or 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23 or 150, wherein preferably, the substitution is a substitution from an I to a N, or a substitution from a G to an R; and / or c. a mutation, preferably a substitution, at position 2 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151 , wherein preferably, the substitution is a substitution from H to G; and / or d. a mutation, preferably a substitution, at position 3 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151 , wherein preferably, the substitution is a substitution from D to V.

9. The genetically altered bacteria of claim 4 to 7, wherein the one or more mutation is selected from one or more of the following, or any combinations thereof: a. a mutation, preferably a substitution, at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and / orb. a mutation, preferably a substitution, at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and / or c. a mutation, preferably a substitution, at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; and / or d. a mutation, preferably a substitution, at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V.

10. The genetically altered bacteria of any of claims 4 to 9, wherein the mutation is a substitution.11 . The genetically altered bacteria of any preceding claim, wherein the sequence of the NLR or NLR-like protein is selected from SEQ ID NO: 35 to 53 or a functional variant or homologue thereof.

12. The genetically altered bacteria of claim 1 , 2 or 3, wherein the bacteria expresses a nucleic acid construct comprising a nucleic acid sequence encoding a mutated NLR or NLR-like protein, preferably the mutated NLR or NLR-like protein of claims 4 to 10.

13. The genetically altered bacteria of claim 12, wherein the nucleic acid sequence encodes a mutated NLR or NLR-like protein as defined in SEQ ID NO: 95 to 108, and wherein preferably the nucleic acid sequence is operably linked to a regulatory sequence.

14. The genetically altered bacteria of any of any preceding claim, wherein the bacteria is selected from the phyla Actinomycetota, Pseudomonadota, Bacillota or Myxococcota, most preferably Actinomycetota, and preferably is selected from the genus Streptomyces.

15. The genetically altered bacteria of claim 14, wherein the bacteria is selected fromS.coelicolor, S.peucetius, S.rapamycinus, S.nodosus, S.avermitilis, S.griseus, S.bingchenggensis, S.scabiei, S.cattleya, S.plantensis, S.roseosporus, S.ribosidificus, S.fradiae, S.kanamyceticus, S. niveus, S.garyphalus,S.lincolnensis, S.pristinaespiralis, S.virginiae, S.vinaceus, S.capreolus,S.noursei, S.aureofaciens, S.fradiae, S.venezuelae, S.clavuligerus, S.griseus and S. cacaoi.

16. A method of producing a metabolite in a bacteria, the method comprising expressing an autoactive nucleotide-binding oligomerisation domain-like receptor (NLR) protein or NLR-like protein in the microorganism.

17. The method of claim 16, wherein the metabolite is a secondary metabolite or specialised metabolite.

18. The method of claim 16 or 17, wherein the NLR or NLR-like protein is selected from AfsR, KY5 AfsR, AfsR-sl, AfsR-sv, AfsR-p, AfsR-g, AfsR-pr, AfsR-L, PteR, Orf2, AveR, NysRI, CdaR, GdmRI, bfmR, pimR, PolY, SanG, NemR, MycG1 , AstG1 , StaR, RebR, NeoR, SCO_0877, MilR, VemR, GdmRII, ttmRI, BafG, HbmRII, amphRI, snorA, fdmRI, pnxR2, sanR1 , pokR1 , IIpRIV, gilS, OzmU, scnRI, MgsA, PlmR1, pnR2, pnR1 , lmR1 , FilR, LcmRIII, ChxA, SCAB1371, SgnR, ORF4, Ict22, Ict23, Orf15, Orf17, sky44, Orf(+2), AcoK, GutR, RslR3 , MalT, BpdS, PteF, CphR, PtnR, Asm18, orf18, SalRI, SalRII, AmphRII, FscRII, FscRIV, AbmH, TrdH, cvm7P, Kmy4, IdmG, OlmRI, Tmn5, CalR2, PoIR, MlaH, BecH, cvm7, ttmRII, aas1 , ttmRIII, MonH, SCO7173, PikD, NbmM, TmcN, fkbN, SlnR, IdnRI , NysRIII, SamR0484, RapH, AmphRIII, FscRIII, Hgc1 , Div8, Cuv20, OlmRII, Tei15, LipReg4, TvaN, TtnG, vioT, NysRII, AtmR, LooR, AmphRVI, RevU, AcuR1 , AcuR2, AcuR3, SelRI, SelRII, SelRIII, SelRV, SeIRVI, CppRI, CppRII, CppRI II, CppRV and ClaR and ClaR.

