Novel antibacterial compound

Abstract

The present invention provides a new antibacterial compound named nidaramycin, which is derived from a novel biosynthetic gene cluster (BGC), and its use. In addition, this specification provides novel genes and nucleic acid molecules encoding the biosynthetic machinery for the production of nidaramycin, as well as constructs, vectors, and host cells for expressing the BGC, and methods for producing the compound.

Description

Technical Field

[0001] The present disclosure and invention relate to a new antibacterial compound named nidaromycin by us, as well as its use and biosynthesis. In particular, a new biosynthetic gene cluster (BGC) has been identified, sequenced, and cloned, and by introducing and expressing the BGC in a host cell, it has become possible to produce the compound. Therefore, the present disclosure and invention also relate to novel genes and nucleic acid molecules encoding the biosynthetic machinery for the production of nidaromycin, as well as constructs, vectors, and host cells for expressing the BGC, and methods for producing the compound.

Background Art

[0002] Natural products produced by bacteria and fungi are very important in view of their potential use as pharmaceutical or animal products, most notably as antibiotics. Actinomycetes, a class of filamentous Gram-positive bacteria with a high GC content, produce the majority of all known antibiotics of microbial origin, and approximately half of them are obtained from the genus Streptomyces.

[0003] For example, genes for the synthesis of secondary metabolites such as antibiotics in actinomycetes tend to be organized as clusters containing genes encoding biosynthetic enzymes, transporter proteins, and other proteins involved in their synthesis or control. Various gene clusters for the synthesis of some antibiotics in different organisms have been reported.

[0004] The increase in antibiotic resistance in a wide range of bacterial pathogens is a major global health problem, and at the same time, the discovery of new antibacterial agents has been decreasing. For this reason, the development of new strategies for antibiotic discovery has been promoted, and one such approach is to identify novel secondary metabolite gene clusters of unknown function that may encode genes for the biosynthesis of clinically useful antibiotics by genomic mining of actinomycetes. There are various strategies for this, which include attempting to induce the expression of potential gene clusters in natural hosts, such as by culturing under different conditions, or heterologous expression of gene clusters in alternative hosts. We adopted the latter approach combined with bioinformatics and phylogenetic analysis to identify new BGCs encoding the biosynthesis of new antibacterial compounds.

Summary of the Invention

Means for Solving the Problems

[0005] For this purpose, we analyzed the genomes of 1200 marine actinobacterial strains isolated from the Trondheim Fjord and 576 actinobacterial type strains retrieved from public databases based on various criteria including phylogenetic novelty of the strains, diversity of gene clusters, and antibacterial activity. Based on this analysis, various strains including the actinobacterial strain P08-G05 were selected for further investigation. The genome sequence of this strain was determined and subjected to bioinformatics analysis to identify and analyze potential new biosynthetic gene clusters (BGCs) containing genes encoding antibiotic-like compounds. This led to the identification of a new BGC, which is named herein as P08-G05-cluster 16 (P08-G05-c16), and its DNA sequence is shown in SEQ ID NO.1. The cluster was cloned and expressed in a heterologous actinomycete host, specifically the strain Streptomyces coelicolor M1152ΔmatAB, which is a modified derivative of the model strain Streptomyces coelicolor A3(2) / M145 (ATCC BAA-471). The preparation of this strain is described in the following examples. The P08-G05-c16 gene cluster was cloned in an inducible bacterial artificial chromosome (BAC) vector and transferred into the Streptomyces coelicolor M1152ΔmatAB strain by tri-parental conjugation to prepare the conjugant strain M1152ΔmatAB(P08-G05_C16). This conjugant expresses the BGC and synthesizes the compound. Its extract has been shown to have antibacterial activity. This new compound, which we named nidaramycin, was extracted from the heterologous host, purified, and subjected to structure analysis and characterization, confirming that it has structural novelty, antibacterial activity, and low cytotoxicity.

[0006] Accordingly, a first aspect provided herein is a compound of formula (I), wherein

[0007]

Chemical formula

[0008] wherein R 1 is -SO2OH, -SO2OR, or -SO2R, and R 2 is H, or R 2 is -SO2OH, -SO2OR, or -SO2R, and R 1 is H, R is a C1-C 20 hydrocarbyl group, each R 3 is independently selected from H or a C1-C 20 hydrocarbyl group, a compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof.

[0009] A second aspect provided herein is a compound of formula I as defined herein for use as a drug, or in other words for therapeutic use.

[0010] In particular, the drug is an antibiotic and the treatment is an antimicrobial treatment, particularly an antibacterial treatment.

[0011] Thus, a third aspect provides a compound of formula I as defined herein for use as an antibacterial drug, or for use in the treatment of a microbial infection, particularly a bacterial infection.

[0012] A fourth aspect provides the use of a compound of formula I as defined herein for the preparation of a drug for use in the treatment of a microbial infection, particularly a bacterial infection.

[0013] The fifth aspect provides a method for treating microbial infections, particularly bacterial infections, in a subject, the method comprising administering to the subject an effective amount of a compound of formula I as defined herein.

[0014] The subject may be any human or non-human animal, particularly a mammal. Thus, the medical uses herein include human clinical and veterinary uses, as well as uses in animal husbandry and agriculture, which include uses in aquaculture and use as a plant protection agent against phytopathogens.

[0015] In certain embodiments, the bacterial infection is an infection by Gram-positive bacteria.

[0016] The sixth aspect provides a pharmaceutical composition comprising a compound of formula I as defined herein together with at least one pharmaceutically acceptable carrier, additive, and / or excipient. In certain embodiments, the pharmaceutical composition is suitable for parenteral, oral, or topical administration.

[0017] Similar to the medical uses outlined above, the compound may also be used non-medically (i.e., non-therapeutically), for example for surface decontamination, disinfection, or sterilization, using its in vitro or ex vivo antimicrobial / antibacterial properties.

[0018] Accordingly, the seventh aspect provides the use of a compound of formula I as defined herein as an antimicrobial agent, particularly an antibacterial agent.

[0019] In other words, this aspect also provides a method for controlling bacteria on a surface, the method comprising the step of applying (or contacting) the compound as defined herein to the surface. Controlling bacteria includes suppressing the growth and / or survival rate of the bacteria. This may further include reducing the number of bacteria (e.g., killing the bacteria), and / or reducing or preventing the growth (replication) of the bacteria.

[0020] The compound may be prepared by biosynthesis in a host, which is a host that has been modified or engineered to express BCG containing the biosynthetic genes for the synthesis of the compound, or in other words, a host into which a BGC, or more specifically a nucleic acid molecule containing the BGC or its constituent genes, has been introduced. In certain embodiments, the host is a heterologous host, i.e., a host that does not naturally contain the BGC. However, in other embodiments, the BGC may be introduced into the organism from which the BGC was obtained, i.e., isolate P08-G05 or more generally a strain that intrinsically contains the BGC. Alternatively, the compound may be prepared in an in vitro transcription and translation (IVTT) system.

[0021] Thus, the eighth aspect is (a) a nucleotide sequence shown in SEQ ID NO.1, or (b) a nucleotide sequence that is a complement of SEQ ID NO.1, or (c) a nucleotide sequence degenerate with SEQ ID NO.1, or (d) a nucleotide sequence having at least 85% sequence identity with SEQ ID NO.1, or (e) a portion of any one of (a) to (d) to provide a nucleic acid molecule, said nucleic acid molecule encoding or being complementary to a nucleic acid molecule encoding one or more polypeptides, or comprising or being complementary to a nucleic acid molecule comprising one or more genetic factors having functional activity in the synthesis of an antibacterial compound.

[0022] The functional activity may be enzymatic activity, or transport or import activity, or regulatory activity (e.g., regulation of gene expression), or any other activity that contributes to the synthesis or transport of a compound or its constituent moieties.

[0023] Thus, more generally, a nucleic acid molecule may be defined as one that includes one or more nucleotide sequences contributing to the biosynthesis of a compound. In other words, a nucleic acid molecule may include one or more nucleotide sequences that constitute or are part of a biosynthetic gene cluster for the synthesis of a compound.

[0024] In particular, the antibacterial compound is a compound of formula I as defined herein or a derivative thereof.

[0025] Particularly in part (e), the portion of the nucleotide sequence includes a sequence corresponding to an open reading frame (ORF) encoding a biosynthetic gene or a protein involved in the biosynthesis of the compound, or a sequence complementary to or degenerate with such a sequence.

[0026] In certain embodiments, the nucleic acid molecule includes nucleotide sequences (a) to (d) and encodes a polypeptide for the synthesis of an antibacterial compound, particularly a compound of formula I as defined herein or a derivative thereof. In other words, the nucleic acid molecule includes nucleotide sequences that together provide the biosynthetic machinery for the production of the compound. Thus, in this embodiment, the nucleic acid molecule may be defined as encoding a biosynthetic system for the synthesis of the compound or as including a BGC for the synthesis of the compound.

[0027] In certain embodiments, the nucleic acid molecule includes a nucleotide sequence for the production of a compound in a host strain that is an actinomycete host, particularly a Streptomyces host, more particularly a Streptomyces coelicolor host, particularly the Streptomyces coelicolor strain A3(2), M145, M1152, or M1152ΔmatAB as defined or described herein.

[0028] As defined in part (e), a nucleic acid molecule may encode for a part of a complete biosynthetic system, such as individual components of a BGC. Individual genes or ORFs of a BGC have been identified and annotated as described in more detail below (see Table 1). Thus, a portion of a nucleic acid molecule may represent or correspond to an individual gene or ORF, such as one that encodes a polypeptide involved in the biosynthesis of a compound, or two or more such genes or ORFs. Thus, in certain embodiments, a nucleic acid molecule comprises a nucleotide sequence shown in any one or more of SEQ ID NOs: 2-29, or a nucleotide sequence that is complementary or degenerate thereto, or a nucleotide sequence having at least 85% sequence identity thereto.

[0029] In another embodiment, a nucleic acid molecule comprises a nucleotide sequence that encodes an amino acid sequence shown in any one or more of SEQ ID NOs: 30-57, or an amino acid having at least 85% sequence identity thereto.

[0030] A ninth aspect provides a polypeptide encoded by a nucleic acid molecule as defined above.

[0031] A tenth aspect provides a recombinant construct comprising a nucleic acid molecule as defined herein.

[0032] In certain embodiments, the recombinant construct comprises one or more other nucleic acid sequences, such as control sequences, expression regulatory sequences, or genetic factors involved in the replication or transfer of the nucleic acid molecule.

[0033] An eleventh aspect provides a vector comprising a nucleic acid molecule or recombinant construct as defined herein.

[0034] In certain embodiments, the vector is a plasmid, cosmid, or artificial chromosome, particularly a bacterial artificial chromosome (BAC).

[0035] Aspect 12 provides a microbial host cell comprising a nucleic acid molecule, recombinant construct, or vector as defined herein. In other words, this aspect provides a modified or engineered microbial host cell into which a nucleic acid molecule, recombinant construct, or vector has been introduced. As noted above, the host may be a heterologous host. By definition, a heterologous host or engineered host cell does not include a natural producer of the compound or a natural strain that inherently contains the BGC. However, as also noted above, it is not excluded that a nucleic acid molecule, recombinant construct, or vector is introduced into the original strain from which the nucleic acid molecule is derived. The nucleic acid molecule may be introduced into the host cell by increasing the copy number, i.e., one or more copies may be introduced.

[0036] The host cell may be a production host cell for the production of a compound, or a host cell generated for the purpose of cloning a nucleic acid molecule (e.g., growing or producing a nucleic acid molecule) or transferring it to another host cell (i.e., it may be a cloning or transfer host cell).

[0037] Aspect 13 provides a method for producing a compound of formula I as defined herein, the method comprising introducing a nucleic acid molecule, recombinant construct, or vector as defined herein into a microbial host cell and enabling the expression of the nucleic acid molecule (in particular, enabling the expression of the individual gene sequences or ORFs of the nucleic acid molecule, i.e., enabling the expression of the genes of the BGC).

[0038] Alternatively defined, this aspect provides a method for producing a compound of formula I as defined herein, the method comprising introducing a nucleic acid molecule, recombinant construct, or vector as defined herein into a microbial host cell and culturing the host cell (or enabling its growth) under conditions under which the biosynthetic system for the synthesis of the compound is expressed.

[0039] More specifically, the condition is a condition that enables a compound to be synthesized by the expressed biosynthetic system.

[0040] This production method may further include a step of recovering (or in other words, collecting or harvesting) the compound. Further, this method may include a step of isolating, separating, or purifying the compound.

