WO2012093192A1 - Coly-mycin derivatives - Google Patents

Abstract

The present invention relates to the isolation, cloning and sequencing of the group of genes involved in the biosynthesis of coly-mycin by Streptomyces spp. CS40, and to the use of said genes to increase the production of coly-mycin and/or associated analogous compounds or derivatives by means of productive strains. Said compounds produced in the present invention can be used to treat several diseases, such as cancer, neuro-degenerative diseases and infectious diseases.

Description

 COLISMYCIN DERIVATIVES

FIELD OF THE INVENTION

The present invention is comprised within the field of biology, pharmacy and medicine. STATE OF THE TECHNIQUE

Within nature, the molecules that have a 2,2'-bipyridyl group in their structure are compounds with a large number of biological activities described (Cristalli et al., 1986); (Gomi et al., 1994); (Tsuge et al. 1999). One of these molecules is colismicin produced by Streptomyces spp. CS40 (FIG. 1A), which presents antibiotic activity against Gram-positive and Gram-negative bacteria, antifungal against a broad spectrum of fungi and cytotoxic against P388 leukemia cells (Gomi et al. 1994); (Tsuge et al., 1999). Additionally, colismicin is described as a binding inhibitor between dexamethasone and glucocorticoid receptors (Shindo et al, 1994) and in patent WO / 2007/017146 the ability of colismicin to inhibit oxidative stress in cells is described.

The development of recombinant DNA technology is becoming a powerful tool when it comes to increasing our knowledge about the genes involved in the biosynthesis of bioactive compounds. This technology can nowadays be applied to the improvement in the production levels of different bioactive compounds and to obtain new derived molecules with better clinical properties through the combination of genes of different bioactive molecule biosynthesis pathways and their expression in microorganisms producing these compounds, in what has been called combinatorial biosynthesis.

Recombinant DNA technology has made it possible to isolate clusters of complete genes for the biosynthesis of different bioactive compounds using, among other strategies, the cloning, selection or analysis of libraries of microorganisms producing molecules with pharmacological interest by probes of DNA This strategy is based on the existence of previous genetic information on the biosynthesis route or biosynthetically related routes, which allows to use or Design genetic probes from the total or partial sequence of a biosynthesis enzyme.

In the scientific literature there is hardly any information about the cellular machinery responsible for the biosynthesis of compounds of the 2,2'-bipyridil type. Only studies have been carried out on the biosynthesis of a molecule of the 2-2'-bipyridil family, caerulomycin. Caerulomycin is a molecule with a structure similar to that of colismicin (FIG. IB) produced by Streptomyces caeruleus (Funk and Divekar, 1959).

Through experiments of incorporation of radioactively labeled precursors, some of the intermediary molecules were identified during the biosynthesis of caerulomycin, one of these molecules is picolinic acid (Vining et al., 1988). This compound has also been described as an intermediate metabolite in the biosynthesis of other bioactive molecules by some Streptomyces species such as nikomycin D (Bruntner and Bormann, 1998). In all cases described in the literature, the precursor for the biosynthesis of picolinic acid or 3-hydroxypicolinic acid (in the case of virginiamycin biosynthesis) is the amino acid lysine and the enzymes responsible for the generation of this compound have been identified, being the first of them an enzyme with lysine activity 2-aminotransferase (Bruntner and Bormann, 1998; Namwat et al., 2002).

The present invention describes the cloning and sequencing of the colismicin biosynthesis gene cluster in the Streptomyces sp. CS40 The genetic information available on enzymes of the lysine-2-aminotransferase type has been used to design oligonucleotides and construct a genetic probe that has allowed the isolation and cloning of the colismicin biosynthesis gene cluster. From the genetic point of view, there are no previous descriptions in the literature related to the isolation and sequencing of the biosynthesis gene cluster of colismicin or another molecule of the 2,2'-bipyridyl family produced by Streptomyces species.

The invention provides an important tool for the genetic manipulation of this gene cluster in the sense of increasing the production of colismicin and / or obtain new derivatives of this molecule or structurally related molecules with improved properties.

DESCRIPTION OF THE INVENTION

The present invention relates to the isolation and identification of a cluster of genes that participate in the biosynthesis of colismicin by Streptomyces spp. CS40, and provides a tool for the genetic manipulation of this gene pool to increase the production of colismicin and obtain new derivatives with improved properties for their isolation and use, among others, in the pharmaceutical or chemical sector.

This invention describes a DNA sequence containing 24 genes involved in the biosynthesis of colismicin and its precursors, including those involved in the regulation of gene pooling. The gene pool includes genes that code for a non-ribosomal polyketide synthase-peptide synthetase (PCS-NRPS) hybrid system responsible for synthesizing the central structure of colismicin, genes that encode enzymes involved in the biosynthesis of the picolinic acid precursor of colismicin, genes which encode enzymes involved in modifications of the molecule such as dehydrogenases, aminotransferases and methyltransferases. The invention therefore relates to new genes and nucleic acid molecules that encode proteins / polypeptides that show functional activities involved in the biosynthesis of colismicin, and its potential application in increasing the levels of colismicin production in Streptomyces spp. CS40 and in the production of new derivatives of colismicin.

Experimental procedures applied to the present invention include conventional molecular biology methods. A detailed description of the methods used and not detailed here can be obtained from Hopwood et al. (1985); Sambrook et al. (1989) and Kieser et al, (2000).

In order to clone the colismicin biosynthesis gene cluster, a Streptomyces spp. Chromosomal DNA library was constructed. CS40 in Escherichia coli, using the cosmid pWE15 (see example 2). For the isolation of the colismicin biosynthesis gene cluster, a genetic probe consisting of a 412 bp PCR fragment from the partial amplification of the gene encoding a 2-aminotransferase lysine involved in the biosynthesis of colismicin was used. For its amplification, the chromosomal DNA of strain CS40 and two oligonucleotides were used as template: L2ATsonl (SEQ ID NO: 31) and L2ATson2 (SEQ. ID NO: 32) designed based on the nucleotide sequence of a lysine 2-aminotransferase whose sequence, of approximately 500 bp, was previously identified using degenerate oligonucleotides L2ATFW2 (SEQ. ID NO: 29) and L2ATRV2 (SEQ ID NO: 30). The use of this probe in the hybridization of the Streptomyces spp. Chromosomal DNA library. CS40 allowed to isolate a cosmid (coslC3) that defines a region of approximately 41 kb on the chromosome. The participation of the cloned DNA in the coslC3 cosmid in the biosynthesis of colismicin was determined using a BamHl fragment of 3128 bp, which contained an internal fragment to a gene encoding an NRPS. This fragment was cloned into plasmid pOJ260 (Bierman et al., 1992). The resulting construct was used for gene disruption in Streptomyces spp. CS40 generating a non-producing colismicin mutant (see example 4) as evidence of the involvement of cloned DNA in the biosynthesis of colismicin.

However, analysis of the coslc3 cosmid sequence showed the need to extend the region to the left to look for other genes not present in the coslC3 cosmid, possibly involved in the biosynthesis of colismicin. A PCR amplified fragment using the oligonucleotides Ic3-5FW (SEQ ID NO: 33) and the c3-5RV (SEQ ID NO: 42) of 1.9 kb from the end of the coslC3 cosmid was used as a probe to re-analyze the library of Streptomyces spp. CS40 isolating a new cosmid overlapping with the cosmid coslC3, cos3Bl 1, used to complete the 46672 bp sequence of the region that contains the gene pool involved in colismicin biosynthesis (FIG. 2).

The assignment of functions in biosynthesis to the different gene products was done by comparing their amino acid sequences with those of other proteins present in databases (see example 3). Some genes encode structural biosynthetic enzymes (including PCSs, NRPSs, etc.), activators transcriptional, proteins involved in the export abroad of the cell, and proteins involved in the contribution of precursors for the biosynthesis of colismicin. The participation of this gene pool in the biosynthesis of colismicin was demonstrated by inactivations of different genes both by disruption and by gene replacement (see examples 4, 6 and 8), generating in some of them new derivatives of colismicin (see examples 7 and 8 ). The antitumor activity of the new compounds generated by manipulation of the colismicin biosynthesis pathway has been assessed by in vitro cytotoxicity assays against different tumor cell lines (see example 9). The neuroprotective activity of the new compounds has been assessed in trials using the zebrafish model (see example 10). Not all genes identified in the DNA region presented in this invention are part of the gene pool involved in the biosynthesis of colismicin. The establishment of the limits of gene clustering was performed by disruption of the orfl and orf27 genes and replacement of the orf25 gene (see example 5). The present invention describes methods for the manipulation of biosynthesis genes, aimed at obtaining new derivatives of colismicin by disruption and / or gene replacement techniques generating mutants in colismicin biosynthesis that accumulate different intermediates. The new strains generated, producing analogs of colismicin by genetic manipulation are called Streptomyces spp. CLM-A, Streptomyces spp. CLM-L, Streptomyces spp. CLM-M2 and have been deposited in the Spanish Type Culture Collection (CECT), with deposit numbers 7755, 7754, 7756 and 7757 respectively. Similarly, Streptomyces spp. CLM-M, Streptomyces spp. CLM-M1, Streptomyces spp. CLM-AH have been deposited in the Spanish Type Crops Collection (CECT) with deposit numbers 8069, 8070 and 8071, respectively.

The present invention also provides analogous compounds of colismicin A and colismicin C obtained by enzymatic acylation catalyzed by a lipase of the oxime or hydroxyl group, respectively.

Within the meaning of the present invention, enzymatic acylation is understood as the transformation of a substrate into an acylated derivative from its reaction with an acylating agent catalyzed by lipase. Lipases useful for acylation can be found in Tetrahedron 2004, 60, 501-519; Chem. Soc. Rev. 2004, 33, 201-209; or Adv. Synth Catal. 2006, 348, 797-812. More specifically, in the present invention Burkholderia cepacia lipase (PS-C) is used to obtain acylated colismicin A and colismicin C derivatives. These lipases are presented in different forms of immobilization on hydrophobic and mechanically resistant supports or on acrylic resins, such as an epoxyacrylic resin activated with deca-octyl groups. Acylating agents useful for the present invention are those that can act as substrates of the lipase used resulting in the acylation of colismicin A and colismicin C, and may be esters, carbonates and anhydrides. The reaction can be carried out in a wide variety of organic solvents. More specifically, in the present invention methyl tertbutyl ether (MTBE) is used. In general, the temperature must keep the enzyme structure intact without denaturing phenomena. The reaction can be carried out between 5 and 60 ° C, preferably between 10 and 60 ° C, more preferably between 20 and 50 ° C. Also, the present invention provides compounds characterized by the following formula (I):

(I) where Ri, R ₂ , R ₃ and R4 are, each and independently, hydrogen or a protecting group. The protecting group may consist of an alkyl group, a cycloalkyl group, a heterocyclic cycloalkyl group, a hydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, a alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, a oxime group, a nitrile group, a urethane group, a silyl group, a sulfo group combining them; Except for compounds with the following formulas:

, also known as Colismicin A and as (E) -5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -carbaldehyde oxime,

, also known as Colismicin B, and as (Z) -5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -carbaldehyde oxime, and

, also known as Colismicin C and as 5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -methanol.

In particular, the present invention provides, among others, the compounds of Formula II to XVI comprised in Formula I:

• Formula II: 6- (hydroxymethyl) -5- (methylthio) - [2,2'-bipyridin-4-ol].

 • Formula III: (E) -4-hydroxy-5- (methylthio) - [2,2'-bipyridin-6-yl] -carbaldehyde oxime.

• Formula IV: 4-hydroxy-5- (methylthio) - [2,2'-bipyridinyl-6-yl] -carbonitrile.

 • Formula V: 4-hydroxy-5- (methylthio) - [2,2'-bipyridin-6-yl] -carboxylic acid.

 • Formula VI: (E) -6'-methyl-5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] - carbaldehyde oxime.

 • Formula VII: (E) -4'-methyl-5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] - carbaldehyde oxime.

• Formula VIII: 4'-methyl-5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -methanol. Formula IX: (E) -4-hydroxy-4'-methyl-5- (methylthio) - [2,2'-bipyridin-6-yl] - carbaldehyde oxime.

 Formula X: 4-hydroxy-4'-methyl-5- (methylthio) - [2,2'-bipyridin-6-yl] -carbonitrile. Formula XI: 5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -methyl acetate.

Formula XII: 5- (methylthio) -4-methoxy- [2,2'-bipyridin6-yl] -methyl butanoate. Formula XIII: 5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -methyl benzoate. Formula XIV: (E) -5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -carbaldehyde O-acetyl oxime.

 Formula XV: (E) -5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -carbaldehyde O-chloroacetyl oxime.

Formula XVI: (E) -5- (methylthio) -4-methoxy- [2,2'-bipyridin-6-yl] -carbaldehyde O-butanoyl oxime.

The present invention also provides the compounds of Formula XVII, XVIII, XIX and XX: Formula XVII: 6- [N- (1-Carboxy-3-methylbutyl) carbamoyl] -5-methylthio [2,2 'bipyridine] -4-ol.