19. The method of claim 18, wherein the NLR or NLR-like protein is AfsR.

20. The method of any of claims 16 to 19, wherein said bacteria comprises at least one mutation in at least one nucleic acid sequence encoding a NLR or NLR-like protein.21 . The method of claim 20, wherein the at least one mutation leads to autoactivation of the NLR or NLR-like protein.

22. The method of any of claims 16 to 21 , wherein the NLR or NLR-like protein comprises at least one nucleotide oligomerisation domain (NOD), and wherein the genetically altered bacteria has at least one mutation in the nucleotide oligomerisation domain.

23. The method of claim 22, wherein the nucleotide oligomerisation domain comprises at least a hhGRExE motif, RNBS-B motif, GLPL motif, or MHD motif, and wherein the genetically altered bacteria has at least one mutation in at least one of the hhGRExE motif, RNBS-B motif, GLPL motif and MHD motif.

24. The method of any of claims 20 to 23, wherein the mutation is selected from at least one or any combination of a. a mutation, preferably a substitution, at position 9 of the RNBS-B motif, for example as defined in one of SEQ ID NOs: 17 to 20 or 149, wherein preferably, the substitution is a substitution from an R to a Q; and / or b. a mutation, preferably a substitution, at position 1 or 6 of the GLPL motif, for example as defined in one of SEQ ID NOs 21 to 23 or 150, wherein preferably, the substitution is a substitution from an I to a N or a substitution from a G to an R; and / or c. a mutation, preferably a substitution, at position 2 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151. More preferably, the substitution is a substitution from H to G; and / or d. a mutation, preferably a substitution, at position 3 of the MHD motif, for example, as defined in on of SEQ ID Nos 24 to 26 or 151 , wherein preferably, the substitution is a substitution from D to V.

25. The method of any of claims 20 to 23, wherein the one or more mutation is selected from one or more of the following, or any combinations thereof: a. a mutation, preferably a substitution, at position 438 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a R to Q; and / or b. a mutation, preferably a substitution, at position 494 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a I to N; and / or c. a mutation, preferably a substitution, at position 603 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a H to G; and / or d. a mutation, preferably a substitution, at position 604 of SEQ ID NO: 44 or a corresponding position in a homologous sequence, wherein preferably the substitution is a D to V.

26. The method of any of claims 20 to 25, wherein the mutation is a substitution.

27. The method of any of claims 16 to 26, wherein the sequence of the NLR or NLR- like protein is selected from SEQ ID NO: 35 to 53 or a functional variant or homologue thereof.

28. The method of any of claims 16 to 19, wherein the bacteria expresses a nucleic acid construct comprising a nucleic acid sequence encoding a. a mutated NLR or NLR-like protein, preferably the mutated NLR or NLR- like protein of claims 4 to 10; or b. a NLR or NLR-like protein as defined in one of SEQ ID NO: 35 to 53 or a functional variant or homologue thereof; or c. an effector domain linked to a protein capable of oligomeristaion, preferably YFP, wherein preferably the nucleic acid sequenc encodes an effector domain as defined in one of SEQ ID NO: 127 to 145 or a functional variant or homologue thereof.

29. The method of claim 28, wherein the nucleic acid sequence encodes a mutated NLR or NLR-like protein as defined in SEQ ID NO: 95 to 108, and wherein preferably the nucleic acid sequence is operably linked to a regulatory sequence.