[0041] In certain embodiments, the host cell is a bacterial host cell such as, for example, actinobacteria. In specific embodiments, the host cell is an actinomycete, particularly a Streptomyces host cell, more particularly Streptomyces coelicolor, particularly the Streptomyces coelicolor strain A3(2), M145, M1152, or M1152ΔmatAB as defined or described herein.

[0042] A fourteenth aspect provides a compound obtainable or obtainable by the production method defined herein. In particular, this compound is obtainable or obtainable by a method comprising expressing a nucleic acid molecule in a heterologous actinomycete host cell, particularly a Streptomyces host cell, more particularly Streptomyces coelicolor, particularly the Streptomyces coelicolor strain A3(2), M145, M1152, or M1152ΔmatAB as defined or described herein.

[0043] Compounds obtainable or obtainable by this method may be used in any of the uses or methods shown above, may be for use therein, or may be included in a pharmaceutical composition. In other words, in any of the embodiments shown above, this compound may be alternatively or additionally defined as a compound obtainable or obtainable by the production method defined herein.

[0044] Furthermore, the production method herein enables modification of the biosynthetic system for synthesis in order to modify the produced compound. Thus, the nucleic acid molecule or the individual nucleotide sequence encoding the biosynthetic enzyme contained therein may be modified, inactivated, or deleted, and / or additional enzyme activity may be introduced. Such modifications may also change the biosynthetic pathway to enable the obtainment of modified derivatives of the compound.

[0045] Accordingly, a fifteenth aspect provides a method for preparing a nucleic acid molecule encoding a modified biosynthetic system for the synthesis of a modified derivative of a compound of formula I as defined herein, said method comprising modifying a nucleic acid molecule as defined herein. The nucleic acid molecule may be modified by introduction, mutagenesis, deletion, substitution, or inactivation of a sequence encoding one or more activities or proteins encoded by said nucleic acid molecule.

[0046] In certain embodiments, one or more of the nucleotide sequences of SEQ ID NOs: 2-29, or a nucleotide sequence complementary or degenerate thereto relative to any one of SEQ ID NOs: 2-29, is modified. BRIEF DESCRIPTION OF THE DRAWINGS

[0047]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0048] The disclosure and invention of this specification relate to nidamycin of a new antibacterial compound having the structure shown in formula I, as defined above. As shown above, this novel compound was discovered by screening and identifying a new biosynthetic gene cluster in strains of Actinobacteria. As detailed in the following examples, by combining the previous bioactivity data for those strains with the results of a wide range of bioinformatics and phylogenetic analyses, including genome sequencing, gene annotation, cluster analysis, and manual curation of the analysis results, we were able to select specific strains and identify novel BGCs in their genomes. Based on the results of our analysis, one such cluster, cluster 16, was selected from an isolate identified as strain P08-G05. Based on bioinformatics and cluster analysis, a hypothesis was proposed that this cluster encodes a novel moenomycin-like compound. This cluster was cloned and transferred into a heterologous host, specifically the strain Streptomyces coelicolor M1152ΔmatAB. The preparation of this strain is further detailed below. The resulting conjugational completion strain, strain M1152ΔmatAB(P08-G05_C16), was cultured and antibacterial activity in the cell-free extract was demonstrated against various Gram-positive bacteria, including Staphyloccus aureus and Enterococcus faecium. Furthermore, in the toxicity tests described in the following examples, it was demonstrated that the compound is not toxic to mammalian cell lines. As detailed in the following examples, the compound was purified and subjected to structure elucidation investigations. This led to the elucidation of the structure of the novel compound.

[0049] As shown in formula I, we consider there to be two possibilities for the position of the sulfate group in this molecule, and those positions are R at C4 and C3, respectively, of ring moiety D 1 and R 2as shown (see below), and are called nidarosomycin D4 and nidarosomycin D3, respectively.

[0050] [Chemical formula]

[0051] In the compound of formula (I), R 1 is -SO2OH, -SO2OR, or -SO2R, and R 2 is H, or R 2 is -SO2OH, -SO2OR, or -SO2R, and R 1 is H, R is a C1-C 20 hydrocarbyl group.

[0052] Preferably, R 1 is -SO2OH or -SO2OR, and R 2 is H, or R 2 is -SO2OH or -SO2OR, and R 1 is H. Most preferably, R 1 is -SO2OH, and R 2 is H, or R 2 is -SO2OH, and R 1 is H. It will be recognized that when the compound is in salt form, the H in any -SO2OH group can be replaced by a non-H cation.

[0053] The carboxylic acid group in the nidarosomycin compound can be modified to form an ester form as shown by the group -COOR 3 in formula I, where R 3 is C1-C 20 hydrocarbyl. As is well known in the art, this modification may be achieved in both chemical and enzymatic ways.

[0054] As used herein, hydrocarbyl typically means alkyl, alkenyl, alkynyl, or aryl, preferably alkyl and aryl, most preferably alkyl. As used herein, C1-C 20 Hydrocarbyl typically refers to C1-C 20 alkyl, C1-C 20 alkenyl, C1-C 20 alkynyl, or C6-C 20 aryl group. The alkyl, alkenyl, and alkynyl groups may be cyclic or acyclic. Additionally, the alkyl, alkenyl, and alkynyl groups may be linear or branched. C1-C 20 Hydrocarbyl groups are typically C1-C 10 hydrocarbyl groups (e.g., C1-C 10 alkyl, alkenyl, alkynyl, or aryl groups), such as C1-C6 hydrocarbyl groups (e.g., C1-C6 alkyl, alkenyl, alkynyl, or aryl groups), most preferably C1-C6 alkyl groups. The above definitions for hydrocarbyl apply to R and especially R 3 .

[0055] Each R 3 group may be independently selected from H or a C1-C 20 hydrocarbyl group. The compound may have all R 3 groups as H, or two R 3 groups as H and the others as C1-C 20 hydrocarbyl, or one R 3 group as H and the other two as C1-C 20 hydrocarbyl, or all three R 3 groups as C1-C 20 hydrocarbyl. Preferably, all R 3 groups are H. When the compound is in salt form, it is clear that H (i.e., R 3 as H) may be substituted with a non-H cation.

[0056] Thus, in one embodiment, the compound is of formula II and

[0057] [Chemical formula]

[0058] Here, R 1 is -SO2OH, -SO2OR, or -SO2R, and R 2 is H, or R 2 is -SO2OH, -SO2OR, or -SO2R, and R 1 is H, R is a C1-C 20 hydrocarbyl group, preferably R 1 is -SO2OH, and R 2 is H, or R 2 is -SO2OH, and R 1 is H, Formula II, or has a structure of its pharmaceutically acceptable salt, solvate, or hydrate.

[0059] In certain embodiments, the compound has Structure I,

[0060] [Chemical formula]

[0061] or has its pharmaceutically acceptable salt, solvate, or hydrate.

[0062] In another certain embodiment, the compound has Structure II,

[0063] [Chemical formula]

[0064] or has its pharmaceutically acceptable salt, solvate, or hydrate.

[0065] It will be appreciated that the compounds can be in the form of pharmaceutically acceptable salts, solvates, or hydrates.

[0066] The compounds may be in the form of metal salts such as, for example, lithium, sodium, potassium, or calcium salts (in this case typically, one or more R 3 groups are lithium, sodium, or potassium).

[0067] In addition, pharmaceutically acceptable salts may be readily prepared by using the desired acid. The salts may be precipitated from solution and collected by filtration, or recovered by evaporation of the solvent. Suitable addition salts are formed from inorganic or organic acids that form non-toxic salts, examples of which are hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, oxalate, oxaloacetate, trifluoroacetate, saccharate, benzoate, alkyl or aryl sulfonates (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, or p-toluenesulfonate), and isethionate.

[0068] One of ordinary skill in organic chemistry will recognize that many organic compounds can form complexes with the solvent in which they reacted or from which they precipitated or crystallized. These complexes are known as "solvates". Complexes with water are known as "hydrates". Solvates and hydrates of the compounds of the present invention are within the scope of the present invention. The salts of the compounds may form solvates and hydrates, and the present invention also includes all such solvates and hydrates.

[0069] As described above, the compound has been shown to have antibacterial activity. In other words, the compound has antimicrobial activity. More specifically, the compound has antibacterial activity. That is, the compound can suppress the growth and / or survival rate of microorganisms, particularly bacteria. In certain embodiments, the compound has antibacterial activity against Gram-positive bacteria.

[0070] As detailed in the following examples, extracts of the conjugant strains producing the compound were tested in a bioassay against a panel of strains to demonstrate activity. Further, the compound was purified and the activity against the purified compound was confirmed. As follows, the minimal inhibitory concentrations (MIC) against Gram-positive indicator organisms were determined. -MIC 70 Staphylococcus aureus ATCC29213: 0.53 μg / ml -MIC 70 Staphylococcus aureus ATCC43300 (MRSA): 0.53 μg / ml -MIC 70 : Enterococcus faecium CTC492: 8.45 μg / ml -MIC 70 : Enterococcus faecium CCUG37832: 2.11 μg / ml Except for the case of Enterococcus faecium CCUG37832, these values are not inferior compared to vancomycin used as a reference compound. In particular, the effectiveness of the compound was higher than that of vancomycin against both methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus.

[0071] Therefore, in certain embodiments, the compound has activity against drug-resistant (or antibiotic-resistant) bacteria, particularly multi-drug resistant (MDR) bacteria. In particular, the compound has activity against drug-resistant (or antibiotic-resistant) Gram-positive bacteria, particularly multi-drug resistant (MDR) Gram-positive bacteria.

[0072] In particular, the compound is effective against methicillin and / or vancomycin-resistant bacteria, especially Gram-positive bacteria.

[0073] More generally, the compound is effective against Gram-positive bacteria that are resistant to any class of antibiotics, especially classes of antibiotics including β-lactams (including penicillins and cephalosporins), glycopeptides, (phospho)glycolipids, macrolides, tetracyclines, sulfonamides, aminoglycosides, carbapenems, and quinolones (including fluoroquinolones). In certain embodiments, the class of antibiotics is a β-lactam or a glycopeptide.

[0074] In particular, the compound is effective against Staphylococcus and / or Enterococcus. In certain embodiments, the compound is effective against Staphylococcus aureus and / or Enterococcus faecium. In more particular embodiments, the compound is effective against methicillin-resistant Staphylococcus aureus (MRSA) and / or vancomycin-resistant Enterococcus faecium.

[0075] However, more generally, the compound may be used against Gram-positive bacteria of any species, especially clinically relevant Gram-positive bacteria including, for example, Micrococcus, Streptococcus, Pneumococcus, Bacillus, Listeria, Clostridium, and the like.

[0076] Antibacterial or antimicrobial activity may be readily evaluated according to methods well known in the art. For example, broth microdilution assays for determining MIC, such as those used in the following examples, are widely used and reported as well as agar plate-based methods (such as disk diffusion assays).

[0077] Notably, it has also been demonstrated that the compound has no or negligible cytotoxicity to mammalian cells, indicating that this compound is suitable for clinical use.

[0078] Thus, as shown above, the compounds herein are used medicinally, particularly as therapeutic antimicrobial agents or more specifically as antibacterial agents, i.e., in the treatment or prevention of microbial or bacterial infections.

[0079] The subject to be treated by the compound may be any subject infected or at risk of infection. As noted above, the subject is typically human, but veterinary use is included, so the subject may be any animal, particularly a vertebrate, such as an animal selected from mammals, birds, amphibians, fish, and reptiles. Thus, the compound is used in both clinical and veterinary and aquaculture environments, for example. In certain embodiments, the subject is a mammal. The animal may be a livestock or a breeding or commercially valuable animal, including laboratory animals or animals in zoos or animal sanctuaries. Thus, representative animals include dogs, cats, rabbits, mice, guinea pigs, hamsters, horses, pigs, sheep, goats, cows, chickens, turkeys, butterflies, ducks, geese, parrots, budgerigars, pigeons, salmon, trout, cod, sea bass, and carp. The subject may be considered a patient.

[0080] Similar to its use in the context of animals, the compound may also be used as an antimicrobial (or antibacterial) agent in the context of plants, i.e., as a plant protection agent against plant pathogens. Thus, the compound generally has benefits in the context of agriculture and horticulture, or put differently, in the treatment or prevention of infections in plants.