 Formula XVIII: 2- (1-Carboxy-3-methylbutyl) -5- (2-pyridyl) -2,3-dihydro-1, 2 thiazo lo [4,5-b] pyridin-3-one.

 Formula XIX: N - ((4-hydroxy-5- (methylthio) - [2,2'-bipyridin] -6-yl) methyl) acetamide. For -hydroxy-5- (methylsulfinyl) - [2,2'-bipyridine] -6-carboxamide.

 (XIX) (XX)

These compounds are derivatives or analogs of colismicin A with a methyl group at 4 'or 6' positions (compounds of formula VI and VII, respectively), colismicin A demethylated at 4 (compound of formula III), colismicin A demethylated at 4 and with a 4 'methyl group (compound of formula IX), 4-methylated colismicin C (compound of formula II), 4' methylated colismicin C (compound of formula VIII), or compounds similar to the 2,2'- unit characteristic bipyridyl and a nitrile or carboxylic acid functional group in position 6 (compounds of formula IV, V and X). Likewise, the compounds of formula XI, XII and XIII are esters derived from colismicin C and the compounds of formula XIV, XV and XVI are oxime esters derived from colismicin A. Also, compounds XVII and XVIII are precursors of colismicin with a residue Additional leucine and compounds XIX and XX are analogous compounds with the characteristic 2,2'-bipyridyl unit and an amide functional group. The present invention also describes the use of the new compounds as antibiotics, antitumor or neuroprotective.

The compounds of the invention are tumor growth inhibitors and are therefore useful in the treatment of cancer. Likewise, the compounds of the invention are inhibitors of lysis or neuronal apoptosis induced by oxidative stress and therefore useful in the treatment of those neurodegenerative diseases caused by oxidizing agents, both exogenous and endogenous, to the patient.

Thus, pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament are object of the present invention.

It is also the object of the present invention to use a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof to inhibit the growth of a tumor or neuronal lysis or apoptosis caused by oxidative stress. As used here, "inhibit" means decrease, slow down, or stop. Therefore, a compound of this invention can decrease, slow down, or stop the growth of a tumor cell or decrease slow down, or stop lysis or neuronal apoptosis under conditions of oxidative stress.

As used herein, "growth" means increase in size, or proliferation, or both. Thus, a compound of this invention may inhibit the increase in size of a tumor cell and / or may prevent the tumor cell from dividing and increase the number of tumor cells. A "tumor cell" is a cell that constitutes a neoplasm (new growth), which can be cancerous (malignant) or non-cancerous (benign). A cancerous tumor cell can invade normal surrounding tissues and blood / lymphatic vessels and metastasize in tissues away from the original tumor. In contrast, a non-cancerous tumor cell can grow and compress adjacent normal tissues but cannot invade normal tissues and blood / lymphatic vessels, nor can it metastasize in tissues away from the original tumor. As used herein, neurodegenerative disease means that type of disease whose symptoms are the result of death from lysis or apoptosis of neurons. Oxidative stress in the context of neurodegenerative disease refers to that anomalous situation in which the cytoplasm of neurons shows an increase in the amount of reactive oxygen species (H ₂ 0 ₂ , H0 ₂ , O ² and OH ^" ). These reactive oxygen species can be generated by the diseased neuron's own metabolism, by other organs of the individual or be exogenous to the individual.

As used herein, a neuroprotective compound against oxidative stress is that compound capable of stopping, minimizing or slowing down lysis or neuronal apoptosis caused by oxidizing agents.

It is also the object of the present invention to use a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof to treat cancer or neurodegenerative diseases caused or aggravated by lysis or neuronal apoptosis induced by oxidative stress. It is also the object of the present invention to use a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament with antitumor or neuroprotective activity against oxidative stress.

It is also the object of the present invention to use a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a drug for the treatment of cancer or neurodegenerative diseases. .

The subject of the present invention is also a method of treating a subject, including a human being, diagnosed with cancer or with a neurodegenerative disease, which consists in treating said subject with a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

As used herein, a "subject" may include domesticated animals (for example, cats, dogs, etc.), livestock (for example, cows, horses, pigs, sheep, goats, etc.), laboratory animals (for for example, mice, rabbits, guinea pigs, etc.) and birds. From preferably, the subject is a mammal such as a primate and, more preferably, a human being.

In general, a "therapeutically effective amount" of a compound is that amount necessary to achieve the desired result. For example, the effective amount of a compound of the present invention treats cancer by inhibiting the growth of the cells that constitute the tumor, thereby preventing the invasion of normal tissues and blood / lymphatic vessels by tumor cells and Therefore, it prevents metastasis, or an effective amount of a compound of the present invention is one capable of slowing down or inhibiting neuronal apoptosis under conditions of oxidative stress. The term "acceptable pharmaceutical composition" refers to a biologically suitable material, that is, that the material can be administered to the subject without causing substantially harmful biological effects.

The doses or amounts of the compounds of the invention must be large enough to produce the desired effect. However, the dose should not be so high that it causes adverse side effects, for example, unwanted cross reactions, anaphylactic reactions, and the like. Generally, the dose will vary with the age, condition, sex and degree of the subject's disease, and can be determined by any person skilled in the art. The dose can be adjusted by each doctor, based on the clinical condition of the subject involved. The dose, dosage regimen and route of administration may be varied.

The compounds of the invention may be useful for research in biochemistry or cell biology.

Any of the compounds of the invention can be used therapeutically as part of an acceptable pharmaceutical composition. Acceptable pharmaceutical compositions may consist of sterile solutions in water, saline solutions, or buffered solutions at physiological pH. Any of the compounds of the invention can be prepared in the form of a pharmaceutical composition. The pharmaceutical compositions may include various transport agents, thickeners, diluents, buffers, preservatives, surfactants, and others, in addition to the compound of the invention. Pharmaceutical compositions may also include active ingredients such as antimicrobial agents, anti-inflammatories, anesthetics, etc.

The compounds of the invention can be administered to the subject in several different ways, depending on whether it is desired that the treatment be local or systemic, and depending on the area to be treated. Thus, for example, a compound of the present invention can be administered in the form of an ophthalmic solution, for application on the surface of the eye. In addition, a compound can be administered to a subject vaginally, rectally, intranasally, orally, by inhalation, or parenterally, either intradermally, subcutaneously, intramuscularly, intraperitoneally, intrarectally, intraarterially, intralymphatically, intravenously, intrathecally and intratracheally. . Parenteral administration, if used, is generally performed by injection. Injectables can be prepared in various forms, such as liquid solutions or suspensions, solid forms suitable to be dissolved or suspended before injection, or as emulsions. Other forms of parenteral administration employ slow or sustained release systems, so that a constant dose is maintained (see, for example, US Patent 3,710,795). Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may also contain buffers and diluent additives and others. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl ethyl acetate. Examples of aqueous solvents are: water, alcoholic-aqueous solutions, emulsions or suspensions, including saline and buffered solutions. Examples of parenteral vehicles are: sodium chloride solution, Ringer's dextrose, sodium chloride and dextrose, etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelators, inert gases, etc. Formulations for topical administration may include creams, lotions, gels, drops, suppositories, sprays, liquids and powders. Certain conventional pharmaceutical carriers, aqueous, oily, or powdered bases, thickeners, etc. may also be necessary or desirable. Compositions for oral administration may include powders or granules, suspensions or solutions in water or non-aqueous medium, capsules, or tablets. It may be desirable to include thickeners, flavorings, diluents, emulsifiers, dispersants, etc. For the purposes of the present invention and its description, the term "derivative" or "analog" of colismicin should be construed as a compound covered by the general Formula (I) that should not necessarily be derived from colismicin but may be synthesized de novo . The present invention is described in detail below with a series of examples without being in any case examples that limit its use to those mentioned.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1.

A. Structure of colismicin A produced by Streptomyces spp CS40.

B. Structure of caerulomycin produced by Streptomyces caeruleus.

Figure 2. Diagram showing a schematic representation of the restriction map, using the restriction enzyme BamHI (numbered positions in the scheme), of the gene pool for biosynthesis of colismicin in Streptomyces spp. CS40 contained in the nucleotide sequence described as SEQ IN NO: 1. The scale is shown in kilobases (kb). coslC3 and cos3Bl l represent the cosmids in which the nucleotide sequence described as SEQ IN NO has been isolated: 1. The genes present in the gene pool are represented by numbers under the arrows. The genes that have been inactivated within the gene cluster are shown as gray arrows and the rest as black arrows. The genes not involved in the biosynthesis of colismicin are shown as white arrows.

Figure 3. Analysis by liquid chromatography (UPLC) and liquid chromatography coupled to mass spectrometry (HPLC / MS). FIG. 3A shows the production of colismicin A (peak at 3,259 min) in the wild strain Streptomyces spp. CS40 analyzed by UPLC. The ordinates represent the time in minutes (min.) And the abscissa as arbitrary units (AU). FIG. 3B shows the absorption spectrum of colismicin A. In the abscissa the wavelength is represented in nanometers (nm) and in the ordinates as arbitrary units. FIG 3C shows purified colismicin A produced in the wild strain Streptomyces spp. CS40 and analyzed by HPLC / MS. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG. 3D shows the mass spectrum of colismicin A, marking the mass value at each peak (without units).

Figure 4. UPLC analysis of the wild strain Streptomyces spp. CS40 producing colismicin A is shown in FIG. 4A. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). In FIG. 4B shows the UPLC analysis of the CLM-L mutant obtained by gene replacement, not producing colismicin A. In the abscissa the time is represented in minutes (min.) And in those ordered as arbitrary units (AU). In FIG. 4C shows the UPLC analysis of the CLM-12 mutant, obtained by gene disruption, not producing colismicin A. In the abscissa the time is represented in minutes (min.) And in those ordered as arbitrary units (AU).

Figure 5. UPLC analysis of the mutant strain CLM-22D producing colismicin A is shown in FIG. 5A. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). In FIG. 5B shows the analysis by UPLC of the mutant strain CLM-24, producing colismicin A. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU).

Figure 6. Schematic of the initial steps in the biosynthesis route of colismicin A. The amino acid lysine, in a reaction catalyzed by the gene product of the orfl9 and orf20 genes, is transformed into picolinic acid. Subsequently, the rest of the genes involved in biosynthesis will continue modifying this compound until producing colismicin A.

Figure 7. FIG. 7 A shows a scheme of the genetic organization of the CLM-L mutant (strain deposited as CECT 7754) that has interrupted the orfl 9 gene that codes for a lysine 2-aminotransferase. In FIG. 7B. The liquid chromatography (UPLC) analysis of the extract of the non-producing colismicin A mutant strain is shown. In the abscissa the time is represented in minutes (min.) and in the ordinates as arbitrary units (AU). FIG. 7C shows the analysis by very high resolution liquid chromatography (UPLC) of the extract of the non-producing strain of colismicin A supplemented with picolinic acid, compound capable of rescue the production of colismicin A. In the abscissa the time is represented in minutes (min.) and in the ordinates as arbitrary units (AU).

Figure 8. Analysis of the production of colismicin C and the compound of formula II by the mutant strain CLM-A (deposited as CECT 7755). In FIG. 8A shows the scheme of the genetic organization of the CLM-A mutant, which has an interrupted orfl 1 gene, which codes for an aminotransferase. FIG. 8B shows the UPLC analysis of the CLM-A mutant extract, the peak at 2.86 min corresponds to the compound of formula II and the peak at 1989 to Colismicin C. In the abscissa the time is represented in minutes (min.) And in those ordered as arbitrary units (AU). FIG. 8C shows the absorption spectrum of the compound of formula II. The abscissa represents the wavelength in nanometer (nm) and in the ordinates as arbitrary units. FIG 8D shows the absorption spectrum of Colismicin C. In the abscissa the wavelength is represented in nanometer (nm) and in the ordinates as arbitrary units. FIG. 8E shows the MS profile corresponding to the compound of formula II with the mass value being marked at each peak (without units). FIG. 8F shows the MS profile corresponding to Colismicin C, marking the mass value at each peak (without units).

Figure 9. Analysis of the production of modified colismicins, compounds of formula III and formula IV by the mutant strain CLM-M2 (deposited as CECT 7756). In FIG. 9A shows the scheme of the genetic organization of the CLM-M2 mutant, which has an interrupted orf9 gene, which codes for a methyltransferase. FIG. 9B shows the UPLC analysis of the CLM-M2 mutant extract, indicating the compounds of formula III and formula IV. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG. 9C shows the absorption spectrum of the compound of formula III. The abscissa represents the wavelength in nanometer (nm) and in the ordinates as arbitrary units. FIG 9D shows the absorption spectrum of the compound of formula IV. The abscissa represents the wavelength in nanometer (nm) and in the ordinates as arbitrary units. FIG. 9E shows the MS profile of the compound of formula III with the mass value being marked at each peak (without units). The FIG. 9F shows the MS profile of the compound of formula IV with the mass value being marked at each peak (without units).