30. The method of any of claims 16 to 29, wherein the bacteria is selected from the phyla Actinomycetota, Pseudomonadota, Bacillota or Myxococcota, most preferably Actinomycetota, and preferably is selected from the genus Streptomyces.31 . The genetically altered bacteria of claim 30 wherein the bacteria is selected fromS.coelicolor, S.peucetius, S.rapamycinus, S.nodosus, S.avermitilis, S.griseus, S.bingchenggensis, S.scabiei, S.cattleya, S.plantensis, S.roseosporus, S.ribosidificus, S.fradiae, S.kanamyceticus, S. niveus, S.garyphalus,S.lincolnensis, S.pristinaespiralis, S.virginiae, S.vinaceus, S.capreolus,S.noursei, S.aureofaciens, S.fradiae, S.venezuelae, S.clavuligerus, S.griseus and S. cacaoi.

32. The method of any of claims 16 to 31 , wherein the metabolite is selected from an antibiotic, antibiotic precursor, antibacterial metabolite, anti-cancer drug, pigment, growth hormone, immunosuppressant, agrochemical, antiviral or antifungal.

33. The method of claim 32, wherein the metabolite is selected from actinorhodin, pikromycin, doxorubicin, a-factor, pristinamycin i, pristinamycin ii, butenyl- spinosyn, filipin, thaxtomin a, lomofungin, avermectin, nystatin, calciumdependent antibiotic, geldanamycin, ebafilomycin, pimaricin, natamycin, polyoxin, nikkomycin, nemadectin, mycotrienin (myc) members, ansatrienins (hydroxymycotrienin a and thiazinotrienomycin g), staurosporine, rebeccamycin, neomycin, milbemycin, venemycin, tetramycin, herbimycin pks, amphotericin, nogalamycin, fredericamycin, fd-594, a-74528, polyketomycin, lysolipin, gilvocarcin (v), oxazolomycin, natamycin, iso-migrastatin, migrastatin, dorrigocin, phospholactomycin, lactomycins a-c, lucensomycin, cycloheximide pyochelin, aranciamycin, lactonamycin, salinomycin, concanamycin a, skyllamycin a, himastatin, rishirilide(s), pentamycin, validamycin a, ansamitocin, ahba,candicidin, neoabyssomicin, tirandamycin, 5s clavam, kendomycin b, indanomycin, oligomycin, tetronomycin, ml-449, be-14106, calcimycin(a23187), clavam, aminoansamycins, monensin pks, methymycin, neomethymycin, narbomycin, pikromycin, tautomycin, fk506, incednine, stambomycin, xcp, rapamycin, amphotericin b, hygrocins, divergolides, cuevaene a, teicoplanin, a-lipomycin, thioviridamide, tautomycetin, viomycin, at2433, loonamycin, reveromycin a, aculeximycin, selvamicin, npp, YM3163-A, avenolides, prodiginine, CDA, WS599B, holomycin, daunorubicin, daptomycin and cladoniamides.

34. The method according to claim 33, wherein the metabolite is doxorubicin.

35. The method of claim 33, wherein the metabolite is actinorhodin.

36. The method of claim 16 to 35, wherein the method comprises culturing the bacteria and extracting the metabolite, wherein the metabolite is extracted at any developmental stage of the microorganism and / or following culture in any form of media, preferably a simple media.

37. The method of claim 16 to 36, wherein the method increases the level of metabolite production in the bacteria compared to the level of metabolite production in a wild-type or control bacteria.

38. A method for identifying and / or selecting bacteria that will have increased secondary metabolism compared to a wild-type or control bacterium, the method comprising detecting in said bacteria at least one mutation in at least one NLR gene.

39. A method of identifying one or more novel metabolites, the method comprising identifying a biosynthetic gene clusters (BGC) and at least one associated NLR or NLR-like protein in a bacteria, expressing an autoactive variant of the BGC- associated NLR or NLR-like protein as defined in any of claims 1 to 15 and screening for the presence of one or more novel metabolites.

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