[0081] The compound is administered to a subject in an effective amount to treat or prevent an infection. The "effective amount" of the compound is, for example, an amount effective to inhibit the growth and / or survival rate of microorganisms such as bacteria, and / or to provide a measurable or recognizable improvement in the clinical condition of the subject, such as in one or more clinical parameters or symptoms of an infection, etc., thereby providing a clinical benefit to the subject.

[0082] One of ordinary skill in the art could readily determine what the effective amount of the compound is based on routine dosage response protocols, and conveniently, routine techniques for evaluating microbial growth inhibition as discussed above.

[0083] The preferred dosage of the compound varies for each subject and can be determined by a physician or veterinarian according to the subject's weight, age, and gender, the severity of the condition, and the mode of administration.

[0084] As used herein, the term "treatment" is used broadly to include any therapeutic effect, i.e., any beneficial effect against or related to an infected condition. Thus, it includes not only the eradication or elimination of an infection, or the cure of a subject or an infection, but also the improvement of an infection or condition in a subject. Thus, for example, it includes any symptom or sign of an infection or condition, or the improvement of any clinically acceptable indicator of an infection / condition. Thus, treatment includes both radical and symptomatic therapies for existing or diagnosed infections / conditions, for example.

[0085] As used herein, the term "prevent" indicates any prophylactic or preventive effect. Thus, this term includes, for example, delaying, limiting, reducing, or preventing infection or its onset, or one or more of its symptoms or signs, with respect to a state or symptom or sign prior to a prophylactic treatment. Thus, prevention explicitly includes both the absolute prevention of the appearance or occurrence of an infection or its symptoms or signs and any delay in the onset or occurrence of an infection or symptoms or signs, or a reduction or limitation in the occurrence or progression of an infection or symptoms or signs.

[0086] For such use, the compound may be formulated as a pharmaceutical composition. The pharmaceutical composition includes one or more pharmaceutically acceptable carriers, additives, or excipients.

[0087] The pharmaceutical composition may be formulated for administration by any convenient or desired means, such as, for example, parenteral, enteral, oral (particularly peroral), or topical administration, or administration by inhalation.

[0088] Conventional galenic preparations include tablets, pills, powders (e.g., inhalable powders), troches, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), sprays (e.g., nasal sprays), compositions for use in nebulizers, ointments, soft and hard (e.g., gelatin) capsules, suppositories, sterile injection solutions, and sterile packaged powders, among others.

[0089] Examples of suitable carriers, excipients, and diluents are lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, inert alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, water, water / ethanol, water / glycol, water / polyethylene, hypertonic saline, glycol, propylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, or fatty substances such as, for example, hard fat, or suitable mixtures thereof. Notable excipients and diluents are mannitol and hypertonic saline (physiological saline).

[0090] The composition may additionally contain additives such as, for example, lubricants, wetting agents, emulsifying agents, suspending agents, preservatives, sweeteners, and flavoring agents. The pharmaceutical composition may contain additional therapeutically active agents.

[0091] For example, parenterally administrable forms such as intravenous solutions should be sterile and free of agents that are not physiologically acceptable and should have a low volume osmolality to minimize irritation or other adverse effects upon administration, and thus the solution should preferably be isotonic or slightly hypertonic, such as, for example, hypertonic saline (physiological saline). Suitable media include aqueous media conventionally used for administering parenteral solutions, such as sodium chloride injection, Ringer’s injection, glucose injection, glucose and sodium chloride injection, lactated Ringer’s injection, and other solutions known in the art. The solution may contain preservatives, antimicrobial agents, buffers, and antioxidants conventionally used in parenteral solutions, excipients, and other additives that are compatible with the compound and do not interfere with the manufacture, storage, or use of the product.

[0092] For topical administration, the compound can be incorporated into creams, ointments, gels, and transdermal patches, etc. Additionally, the compound can be incorporated into medical dressings such as wound dressings like woven fabric (e.g., textile) dressings or non-woven fabric dressings (e.g., dressings with gels or gel components).

[0093] Further delivery systems include in situ drug delivery systems, such as gels, where a solid, semi-solid, amorphous, or liquid crystal gel matrix is formed in situ and may contain the compound. Such matrices can be conveniently designed to control the release of the compound from the matrix, for example, to delay the release and / or sustain it over a selected period. Such systems may form a gel only when in contact with biological tissues or fluids. Typically, the gel is bioadhesive. Delivery to any body site that can hold or be adapted to hold a pre-gel composition can be targeted by such delivery techniques.

[0094] For applications to the oral cavity, buccal, and tooth surfaces, for example, for oral health care or hygienic purposes, toothpastes, dental gels, dental foams, and mouthwashes are specifically mentioned.

[0095] Inhalable compositions may take the form of, for example, inhalable powders, solutions, or suspensions. These may include, for example, sprayable solutions without propellants.

[0096] The compound may be used with other therapeutically active agents including other antibiotics or antimicrobials, such as antifungal or antiviral agents, etc. These agents may be used separately, together in the same composition, or simultaneously, sequentially, or separately, for example, at any desired time intervals.

[0097] Accordingly, provided herein are products, such as kits, that include the compounds defined herein together with a second therapeutically active agent for separate, sequential, or simultaneous use in the treatment of a subject, particularly the treatment of an infection in a subject.

[0098] Other possible therapeutically active agents include, for example, immunomodulators such as immunostimulants like cytokines or interferons, growth factors, enzymes, mucolytics, analgesics, anti-inflammatory agents, bronchodilators, or steroids.

[0099] As noted above, the compound and the second therapeutically active agent may be formulated together in the same composition or may be formulated in separate compositions for simultaneous or separate administration, for example, according to a defined dosing schedule.

[0100] In addition to medical use, the antimicrobial properties of the compounds may also be utilized in a non-clinical environment. Thus, in addition to the plant protection uses discussed above, the compounds may be used in an abiotic environment, such as on abiotic (or in other words, inanimate) surfaces or in abiotic locations, for purposes of disinfection or decontamination, or to prevent or reduce bacterial colony formation.

[0101] Thus, the compound may be used as an antibacterial agent against bacteria on any surface. The surface is not limited and includes any surface on which bacteria can occur. Inanimate (or abiotic) surfaces include any such surface that is exposed to the risk of microbial contact or contamination. Thus, specifically included are the surfaces of medical devices or machines such as industrial machines, or any surface exposed to an aquatic environment (e.g., marine equipment, or ships or boats or their parts or components), or any surface exposed to any part of the environment such as on pipes or buildings. Such inanimate surfaces exposed to microbial contact or contamination specifically include machines or equipment for food or beverage processing, preparation, storage, or distribution, air conditioners, industrial machines in, for example, chemical or biotechnology processing plants, storage tanks, medical or surgical equipment, and any part of cell and tissue culture equipment. Any device or equipment for transporting or shipping or delivering materials is susceptible to microbial contamination. Such surfaces will specifically include pipes (as used herein, this term is used broadly to include any conduit or line). Representative inanimate or abiotic surfaces include, but are not limited to, equipment or surfaces for food processing, storage, distribution, or preparation, tanks, conveyors, floors, drain pipes, coolers, freezers, equipment surfaces, walls, valves, belts, pipes, air conditioning ducts, cooling devices, food or beverage distribution lines, heat exchangers, boat hulls, or any part of the boat structure exposed to water, dental waterlines, oil drilling conduits, contact lenses, and storage cases.

[0102] As described above, medical or surgical devices or instruments represent certain classes of surfaces where bacterial contamination can form. This may include any type of line, including catheters (e.g., central venous and urinary catheters), prosthetic devices such as heart valves, artificial joints, dentures, dental crowns, dental caps, and soft tissue implants (e.g., chest, hip, and lip implants). Any type of implanted (or "indwelling") medical device is included (e.g., stents, intrauterine devices, pacemakers, indwelling tubes (e.g., endotracheal or tracheostomy tubes), orthotics or prosthetic devices, lines or catheters). An "indwelling" medical device may include a device where any part of it is included within the body, i.e., the device may be wholly or partially indwelling.

[0103] The surface can be made of any material. For example, the surface may be a metal such as aluminum, steel, stainless steel, chromium, titanium, iron, and alloys thereof. In addition, the surface can be plastic, glass, brick, tile, ceramic, porcelain, wood, vinyl, linoleum, or carpet, and combinations thereof. In addition, the surface can be a food such as beef, chicken, pork, vegetables, fruits, fish, crustaceans, and combinations thereof.

[0104] In addition, compounds may be incorporated into materials and products for such use of disinfection.

[0105] In addition, for such in vitro use, the compound may be used together with other agents such as other antimicrobial agents, disinfectants, detergents, etc., or incorporated into paints and coatings, etc.

[0106] The compound is biosynthetically prepared by the expression of the BGC in a suitable host. For this purpose, provided herein is a nucleic acid molecule comprising a nucleotide sequence corresponding to the BGC (or in other words, a nucleic acid molecule comprising the nucleotide sequences of the components of the BGC).

[0107] The nucleotide sequence of the BGC is shown in SEQ ID NO.1, which represents the sequence of the BGC cloned from the marine actinomycete isolated strain PG08-G05. SEQ ID NO.1 has been annotated and is shown to contain several genes or ORFs encoding various polypeptides responsible for the activities required for the synthesis of the compound.

[0108] In particular, the putative functions of the genes were predicted using antiSMASH software in combination with manual analysis and curation. antiSMASH is a software package used for the identification, annotation, and analysis of secondary metabolite biosynthetic gene clusters in microbial genome sequences (Medema et al., Nucleic Acids Research, 2011, 39, Web Server Edition, W339-W346).

[0109] The BGC encodes the components necessary for the production of the compound in the host strain or IVTT system. However, since the roles of all the encoded polypeptides in biosynthesis have not yet been assigned, not all ORFs may be essential. The various genes / ORFs may encode enzymes that catalyze one or more reactions in the biosynthetic pathway, or proteins that do not have enzymatic activity but instead are involved in other processes, for example, the control of the synthetic process such as a transcription factor, or the transport of a compound or intermediate or substrate compound, for example, intracellularly or into or out of the cell, or conferring resistance (or "immunity") to the synthesized compound. Several sequences encoding transporter proteins have been identified. The BGC contains 28 ORFs shown in Table 1 below.

[0110]

Table 1

[0111] The amino acid sequences corresponding to the translations of SEQ ID NOs. 2 to 29 are shown in SEQ ID NOs. 30 to 57, respectively.

[0112] As described above, the nucleic acid molecule may contain a nucleotide sequence corresponding to all or part of the BGC. Thus, the nucleic acid molecule may contain a nucleotide sequence corresponding to all 28 of SEQ ID NOs. 2 to 29 (or a sequence having at least 85% sequence identity thereto), or may contain a subset thereof. In other words, the nucleic acid molecule may contain all or part of SEQ ID NO. 1 (or a sequence having at least 85% sequence identity thereto). The part may correspond to or represent an individual ORF or gene. For example, it may encode a polypeptide involved in the biosynthesis of a compound. Such polypeptides and their coding sequences may themselves represent individual useful products and may be used for other things beyond the biosynthesis of the compound.

[0113] In another embodiment, the part may contain a plurality or two or more ORFs, but less than the total number of 28 ORFs, i.e., it may contain any 2 to 27 of SEQ ID NOs. 2 to 29. The part may contain the ORFs necessary and sufficient for the biosynthesis of the compound.

[0114] The nucleotide sequence encoding the polypeptide is a specific embodiment herein. In another embodiment, the nucleotide sequence may contain functional genetic elements such as, for example, a promoter, an operator, a promoter-operator, an enhancer, or other regulatory regions.

[0115] The nucleic acid molecule need not include the entire cluster shown in Table 1 and may include a portion or part thereof. This nucleic acid molecule may include one or more genes and / or regulatory sequences, or non-coding or coding functional genetic factors, etc. Generally speaking, the nucleic acid molecule for the production of a compound will include several different genes and / or regulatory molecules that result in the synthesis of an antibacterial compound such as niddamycin or its derivatives. For example, in order to produce esters included in Formula I, or to produce derivatives or analogs, etc., it is also possible to include in the nucleic acid molecule one or more additional nucleotide sequences encoding other activities, such as polypeptides having activity for modifying the structure of niddamycin. As further described below, it is also possible to modify the sequences encoding the enzymes to change their activities, or to delete or inactivate them, to change the structure of the synthesized compound and generate derivative molecules.

[0116] In certain embodiments, the nucleic acid molecule includes a nucleotide sequence sufficient for the synthesis of a compound in a suitable host cell. In other words, the nucleic acid molecule encodes a biosynthetic system or biosynthetic machinery for the synthesis of a compound. Stated another way, the nucleic acid molecule includes a BGC for the synthesis of a compound.