Figure 10. Analysis of the production of modified colismicin, compound of formula V by the mutant strain CLM-G. In FIG. 10A shows the scheme of the genetic organization of the CLM-G mutant, which has uninterrupted genes orfl3 and orfl4, which encode similar GriD (YP 001825756) and GriC (YP 001825755) proteins. FIG. 10B shows the HPCL / MS analysis of the excision of the silvesíre Sírepíomyces spp. CS40 In the abscissa the time was repressed in minutiae (min.) And in the ordinates as arbitrary units (AU). FIG. 10C shows the HPCL / MS analysis of the excision of the mumanie strain CLM-G. In the abscissa the time was repressed in minutiae (min.) And in the ordinates as arbitrary units (AU). FIG. 10D shows the absorption specimen of the compound of formula V. In the abscissa the wavelength was represented in nanomer (nm) and in the ordinates as arbitrary units. FIG. 10E shows the MS profile of the compound of formula V, marking at each peak the mass value (without units).

Figure 11. Analysis of the production of modified colismicins, compounds of formula VI and formula VII, produced by the CLM-L muieie strain (deposited as CECT 7754) supplemented with 6-methyl picolinic acid and 4-methylpyridine-2-carboxylic acid respectfully. In FIG. 1 1 A UPLC analysis of the excision of the CLM-L muierie supplemented with 6-methyl picolinic acid is shown, indicating the compound of formula VI. In the abscissa the time was repressed in minutiae (min.) And in the ordinates as arbitrary units (AU). FIG. 1 IB shows the absorption specimen of the compound of formula VI. In the abscissa, the wavelength was represented in nanomer (nm) and in the ordinates as arboreal units. FIG. 1 1C shows the MS profile of the compound of formula VI, marking at each peak the mass value (without units). In FIG. 1 ID shows the analysis by UPLC of the excision of the CLM-L muierie supplemented with 4-methylpyridine-2-carboxylic acid, indicating the compound of formula VIL In the abscissa the time was represented in minutes (min.) And in the ordinates as Arbitrary units (AU). FIG. 11E shows the absorption specimen of the VIL compound. In the abscissa, the wavelength was represented in nanomer (nm) and in the ordinates as arboreal units. FIG. 11F shows the MS profile of the compound of formula VII with the mass value being marked at each peak (without units).

Figure 12. Analysis of the production of modified colismicin, compound of formula VIII, produced by the mutant strain CLM-A (deposited as CECT 7755) supplemented with 4-methylpyridine-2-carboxylic acid. In FIG. 12A shows the UPLC analysis of the CLM-A mutant extract supplemented with 4-methylpyridine-2-carboxylic acid, indicating the compound of formula VIII. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG. 12B shows the absorption spectrum of the compound of formula VIII. The abscissa represents the wavelength in nanometer (nm) and in the ordinates as arbitrary units. FIG. 12C shows the MS profile of the compound of formula VIII with the mass value being marked at each peak (without units).

Figure 13. Analysis of the production of modified colismicins, compounds of formula IX and X, produced by the mutant strain CLM-M2 (deposited as CECT 7756) supplemented with 4-methylpyridine-2-carboxylic acid. In FIG. 13A, the UPLC analysis of the excision of the CLM-M2 muierie supplemented with 4-methylpyridine-2-carboxylic acid is shown showing the compounds of formula IX and formula X. In the abscissa the time is represented in minutes (min.) And in those ordered as arbitrary units (AU). FIG. 13B shows the absorption spectrum of the compound of formula IX. The abscissa represents the wavelength in nanometer (nm) and in the ordinates as arbitrary units. FIG. 13C shows the absorption spectrum of the compound of formula X. In the abscissa the wavelength is represented in nanometer (nm) and in the ordinates as arbitrary units. FIG. 13D shows the MS profile of the compound of formula IX with the mass value being marked at each peak (without units). FIG. 13E shows the MS profile of the compound of formula X with the mass value being marked at each peak (without units).

Figure 14. Neuroprotective capacity of colismicin A and the compounds of formula VI and VIL In all cases of FIG 14 the presence of apoptotic cells is shown by intensely stained spots in the brain of zebrafish embryos. FIG 14A shows the brain of untreated zebrafish embryos. FIG 14B shows the brain of zebrafish embryos treated with retinoic acid 10 μΜ. FIG 14C shows the brain of zebrafish embryos treated with 10 μΜ retinoic acid and 1 μΜ colismicin. FIG 14D shows the brain of zebrafish embryos treated with 10 μΜ retinoic acid and the compound of formula VI 1 μΜ. FIG 14E shows the brain of zebrafish embryos treated with 10 μΜ retinoic acid and the compound of formula VII 1 μΜ. FIG 14F shows the brain of zebrafish embryos treated with 10 μΜ retinoic acid and lipoic acid

I μΜ. A graphical representation of the neuroprotective capacity of colismicin A and the compounds of formula II, XI, VI and VII respectively is shown in FIG 14G. The ordinate axis shows the percentage of apoptosis in the brain of zebrafish embryos. 0% of apoptosis was considered as the level of basal apoptosis of untreated embryonic brains (EW) and 100% of apoptosis as the level of apoptosis present in the brains of embryos treated with 10 μtino retinoic acid (RA). Neuroprotection assays were performed by treating zebrafish embryos with retinoic acid in the presence of the various compounds to be tested. As a neuroprotection control, lipoic acid (AR + LP) was used. The compounds tested as neuroprotectors were colismicin A (AR + Col.A), the compound of formula

II (AR + II), colismicin C (AR + Col.C), the compound of formula VI (AR + VI) and the compound of formula VII (AR + VII). As can be seen in the graph, the compound of formula VII has an improved neuroprotective activity with respect to colismicin A and colismicin C.

Figure 15. Analysis of the production of the compound of formula XVIII by the mutant strain CLM-Ml (deposited as CECT 8070). In FIG. 15A shows the scheme of the genetic organization of the CLM-Ml mutant, which has an interrupted orfl2 gene, which codes for a methyltransferase. FIG. 15B shows the UPLC analysis of the CLM-Ml mutant extract, the peak at 3.84 minutes corresponds to the compound of formula XVIII. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG. 15C shows the absorption spectrum of the compound of formula XVIII. In the abscissa the wavelength is represented in nanometers (nm) and in the ordinates as arbitrary units. FIG 15D shows the MS profile corresponding to the compound of formula XVIII with the mass value being marked at each peak (without units). Figure 16. Analysis of the production of the compound of formula XIX by the mutant strain CLM-M (deposited as CECT 8069). In FIG. 16A shows the scheme of the genetic organization of the CLM-M mutant, which has an interrupted orfl5 gene, which codes for a monooxygenase. FIG. 16B shows the HPLC analysis of the CLM-M mutant extract, the peak at 12.62 min corresponds to the compound of formula XIX. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG. 16C shows the absorption spectrum of the compound of formula XIX. In the abscissa the wavelength is represented in nanometers (nm) and in the ordinates as arbitrary units. FIG 16D shows the MS profile corresponding to the compound of formula XIX with the mass value being marked at each peak (without units).

Figure 17. Analysis of the production of the compound of formula XVII by the mutant strain CLM-AH (deposited as CECT 8071). In FIG. 17A shows the scheme of the genetic organization of the CLM-AH mutant, which has an interrupted orfl6 gene, which codes for an amidohydrolase. FIG. 17B shows the UPLC analysis of the CLM-AH mutant extract, the peak at 3.67 min corresponds to the compound of formula XVII. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG. 17C shows the absorption spectrum of the compound of formula XVII. In the abscissa the wavelength is represented in nanometers (nm) and in the ordinates as arbitrary units. FIG 17D shows the MS profile corresponding to the compound of formula XVII with the mass value being marked at each peak (without units).

Figure 18. Analysis of the production of the compound of formula XX by the mutant strain CLM-M2 (deposited as CECT 7756). In FIG. 18A shows the UPLC analysis of the CLM-M2 mutant extract, the peak at 2.22 min corresponds to the compound of formula XX. In the abscissa the time is represented in minutes (min.) And in the ordinates as arbitrary units (AU). FIG 18B shows the MS profile corresponding to the compound of formula XX with the mass value being marked at each peak (without units). FIG. 18C shows the absorption spectrum of the compound of formula XX. In the abscissa the wavelength is represented in nanometers (nm) and in the ordinates as arbitrary units. Therefore, a first aspect of the present invention refers to a method of isolation and purification of a DNA fragment, which contains the gene pooling of the colismicin biosynthesis pathway of the Streptomyces spp. CS40, which is included in a 46672 bp fragment of the genome of Streptomyces spp. CS40 The process comprises the following steps: (a) obtaining a genomic DNA library from a colismicin-producing microorganism; (b) transfection of clones of said library into host cells; (c) oligonucleotide design for the isolation of the colismicin biosynthesis gene cluster; (d) construction of a probe comprising a nucleotide sequence of a cluster of colismicin biosynthesis genes; (e) use of heterologous probes for the isolation of the group of colismicin biosynthesis genes; (f) hybridization of said probe with a genomic DNA library obtained from said microorganism and (g) isolation of said gene pool from clones with positive hybridization.

The second aspect of the present invention refers to a nucleic acid molecule consisting of a nucleotide sequence described as SEQ ID NO: 1; or a nucleotide sequence complementary to SEQ ID NO: 1; or a degenerated nucleotide sequence with respect to SEQ ID NO: 1; or a nucleotide sequence capable of hybridizing under restrictive conditions with SEQ ID NO: 1, with the complementary strand of SEQ ID NO: 1, or with a hybridization probe derived from SEQ ID NO: 1 or its complementary strand; or a nucleotide sequence that has at least 80% sequence identity with SED ID NO: l; or a nucleotide sequence that has at least 65% sequence identity with SEQ ID NO: l and that preferably encodes or is complementary to a sequence that encodes at least one biosynthetic colismicin enzyme or a part thereof. In a preferred embodiment of the invention the nucleic acid molecule is characterized in that it is at least 15 nucleotides in length. In another preferred embodiment of the invention the nucleic acid molecule encodes one or more polypeptides, or that includes one or more genetic elements, which possess a functional activity in the synthesis of a 2,2'-bipyridyl antibiotic or a precursor of a 2 , 2'-bipyridyl. In another preferred embodiment of the invention said 2,2'-bipyridyl antibiotic is colismicin or a precursor of colismicin. In another preferred embodiment of the invention the nucleic acid molecule encodes one or more polypeptides, or includes one or more genes and / or one or more sequences. regulators, and / or one or more coding or non-coding genetic elements, which have functional activity in the synthesis of a 2,2'-bipyridyl or a precursor of a 2,2'-bipyridyl. In another preferred embodiment of the invention the nucleic acid molecule is characterized in that said 2,2'-bipyridyl antibiotic or precursor of a 2,2'-bipyridyl is colismicin or a precursor of colismicin. In another preferred embodiment of the invention, the nucleic acid molecule is characterized by including a nucleotide sequence encoding one or more amino acid sequences of those described in SEQ ID NOs: 2 to 28, or a nucleotide sequence that is complementary or degenerate. with respect to a nucleotide sequence encoding one or more amino acid sequences that have at least 60% sequence identity with any of SEQ ID NOs: 2 to 28. In another preferred embodiment of the invention the nucleic acid molecule encodes one or more amino acid sequences that have at least 85% sequence identity with any of SEQ ID NOs: 2 to 28.

The third aspect of the present invention refers to a polypeptide encoded by the nucleic acid molecule described above. In a preferred embodiment of the invention the polypeptide includes: one or more complete amino acid sequences, or parts thereof, described in SEQ ID NOs: 2 to 28; or one or more complete amino acid sequences, or parts thereof, that have at least 60%> sequence identity with any of SEQ ID NOs: 2 to 28. In another preferred embodiment of the invention the polypeptide is characterized in that the amino acid sequences mentioned above possess at least 85% sequence identity with any of SEQ ID NOs: 2 to 28. In another preferred embodiment of the invention the polypeptide has a functional activity in the synthesis of a 2,2'-bipyridyl antibiotic . The fourth aspect of the present invention refers to a recombinant DNA molecule, which includes the aforementioned DNA fragment, or a part with similar characteristics, cloned into a vector that replicates in Streptomyces or in E. coli. In a preferred embodiment of the invention the recombinant DNA is the coslc3 cosmid or the cos3bl l cosmid. The fifth aspect of the present invention refers to a host cell or a non-human transgenic organism that contains a nucleic acid molecule mentioned above.

Thus, the genes encoded by the aforementioned DNA fragment, and host cells or transgenic organisms comprising said DNA fragment, can be used in the production of 2,2'-bipyridyl metabolites, particularly in the production of colismicin, derived from colismicin or colismicin precursors; to increase the production of 2,2'-bipyridyl metabolites; to increase the production of colismicin, colismicin derivatives or colismicin precursors; in the inactivation of genes involved in the biosynthesis of colismicin; in PCR amplification techniques aimed at the isolation and / or use of genes involved in the biosynthesis of colismicin.

The sixth aspect of the present invention refers to a process to increase the production of 2,2'-bipyridyls in a bacterial host, which comprises the transfer of the above-mentioned DNA fragment to a Streptomyces host, culture of the recombinant strain obtained , and isolation of the 2,2'-bipyridyl produced. In a preferred embodiment of the invention the host is of the genus Streptomyces is Streptomyces spp. CS40 In another preferred embodiment of the invention the host Streptomyces spp. CS40 is a mutant derived from Streptomyces spp. CS40 In another preferred embodiment of the invention, the 2,2'-bipyridyl compound is colismicin, a derivative of colismicin or a precursor of colismicin.

The seventh aspect of the present invention refers to a process for generating colismicin derivatives or colismicin precursors by inactivating genes encoded by the aforementioned DNA fragment. In a preferred embodiment of the invention, the process focuses on the use of colismicin intermediates or colismicin derivatives as starting compounds in the chemical synthesis of 2,2'-bipyridyl products. The eighth aspect of the present invention refers to a process for generating derivatives or analogs of colismicin A and colismicin C by enzymatic acylation catalyzed by a lipase.

The ninth aspect of the present invention refers to the mutant strain Streptomyces spp. CLM-A deposited in the Spanish Type Crops Collection with identification number 7755 and the products accumulated by it.

The tenth aspect of the present invention refers to the mutant strain Streptomyces spp. CLM-M deposited in the Spanish Type Crops Collection with the identification number 7756 and the products accumulated by it. The eleventh aspect of the present invention refers to a compound of Formula (I), except for compounds of formula:

where Ri, R ₂ , R ₃ and R4 are, each and independently, hydrogen or a protecting group. The protecting group may consist of an alkyl group, a cycloalkyl group, a heterocyclic cycloalkyl group, a hydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, a alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or a combination from them. In a preferred embodiment of the present invention the compound of Formula (I) is selected from the compounds of Formula II to XX. The twelfth aspect of the present invention refers to the use of the compounds of Formula I for the preparation of a pharmaceutical composition intended for the treatment of cancer, the treatment of neurodegenerative diseases, or their use as a neuroprotector, or the treatment of infectious diseases, or their use as an antibiotic In addition, this aspect also refers to the compounds of Formula I for use in the treatment of cancer, in the treatment of neurodegenerative diseases, or as a neuroprotector, or in the treatment of infectious diseases, or as an antibiotic.

The thirteenth aspect of the present invention refers to a pharmaceutical composition comprising at least one of the compounds of Formula I and at least one pharmaceutically acceptable excipient.

The last aspect of the present invention refers to a method for the treatment of cancer, for the treatment of neurodegenerative diseases or for the treatment of infectious diseases comprising the administration to the patient of a therapeutically effective amount of a compound of Formula I or of a pharmaceutical composition comprising at least one compound of Formula I.

EXAMPLES

Example 1. Isolation of the Streptomyces spp. Colismicin producing strain. CS40 Streptomyces spp. CS40 was isolated from some leaf-cutting ants of Acromyrmex octospinosus species collected in the Lambayeque department in Peru. The sequencing and analysis of its 16SrDNA showed its location within the genus Streptomyces without conclusive identity levels at the species level. Streptomyces spp. CS40 is deposited in the Spanish Type Crops Collection with deposit number 7757.

Example 2. Cloning of the gene pool involved in the biosynthesis of colismicin.

2.1. Microorganisms, plasmids and culture conditions. The microorganisms and plasmids used are described in Table 1. Streptomyces spp. CS40 and the mutants generated from it were cultured for sporulation in medium A (Fernández et al, J. Bacteriol, 180, 4929-4937, 1998); for the production of antibiotic, R5A was grown in liquid medium (Fernández et al, J. Bacteriol, 180, 4929-4937, 1998); using an inoculum previously cultured in liquid medium TSB (Tryptone Soya Broth, Merck). Intergeneric conjugation from Escherichia coli ET12567 (pUB307) to Streptomyces spp. CS40 was performed according to Sambrook et al, Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory press (1989). E. coli strains were grown and transformed as described by Sambrook et al, (1989). Culture media were supplemented with appropriate antibiotics to each resistance marker at the following concentrations: 100 μg / ml ampicillin, 20 μg / ml tobramycin, 25 μg / ml apramycin, 50 μg / ml thiostreptone, 25 μg / ml kanamycin 10 μg / ml tetracycline, 25 μg / ml chloramphenicol and 50 μg / ml nalidixic acid. Table 1. Bacterial strains and plasmids used in this study.

Strain, plasmid Properties Source or reference

E. coli DH10B General Cloning Guest Invitrogen

 E. coli XLI Blue Guest for the construction of the Stratagene

 MR library

 E. coli ET12567 Strain for conjugation Kieser et al., Practical

(pUB307) intergeneric Streptomyces Genetics.

 The John Innes

 Foundation Norwich,

2000

 Streptomyces Producer of colismicin A Isolated on our spp. CS40 laboratory

 pWE15 Cosmetic for construction Stratagene

 from the pOJ260 library Plasmid for gene disruption Bierman et al., Gene 116,

 43-49, 1992

 pEM4T Plasmid for gene expression in Menéndez et al., Appl.

 Streptomyces Environ. Microbiol 72,

 167-177, 2006 pCR-BLUNT Plasmid for the cloning of Invitrogen products

 PCR

 pU09090 Plasmid source of the resistance gene to Prado et al., Mol Gen apramycin Genet. 201, 216-225,

 1999

 pBluescript SK + Cloning vector in E. coli Stratagene

 pHZ1358 Plasmid for gene replacement Sun et al.,

 Appl. Microbiol Biotech nol 82, 303-310, 2009 pGemT Plasmid for the cloning of Promega products

 PCR

2.2. Analysis of colismicin A production. Colismicin A production was routinely performed in 1.5 mL of solid R5A medium (Fernández et al., J. Bacteriol, 180, 4929-4937, 1998) in 25-well plates . For its inoculum, spores of Streptomyces spp. CS40 and the cultures were maintained for 7 days at 30 ° C, extracted after that time with 1 ml of ethyl acetate. Colismicin A production was also performed in liquid cultures of 5 to 7 days grown in an orbital shaker at 30 ° C and 250 rpm. Flasks were used for this. 250 ml Erlenmeyer containing 50 ml of liquid R5A medium (Fernández et al., J Bacteriol, 180: 4929-4937, 1998). For its inoculum a volume of 2% of a pre-circle of Streptomyces spp. CS40 performed on TSB medium (50 ml in 250 ml flasks) that was collected after two days of incubation in an orbital shaker at 30 ° C and 250 rpm. The colismicin A present in the liquid cultures was extracted with varying volumes of ethyl acetate. The ethyl acetate extracts were evaporated using a vacuum coupled centrifuge (Speed-Vac) and once evaporated the samples were resuspended in methanol for analysis.

The identification and quantitative analysis of colismicin A was carried out by reverse phase chromatography in an Acquity UPLC equipment using a BEH C18 column (2.1 x 100 mm Waters) and using acetonitrile and 0.1% TFA in water as solvents. The samples were eluted with 10% acetronitrile for 1 min followed by a linear gradient of acetonitrile from 10% to 80% for 7 min. The flow used was 0.5 ml / min and the column temperature was 35 ° C. For the HPLC-coupled mass analysis (HPLC / MS) an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a Symmetry C18 column (2.1 x 150 mm, Waters) was used. The solvents used were the same as those described above and elution was performed initially maintaining 10% acetonitrile for 4 min, followed by a linear gradient from 10%> to 88% acetonitrile for 26 min, using a flow 0.25 ml / min. The mass analysis was performed by electrospray ionization in positive mode, with a capillary voltage of 3 kV and a cone voltage of 20 V. The detection of the peaks and the characterization of their absorption spectrum was performed in both cases with a online photodiode system and using Waters Empower software, extracting two-dimensional chromatograms at a wavelength of 332 nm.

For the structural characterization of the derivatives of colismicin A mentioned in this invention, cultures of the Streptomyces spp. Corresponding CS40. After centrifugation to remove the cells, the resulting broths were subjected to solid phase extraction in extraction cartridges with C18 filler (Sep-Pak Vac, Waters). The compounds sought were purified from the resulting extracts using preparative reverse phase HPLC. All purifications are they were made under isocratic conditions with mixtures of acetonitrile or methanol and 0.05% TFA in water, in optimized proportions for each compound. The columns used were an XTerra PrepRP18 (19 x 300 mm, Waters) and a Symmetry C18 (7.8 x 300 mm, Waters). The solutions with the purified peaks were partially evaporated in a rotary evaporator to reduce their organic solvent content and subsequently loaded into a solid phase extraction cartridge (Sep-Pak C18, Waters). The retained compounds were washed successively with water, 0.1% ammonia in water and again water, to completely eliminate TFA. Finally, they were eluted with methanol, dried in vacuo and finally lyophilized. The identification and quantitative analysis of colismicin A was carried out by reverse phase chromatography in an Acquity UPLC equipment using a BEH C18 column (2.1 x 100 mm, Waters) and using acetonitrile and 0.05% TFA as solvents. The samples were eluted with 10% acetronitrile) for 1 min followed by a linear gradient of acetonitrile from 10%> to 80% for 7 min. The flow used was 0.5 ml / min and the column temperature 30 ° C. For the HPLC-coupled mass analysis (HPLC / MS) an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a Symmetry C18 column (2.1 x 150 mm, Waters) was used. The solvents used were the same as those described above and the elution was performed with an initial isocratic gradient of 10% acetonitrile maintained for 4 min and followed by a linear gradient of acetonitrile from 10% to 88% for 26 min, using for it a flow of 0.25 ml / min. The mass analysis was performed by electrospray ionization in positive mode, with a capillary voltage of 3 kV and a cone voltage of 20 V. The detection of the peaks and the characterization of their absorption spectrum were performed in both cases with a online photodiode system and using Waters Empower software, extracting two-dimensional chromatograms at a wavelength of 360 nm.

The colismicin A analyzed in UPLC has a retention of 3.30 min and an absorption spectrum with maximums at 250 and 332 nm. Colismicin A analyzed in HPLC / MS has a retention of 15.13 min and shows a molecular ion with a mass of 276 m / z [M + H] +. (FIG. 3) For the structural characterization of the colismicin derivatives mentioned in this invention, cultures of the Streptomyces spp. Corresponding CS40 and the extracts were dissolved in 5 ml of a mixture of DMSO and methanol in equal parts. They were then centrifuged and the upper layer corresponding to the lipid fraction was removed. The first purification step was performed by chromatography on an XTerra PrepRP18 column (19 x 300 mm, Waters) using acetonitrile and 0.05% acetic acid trifluoro (TFA) as solvents dissolved in water. A linear gradient from 30% to 100% acetonitrile was used for 7 min followed by 3 min of 100% acetonitrile. The flow used was 15 ml / min. The peaks of interest were collected on 0.1 M phosphate buffer, pH 7.0. The solutions obtained were partially evaporated in a rotary evaporator to reduce the concentration of acetonitrile and subsequently applied to a solid phase extraction cartridge (Sep-Pak C18, Waters), subsequently washed with water to remove salts and eluted with methanol . Subsequent purifications were performed under isocratic conditions, optimized for each peak, using a Symmetry C18 column (7.8 x 300 mm, Waters) and mixtures of acetonitrile and 0.05% TFA> dissolved in water, using a flow of 7 ml / min As mentioned above, the peaks were always collected on 0.1 M phosphate buffer, pH 7.0, desalted using solid phase extraction and finally lyophilized. 2.3. DNA manipulation.

The preparation of plasmids, total DNA, restriction enzyme digestions, DNA linkages, etc., was carried out following standardized methods previously described (Sambrook et al., Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory press, 1989; Kieser et al, Practical Streptomyces Genetics. The John Innes Foundation. Norwich, 2000). The DNA fragments were isolated from agarose gels using the QIAGEN QUIAquick Gel Extraction Kit (Hilden, Germany) and labeled using the DIG DNA Labelling and Detection Kit from Roche Diagnostics (Manheim, Germany) used for Southern analysis according to the manufacturer's instructions. Sequencing was performed on double stranded DNA using the method described by Sanger et al, Proc. Nati Acad. Sci. USA. 74, 5463-5467 (1977) and the Cy5 Autocycle Sequencing Kit sequencing kit (Pharmacia Biotech). The electrophoresis of the samples was performed in an automatic Alf-express sequencer (Pharmacia Biotech). The sequences obtained were analyzed using the GCG software package, from the Genetics Computer Group of the University of Wisconsin (Devereux et al, Nucleic Acids Res. 12, 387-395, 1984) and the BLAST program (Altschul et al, J Mol. Biol. 215, 403-410, 1990). The analysis of the transmembrane regions of possible transmembrane proteins was performed using the TMHMM v program. 2.0 (Krogh et al, J. Mol. Biol. 305, 567-580, 2001). The analysis of PCS and NRPS was performed using the ASMPKS programs (Tae et al, BMC Bioinformatics. 8, 327-335, 2007) and NRPSpredictor (Rausch et al, Nucleic Acids Res. 33, 5799-5808, 2005).

2.4. Amplification of DNA fragments by PCR and cloning of a DNA fragment encoding part of a 2-aminotransferase plant of the genome of Streptomyces spp. CS40 The strategy used for cloning the colismicin biosynthesis gene cluster was the use of genetic homology with some proteins encoded by previously characterized genes and for which there should be a homologue in the colismicin biosynthesis pathway.