[0117] The nucleotide sequence included in the nucleic acid molecule may be defined as a biosynthetic gene or ORF, i.e., it is a gene or ORF encoding a polypeptide that functions in the biosynthetic process for a compound of Formula I (or more specifically Formula II), or niddamycin or its derivatives (shown in Structures I and II above), or related molecules.

[0118] In certain embodiments, the portion of the nucleic acid molecule may be at least 300 bases in length, such as at least 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bases, particularly as far as individual genes / ORFs are concerned. However, when the molecule contains sequences corresponding to all or most of BCG, the portion becomes quite large, such as at least 10,000, 20,000, 25,000, or 30,000 bases.

[0119] The nucleic acid molecule may be an isolated molecule, i.e., a molecule separated from the components that are normally found together in nature, or it may be a recombinant or synthetic nucleic acid molecule. Generally speaking, since the BGC is cloned from its natural host, the molecule becomes an artificial molecule.

[0120] The molecule may be any nucleic acid, but generally speaking it is DNA.

[0121] As defined above, the nucleic acid molecule may include a nucleotide sequence that is a variant of the sequences of SEQ ID NOs. 1 - 29, or a nucleotide sequence that encodes a variant, such as a functionally equivalent variant, of the amino acid sequences of SEQ ID NOs. 30 - 57. Such variants may include portions, degenerate sequences, or homologs defined by % sequence identity to any one or more of SEQ ID NOs. 1 - 57. The activity of the variant polypeptide or the polypeptide encoded by the variant nucleotide sequence may be as defined above.

[0122] The term "biosynthetic gene or ORF" as used above includes such variant sequences. The variant sequence retains at least one function of the entity from which it is derived, and encodes a polypeptide that has, for example, substantially the same properties or activities as the original / origin / parent polypeptide, or at least the same general type of properties or functions.

[0123] Generally, the term "gene" includes an ORF encoding a polypeptide and may also include regulatory sequences such as a promoter. The term "ORF" refers only to the part of a gene that is responsible for encoding a polypeptide.

[0124] As described above, the nucleic acid molecule may comprise a nucleotide sequence selected from SEQ ID NO.1, or any one or more of SEQ ID NOs.2-29, or a nucleotide sequence showing at least 85% sequence identity with any of the foregoing sequences. More specifically, this may be at least 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity, or a sequence that is complementary to or degenerate with respect thereto.

[0125] Furthermore, the nucleic acid molecule may comprise one or more amino acid sequences selected from SEQ ID NOs.30-57, or a nucleotide sequence encoding an amino acid sequence showing at least 85% sequence identity thereto.

[0126] Similarly, a polypeptide herein, i.e., a polypeptide encoded by a nucleic acid molecule defined and described herein, may comprise any one of the amino acid sequences shown in SEQ ID NOs.30-57, or all or part of an amino acid sequence having at least 85% sequence identity thereto.

[0127] More specifically, in the context of the amino acid sequences shown above, this sequence identity may be at least 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with any one of the amino acid sequences shown in SEQ ID NOs.30-57.

[0128] % sequence identity may be readily determined using commercially available sequence comparison programs capable of calculating the percentage homology or identity between two or more sequences.

[0129] Percentage identity or sequence identity may be calculated for a continuous sequence, i.e., one sequence is aligned with the other, and each amino acid of one sequence is directly compared with the corresponding amino acid of the other sequence, one residue at a time. This is called "ungapped" alignment. Typically, such ungapped alignments are performed only for a relatively small number of residues.

[0130] This is a very simple and consistent method, but it does not take into account, for example, that in an otherwise identical pair of sequences, one insertion or deletion in the nucleotide sequence can shift the alignment of subsequent codons, with the result that the percent identity can be significantly reduced when the overall alignment is performed. Thus, most sequence comparison methods are designed to generate an optimal alignment taking into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to attempt to maximize local homology.

[0131] However, these more complex methods make the sequence alignment with fewer gaps, which reflect a higher relatedness between the two comparison sequences for the same number of identical amino acids, achieve a higher score than those with many gaps, by assigning a "gap penalty" to each gap that occurs in the alignment. An "affine gap cost", which gives a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue within the gap, is typically used. This is the most commonly used gap scoring system. A high gap penalty will, of course, result in an optimized alignment with fewer gaps. Most alignment programs allow modification of the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalties for amino acid sequences are -12 for the gap and -4 for each extension.

[0132] Therefore, to calculate the maximum percentage homology / sequence identity, it is necessary first to generate an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984), Nucleic Acids Res. 12:387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403 - 410), and a series of comparison tools in GENEWORKS. Both BLAST and FASTA are available for both offline and online searches (see Ausubel et al. (1999) ibid, pp. 7 - 58 to 7 - 60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool called BLAST2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247 - 50; FEMS Microbiol. Lett. (1999) 177:187 - 8).

[0133] While the final percentage identity can be measured from the perspective of identity, typically the alignment process itself is not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance is commonly used. An example of such a commonly used matrix is the BLOSUM62 matrix, which is the default matrix for a series of BLAST programs. The GCG Wisconsin program generally uses either the public default values or, when supplied, a custom symbol comparison table (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, it is preferred to use a default matrix such as BLOSUM62. Preferably, the percentage identity is determined over the entire reference and / or query sequence. Once the software generates an optimal alignment, it becomes possible to calculate the percentage homology, preferably the percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0134] Such variants of a defined sequence may be readily prepared using, for example, site-directed mutagenesis, or recombinant DNA techniques such as gene substitution or gene editing techniques, homologous recombination. In the art, various such methods are known and described.

[0135] As described above, the nucleic acid molecules and nucleic acid sequences defined herein may include any nucleic acid, which may be DNA or RNA. These nucleic acids may be single-stranded or double-stranded. As a result of the degeneracy of the genetic code, those skilled in the art will understand that a number of different nucleic acid molecules / nucleotide sequences may encode the same polypeptide. In addition, those skilled in the art may use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule / polynucleotide / nucleotide sequence defined herein so as to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is to be expressed.

[0136] For example, nucleic acid molecules / nucleotide sequences such as DNA nucleic acid molecules / sequences may be produced by recombinant, synthetic, or any means available to those skilled in the art. In addition, those nucleic acid molecules / nucleotide sequences may be cloned by standard techniques.

[0137] Longer nucleic acid molecules / polynucleotides / nucleotide sequences will generally be produced using recombinant means such as polymerase chain reaction (PCR) cloning or other cloning techniques.

[0138] The present nucleic acid molecule may further include a nucleotide sequence encoding a selectable marker. Suitable selectable markers are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. Preferably, the selectable marker may be a fluorescent protein such as GFP, YFP, RFP, tdTomato, dsRed, or variants thereof.

[0139] The nucleic acid molecule may be provided as part of a nucleic acid construct that includes one or more other nucleotide sequences together with the nucleic acid molecule. These other nucleotide sequences may encode a selectable marker or other polypeptide, and this other polypeptide may be any other polypeptide that is desired to be introduced into the host cell together with the BGC.

[0140] In another embodiment, the nucleic acid molecule may be provided in the form of a recombinant construct that includes a nucleic acid molecule operably linked to one or more expression regulatory sequences, such as a promoter, optionally with one or more additional control sequences. Thus, for example, a nucleotide sequence corresponding to an individual ORF from a BGC or a selected ORF may be provided in a construct having a heterologous control sequence for the regulation of gene expression. A construct that includes a nucleic acid molecule containing one or more coding sequences and one or more expression regulatory sequences may be referred to herein as an expression construct.

[0141] The nucleic acid molecule or recombinant construct may be contained within a vector, and the vector may be for purposes of cloning, transfer, or expression. Thus, in embodiments, the vector may be a cloning vector, a transfer vector, or an expression vector. As used herein, the term "vector" refers to any genetic element that can serve as a vehicle for the transfer, expression, or replication of a foreign nucleic acid sequence in a host strain.

[0142] The vector may exist as a single nucleic acid molecule or as two or more separate nucleic acid molecules. The vector may be a single-copy vector or a multi-copy vector when present in the host strain.

[0143] The specific vectors for use herein are expression vectors. In such vectors, one or more genes / coding sequences can be inserted into the vector molecule in an appropriate orientation and in proximity to the expression regulatory elements such that when the vector molecule is present in a host strain, the expression of one or more proteins is directed. The expression regulatory elements may be provided by the vector, but particularly when the nucleic acid molecule contains a nucleotide sequence corresponding to a complete or substantially complete BGC or a substantial portion thereof, conveniently the expression regulatory elements are part of the nucleic acid molecule inserted into the vector (i.e., the nucleic acid molecule derived from the BGC defined and described herein). In other words, in certain embodiments, the nucleic acid molecule within the vector may contain regulatory and control sequences for the expression of the coding nucleotide sequence, and the vector may be merely a vehicle for the molecule for purposes such as cloning of the molecule, or introduction into cells, reproduction in cells, etc.

[0144] The construction of suitable vectors and other recombinant or genetic modification techniques for use herein are well known in the art (see, for example, Green and Sambrook, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y.) (2012), and Ausubel et al., "Short Protocols in Molecular Biology, Current Protocols", John Wiley and Sons (New Jersey) (2002), etc.).

[0145] The vector can be a plasmid, cosmid, phagemid or other phage vector, viral vector, episome, such as an artificial chromosome like a bacterial artificial chromosome (BAC) or a P1 artificial chromosome (PAC), or other polynucleotide construct.

[0146] Advantageously, the vector is an artificial chromosome, particularly a BAC. This is especially the case when the nucleic acid molecule contains a sequence corresponding to the whole or a substantial part or majority of the BGC, in view of the size of the molecule. As described in the following examples, the BGC is cloned in a BAC and generally speaking, artificial chromosomes, particularly BACs, will be used for the cloning of nucleic acid molecules for the synthesis of compounds in a host. An exemplary BAC for this purpose is the BAC vector pDualP from Varigen Biosciences, Madison, WI, USA.

[0147] For the cloning and expression of smaller nucleic acid molecules, a wide range of plasmids suitable for use in selected or desired host strains of bacteria are available and known in the art.

[0148] Generally, regulatory sequences are operably linked to the coding nucleic acid sequence and it includes configurations, controls, and inducible promoters, transcriptional enhancers, as well as transcriptional terminators, etc., well-known in the art. The coding nucleic acid sequence can be operably linked to one common expression regulatory sequence or linked to different expression regulatory sequences. However, as shown above, particularly in the case of vectors containing nucleic acid molecules for the synthesis of compounds, advantageously the native regulatory sequences of the genes of the BGC are used.

[0149] Suitable promoter sequences for expression in bacteria, particularly actinomycetes, are known in the art. When a heterologous promoter is used, it may be advantageous to use a strong promoter. In particular, a strong inducible promoter may be used. This may be the case, for example, for the expression of individual genes or ORFs.

[0150] The choice of vector will typically depend on the size of the nucleic acid molecule and the compatibility between the host strain into which the vector is to be introduced and the vector. The vector may be a linear or circular plasmid. In addition, the vector may be a self-replicating vector, i.e., an extrachromosomal entity that exists as such and whose replication is independent of chromosomal replication, which may be, for example, a plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may include any means for ensuring self-replication. Alternatively, the vector may be integrated into the genome when introduced into the host strain and replicated together with the chromosome(s) into which it has been integrated. Integrating plasmids are known in the art. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or a transposon that together contain the total nucleic acid to be introduced into the genome of the host strain may be used.

[0151] As described above, the vector may include one or more selectable markers that allow for easy selection of transformed cells. For example, a selectable marker gene can perform functions such as encoding a detectable product such as a fluorescent protein, providing resistance to an antibiotic or toxin, complementing an auxotrophic defect, or supplying an essential nutrient not present in the medium, and / or providing regulation of chromosomal integration. Examples of bacterial selectable markers are markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, apramycin, or tetracycline resistance.

[0152] In addition, the vector may contain one or more elements that enable integration of the vector into the host strain genome or autonomous replication of the vector in a host independent of the genome. For integration into the host strain genome, the vector may rely on coding nucleic acid sequences or other elements of the vector for integration into the genome by homologous or non-homologous recombination. To achieve integration, a CRISPR-based system may also be used. For autonomous replication, the vector may further contain an origin of replication that enables autonomous replication of the vector in the strain in question. The origin of replication may be any plasmid replicon that mediates autonomous replication functioning in the cell. As used herein, the terms "origin of replication" or "plasmid replicon" are defined as nucleotide sequences that enable a plasmid or vector to replicate in vivo.