The information available on the biosynthesis of picolinic acid (Bruntner and Bormann, 1998; Namwat et al, 2002), an intermediary in the biosynthesis pathway of caerulomycin (Vining et al, 1988) and possibly also in that of colismicin was used to design degenerate oligonucleotides L2ATFW2 (SEQ ID NO: 29) and L2ATRV2 (SEQ ID NO: 30) and try to amplify a sequence belonging to a gene homologous to lysine 2-aminotransferases. To obtain the total DNA of Streptomyces spp. CS40 the microorganism was cultured in TSB liquid medium and the total DNA was isolated as described by Kieser et al, (2000). Total DNA of Streptomyces spp. CS40 was used as a template for the polymerase chain reaction using oligonucleotides L2ATFW2 (SEQ ID NO: 29) and L2ATRV2 (SEQ ID NO: 30). It was assumed that, as a consequence of the amplification, a DNA fragment of approximately 0.6 kb would be obtained which It would contain the inner part of the gene that codes for a lysine 2-aminotransferase. The polymerase chain reaction was carried out in a total volume of 50 μΐ and the reaction mixture contained 0.1 μg of total Streptomyces spp. DNA. CS40, 2.5% dimethylsulfoxide (DMSO), 200 pmoles of each primer, dNTPs (final concentration 200 μΜ), lxPCR of the enzyme Taq DNA polymerase (Invitrogen). The chain reaction was carried out in an Applied Biosystems GeneAmp ^® PCR System9700 thermocycler with the following program: 1 cycle of denaturation at 98 ° C (5 min), 30 cycles of denaturation / ringing / synthesis at 94 ° C (1 min) / 55 ° C (1 min) / 72 ° C (1 min) and 1 final extension cycle at 72 ° C (10 min). The DNA fragment obtained with this procedure was cloned into the E. coli pGemT vector using the procedure recommended by the manufacturer and was subjected to sequencing using standardized techniques. Sequence analysis of the PCR amplified fragment revealed that it contained part of a protein that showed clear similarities at the amino acid level with several previously characterized lysine 2-aminotransferases: NikC of Streptomyces tendae, accession number CAA75797 (Bruntner and Bormann, 1998) and StAptomyces virginiae VisA, accession number BAB83671 (Namwat et al, 2002).

From this sequence homologous to amplified lysine 2-amino transferases, two oligonucleotides were designed to construct a genetic probe. The synthetic oligonucleotides L2ATsonl (SEQ ID NO: 31) and L2ATson2 (SEQ ID NO: 32) were used as primers for the amplification of the genetic probe by means of the polymerase chain reaction (PCR) and using as template DNA the Streptomyces spp. Chromosomal DNA CS40 The polymerase chain reaction was carried out in a total volume of 50 μΐ and the reaction mixture contained 0.1 μg of total Streptomyces spp. DNA. CS40, 2.5% dimethylsulfoxide (DMSO), 200 pmoles of each primer, dNTPs (final concentration 200 μΜ), lxPCR of the enzyme pfx DNA polymerase (Invitrogen). The chain reaction was carried out in an Applied Biosystems GeneAmp ^® PCR System9700 thermocycler with the following program: 1 cycle of denaturation at 98 ° C (5 min), 30 cycles of denaturation / ringing / synthesis at 94 ° C (1 min) / 60 ° C (1 min) / 68 ° C (1 min) and 1 final extension cycle at 68 ° C (10 min). The DNA fragment obtained with this procedure was cloned into the Escherichia coli pCR-Blunt vector and subjected to sequencing using standardized techniques. Once confirmed that the amplified fragment was part of the coding region of lysine 2-aminotransferase, this fragment was used as a genetic probe for the analysis of a Streptomyces spp chromosomal DNA library. CS40 2.5 Construction and analysis of the Streptomyces spp. Chromosomal DNA library. CS40

The chromosomal DNA library of Streptomyces spp. CS40 was built on the cosmid pWE15 that is able to replicate in E. coli. The genomic DNA of Streptomyces spp. CS40 was isolated as described above, partially digested with Sau3AI and the fragments obtained, approximately 35-40 kb in size, were dephosphorylated by treatment with alkaline phosphatase (Roche Diagnostics, Mannheim). The cosmid pWE15, used as a vector, was linearized with BamHI. The DNA fragments and the vector were ligated and packaged in vitro using a commercial packaging kit Gigapack III Gold packaging Extract kit following the instructions of the commercial house (Stratagene). The recombinant DNA particles were used to infect E.coli XLI Blue MR cells and the transductants were selected on plates with LA medium (Luria-Bertani agar) containing as an ampicillin selection antibiotic. Approximately 1000 transducer colonies were cultured in microtiter plates containing LB medium (Luria-Bertani broth) and the selection antibiotic. After incubation at 37 ° C for 24 h, they were maintained in the presence of 25% glycerol at -70 ° C for preservation.

In order to clone the cluster of colismicin biosynthesis genes, the analysis of the Streptomyces spp library was carried out. CS40 by in situ hybridization of colonies with the probe mentioned above. The transductants were transferred from the microtiter plates to LA medium plates containing as an ampicillin selection antibiotic and after a night of growth at 37 ° C the colonies were transferred to nylon filters for in situ hybridization following the protocols described by Sambrook et al, (1989). The filters were analyzed with the probes labeled using the commercial DIG DNA labeling and detection kit from Roche. In this way (and as detailed above) the coslC3 cosmid was isolated. Example 3. Obtaining and analyzing the nucleotide sequence of the gene pool responsible for the biosynthesis of colismicin and deduction of gene functions.

The coslC3 cosmid and the 7.7 kb EcoRV / HindIII fragment from the cos3B l l cosmid subcloned into the same sites in the pBluescriptSK + vector were sequenced in their entirety. Sequencing was performed on double stranded DNA using the method described by Sanger et al, (1977) and using the Cy5 Autocycle Sequencing Kit (Pharmacia Biotech). The electrophoresis of the samples was performed in an automatic Alf-express sequencer (Pharmacia Biotech) and the data obtained were analyzed using the GCG software package, from the Genetics Computer Group of the University of Wisconsin (Devereux et al, 1984) and the BLAST program (Altschul et al., 1990). The analysis of the transmembrane regions of possible transmembrane proteins was performed using the TMHMM v program. 2.0 (Krogh et al, J. Mol. Biol. 305, 567-580, 2001). The analysis of PCS and NRPS was performed using the ASMPKS (Tae et al, 2007) and NRPSpredictor (Rausch et al, 2005).

Computer analysis of the 46668 bp DNA sequence (SEQ ID NO: 1) showed the presence of 27 open reading patterns (ORFs) (FIG. 2 and Table 2) with a high G + C content characteristic of the DNA of Streptomyces In addition, a particularly high content in high G + C is shown in the third position of the codons characteristic of Streptomyces genes. The gene functions were deduced by comparison of the amino acid sequences, conceptually translated from the nucleotide sequence, with known sequences available in databases. The results obtained are shown in Table 2 referring to the sequence data included in the application. 23 of the 27 ORFS found, which occupy a 37.5 kb region, are probably involved in the biosynthesis of colismicin. This region is flanked by 4 ORFs probably not involved in the biosynthesis of colismicin (blank in FIG. 2). Of these ORFs, orfl, showed great similarity with a possible S. viridochromogenes DSM 40736 helicase (ZP_05534927) and a possible S. scabiei 87.22 kinase, (YP 003487313). Of the 23 proposed ORFs involved in the biosynthesis of colismicin A, 16 ORFs are involved in the biosynthesis of the structural part of colismicin, 5 of them {orfl -7) are involved in the transport of colismicin, 2 ORFs {orfl and orfó) are probably involved in regulatory processes. Table 2. Genes identified in the chromosome region of Streptomyces spp. CS40 involved in the biosynthesis of colismicin.

Gene Position Amino acids Function deducted Notes

 549-2237 Unknown

 orfl 563 SeqID.NO:2 compl.

 Homologous Transcriptional Regulator

 orf2 2857-3414 186 SeqID.NO:3 to TetR

 3514-5247

 orfl 578 Conveyor SeqID.NO:4 compl.

 5256-7016

 orf4 587 Conveyor SeqID.NO:5 compl.

 orfl 7225-8193 323 Conveyor SeqID.NO:6 orfó 8322-9305 328 Conveyor SeqID.NO:7 orfl 9314-10099 262 Conveyor SeqID.NO:8

 Homologous Transcriptional Regulator

 orfl 10363-10869 169 SeqID.NO:9 to LuxR

 11042-12070

 orfl 343 Methyl transferase SeqID.NO: 10 compl.

 1210-13-13703

 orflO 533 Dehydrogenase SeqID.NO: l l compl.

 13861-15495

 Orfll 545 Aminotransferase SeqID.NO: 12 compl.

 orfl 2 15620-16606 329 Methyltransferase SeqID NO: 13

 GriD-like protein

 16688-18076 (YP 001825756)

 orfl 3 463 SeqID.NO: 14 compl. Arylcarboxylate reductase

component Gene Position Amino acids Function deducted Notes

GriC-like protein

 18078-19109 (YP_001825755)

 orfl4 344 SeqID.NO: 15 compl. Arylcarboxylate reductase

 component

 orfl5 19405-20613 403 Monoxygenase SeqID NO: 16

20680-21921

 Orphlous 414 Amidohydrolase SeqID.NO: 17 compl.

 21926-23614

 orfl 7 563 Adenylate forming enzyme SeqID.NO: 18 compl.

 23611-23871

 orfl8 87 Phosphopantetein binding protein SeqID.NO: 19 compl. orfl9 24108-25484 459 Lysine 2-aminotransferase SeqID.NO:20 orfiO 25481-26662 394 Sarcosine oxidase monomeric SeqID.NO:21 orf21 26548-34206 2553 PCS / NRPS hybrid protein SeqID.NO:22 orf22 34203-37478 1092 NRPSq SeqID. NO: 23 orfi3 37475-38659 395 Dehydrogenase SeqID NO: 24 orf24 38652-39380 243 Thioesterase SeqID NO: 25

39442-40665

 orfi5 408 Unknown SeqID.NO:26 compl.

 orf26 41103-41708 202 Unknown SeqID.NO:27 orfi7 42017-46105 1363 Unknown SeqID.NO:28

Example 4. Inactivation of the colismicin biosynthesis gene cluster by gene disruption. In order to demonstrate the involvement of cloned gene clustering in the biosynthesis of colismicin, inactivation of the orf21 gene was carried out. For the inactivation of the orfll a BamHI fragment of the coslC3 cosmid was isolated. This 3128 bp fragment is internal to orfll (BamHI sites 13 and 14, FIG. 2) and was cloned into plasmid pOJ260 digested with BamHI. The resulting construct, pOJB12 was introduced in Streptomyces spp. CS40 by intergener conjugation from E. coli ET12567 (pUB307) to generate the mutant strain CLM-12 that was selected for its apramycin resistance.

The mutation in the orfll gene was checked by Southern analysis. The mutant was shown not to produce colismicin A by UPLC analysis of culture samples from Streptomyces spp. CS40 and CLM-12 extracted with ethyl acetate (FIG. 4), thereby confirming the implication of gene clustering in colismicin biosynthesis.

Example 5. Establishment of the limits of the clustering of colismicin biosynthesis genes by gene replacement.

To establish the limits of the gene pooling involved in the biosynthesis of colismicin, two mutants were made by gene disruption and one mutant by gene replacement in the genes orfl (mutant CLM-H) and orfl7 (mutant CLM-24) and orfl5 (mutant CLM- 22D) respectively In the case of the CLM-H and CLM-24 mutants the ORFs are interrupted by the introduction of an apramycin resistance gene and in the case of the CLM-22D mutant the ORF is replaced by the apramycin resistance gene.

To obtain the mutant in the orfl a region of 910 bp corresponding to a fragment internal to the orfl was amplified with the oligonucleotides HelDisrl (SEQ ID NO: 34) and HelDisr2 (SEQ ID NO: 35). Said fragment was cloned into the pCR-Blunt vector to generate plasmid pCRBH and subjected to sequencing using standardized techniques. Subsequently, this fragment was obtained from plasmid pCRBH by digesting it with EcoRI and was cloned into the same restriction site of vector pOJ260, thus generating plasmid pOJBH that was used for gene disruption in Streptomyces spp. CS40 In no case were we able to obtain mutants that would interrupt the orfl suggesting that this gene is essential for cell viability. This data rules out the participation of orfl in colismicin biosynthesis, since the different mutants (CLM-L, CLM-12) that have affected colismicin biosynthesis are perfectly viable, thus excluding this gene from the colismicin biosynthesis cluster.

To obtain the CLM-24 mutant, a Clal-BamHI fragment of 2271 bp was obtained, from the coslC3 cosmid corresponding to a fragment internal to orf27. Said fragment became blunt and was cloned into vector pOJ260 at the EcoRV site thereby generating plasmid pOJB23P that was used for gene disruption in Streptomyces spp. CS40 generating the CLM-24 mutant.

The gene replacement technique was used to obtain the CLM-22D muierie. A 1526 bp fragment that is flanking upstream to the orf25 gene was amplified by PCR. For this, the oligonucleotides ORF22dell (SEQ ID NO: 36) and ORF22del2 (SEQ ID NO: 37) were used to which the EcoRI and HindIII restriction targets were introduced respectively, said fragment was cloned into the pCR-Bluní neighbor and was subjected to sequencing using standardized techniques. Subsequently, said fragment was cloned into the neighbor pUO9090 at the EcoRI and HindIII resiriction sites generating plasmid pUO909022A. Additionally, a second 1496 bp fragment that is flanking runs down to the orf25 gene was amplified by PCR. To do this, oligonucleotides ORF22del3 (SEQ ID NO: 38) and ORF22del4 (SEQ ID NO: 39) were used, which were inroduced with the EcoRV target, which fragment was cloned into the pCR-Bluní neighbor and subjected to sequencing using techniques They are standardized. Subsequently, said fragment was cloned into the neighbor pUO909022A using the EcoRV resync site to generate the neighbor pUO909022AB.

For gene replacement the replacement case was obtained from the neighbor pUO909022AB digested with Spel and was cloned into the neighboring pHZ1358. The resulting construction, pHZ22AB, was used for gene replacement in Streptomyces spp. CS40 generating the CLM-22D muierie. The integration of the apramycin resistance gene in the case of mutants generated by disruption as well as the replacement on the chromosome of the wild copy of the gene by the mutated was confirmed in the transconjugants by Southern analysis. Each of the mutants was analyzed to determine the production of colismicin A, in parallel with the parental strain Streptomyces spp. CS40 via UPLC. The CLM-22D and CLM-24 mutants were shown as producers of colismicin A (FIG. 5), indicating that the mutated genes do not participate in the biosynthesis of colismicin and thus confirming the limits of the gene pooling involved in the biosynthesis of colismicin

Example 6. Generation of a mutant in the initial steps of the biosynthesis of colismicin.

The first of the proposed steps for biosynthesis of colismicin (FIG 6) consists of the formation of picolinic acid from lysine and the first gene involved in this process would be a lysine 2-aminotransferase encoded by orfl9. To confirm this data, the orfl9 gene was inactivated.

The gene replacement technique was used to inactivate orfl9. A 1589 bp fragment that is flanking upstream to the orfl9 gene was amplified by PCR. For this, the oligonucleotides ORFlódell (SEQ ID NO: 40) and ORF16del2 (SEQ ID NO: 41) were used to which the EcoRI and HindIII restriction targets were introduced respectively, said fragment was cloned into the pCR-Blunt vector and was Sequenced using standardized techniques. Subsequently, said fragment was cloned into the vector pUO9090 at the EcoRI and HindIII restriction sites generating plasmid pUO909016A. Additionally, a second 1559 bp fragment that is flanking downstream to the orfl9 gene was amplified by PCR. For this, oligonucleotides ORF16del3 (SEQ ID NO: 43) and ORF16del4 (SEQ ID NO: 44) were used to which the BamHI and EcoRV restriction targets were introduced respectively, this fragment was cloned into the pCR-Blunt vector and was subjected to sequencing using techniques Standardized Subsequently said fragment was cloned into the vector pUO909016A using the EcoRV and BamHI restriction sites to generate the vector pUO909016AB.

For gene replacement the replacement cassette was obtained from the vector pUO909016AB digested with Spel and was cloned into the vector pHZ1358. The resulting construct, pHZ16AB, was introduced in Streptomyces spp. CS40 by intergener conjugation from E. coli ET12567 (pUB307) to generate the mutant strain CLM-L. The strain CLM-L is deposited in the Spanish Type Culture Collection with deposit number 7754.

The mutant was shown not to produce colismicin through UPLC analysis of culinary samples of Streptomyces spp. CS40 and CLM-L exirated with eyl oily (FIG. 7B), thereby confirming the involvement of this gene in the biosynthesis of colismicin.

In order to corroborate the participation of the orfl9 gene in the initial steps of biosinitis, the production of colismicin A per parle of the CLM-L muierie in the presence of picolinic acid (1 mM final concentration), a possible inbred meiabolium, was analyzed by UPLC The culinary medium. The exirated notches with eyl oily showed that the CLM-L muierie in the presence of picolinic acid regains the ability to produce colismicin A (FIG 7C). This data confirms the participation of orfl9 in the initial steps of biosinitis and the existence of picolinic acid as an inmedial meliaboliío in the roller.

Example 7. Generation of mutants producing new derivatives of colismicin by gene replacement.

To generate mules of Streptomyces spp. CS40 capable of producing chemically modified colismicins, the genes orfll and orfi ⁾ were selected individually and jointly orf! 3 and orfl4 (FIG. 2), all of them possibly involved in final steps of colismicin biosinitis. For this purpose, plasmids plasmids pHZ6AB, pHZ8AB and ρΗΖΙΟΙΑΒ were constructed. It was first amplified by PCR, using the synthetic oligonucleotides orfódeB (SEQ ID NO: 45) and orf del4 (SEQ ID NO: 46) a fragment of 1499 bp corresponding to the downstream region of the orfll gene, which was cloned into the pCR-Blunt vector to give plasmid pCRB8B that was subjected to sequencing using standardized techniques. This fragment was extracted from plasmid pCRB8B using the EcoRI sites existing in the polilinker of the pCRB-Blunt vector, made blunt and subcloned into plasmid pU09090 to give plasmid pU08B.

It was subsequently amplified by PCR using the synthetic oligonucleotides orfódell (SEQ ID NO: 47) orf8del2 (SEQ ID NO: 48) that included EcoRI and HindIII restriction sites respectively to facilitate subcloning, a fragment of 1516 bp corresponding to the upstream region of the orfll gene, which was cloned into the pCR-Blunt vector to give plasmid pCRB8A that was subjected to sequencing using standardized sequencing techniques. This fragment was extracted from plasmid pCRB8A using the EcoRI and HindIII restriction sites and subcloned into the same sites of plasmid pU08B to generate plasmid PU08AB.

The replacement cassette was extracted from plasmid pU08AB using the Spel restriction sites and cloned into the pHZ1358 vector to generate the plasmid pHZ8AB that was introduced into Streptomyces spp. CS40 by intergener conjugation from E. coli ET12567 (pUB307) to generate the mutant strain CLM-A. The strain CLM-A is deposited in the Spanish Type Culture Collection with deposit number 7755.

For the generation of the CLM-M2 mutant strain, a 1504 bp fragment was amplified by PCR to the region above the orf9 gene. For this purpose, the oligonucleotides orfódell (SEQ ID NO: 49) and orf6del2b (SEQ ID NO: 50) that carried the EcoRI and HindIII resyriction sites were used respectively. This fragment was cloned into the pCR-Bluní neighbor thus generating the plasmid pCRB6A that was sequenced using standard techniques. Subsequently, the fragment was obtained by digesting the plasmid pCRB6A with EcoRI and HindIII and was cloned into the same sites of the neighbor pUO9090 generating the neighbor pU06A. A 1504 bp fragment belonging to the downstream region of the orf9 gene was then amplified by PCR. For this purpose, the orfódeB oligonucleotides (SEQ ID NO: 51) and orf6del4 (SEQ ID NO: 52) were used. This fragment was cloned into the pCR-Blunt vector thus generating plasmid pCRB6B that was sequenced using standardized techniques. Subsequently, the fragment was obtained by digesting plasmid pCRB6A with EcoRI, became blunt and was cloned into the EcoRV site of plasmid pU06A thereby generating plasmid pU06AB.

The replacement cassette was extracted from plasmid pU06AB using the Spel restriction sites and cloned into the pHZ1358 vector to generate the plasmid pHZ6AB that was introduced into Streptomyces spp. CS40 by intergener conjugation from E.coli ET12567 (pUB307) to generate the mutant strain CLM-M2. The strain CLM-M2 is deposited in the Spanish Type Culture Collection with deposit number 7756.

For the generation of the double mutant in the orfl3 and orfl4 genes, a 1496 bp fragment was amplified by PCR from the region running above the orfl4 gene. For this purpose, the oligonucleotides orflOldell (SEQ ID NO: 53) and orfl01del2 (SEQ ID NO: 54) were used, which carried the EcoRI and HindIII restriction sites respectively (underlined sequence). This fragment was cloned into the pCR-Bluní vector thus generating the plasmid pCRBIOlA that was sequenced using standard techniques. Subsequently, the fragment was obtained by digesting the plasmid pCRBIO lA with EcoRI and HindIII and cloned into the same sites of the vector pUO9090 generating the vector pUOlOlA.

A 1522 bp fragment was then amplified by PCR to the region running down the orfl3 gene. For this purpose, the oligonucleotides orflOldeB (SEQ ID NO: 55) and orflOldeM (SEQ ID NO: 56) were used which bore the EcoRV resyriction site (underlined sequence). This fragment was cloned into the pCR-Bluní neighbor thus generating the plasmid pCRBlOlB that was sequenced using standardized techniques. Subsequently, the fragment was obtained by digesting the plasmid pCRBlOlB with EcoRV and was cloned into the same site of the plasmid pUOlOlA thus generating the plasmid pUOlOlAB. The replacement cassette was extracted from plasmid pUOlOlAB using Spel restriction sites and cloned into vector pHZ1358 to generate plasmid ρΗΖΙΟΙΑΒ that was introduced into Streptomyces spp. CS40 by intergener conjugation from E. coli ET12567 (pUB307) to generate the mutant strain CLM-G. The strain CLM-G is deposited in the Spanish Type Culture Collection with deposit number CECT7861.

In all cases the transconjugants in which a double overcrossing event had occurred were selected for their resistance to apramycin and their sensitivity to thioestreptone. Chromosome replacement was confirmed in transconjugants by Southern analysis.

The analysis of cultures of the CLM-A strain by UPLC showed two peaks with characteristic absorbance of colismicin A but with mobility of 2.86 min and 2.99 min corresponding to compounds of formula II and colismicin C (FIG. 8). Subsequent analysis by HPLC / MS determined that the compound of formula II has a mobility of 13.42 min and an ion of 249 m / z [M + H] ⁺ . Colismicin C has a mobility of 13.56 min and an ion of 263 m / z [M + H] ⁺ .

The analysis of cultures of the CLM-M2 strain by UPLC showed two peaks with characteristic absorbance of colismicin A but with mobility of 3.05 min and 4.1 min corresponding to the compounds of formula III and formula IV (FIG. 9) . Subsequent analysis by HPLC / MS determined that the compound of formula III exhibits a mobility of 15.16 min and an ion of 262 m / z [M + H] ⁺ . The compound of formula IV has a mobility of 19.12 min and an ion of 244 m / z [M + H] ⁺ .

Culture analysis of the CLM-G strain by HPLC / MS showed a peak with characteristic absorbance of colismicin A absent in the wild strain (FIG 10A and B) corresponding to the compound of formula V and exhibiting a mobility of 6.15 min (FIG 10C) and an ion of 263 m / z [M + H] ⁺ . Example 8. Generation of new derivatives of colismicin by adding different precursors to the mutant strains CLM-L, CLM-A and CLM-M2.

In order to generate new molecules derived from colismicin, the culture media in which the CLM-L, CLM-A and CLM-M2 mutants were inoculated were supplemented with two structural analogs of the picolinic acid waiting for said compounds to be incorporated into the pathway. of colismicin biosynthesis.

For this, 6-methyl picolinic acid and 4-methylpyridine-2-carboxylic acid were added at a final concentration of 0.7 mM to the R5A medium in which the CLM-L, CLM-A and CLM-M2 mutants were inoculated. After 6 days of growth at 30 ° C, the new compounds generated with ethyl acetate were extracted.

The analysis of the culture of strain CLM-L to which 6-methyl picolinic acid was added, by UPLC showed a peak with characteristic absorbance of colismicin but with mobility 3.06 min corresponding to the compound of formula VI. Subsequent analysis by HPLC / MS determined that compound VI has an ion of 290 m / z [M + H] ⁺ (FIG. HA, B and C).

Analysis of the culture of strain CLM-L to which 4-methylpyridine-2-carboxylic acid was added, by UPLC showed a peak with characteristic absorbance of colismicin but with mobility 3.14 min corresponding to the compound of formula VIL. Further analysis by HPLC / MS determined that the compound of formula VII has an ion of 290 m / z [M + H] ⁺ (FIG 1 ID, E and F).

UPLC culture analysis of strain CLM-A to which 4- methylpyridine-2-carboxylic acid was added, showed a peak with characteristic absorbance of colismicin but with mobility 3.14 min corresponding to the compound of formula VIII ( FIG. 12). Subsequent analysis by HPLC / MS determined that the compound of formula VIII has an ion of 277 m / z [M + H] ⁺ .