[0153] The vector may be introduced into the host cell by any convenient or desired means, which may depend on the nature of the vector. As used herein, the term "introduced" refers to a method for inserting a foreign nucleic acid, such as DNA or RNA, into a cell. This includes both methods of conjugation and transformation, or in fact any method suitable for introducing a nucleic acid molecule or vector into a host cell. A host cell modified by the introduction of a nucleic acid molecule or vector may sometimes be referred to as a manipulated host cell. Thus, a manipulated host cell is distinguished from a natural or wild-type host cell by the presence of a nucleic acid molecule in the cell that is not present in the natural or wild-type host cell.

[0154] Methods of transformation for plasmid vectors and such analogs are known in the art. However, for larger vectors such as those that would be used to transfer nucleic acid molecules for, for example, the synthesis of compounds, methods for transferring plasmids into host cells by conjugation would typically be used. This method may involve transferring the vector into an intermediate host, i.e., a transfer host, prior to introduction into the host for expression, for example, for the production of compounds. Conveniently, as known and reported in the art, the method of triparental conjugation may be used to transfer vectors such as BACs into production hosts for the production of molecules. Thus, a vector containing a nucleic acid molecule for the synthesis of a compound may be transferred into a transfer host together with a driver plasmid by triparental conjugation of a cloning host containing the vector, a host containing the driver plasmid, and the transfer host. Such processes are described in the following examples. Thereafter, the transfer host containing the vector and the driver plasmid is conjugated with the intended production host cell to transfer the vector into the production host cell for the synthesis of the compound.

[0155] As described above, once introduced, the vector may be maintained as an integrated chromosomal entity or as an extrachromosomal vector that self-replicates. Transformation can be confirmed using methods well known in the art. Such methods include, for example, PCR or genomic sequencing at the integration site (primers in the vector and host chromosome). Alternatively or additionally, analysis at the gene expression level may be performed using, for example, Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for the expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. In the case of a vector containing a nucleic acid molecule for the synthesis of a compound, expression will be confirmed by the production and detection of the compound. The expression level can be further optimized to obtain sufficient expression using methods well known in the art.

[0156] The host cell into which the vector is introduced depends on the purpose of the host cell, i.e., whether the host cell is for cloning or introduction, or the expression of one or more polypeptides, or the production (synthesis) of a compound.

[0157] Suitable host cells for cloning hosts may be any cells known in the art for such purposes, which will depend on the size of the nucleic acid molecule or vector. For example, for the cloning of smaller nucleic acid molecules containing nucleotide sequences encoding a single polypeptide or smaller selections thereof, a wide range of host cells may be used, including various strains of Escherichia coli (E. coli). When the nucleic acid molecule is a large molecule, especially for the synthesis of a compound, the host cell needs to be suitable for the growth of large constructs, and such hosts are also known in the art, including, for example, strain 10Beta of Escherichia coli (E. coli).

[0158] Suitable hosts for transfer by conjugation are also known in the art, including, for example, Escherichia coli (E. coli) ET12567.

[0159] Suitable hosts for the expression of individual polypeptides are also known in the art, including many strains of Escherichia coli (E. coli), Bacillus, and other bacteria including actinomycetes and especially Streptomyces.

[0160] The host cell for the production of a compound may be any suitable host cell in which the nucleic acid molecule can be expressed and the compound can be synthesized. Production hosts are typically bacteria, conveniently actinomycetes, more specifically bacteria of the genus Streptomyces.

[0161] The production host cell may be a heterologous host cell, i.e., a host cell that does not naturally contain or synthesize a BGC. However, in an alternative embodiment, the nucleic acid molecule may be introduced into an organism in which the BGC has been cloned, i.e., isolate P08-G05, or more generally, a strain that intrinsically contains the BGC.

[0162] A wide range of different Streptomyces hosts are known in the art and are available for use. In certain embodiments, the host is Streptomyces coelicolor. Again, various strains and isolates of S. coelicolor are available for use. Strain A(3)2 is a well-known model strain.

[0163] The Streptomyces coelicolor strain M145 is also known as Streptomyces violaceoruber (Waksman and Curtis) Pridham, which is a prototrophic derivative of strain A(3)2 and is available from the ATCC under the number BAA-471. The Streptomyces coelicolor strain M145 (ATCC BAA-471) is described in Bentley et al., 2002, Nature, 417, 141-147, and in particular lacks the plasmids SCP1 and SCP2 of the parental A3(2) strain. As described in Gomez-Escribano and Bibb, Microbial Biotechnology, 2011, 4(2), 207-215, derivatives of the Streptomyces coelicolor strain M145 (ATCC BAA-471) have been engineered for use in heterologous expression of secondary metabolite gene clusters. Any of the strains described in this document may be used. This strain has been modified to lack all or some of the four antibacterial gene clusters (the act, red, cda, and cpk gene clusters) of strain M1146, and has further been modified to introduce point mutations into the genes rpoB and / or rpsL, which encode the RNA polymerase β-subunit and ribosomal protein S12, respectively. Each mutation has been shown to increase the level of antibiotic production in Streptomyces without accompanying growth impairment. As described in the above document, the mutagenic genes were incorporated into a suicide plasmid and replaced with the wild-type genes by homologous recombination. Any of the strains M1141 - M1146 or M1151 - M1156 described in this document may be used. Strain M1152 (Δact Δred Δcpk Δcda rpoB(C1298T)) may be specifically mentioned.Furthermore, as described in the following examples, mutants of strain M1152 are generated, which are specifically in-frame deletion mutants for SCO2963 and SCO2962, where the matAB locus is deleted. The strain Streptomyces coelicolor M1152ΔmatAB described in Example 1 below represents a preferred host cell for the production of compounds.

[0164] When a nucleic acid molecule is introduced into a host strain for the production of a compound, the modified bacterium is grown or cultured under conditions suitable for the expression of the encoded polypeptide and the synthesis of the compound. Again, the procedures and conditions for this are known in the art and can be readily achieved according to techniques and principles well-known in the art.

[0165] Suitable growth media for Streptomyces bacteria are known in the art, and in the following examples, for example, the MG-2.5w / NaCl medium is described.

[0166] Alternatively, the compound may be prepared in an in vitro transcription and translation (IVTT) system, i.e., a cell-free system, according to principles and techniques known in the art. This system may include cell extracts of the various S. coelicolor strains discussed above, or more generally, the host cells described above including Streptomyces or actinomycete cells.

[0167] After the compound is synthesized or the desired polypeptide is expressed, it may be harvested from the culture or, in other words, recovered or collected.

[0168] In particular, the compound or polypeptide may also be extracted or separated from bacterial cells by cell lysis procedures well-known in the art. Thus, a crude extract containing the compound or polypeptide may be obtained.

[0169] Furthermore, to isolate or purify the compound or polypeptide, separation and purification procedures known in the art may be used. These procedures include, for example, methods of precipitation, chromatography, or filtration, such as ammonium sulfate precipitation, ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, gel filtration, HPLC, and the like. Any desired or convenient combination of purification methods may be used. For the isolation of the compound, extraction with solvents and / or acids, as well as preparative chromatography methods such as HPLC, may be used. Suitable procedures are described in the following examples.

[0170] Thus, the methods herein may include an additional step of purifying the compound or polypeptide.

[0171] In certain aspects and embodiments herein, the compound is a compound that can be obtained by the expression of a nucleic acid molecule comprising SEQ ID NO.1 in Streptomyces coelicolor strain M1152ΔmatAB. More specifically, the compound can be obtained by the expression of a nucleic acid molecule comprising SEQ ID NO.1 in Streptomyces coelicolor strain M1152ΔmatAB according to the method described in the following examples.

[0172] Thus, the compound may have the structure shown in Formula I above, which more specifically includes the structure of Formula II above or Structures I or II. However, as shown above, modifications of the compound may be obtained, i.e., derivatives may be produced by chemical modification of the compound or by modifying one or more of the coding sequences of the nucleic acid molecule. Such modifications may alter one or more enzyme activities, thereby resulting in a modification of the compound obtained as a result of the synthesis. Modifying the genes of an antibacterial gene cluster to modify the antibacterial compound produced is described in the art, for example, in WO 2001 / 059126 (nisin) or WO 2009 / 115822 (BE-14106).

[0173] Hereinafter, the present invention will be described in more detail in the following non-limiting examples with reference to the following drawings.

Example

[0174] [Example 1] Preparation of the host strain Streptomyces coelicolor M1152ΔmatAB The S. coelicolor strain M1152 described in Gomes-Escrivano 2010 (supra) was obtained from the John Innes Centre, Norwich, UK.

[0175] As previously described (van Dissel et al., 2015, Microbial Cell Factories, 14(1), pp. 1-10), an in-frame deletion mutant of SCO2963 / SCO2962 in S. coelicolor M1152 was generated. Briefly, the upstream region of SCO2963 from -1326 to +43 relative to the start codon and the downstream region of SCO2962 from +2190 to +3610 were amplified by PCR from the S. coelicolor genome using the primers listed in Table 2. The amplified flanks were cloned into the unstable shuttle vector pWHM3-oriT (Wu et al., 2019, Angewandte Chemie, 131(9), pp. 2835-2840) using EcoRI and HindIII restriction sites. An apramycin resistance cassette aacC4 flanked by loxP sites was inserted between the flanking regions using an XbaI site characterized in both amplified regions. The completed vector (pMAT1) was introduced into E. coli ET12567+PUZ8002, which enabled the transfer of pMAT1 into S. coelicolor M1152 by conjugation. Mutants in which the matAB locus was replaced by the aacC4 cassette and the pWHM3 vector was lost were selected by replica plating for the Thio- / Apra+ phenotype. Introduction of the pUWLcre plasmid expressing Cre recombinase, which cleaves the loxP sites flanking the apramycin resistance gene, yielded the marker-free S. coelicolor M1152ΔmatAB strain.

[0176] [Table 2]

[0177] [Example 2] Identification of the biosynthetic gene cluster (cluster 16) from the marine actinobacterial isolate P08-G05 Origin of the marine isolate strain P08-G05 The marine isolate P08-G05 was obtained from the SINTEF / NTNU marine actinobacterial strain collection constructed from water, sediment, and sponge samples collected from the Trondheim fjord. This strain was selected based on an extensive evaluation of the draft genomes of 1200 isolates from the strain collection based on different criteria such as phylogenetic novelty, gene cluster diversity, and bioactivity observed in the past as described below.

[0178] Frozen glycerol cultures from the collection were streaked onto TSA (Trypton soya broth agar) supplemented with 0.5x artificial seawater (Engelhardt et al., 2010, Applied and Environmental Microbiology 76(15):4969-4976). Pure isolates were cultured in TSB and artificial seawater to produce mycelia for the working cell bank.

[0179] Illumina sequencing and de novo assembly of the genome of strain P08-G05 Biomass of strain P08-G05 for genome sequencing was produced at 30 °C in TSB medium supplemented with 50% artificial seawater. The biomass was collected by centrifugation and sent to BaseClear BV for sequencing, where DNA extraction, sequencing, and post-sequencing data processing were performed.

[0180] Paired-end sequence reads were generated using the Illumina HiSeq2500 system. FASTQ sequence files were generated using the Illumina Casava pipeline version 1.8.3. Initial quality assessment was based on data passing Illumina Chastity filtering. Subsequently, reads containing adapters and / or PhiX control signals were excluded using the in-house filtering protocol of BaseClear. The second quality assessment was based on the remaining reads using the FASTQC quality control tool version 0.10.0. The quality of the FASTQ sequences was improved by trimming low-quality bases using the "Trim sequences" option of CLC Genomics Workbench version 8.0.

[0181] For genome assembly and scaffolding, quality-filtered array reads were assembled into contig arrays. Analyses were performed using the "de novo assembly" option of CLC Genomics Workbench version 8.0. Errors in assembly and nucleotide mismatches between Illumina data and the contig arrays were corrected by Pilon version 1.11. Contigs were linked and placed into scaffolds or supercontigs, resulting in an assembly of 7,315,765 bp and 980 scaffolds. The insert sizes between paired-end and / or mate-pair reads were used to estimate the orientation, order, and distances between the contigs. Analyses were performed using SSPACE Premium scaffolder version 2.3. The gap regions within the scaffolds were (partially) closed in an automated manner using GapFiller version 1.10, utilizing the insert sizes between paired-end and / or mate-pair reads. The resulting draft genome was used for subsequent phylogenetic analysis and genome annotation. The quality of the de novo genome assembly of the Illumina sequencing of P08-G05 was evaluated by the checkM software (version 1.07), showing a high completeness of 95.9% with a low contamination of 1.6%.