The analysis of the culture of the strain CLM-M2 to which 4-methylpyridine-2-carboxylic acid was added, by UPLC showed two peaks with characteristic absorbance of colismicin but with mobility 3.16 min and 3.51 min corresponding to the compound of formula IX and formula X. Subsequent analysis by HPLC / MS determined that the Compounds of formula IX and formula X have an ion of 276 m / z [M + H] and 258 m / z [M + H] ⁺ respectively (FIG. 13).

Example 9. Generation of new derivatives of colismicin by enzymatic acylation catalyzed by a lipase. Enzymatic acylations of colismicin A and colismicin C catalyzed by PS-C were carried out by incubating at 45 ° C and 250 rpm a suspension of 20 mg of lipase in a solution of 5 mg of colismicin A or colismicin C in 2 ml of ether methyl tertbutyl and 1 ml of the corresponding acylating agent. The biotransformation conversion was monitored by HPLC, using an Agilent Technologies 1200 Series chromatographic equipment, using acetonitrile and water (without TFA) as solvents and a reverse phase column (Zorbax Eclipse XDB-C18, RR, 1 .8 μιη, 4.6 x 50 mm, Agilent). The samples were eluted using a method of three linear gradients, the first from 10% to 60% acetonitrile over 5.7 minutes, then another gradient from 60% to 100% acetonitrile for 0.4 minutes, then 0.55 minutes in isocratic at 100% acetonitrile and a final gradient from 100% to 10% acetonitrile for 0.35 minutes, at a flow of 2 ml / min. The wavelength at which the chromatograms were obtained was 332 nm. In this method, colismicin A and colismicin C have a mobility of 2.29 and 1.90 min respectively. When the conversion reached a value close to 90%, the enzyme was filtered under vacuum in a filter plate and washed with abundant methyl tertbutyl ether and methanol. In the case of Colismicin C, the filtrate was concentrated in vacuo and the resulting residue, previously dissolved in 1 ml of methanol, was chromatographed on an XBridge Prep C18 column (30x150 mm, Waters), using acetonitrile mixtures as the mobile phase. water at a flow of 20 ml / min. The peaks of interest were diluted four times with water and subsequently concentrated by solid phase extraction. In the case of Colismicin A, the filtrate was concentrated in vacuo and the resulting residue was purified by column chromatography using hexane: ethyl acetate mixtures. Finally, the compounds obtained were lyophilized for preservation. The new acylated derivatives were initially identified by HPLC analysis, comparing the absorption spectrum and retention time. Then it characterized by analysis of MS and NMR according to the previous examples.

- For the particular case of using colismicin C and vinyl acetate as the acylating agent, the reaction time was 6 hours and a conversion of 95% was achieved. A new compound with a retention time of 4.51 minutes was identified by HPLC which was purified using a mixture of acetonitrile and water (50:50) at a flow rate of 20 ml / min. Once isolated, subsequent NMR analysis confirmed the structure of the compound according to formula XI.

- For the particular case of using colismicin C and vinyl butanoate as the acylating agent, the reaction time was 10 hours and a conversion of 95% was achieved. By

HPLC identified a new compound with a retention time of 5.56 minutes which was purified using a mixture of acetonitrile and water (55:45) at a flow rate of 20 ml / min. Once isolated, subsequent NMR analysis confirmed the structure of the compound according to formula XII. - For the particular case of using colismicin C and vinyl benzoate as the acylating agent, the reaction time was 14 hours and a conversion of 90% was achieved. A new compound with a retention time of 6.22 minutes was identified by HPLC which was purified using as a mobile phase a mixture of acetonitrile and water (60:40) at a flow of 20 ml / min. Once isolated, subsequent NMR analysis confirmed the structure of the compound according to formula XIII.

- For the particular case of using colismicin A and vinyl acetate as the acylating agent, the reaction time was 5 hours and a conversion of 95% was achieved. A new peak with characteristic absorbance of colismicin and with mobility of 2.72 minutes corresponding to the compound of formula XIV was identified by HPLC. Subsequent analysis by HPLC / MS determined that the compound of formula XIV has an ion of 318 mz [M + H] ⁺ .

- For the particular case of using colismicin A and vinyl chloroacetate as the acylating agent, the reaction time was 12 hours and a conversion of 90% was achieved. A new peak was identified by HPLC with characteristic absorbance of colismicin and with mobility of 2.60 minutes corresponding to the compound of formula XV. Subsequent analysis by HPLC / MS determined that the compound of formula XV has an ion of 352 m / z [M + H] ⁺ .

- For the particular case of using colismicin A and vinyl butanoate as the acylating agent, the reaction time was 9 hours and a conversion of 95% was achieved. A new peak with characteristic absorbance of colismicin and with mobility of 3.66 minutes corresponding to the compound of formula XVI was identified by HPLC. Subsequent analysis by HPLC / MS determined that the compound of formula XVI has an ion of 346 m / z [M + H] ⁺ . NMR spectra data of the compound of formula II:

 1 H NMR (DMSO-dg, 600 MHz): 2.35 (s), 4.75 (s), 6.30 (sa), 7.10 (s), 7.55 (sa), 8.00 (sa), 8.25 (sa), 8.80 (sa) , 11.10 (sa)

¹³ C NMR (DMSC / e, 150 MHz): 15.9, 59.1, 1 12.1, 1 17.9, 121.2, 125.8, 138.7, 142.2, 148.5, 149.7, 152.0, 177.2

NMR spectra data of the compound of formula III:

1 H NMR (acetone-í / ₆ , 600 MHz): 2.48 (s), 4.00 (s), 7.68 (dd, J = 7.6, 4.4 Hz), 7.89 (s), 8.16 (dt, J = 7.6, 1.6 Hz ), 8.29 (d, J = 7.6 Hz), 8.82 (d, J = 4.4 Hz), 8.86 (s), 10.80 (s)

1 ³ C NMR (acetone- ^, 150 MHz): 16.0, 110.0, 121.3, 122.9, 126.1, 138.6, 143.7, 145.9, 147.3, 149.5, 150.2, 174.1

NMR spectra data of the compound of formula IV:

1 H NMR (acetone-í / ₆ , 600 MHz): 2.57 (s), 3.50 (s), 7.50 (dd, J = 7.6, 4.4 Hz), 7.99 (dt, J = 7.6, 1.6 Hz), 8.26 (s ), 8.41 (d, J = 7.6 Hz), 8.70 (d, J = 4.4 Hz)

¹³ C NMR (acetone- ^, 150 MHz): 16.9, 110.2, 116.3, 121.0, 124.9, 125.3, 137.5, 137.8, 149.3, 153.6, 157.7, 166.3

NMR spectra data of the compound of formula V:

1 H NMR (DMSO-dg, 600 MHz): 2.36 (s), 7.47 (dd, J = 7.8, 4.2 Hz), 7.94 (t, J = 7.8 Hz), 7.96 (s), 8.29 (d, J = 7.8 Hz), 8.68 (d, J = 4.2 Hz), 11.7 (sa) NMR C (OMSO-de, 150 MHz): 17.7, 108.4, 116.1, 121.2, 125.1, 137.9, 149.8,

154.5, 156.0, 157.6, 166.7, 168.4

NMR spectra data of the compound of formula VI:

1 H NMR (acetone-í / ₆ , 600 MHz): 2.43 (s), 2.67 (s), 4.19 (s), 7.45 (d, J = 7.8 Hz), 7.96 (t, J = 7.8 Hz), 8.41 ( d, J = 7.8 Hz), 8.18 (s), 8.88 (s), 10.70 (s)

1 ³ C NMR (acetone- ^, 150 MHz): 17.2, 23.0, 56.2, 103.3, 118.8, 122.5, 124.7, 138.5, 146.7, 152.3, 153.0, 155.1, 157.8, 168.1 NMR spectra data of the compound of formula VII :

 1 H NMR (DMSO-dg, 600 MHz): 2.36 (s), 2.47 (s), 4.08 (s), 7.45 (d, J = 4.8 Hz), 8.03 (s), 8.32 (s), 8.61 (d, J = 4.8 Hz), 8.73 (s), 10.70 (s)

¹³ C NMR (DMSO- e, 150 MHz): 17.3, 20.4, 56.1, 103.9, 121.9, 122.4, 126.2, 147.2,

148.6, 150.0, 153.0, 153.8, 155.4, 167.4

NMR spectra data of the compound of formula VIII:

 1 H NMR (acetone- ^, 600 MHz): 2.39 (s), 2.49 (s), 4.59 (s), 4.86 (s), 7.30 (d, J = 4.8 Hz), 8.12 (s), 8.48 (s) , 8.56 (d, J = 4.8 Hz)

¹³ C NMR (acetone- ^, 150 MHz): 16.5, 20.2, 62.3, 102.6, 117.9, 121.6, 125.1, 148.2, 149.0, 154.8, 155.7, 160.8, 167.2

NMR spectra data of the compound of formula XI:

 1 H NMR (DMSO-dg, 600 MHz): 2.14 (s), 2.35 (s), 4.06 (s), 5.40 (s), 7.49 (dd, J = 7.3, 4.6 Hz), 7.98 (dt, J = 7.3 , 1.8 Hz), 8.02 (s), 8.35 (d, J = 7.3 Hz), 8.71 (dd, J = 4.6, 1.8 Hz)

¹³ C NMR (DMSO- e, 150 MHz): 17.7, 21.1, 56.8, 65.8, 103.3, 120.3, 121.1, 125.1, 137.9, 149.7, 154.9, 156.1, 157.5, 167.3, 170.6

NMR spectra data of the compound of formula XII:

1 H NMR (DMSO-dg, 600 MHz): 0.91 (t, J = 7.2 Hz), 1.60 (sext, J = 7.2 Hz), 2.35

(s), 2.40 (t, J = 7.2 Hz), 4.06 (s), 5.41 (s), 7.48 (dd, J = 7.8, 4.5 Hz), 7.97 (dt, J = 7.8, 1.8 Hz), 8.01 ( s), 8.35 (d, J = 7.8 Hz), 8.71 (dd, J = 4.5, 1.8 Hz) ¹³ C NMR (DMSO-dg, 150 MHz): 14.0, 17.7, 18.5, 35.8, 56.8, 65.8, 103.3, 120.4, 121.1, 125.1, 137.8, 149.7, 154.9, 156.1, 157.6, 167.2, 172.9

NMR spectra data of the compound of formula XIII:

 1 H NMR (DMSO-dg, 600 MHz): 2.38 (s), 4.08 (s), 5.68 (s), 7.44 (dd, J = 7.8, 4.4 Hz, 7.57 (t, J = 7.2 Hz), 7.70 (t , J = 7.2 Hz), 7.80 (dt, J = 7.8, 1.2 Hz), 8.03 (overlapped d, J = 7.2 Hz), 8.02 (s), 8.18 (d, J = 7.8 Hz), 8.69 (dd , J = 4.4, 1.2 Hz)

1 ³ C NMR (DMSO- e, 150 MHz): 17.6, 56.8, 66.6, 103.4, 120.4, 121.0, 125.1, 129.3, 129.7, 130.2, 133.9, 137.7, 149.7, 154.9, 156.0, 157.3, 166.1, 167.3

Example 10. Antitumor activity of the new compounds generated.

Several tumor cell lines were used for antitumor activity tests: A549 (lung), HT29 (colon), MDA-MB-231 (breast) and HCT116 (colon), as well as the non-tumor control line of NIH373 fibroblasts. The cells were distributed in 96-well plates at a rate of 5,000 cells / well in 100 mL per well of DMEM medium supplemented with 10% FBS and 2 mM glutamine. They were then pre-incubated for 24 h without drug assay so that the cells adhered to the plaque. The following day the appropriate concentrations of the various compounds diluted in DMEM medium and always in a volume of 10 mL were added. As controls, medium without compound was added to quantify the viability of the cell line and 0.1% SDS as a 100% control of death. After 24 h of incubation in the presence of each compound, 10 mL of the WST-8 reagent (Cell Counting Kit-8, Dojindo, Probior, Germany) was added to each well and incubated for 2 h at 37 ° C after which it was determined absorbance at 450 nm in an ELISA reader (ELx800, BioTek). The activity of the different compounds is shown in table 3.

Table 9: Antitumor activity (IC50) of the different compounds against different cell lines.

Some compounds tested against certain cell lines showed IC50 values of micromolar order, as is the case of compounds of formula VI and formula VII against HCT116. However, some compounds showed less elevated cytotoxicity values, of an order greater than 100 micromolar against certain cell lines.

Example 11. Neuroprotective activity of the new compounds generated.

Neuroprotective activity tests were carried out using the zebrafish model. Embryo management, maintenance and breeding was carried out following standardized procedures (Westerfield, 1993; Kimmel et al., 1995). Once collected, they were kept in embryo water (135 μΜ CaCl ₂ , 623 μΜ MgS04, 1.14mM NaHC0 ₃ , 402.41 μΜ KC1, 10.7 mM NaCl, 348.5 μΜ CaS0 ₄ .2H20, Scharlau Chemie, SA, 08016 Barcelona, Spain).