[0182] PacBio Sequencing and Hybrid De Novo Genome Assembly The cytoplasmic mass for PacBio sequencing and direct cloning was prepared in a 500 ml shaking flask containing 3 g of 3 mm glass beads and 120 ml of TSB medium supplemented with 0.5x artificial seawater at 30 °C and 200 rpm with 2.5 orbital motion, OD 600Production was carried out up to 5.6. The cytoplasmic mass was collected by centrifugation, kept at -40°C until shipment, and shipped on dry ice to BaseClear BV in the Netherlands (The Netherlands). At BaseClear, long-read PacBio sequencing was performed using a PacBio Sequel instrument, and the obtained data was processed and filtered using the SMRT Link software suite, where subreads shorter than 50 bp were discarded. As a result, 622,557 reads and a yield of 2,886,923,195 bp were obtained.

[0183] The quality of the Illumina HiSeq reads was improved by trimming low-quality bases using BBDuk, which is part of the BBMap suite version 36.77. High-quality reads were assembled into contigs using ABySS version 2.0.2. Long reads were mapped to the draft assembly using BLASR version 1.3.1. Based on these alignments, the contigs were linked together and placed into scaffolds. The orientation, order, and distances between the contigs were estimated using SSPACE-LongRead version 1.0. The Illumina reads were used to (partially) close the gap regions within the scaffolds using GapFiller version 1.10. Finally, Pilon version 1.21 was used to correct assembly errors and nucleotide mismatches between the Illumina reads and the scaffold sequences. As a result, an assembly of 7,840,734 bp with 22 scaffolds was obtained.

[0184] Phylogenetic positioning of strain P08-G05 As part of a comprehensive phylogenetic analysis performed on the Illumina HiSeq2500 sequenced genomes of 1200 selected strains from the SINTEF / NTNU marine actinobacterial strain collection, generated in the same way as that of P08-G05 above, and 576 actinobacterial-type strains retrieved from public databases, the phylogenetic position of isolate P08-G05 was determined. The analysis was carried out using the IQTREE software (IQ-TREE MPI multicore version 1.6.7.1) together with 92 housekeeping genes as a reference to identify the phylogenetic novelty of strain P08-G05. Since the P08-G05 strain was placed among other strains in the actinobacterial strain collection and not among other types of strains, it was shown that this strain is likely to represent a new actinobacterial species.

[0185] Identification of the biosynthetic gene cluster P08-G05_c16 Based on a collection of profile Hidden Markov models (pHMMs) of BGCs, an in-house Python script was used to evaluate the abundance and diversity of different biosynthetic gene cluster (BGC) classes of 1200 strains from the actinobacterial strain collection. Using the resulting matrix containing the counts of pHMM hits from the corresponding strains, the strains were clustered into different groups by using an implementation in the programming language R of the t-distributed stochastic neighbor embedding (t-SNE) algorithm by AFG. The P08-G05 strain was clustered into cluster 34 (out of 40 t-SNE clusters) together with other strains. This strain was selected together with other strains from different t-SNE clusters for further characterization including long-read PacBio sequencing of a shortlist of 86 strains.

[0186] Based on manual curation of the PacBio sequenced genome of P08-G05 according to the results of antiSMASH, a novel cluster encoding (at least) the (core) metabolic machinery for the synthesis of the niddamycin compound was identified. Using in-house scripts, the resistance genes in the gene cluster were analyzed. No resistance genes were identified in cluster P08-G06_c16.

[0187] [Example 3] Cloning and expression of the gene cluster P08-G06_c16 Cloning and conjugation of gene cluster P08-G06_c16 Based on the results of antiSMASH, a hypothesis was proposed that gene cluster P08-G06_c16 encodes a novel moenomycin-like compound. Moenomycin has the molecular formula C 68 H 106 N5O 34 P and a mass of 1567.645683 g / mol.

[0188] Cloning of cluster P08-G05_c16 in the inducible bacterial artificial chromosome (BAC) vector (pDualP, owned by Varigen Bioscience (Madison, WI, USA)) was purchased from Varigen Bioscience based on the chromosomal DNA of strain P08-G05. The construct was received from Varigen Bioscience in an Escherichia coli (E. coli) strain (10Beta) suitable for the propagation of large constructs. The cluster was transferred into S. coelicolor M1152ΔmatAB prepared according to Example 1 by triparental conjugation following a procedure similar to the method described previously (Jones et al., 2013, PLoS ONE 8(7):e69319.doe:10.1371). Briefly, by triparental conjugation, the construct containing the BGC was transferred into E. coli ET12567 together with the driver plasmid pR9406. For this, each strain was first cultured overnight on LB agar without selection. For all three strains, several colonies were scooped using a inoculation loop and streaked together on a patch on LB agar containing apramycin, chloramphenicol, and ampicillin. As a control, each strain was also patch-applied individually for sample selection. A single colony of ET12567 + pR9406 + DualP-BGC was grown to OD0.6 in a 13 ml culture tube containing 5 ml of LB + ampicillin, chloramphenicol, and apramycin, after which the culture was pelleted and washed twice with cold LB medium. In parallel, the spores of S. coelicolor were pre-germinated by heat shock at 50 °C for 10 minutes and incubation at 30 °C for 2 - 3 h. The E. coli and the spores of S. coelicolor were mixed, spread on a soy flour mannitol (SFM) agar plate, incubated at 30 °C for 18 - 24 h, and then covered with apramycin + nalidixic acid to select for the conjugated Streptomyces colonies.Subsequently, for spore collection and storage according to standard procedures, a single colony was patch-applied to a selective SFM plate, spread, and made into a confluent plate. The new conjugant strain with the nidamycin gene cluster was given the short name M1152ΔmatAB(P08-G05_C16).

[0189] Culture of the conjugant M1152ΔmatAB(P08-G05_C16) and expression of the gene cluster P08-G05_c16 Well plate cultures of M1152ΔmatAB(P08-G05_C16) and M1152ΔmatAB (control) were performed as follows. Seed cultures were produced for 2 days at 30 °C and 225 rpm to an OD600 = 5 - 7 in 250 ml shake flasks with 50 ml of 0.5x tryptone soya broth (TSB) and 1.5 g of 3 mm glass beads (without antibiotics). Production in 24-well plates (AXYGP-DW10ML24C) was carried out in both 5254SW medium (Kralova et al., 2021, Frontiers in Microbiology 12:2131) and MG-2.5 medium (Doull and Vining, 1990, Applied Microbiology and Biotechnology, 32, 449 - 454; Martinez-Castro et al., 2013, Applied Microbiology and Biotechnology, 97, 2139 - 2152) supplemented with 1 g / L NaCl. The wells were filled with 2.5 ml of medium and 4 x 3 mm glass beads, and 1.3% was seeded from the seed culture. The plates were incubated at 800 rpm and 85% humidity for 6 days in a New Brunswick incubator at 30 °C. The broth was lyophilized and extracted with 1 broth volume of DMSO for 1 hour.

[0190] The cell-free extract was analyzed by an Agilent LC-DAD-QTOF equipped with Zorbax Bonus RP 2.1 x 50 mm, 3.5 μL. As the mobile phase, 50 mM ammonium acetate [A] and acetonitrile [B] were used. The gradient was increased from 5% acetonitrile in 0 - 2 minutes and then to 95% over the next 25 minutes. The QTOF was operated in positive and negative ionization modes with a capillary voltage of 3.5 kV, fragmentor voltage of 150 V, skimmer of 65 V, gas temperature of 325 °C, drying gas of 10 l / min, and nebulizer of 50. The data was processed using Agilent's Mass Hunter and Mass Profiler Professional software.

[0191] The LC-DAD-iso plot showed that two peaks were observed in the joined-completed extract but not in the control (Figure 1). The abundance of these two peaks was higher in MG-2.5w / 0.5x seawater than in 5254SW medium. The MS data showed that a mass cluster was observed at the retention time corresponding to the major UV peak. The three major masses were M+H = 1349.5668 and its adduct was M+Na = 1371.5496 (Figure 2). These masses were not found in the extract of the control M1152ΔmatAB. This compound was concluded to be related to the heterologous expression of the introduced P08-G05_c16 and was named nidaramycin.

[0192] [Example 4] Large-scale production and purification of heterologously expressed compounds The large-scale production of the active compound was carried out in a 500 ml shaking flask with 125 ml of MG-2.5w / NaCl. The medium was inoculated with 3% from the seed culture and incubated at 30 °C at 200 rpm with a 2.5 cm orbital motion for 6 days.

[0193] The broth was freeze-dried and homogenized in a mortar. This material was extracted with DMSO acidified with trifluoroacetic acid (TFA) to a final concentration of 0.1%. The amount of the organic solvent was 0.4 times the original broth volume. The DMSO extract was fractionated using an Agilent preparative HPLC equipped with a Zorbax Bonus RP, 9.4 x 250 mm, 7 μm column (Agilent), a diode array detector (DAD), and a fraction collector. The mobile phase was water and 20 mM ammonium acetate [A] and acetonitrile [B]. The gradient was 5% [B] at the time of injection, and then the gradient was increased from 55% to 75% [B] over 10 minutes. The column was washed with 95% [B] for 1 minute and then equilibrated with 5% [B]. The acetonitrile in the HPLC fraction was removed by a rotary evaporator, and the aqueous phase was further purified and concentrated using a 500 mg HLB solid phase extraction column (Waters). The compound was eluted from the SPE column with methanol. Methanol was removed by evaporation using a Speedvac (ThermoFisher) at 50 °C. Water was added to the sample, and it was frozen at -80 °C and freeze-dried.

[0194] It was confirmed by the DAD plot in Figure 3 that the purified compound was obtained. Using the obtained material, the inhibition and toxicity assays described in Example 5, the determination of the molecular formula of nidaramycin described in Example 6, and the structural elucidation by NMR and the determination of the position of the sulfate group described in Examples 7 and 8 were carried out.

[0195] [Example 5] Assay of the activities of the crude extracts and purified compounds Bioassay of the crude extract A cell-free extract was prepared from a culture of strain M1152ΔmatAB (P08-G05_C16) prepared as described in Example 3 and tested in a bioassay against a panel of strains, namely Enterococcus faecium CCUG37832, M. luteus TO-09 ATCC9341, Pseudomonas aeruginosa ATCC15692, and C. albicans CCUG. The extract showed activity against E. faecium CCUG37832.

[0196] Specifically, the extract of the conjugation-completed cells inhibited the growth of E. faecium CCUG37832 at 16x dilution (MG-2.5w / NaCl) and 4x dilution (5254SW), whereas the control extract did not inhibit any of the strains.

[0197] in vitro MIC bioassay The minimal inhibitory concentrations (MICs) against selected Gram-positive indicator organisms were determined by microdilution tests using the 384-well format according to the protocol of the Clinical and Laboratory Standards Institute. The indicator strains were incubated overnight in TSB medium until OD600 = 0.4, then diluted in TSB medium to OD600 = 0.1 and further diluted 45-fold in assay medium (Mueller-Hinton broth, Difco). The seeded medium was dispensed into assay plates. A series of two-fold dilutions of the isolated compound or vancomycin (reference) diluted in DMSO were added to the seeded wells such that the final DMSO concentration in each well was 2.7% and the final concentration of the active compound was 0 - 540 μg / ml (23 different concentrations). Four parallel assays were performed for each compound and concentration. 0.5 mg of the compound produced by strain M1152ΔmatAB (P08-G05_C16) was purified by preparative HPLC and the pure compound was tested in a bioassay against a panel of strains. The indicator organisms were M. luteus ATCC9341, Staphylococcus aureus ATCC29213, Staphylococcus aureus ATCC43300 (MRSA), Enterococcus faecium CCUG37832, Enterococcus faecium CTC492. The results are shown in Table 3 below.

[0198]

Table 3

[0199] in vitro toxicity assay The cytotoxicity of nidamycin against human cell lines was evaluated using human cell lines HepG2, LLC-PK1, and L929 cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U / ml Pen-Strep (HepG2), Medium 199 supplemented with 3% FBS, 2 mM L-glutamine, 100 U / ml Pen-Strep (LLC-PK1), and Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 100 U / ml Pen-Strep (L929). Cells were subcultured according to standard protocols and a Tecan EVO robotic workstation with a disposable tip MCA384 pipetting unit (Tecan MCA125 μl, Cat No. 300-5-1-808) was used to transfer cell suspensions from a stirred reservoir and seed them into 384-well plates (Corning assay plates, 3712). The reservoir (flat bottom, 300 mL, Thermo Scientific, 10723363) was equipped with a sterile magnetic stir bar (15x4.5 mm VWR 442-4522) stirring at 350 rpm. The number of cells in each well was 50,000 (HepG2), 25,000 (LLC-PK1), and 10,000 (L929). After seeding, the microplates with cell suspensions were shaken at 1600 rpm for 20 s at an amplitude of 2.5 mm (Bioshake). The microplates with cells were incubated at 37 °C in a 5% CO2 atmosphere. On the day of cell exposure, serial dilutions were made in DMSO. The serial dilutions with the compound were further diluted with cell culture medium and transferred to assay wells, with the total DMSO concentration in the assay wells set at 0.6%. After exposure, the plates were incubated for an additional 48 h at 37 °C in a 5% CO2 atmosphere.The viability of cells after 24-hour and 48-hour incubations was measured using the Promega CellTiter-GLO 2.0 viability assay. The highest concentration tested was 50 μg / ml of nidarosamycin, and no toxic effects were detected below this concentration. The data are shown in Figure 4.

[0200] [Example 6] Determination of the molecular formula of nidarosmycin MS1 analysis of isotope-labeled broth To determine the molecular formula of nidarosamycin, strain M1152ΔmatAB (P08-G05_C16) was cultured as follows in an isotope-labeled medium where all carbon sources were 13 13C-labeled and all nitrogen sources were 15 15N-labeled. A 3-ml seed culture produced in 0.5x TSB medium was washed once with 10 ml of sterile 0.9% NaCl and resuspended in 0.9% NaCl to an OD = 5 for use in inoculating the production culture (0.5% into 20 ml of medium). Production was carried out in a 250-ml shake flask with 1.5 g of 3-ml glass beads and 20 ml of the following medium of Silante. 1 g / 100 ml of 13C Silex Media Powder for Escherichia coli (E. coli) (115204100), Escherichia coli (E. coli) OD2N (110301402), or Escherichia coli (E. coli) OD2CN (110601402). The cultures were harvested after 6 days.

[0201] The freeze-dried isotope-labeled broth was extracted for 1 hour with 2 ml of DMSO to which 0.1% trifluoroacetic acid per 20 ml of the original broth volume was added. The yield by volume measurement of nidarosamycin in these media was very low, but was high enough to detect the mass of isotope-labeled nidarosamycin. 13 13C, 15 15N, as well as 13 13C and 15The masses of the N-labeled nidaramycin were 1410.7769 Da, 1351.5562 Da, and 1412.7585 Da, respectively, indicating that the molecular formula of nidaramycin contains 61 carbons and two nitrogens (Figure 5). Based on the MS1 data (Figure 2), the most likely molecular formula is C 61 H 92 N2O 29 S, which gave a theoretical monoisotopic mass of 1348.5507 Da.

[0202] MS2 Analysis of Purified Nidaramycin Purified nidaramycin was analyzed by LC-MSMS with fragmentation of the precursor mass m / z = 1349.567. The LC conditions were the same as those described in Example 4, and the MSMS data were generated using a positive-mode Bruker Impact II QTOF. The MS conditions were as follows. Spectral rate: 12 Hz, capillary voltage: 4500 V, endplate offset: 500 V, drying gas: 10 L / min, nebulizer gas: 220, data acquisition control: dynamic MSMS, collision energy 5 V as well as multiCE 20, 50, and 100. From the molecular mass and the MSMS fragmentation pattern (Figure 6), the molecular formula was C 61 H 92 N2O 29 S, suggesting that 29 MSMS fragments could be explained by this formula (data not shown).

[0203] [Example 7] Structure determination by NMR The compound produced by the conjugant M1152ΔmatAB (P08-G05_C16) was subjected to structure determination by 1D and 2D NMR spectroscopy by Red Glead Discovery AB.

[0204] The determined structure is shown in Figure 7, which also shows the atom numbering for the atom-specific assignment performed. This structure consists of four substituted sugar moieties A - D, the linking 2,3-dihydroxypropionic acid (E), and a hydrocarbon moiety (F) having the formula C 30 H 45 . There are several alternative positions that could be reasonable for the proposed sulfate group shown at position 4 on the uronic acid unit "D". As shown below, the alternative positions are position 3 of unit D and position 4 of unit A.

[0205]

Chemical formula

[0206] The NMR investigations performed are detailed below.

[0207] Sample information The samples investigated were provided as a solid material on a ReadGlead. The material was stored at -20 °C upon receipt. The prepared NMR samples were stored at 4 - 8 °C in the dark between measurements. The following sample information was provided. Sample ID: P08 - G05_c16 Monoisotopic mass: 1348.5604 Sum of formula: C 61 H 92 N2O 29 S Amount obtained: 5.13 mg Solubility: 10 mg / mL in DMSO by sonication

[0208] Materials and methods NMR samples PN102-62-01 2.899 mg of sample P08-G05_c16 was weighed into a screw-cap vial, and an NMR sample was prepared by adding 540 μL of DMSO-d6. The sample was slowly dissolved and heated at 40 °C for 1 - 2 minutes and then sonicated for 3 x 10 seconds. The sample still showed a small amount of finely dispersed undissolved particles controlled by visual inspection, but was transferred to a 5 mm NMR tube.

[0209] PN102-62-01B An NMR sample was prepared by adding 20 μL of D2O to the NMR tube of the above sample PN102-62-01.

[0210] PN102-62-01C An NMR sample was prepared by adding 2 μL of TFA-d to the NMR tube of the above sample PN102-62-01B.

[0211] PN102-62-02 An NMR sample was prepared by directly adding 540 μL of CD3OD to an Eppendorf tube containing the remaining P08-G05_c16 (about 2.2 mg). The sample was slowly dissolved and heated at 40 °C for 1 - 2 minutes and then sonicated for 3 x 10 seconds. The sample still contained a significant amount of undissolved material controlled by visual inspection, but its supernatant was transferred to a 5 mm NMR tube.

[0212] Chemicals and materials Equipment : Mettler Toledo MT5 balance Bandelin Sonorex ultrasonic bath, model no. RK31 Agilent 2 mL clear screw-neck vial, part No. 5190-9062 Agilent Technologies screw cap, with 9 mm PTFE / silicone septum, part No. 5190-9068 Hilgenberg standard NMR tube, 5 mm in diameter, item No. 2001745 Closed cap for Hilgenberg NMR tube, 5 mm in diameter, item No. 9400312

[0213]

Table 4

[0214] NMR spectroscopy For the NMR experiments performed, a 500 MHz Bruker Avance Neo spectrometer equipped with a 5 mm iProbe BBF / H / D probe and a 500 MHz Varian Inova spectrometer equipped with a 5 mm 1 H / 13 C / 15 N triple resonance probe were used. Data were recorded at 25 °C or 40 °C. The recorded spectra are listed in Table 4 below.

[0215]

Table 5-1

[0216]

Table 5-2

[0217] 1 H and 13 C chemical shifts, the solvent residual signals of DMSO-d6 (2.50 / 39.52 ppm) and CD3OD (3.31 / 49.00 ppm) were used. The NMR data were processed and analyzed using MestreNova 12.0.1 (Mestrelab Research S.L.). Chemical shift prediction was performed by the "NMRPredict" plugin in MestReNova 12.0.1 using the predictor "Mnova Best" with the solvent set to DMSO-d6.

[0218] Results and conclusions NMR experiments and conditions NMR experiments were carried out on the obtained material dissolved in DMSO-d6 or CD3OD. NMR data were recorded on a 500 MHz Bruker Avance NMR spectrometer and a 500 MHz Varian Inova spectrometer.

[0219] For the provided sample material in DMSO-d6, 1D and 2D 1 H / 13 C / 1 5 N NMR spectral data were acquired. A significant number of low-intensity signals were observed, which may indicate impurities and / or minor conformational isomers that are structurally related to the major species in solution. The spectral region of the sugar moiety was complicated to such an extent that in some cases, due to signal overlap and signal broadening, 1 H- 13 C HSQC cross-peaks could not be easily observed. Heating the DMSO-d6 sample to 40 °C did not result in, or only very slightly resulted in, signal sharpening, so all the data used for structure elucidation were recorded at 25 °C. However, acidification of the DMSO-d6 sample with TFA-d resulted in a significant sharpening of some signals, as well as a change in the chemical shifts of the sugar and sugar derivatives (while the chemical shifts of the hydrocarbon tails remained essentially unaffected).

[0220] The overall appearance of the CD3OD spectrum is slightly clearer and sharper than that of the DMSO-d6 spectrum. However, due to solubility limitations, the intensity of the signal-to-noise ratio in methanol is too low to achieve useful 2D long-range and through-space NMR data that are important for the structure discrimination process. The CD3OD data set could still provide useful information in some cases where the DMSO-d6 data were not clear.

[0221] For structure elucidation, four different NMR data sets were used in total. For completeness, the chemical shift assignments of the proposed structure are reported for both the DMSO-d6 sample (PN102-62-01) and the DMSO-d6 / TFA-d sample (PN102-62-01C), excluding the hydrocarbon tail "F" where the chemical shifts in the two samples are very similar. In addition to the major compounds, the samples were also shown to contain significant amounts of unassignable small molecules.

[0222] Elucidation of the chemical structure and atom-specific assignment The proposed structure of P08-G05_c16 (Figure 7) shares some structural features with related moenomycin compounds, i.e., they all contain a substituted tetrasaccharide linked to a hydrocarbon tail. However, in contrast to moenomycin, P08-G05_c16 lacks the linking phosphodiester and the hydrocarbon tail contains 30 rather than 25 carbon atoms. The structural evidence for P08-G05_c16 is strong. Because essentially all 1 H / 13 C / 15 H, C, and N atoms have been observed and assigned, and their atom connectivity is in complete agreement with the 2D data obtained. Furthermore, a fairly good degree of agreement is observed between the experimental and predicted chemical shift values.

[0223] The sugar units designated as 「A」 and 「D」 are assigned as uronic acids, and 「B」 and 「C」 are assigned as N-acetyl-glucosamine. Stereospecific assignments are not included in this study. The connectivity of the sugar moieties was determined by the correlation of the anomeric proton signal through the glycosidic bond with the corresponding carbon signal (C-4 of sugars 「B」 and 「C」 and C-2 of sugar 「D」) in the HMBC spectrum, and / or the NOE correlation of the anomeric proton signal with the corresponding proton in the next sugar moiety. The connectivity between sugar 「D」 and 2,3-dihydroxypropionic acid 「E」 that links to it was confirmed by the NOE correlation between the anomeric proton of 「D」 and the methylene proton of 「E」 (observed only for the acidified sample PN102-62-01C). The connectivity between 「E」 and the hydrocarbon tail 「F」 was also established through the NOE observed between both the CH and CH2 protons of 「E」 and the two closest CH protons of 「F」 (atom nos. 47 and 48 in Table 6).

[0224] In Tables 5 and 6 below, the chemical shifts assigned to P08-G05_c16 are presented together with the corresponding chemical shifts predicted from the proposed chemical structure. In its entirety, the predicted chemical shift values fully support the molecular structure suggested, and a slight deviation from the predicted values is observed only for the 「D」 moiety.

[0225] Based on the sum of the suggested formulae, a sulfate substituent was assumed at one of the oxygens of the sugar. There are several available positions for this, i.e., it is the sugar position where the OH proton is not detected and no other substituents / bonds are determined. For C-3 and C-6 in moiety 「B」 13The C chemical shifts are very similar compared to moiety "C", so it is unlikely that any of their positions have sulfuric acid. Thus, position C-4 of unit "A", C-3 of unit "D", and C-4 of unit "D" remain as promising candidates. The position of sulfuric acid cannot be defined based on NMR data alone. However, due to the deviation from the predicted chemical shift values, ring system "D" is considered to be more sensitive to pH changes than system "A" and, in addition, has a more complex structure, so it is regarded as a more promising option. O-sulfation can be expected to cause a slightly downfield shift for both the O-sulfated carbon and the proton bonded to it, and thus position C-4 of unit "D" was tentatively assigned.

[0226] Table 5 Chemical shift (δ) values for P08-G05_c16, parts A - D in Figure 8, predicted by "Mnova Predict" and experimentally determined in DMSO-d6 (NMR sample PN102-62-01) and DMSO-d6 (NMR sample PN102-62-01C) after addition of D2O and TFA-d. 1 H / 13 The experimental shifts relative to the solvent residual signals for H / C (2.50 / 39.52 ppm) are reported, while 15 an indirect reference for N is applied. The data was recorded at 25 °C. NO = not observed.

[0227]

Table 6

[0228] Amide nitrogen no.15 in sugar "C" was not observed from the recorded 1 H- 15 N HSQC data. This is because the 1D 1This is a result expected from the observed broadening of the relevant amide proton signals in the H spectrum data. Nevertheless, along with the characteristic chemical shifts of both the amide proton and the adjacent C-2 carbon, the overall fit of the sum of the formulas expected for parts "B" and "C" makes the structural assignment of acetamidosugar "C" very likely. Similarly, carbonyl carbon no.85 in part "E" is not observed from the recorded 1 H- 13 C HMBC data. The predicted and experimental 13 C chemical shift values for methine no.75 match well, it is known that this structural motif exists in the related moenomycin, and by the overall sum of the formulas, the partial structure suggested for "E" is still possible. The large number of quaternary carbon atoms and the splitting of the methylene proton signals observed for fragment "F" explain a cyclic subunit, which also agrees with the total number of rings and double bonds expected for the sum of the suggested formulas. The structural unit "F" deviates significantly from the moenomycin structure(s) and has not been evaluated from a biosynthetic perspective.

[0229] Structural parts "D" and "E" show significantly broadened 1 H signals in DMSO-d6 and 1 H- 13 C HSQC data cross-peaks broaden so much that they cannot be recognized. Adding TFA-d to the sample sharpens the signals and 13 allows the assignment of the

[0230] Table 6 The chemical shift (δ) values for P08-G05_c16, parts E - F in Figure 8, predicted by 「Mnova Predict」 and experimentally determined in DMSO-d6 (NMR sample PN102-62-01). * Shifts reported in DMSO-d6 (NMR sample PN102-62-01C) after the addition of D2O and TFA-d. 1 H / 13 The experimental shifts relative to the solvent residual signals for H / C (2.50 / 39.52 ppm) are reported, while 15 an indirect reference for N is applied. The data was recorded at 25 °C. NO = not observed.

[0231]

Table 7

[0232] [Example 8] Determination of the position of the sulfate group in nidarosmycin Background: The lead lead determined the structure of nidarosomycin (the active compound produced by P08-G05_c16). However, there were some uncertainties regarding the location of the sulfate group (SO4 group). Here, we used in silico fragmentation following MSMS fragmentation with the aim of determining the position of the SO4 group in nidarosomycin.

[0233] Based on the sum of the suggested formulas, a sulfate substituent at one of the oxygens of the sugar was assumed. There were several available positions for this, i.e., sugar positions where the OH proton was not detected and no other substituents / bonds were determined. For C-3 and C-6 in part 「B」 13The C chemical shifts are very similar compared to moiety "C", so it is unlikely that any of their positions have sulfuric acid. Thus, position C-4 of unit "A", C-3 of unit "D", and C-4 of unit "D" remain as promising candidates. The position of sulfuric acid cannot be defined based only on NMR data. However, due to the deviation from the predicted chemical shift values, ring system "D" is considered to be more sensitive to pH changes than system "A" and, in addition, has a more complex structure, so it is regarded as a more promising option. O-sulfation can be expected to cause a slightly downfield shift for both the O-sulfated carbon and the proton bonded to it, and thus position C-4 of unit "D" was tentatively assigned.

[0234] Three possible structures were given by SMILES. Nidaromycin A4 C / C(C)=C\CC1CCC(C)( / C=C / C2=CCC(C)C(C)(C\C=C(\C) / C=C / OC(COC3OC(C(=O)O)C(O)C(O)C3OC3OC(CO)C(OC4OC(CO)C(OC5OC(C(=O)O)C(OS(=O)(=O)O)C(O)C5O)C(O)C4 / N=C(\C)O)C(O)C3\N=C( / C)O)C(=O)O)C2=C)C1(C)C Nidaromycin D3 CC(CC=C1 / C=C / C2(C)C(C)(C)C(CC=C(C)C)CC2)C(C)(C / C=C(\C) / C=C / OC(COC(C(C(C2O)OS(O)(=O)=O)OC(C(C3O)NC(C)=O)OC(CO)C3OC(C(C3O)NC(C)=O)OC(CO)C3OC(C(C(C3O)O)O)OC3C(O)=O)OC2C(O)=O)C(O)=O)C1=C Nidaromycin D4 CC1CC=C(\C=C\C2(C)CCC(CC=C(C)C)C2(C)C)C(=C)C1(C)C\C=C( / C)\C=C\OC(COC1OC(C(OS(O)(=O)=O)C(O)C1OC1OC(CO)C(OC2OC(CO)C(OC3OC(C(O)C(O)C3O)C(O)=O)C(O)C2NC(C)=O)C(O)C1NC(C)=O)C(O)=O)C(O)=O

[0235] Materials and Methods: LC-MS method. Cell-free extracts were analyzed using an Agilent LC-DAD system connected to a Bruker Impact II QTOF. LC was run with 10 mM ammonium acetate buffer [mobile phase A] and 90:10 acetonitrile: water and 10 mM ammonium acetate [mobile phase B]. The gradient was 5% B for 2 minutes, then 5 - 100% B for 2 - 25 minutes. MS was performed in positive mode electrospray ionization with the following MS parameters. Mass range 100 - 1800, spectral rate: 12 Hz, absolute threshold: 25 counts, threshold for fragmentation: 100 counts, capillary voltage: 4500 V, endplate offset: 500 V, drying gas: 10 L / min, nebulizer: 31.9 psi, drying temperature: 220 °C, precursor ion list: 1000 - 1500, data collection control: dynamic MSMS or fixed MSMS, collision energy: 5 V, CID: acqCtr+MultiCe, MultiCe20. In silico fragmentation. In silico fragmentation was performed using MetFrag (Schymanski et al., 2015, Analytical and Bioanalytical Chemistry 407(21): 6237 - 6255).

[0236] Results: Investigation of fragments. In silico fragmentation strongly suggested that the SO4 group was located at either D3 or D4 rather than A4. As shown in Table 7, there were several fragments that should not have been formed if the SO4 group was located at A4. Furthermore, since the structures of D3 and D4 generally gave the same fragments, it was difficult to distinguish between them. In addition, the SO4 group was often lost during fragmentation, and only some low-abundance fragments contained the SO4 group. However, we observed one fragment (M+H = 398.1976) that could be explained by D3 but not by D4. This indicated that the SO4 group was located at D3, but this was supported by only one matching fragment.

[0237] To support the QTOF data, data from FT-ICR MSMS fragmentation in negative ionization were investigated. The FT-ICR fragmentation pattern could not even distinguish between positions D3 and D4. However, some fragments could only be explained by sulfuric acid at either position D3 or D4 rather than position A4 (Table 5).

[0238] Table 7 Fragments obtained by MSMS fragmentation using a Bruker Impact II QTOF were compared with in silico fragmentation of the three suggested structures. Some fragments could not be explained by the structure with the SO4 group at the A4 position.

[0239]

Table 8

[0240] Table 8 The fragments obtained by MSMS fragmentation using a Bruker FT-ICR were compared with the in silico fragmentation of three proposed structures. The fragments shown here could not be explained by the structure having an SO4 group at the A4 position.

[0241]

Table 9

Claims

1. A compound of formula (I), 【Chemistry 1】 Here, R １ is -SO ２ OH, -SO ２ OR, or -SO ２ R, and R ２ is H, or R ２ is -SO ２ OH, -SO ２ OR, or -SO ２ R, and R １ is H, R is C １ ~C ２０ It is a hydrocarbyl group, Each R ３ is H or C １ ~C ２０ A compound of formula (I) independently selected from the hydrocarbyl group, A compound that is either a pharmaceutically acceptable salt, solvate, or hydrate thereof.

2. The compound has the following structure: 【Chemistry 2】 Here R １ ga-SO ２ OH, -SO ２ OR, or -SO ２ It is R and R ２ Is H or R ２ ga-SO ２ OH, -SO ２ OR, or -SO ２ It is R and R １ H is, R is C １ ~C ２０ The structure is a hydrocarbyl group. The compound according to claim 1, or having the structure of a pharmaceutically acceptable salt, solvate, or hydrate thereof.

3. R １ ga-SO ２ OH or -SO ２ OR and R ２ Is H or R ２ ga-SO ２ OH or -SO ２ OR and R １ H is C １ ~C ２０ It is a hydrocarbyl group, Preferably R １ ga-SO ２ OH and R ２ Is H or R ２ ga-SO ２ OH and R １ The compound according to claim 1, wherein is H.

4. The compound has the following structure (A), 【Transformation 3】 or having the structure of a pharmaceutically acceptable salt, solvate, or hydrate thereof, The compound has the following structure (B), 【Chemistry 4】 or having the structure of a pharmaceutically acceptable salt, solvate, or hydrate thereof, The compound according to claim 1.

5. (a) The nucleotide sequence shown in SEQ ID NO. 1, or (b) A nucleotide sequence that is the complement of SEQ ID NO. 1, or (c) A nucleotide sequence that is degenerate with SEQ ID NO. 1 and encodes an amino acid sequence shown in SEQ ID NO. 30-57, or (d) A nucleotide sequence having at least 95% sequence identity with the sequence of SEQ ID NO. 1, A nucleic acid molecule containing, The nucleic acid molecule is a nucleic acid molecule that encodes a biosynthetic system for the synthesis of an antimicrobial compound or is complementary to a nucleic acid molecule that encodes such a system, and the antimicrobial compound is a compound that can be obtained by the expression of SEQ ID NO. 1 in the Streptomyces coelicor strain M1152ΔmatAB.

6. The nucleic acid molecule according to claim 5, wherein the compound is as defined in any one of claims 1 to 4.

7. A recombinant construct comprising a nucleic acid molecule as defined in claim 5.

8. A nucleic acid molecule as defined in claim 5, or A recombinant construct comprising the nucleic acid molecule defined in claim 5 A vector containing this.

9. (i) A nucleic acid molecule as defined in Claim 5, (ii) A recombinant construct comprising the nucleic acid molecule of (i), or (iii) A vector comprising the nucleic acid molecule of (i) or the recombinant construct as defined in (ii). Microbial host cells containing these cells.

10. The aforementioned host cells (i) Actinomycete production host cells for the production of the antimicrobial compound, (ii) Streptomyces species, (iii) Streptomyces coelicor (iv) Streptomyces coelicor strain M145 (ATCC BAA-471), (v) Δact Δred Δcpk Δcda rpoB (C1298T) is a derivative of (iv) Streptomyces coelicolor strain M1152, or The host cell according to claim 9, wherein the Streptomyces coelicolor strain M1152ΔmatAB is a derivative of (v) further comprising a deletion of locus matAB.

11. A method for producing an antimicrobial compound, wherein the method is (i) A nucleic acid molecule as defined in claim 5, (ii) A recombinant construct comprising the nucleic acid molecule of (i), or (iii) A vector comprising the nucleic acid molecule of (i) or the recombinant construct of (ii). Introducing into microbial host cells, This includes expressing the nucleic acid molecule and enabling the synthesis of the antimicrobial compound by the expressed biosynthetic system, The antimicrobial compound is a compound that can be obtained by the expression of SEQ ID NO. 1 in the Streptomyces coelicor strain M1152ΔmatAB, and is described as a method for obtaining the antimicrobial compound.

12. The aforementioned host cells (i) Actinomycete production host cells for the production of the antimicrobial compound, (ii) Streptomyces species, (iii) Streptomyces coelicor (iv) Streptomyces coelicor strain M145 (ATCC BAA-471), (v) Δact Δred Δcpk Δcda rpoB (C1298T) is a derivative of (iv) Streptomyces coelicolor strain M1152, or (vi) is the Streptomyces coelicolor strain M1152ΔmatAB, which is a derivative of (v) that further includes a deletion of the seat matAB. The method according to claim 11.

13. The method described above is (i) recovering the compound, and / or (ii) Purifying the compound. The method according to claim 11, further comprising:

14. A compound that can be obtained or obtained by the method of claim 11.

15. (i) for use as an antimicrobial agent, and / or (ii) For use as an antimicrobial agent against Gram-positive bacteria, optionally the bacteria are Staphylococcus aureus or Enterococcus faecium, including antibiotic-resistant strains. The compound according to any one of claims 1 to 4 or claim 14.

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or 14, and further comprising at least one carrier, additive, and / or excipient.

17. Use of the compound according to any one of claims 1 to 4 or claim 14 as an in vitro antibacterial agent or as an antibacterial agent for plants.

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