The embryos were treated after 3 days postfertilization for 24 hours with 10 μΜ retinoic acid (CAS # 302-79-4, Sigma-Aldrich) (Selderslaghs et al, 2009) in the presence of each of the neuroprotective compounds to be tested and used as positive control α-lipoic acid (CAS 1077-28-7, Sigma-Aldrich) at 1 μΜ. Dimethyl sulfoxide (DMSO; 1%) was incorporated into the tests as it was the solvent in which the compounds to be tested were dissolved. Retinoic acid and potentially neuroprotective compounds were added to the embryo water described above, using 15 zebrafish embryos. At the end of treatment 4 day embryos Post-fertilization were washed three times with water and immersed in a solution of 2 μg / ml of acridinium chloride (Sigma-Aldrich) for 60 min. They were then washed thoroughly three times (5 min per wash) and anesthetized with MS222 anesthetic (tricaine methanesulfonate). They were finally properly positioned for observation by fluorescence microscopy. This was carried out using a Leica DMIL LED fluorescence microscope (Leica Microsistemas, SA, Barcelona), equipped with a green FITC filter (excitation: 488 nm, emission: 515 nm), and an EC3 digital camera (Leica Microsistemas, SA , Barcelona). The images were processed with LAS EZ VI .6.0 (Leica Microsistemas, SA, Barcelona) and Adobe Photoshop 7.0 software (Adobe, San José, CA). Particle analysis (Wasabi, Hamamatsu Photonics Germany GmbH) was used to quantify fluorescence. To compare the efficacy between the different compounds, the analysis of variance (ANO VA) was used to detect if there were significant differences between compounds in the experiments. Dunnett's t-test was used to identify compounds that exhibited significant differences compared to the control (n = 3). All calculations were made using the GraphPad Prism statistical software (GraphPad Software, Inc., San Diego, CA). A P-value less than 0.05 was considered statistically significant.

The neuroprotectors were tested at the NOEC (Non-observed effect concentration) concentration. This NOEC, after a previous study, was determined as 1 μΜ for all the compounds tested. The control, without treatment with retinoic acid showed few or no apoptotic cells (FIG. 14A). In contrast, embryos treated with retinoic acid showed a significant increase in brain apoptosis (FIG. 14B). The images shown in FIG 14C, D and E) are examples of co-treatment with retinoic acid and each of the compounds tested, including lipoic acid as a positive control (Packer et al., 1997. Free Radie Biol Med. ; 22 (1-2): 359-78.) (FIG. 20F) and using the NOEC concentration. The compounds of formula VI and VII showed protection against retinoic acid, the effect of the compound of formula VII being clearly significant (FIG. 14E). Example 12. Additional generation of mutants producing new derivatives of colismicin by gene replacement of the orfl2 orfl5 and orphlo genes.

For the inactivation of the orfl2, a 1542 bp fragment belonging to the downstream region of the orfl2 gene was amplified by PCR. For this purpose the oligonucleotides orf9del3 (SEQ ID NO: 57) and orf9del4 (SEQ ID NO: 58) were used. This fragment was cloned into the pCR-Blunt vector thus generating plasmid pCRB9B that was sequenced using standardized techniques. The fragment was then obtained by digesting plasmid pCRB9B with EcoRI and cloned into the same site of vector pUO9090 to generate plasmid pU09B. A 1598 bp fragment belonging to the upstream region of the orfl2 gene was then amplified by PCR. For this purpose, the oligonucleotides orf9dell (SEQ ID NO: 59) and orf9del2 (SEQ ID NO: 60) were used. This fragment was cloned into the pCR-Blunt vector thus generating plasmid pCRB9B that was sequenced using standardized techniques. Subsequently, the fragment was obtained by digesting plasmid pCRB9A with EcoRI, became blunt and cloned into the EcoRV site of plasmid pU09A thereby generating plasmid pU09AB.

The replacement cassette was extracted from plasmid pU09AB using the Spel restriction sites and cloned into the pHZ1358 vector to generate the plasmid pHZ9AB that was introduced into Streptomyces spp. CS40 by intergener conjugation from E.coli ET 12567 (pUB307) to generate the mutant strain CLM-M1. The strain CLM-M1 is deposited in the Spanish Type Culture Collection with deposit number 8070.

For the inactivation of the orfl5, a 1564 bp fragment belonging to the upstream region of the orfl5 gene was amplified by PCR. For this purpose, the oligonucleotides orfl2dell (SEQ ID NO: 61) and orfl2del2 (SEQ ID NO: 62) were used. This fragment was cloned into the pCR-Blunt vector thus generating plasmid pCRB12A that was sequenced using standardized techniques. Subsequently, the fragment was obtained by digesting plasmid pCRB9B with HindIII and cloned into the same site of vector pUO9090 to generate plasmid pU012A. Then, a 2250 bp fragment belonging to the downstream region of the orfl2 gene (sites 10 and 11 in FIG. 2) was obtained by digestion of the coslC3 cosmid with BamHI. Said fragment was cloned into the EcoRV site of plasmid pU06A thereby generating plasmid pU012AB. The replacement cassette was extracted from plasmid pU012AB using the Spel restriction sites and cloned into the pHZ1358 vector to generate the plasmid pHZ6AB that was introduced into Streptomyces spp. CS40 by intergener conjugation from E.coli ET12567 (pUB307) to generate the CLM-M mutant strain. The strain CLM-M is deposited in the Spanish Type Culture Collection with deposit number 8069.

To generate the mutant strain in the orphlous gene, a 1488 bp fragment run down the gene was amplified by PCR, using the oligonucleóíidos orfl 3dell alt (SEQ ID NO: 63) and orfl 3del2 (SEQ ID NO: 64). This fragment was introduced in the neighboring pCR-Bluní to give rise to plasmid pCRB13A which was sequenced following standard techniques. Subsequently, the fragmenio was obtained by digesting the plasmid pCRB13A with EcoRI and was cloned into the same site of the neighbor pUO9090 generating the neighbor pU013A.

Subsequently, digestion with resynthesis enzymes was obtained by a fragmentation of 1975 bp running above the orphloid gene called B13B. This fragmenio corresponds to the region of the cluster comprised of an EcoRI site and a Pvul site (positions 19042 and 21016 of SEQ ID NO: 1). Esmen fragmenio became blunt and was subcloned in the EcoRV site of plasmid pU013A to generate plasmid PU013AB.

The replacement cassette was excised from plasmid pU013AB using the Spel restriction sites and cloned into the neighboring pHZ1358 to generate the plasmid pHZ13AB, which was introduced in Streptomyces spp. CS40 by inertomeric conjugation from E.coli ET12567 (pUB307) to generate the mumanie strain CLM-AH. The strain CLM-AH is deposited in the Spanish Type Culinary Collection with deposit number 8071. In all cases the transconjugants in which a double overcrossing event had occurred were selected for their resistance to apramycin and their sensitivity to thioestreptone. Chromosome replacement was confirmed in transconjugants by Southern analysis. The analysis of cultures of the CLM-Ml strain by UPLC showed a peak with characteristic absorbance of colismicin A but with 3,841 min mobility corresponding to the compound of formula XVIII. Subsequent analysis by HPLC / MS determined that the compound of formula XVIII has a mobility of 17,442 min and an ion of 360 m / z [M + H] ⁺ . (FIG. 15). The analysis of cultures of the CLM-M strain by UPLC showed a peak with characteristic absorbance of colismicin A but with 2,249 min mobility corresponding to the compound of formula XIX. Subsequent analysis by HPLC / MS determined that the compound of formula XIX has a mobility of 12,617 min and an ion of 290 m / z [M + H] ⁺ . (FIG. 16). Culture analysis of the CLM-AH strain by UPLC showed a peak with characteristic absorbance of colismicin A but with 3,673 min mobility corresponding to the compound of formula XVII. Subsequent analysis by HPLC / MS determined that the compound of formula XVII exhibits a mobility of 16,626 min and an ion of 376 m / z [M + H] ⁺ . (FIG. 17) NMR spectra data of the compound of formula XVII:

 1 H NMR (DMSO-dg, 600 MHz): 0.94 (d, J = 6.0 Hz), 0.95 (d, J = 5.4 Hz), 1.60-1.85 (m), 2.34 (s), 4.48 (m), 7.49 ( t, J = 5.4 Hz), 7.94 (s), 7.99 (t, J = 7.8 Hz), 8.34 (d, J = 7.8 Hz), 8.69 (d, J = 3.6 Hz), 8.70 (brs)

¹³ C NMR (DMSO- e, 150 MHz): 17.7, 21.9, 23.4, 24.8, 40.0, 50.8, 108.3, 1 18.0, 121.3, 125.0, 138.1, 149.5, 154.4, 154.5, 158.4, 166.6, 167.0, 174.3

NMR spectra data of the compound of formula XVIII:

1 H NMR (DMSO-dg, 600 MHz): 0.93 (d, J = 6.6 Hz), 0.94 (d, J = 6.6 Hz), 1.47 (m), -1.88 (m), 2.02 (m), 2.34 (s ), 5.25 (dd, J = 11.4, 4.8 Hz), 7.52 (dd, J = 7.8, 4.8 Hz), 8.01 (dt, J = 7.8, 1.8 Hz), 8.06 (s), 8.43 (d, J = 7.8 Hz), 8.72 (dd, J = 4.8, 0.6 Hz) ¹³ C NMR (DMSO- e, 150 MHz): 21.3, 23.3, 25.0, 39.8, 54.6, 107.9, 121.4, 125.3, 127.5, 138.1, 149.7, 154.4, 157.0, 158.5, 163.6, 167.0, 171.9

NMR spectra data of the compound of formula XIX:

 1 H NMR (DMSO-dg, 600 MHz): 1.95 (s), 2.35 (s), 4.64 (s), 7.53 (m), 7.66 (s), 8.02 (t, J = 7.8 Hz), 8.36 (brs) , 8.39 (d, J = 7.8 Hz), 8.72 (d, J = 3.0 Hz)

1 ³ C NMR (DMSO- e, 150 MHz): 17.1, 23.0, 42.6, 108.5, 119.5, 121.6, 125.4, 138.5, 149.5, 152.5, 152.7, 158.4, 169.5, 170.34 Example 13. Identification of a new colismicin derivative produced by strain CLM-M2.

The detailed analysis of the compounds produced by the CLM-M strain allowed us to identify a new compound accumulated by this strain to older than the compounds of formulas III and IV previously described. UPLC analysis showed a new peak with characteristic absorbance of colismicin A but with 2,249 min mobility corresponding to the compound of formula XX. Subsequent analysis by HPLC / MS determined that the compound of formula XX has a mobility of 12,101 min and an ion of 278 m / z [M + H] ⁺ . (FIG. 18) NMR spectra data of the compound of formula XX:

1 H NMR (DMSO-dg, 600 MHz): 3.08 (s), 7.40 (brs), 7.59 (dd, J = 6.9, 4.8 Hz), 8.04 (dt, J = 7.8, 1.5 Hz), 8.35 (d , J = 7.8 Hz), 8.73 (d, J = 3.9 Hz), 9.0 (brs), 12.5 (brs) 1 ³ C NMR (DMSO- e, 150 MHz): 40.9, 111.6, 120.0, 121.7, 126.2, 138.6 , 140.5, 149.9, 158.4, 158.5, 166.0, 174.1

Deposited microorganisms.

The following microorganisms were deposited on the dates indicated below in the Spanish Type Culture Collection (CECT) / University of Valencia / Pare Científic Universitat de Valencia / Professor Agustín Escardino, 9/46980 Paterna (Valencia), complying with the Budapest Treaty: Microorganism Identification Number Deposit date

Streptomyces spp. CS40 7757 06-18-2010

Streptomyces spp. CLM-L 7754 06.18.2010

Streptomyces spp. CLM-A 7755 06.18.2010

Streptomyces spp. CLM-M2 7756 06.18.2010

Streptomyces spp. CLM-G 7861 16.12.2010

Streptomyces spp. CLM-Ml 8070 28.12.2011

Streptomyces spp. CLM-M 8069 28.12.2011

Streptomyces spp. CLM-AH 8071 28.12.2011

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Claims

1. Nucleic acid molecule consisting of at least one fragment of SEQ ID NO: 1 capable of encoding at least one of the sequences SEQ ID NO: 2 to SEQ ID NO: 28. 2. Nucleic acid molecule, according to claim 1, consisting of SEQ ID NO: 1.

3. Expression vector comprising the nucleic acid molecule of claims 1 or 2.

4. Expression vector according to claim 3, characterized in that it is the cosmid coslc3 or cos3bl l.

5. Non-human transgenic cell or organism comprising the expression vector of claims 3 or 4.

6. Streptomyces spp. with identification number CECT 7754, CECT 7755, CECT 7756, CECT 7757, CECT 7861, CECT 8069, CECT 8070 or CECT 8071. 7. Compound of Formula (I):

where Ri, R ₂ , R ₃ and R4 are, each and independently, hydrogen or a protecting group; wherein the protecting group comprises an alkyl group, an alkyl cyclo group, a heterocyclic cycloalkyl group, a hydroxyalkyl hydro group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, an alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, a oxime group, a nitrile group, a urethane group, a silyl group, a sulfo group combining them; Except for compounds with the following formulas:

8. Compound according to claim 7, selected from the following compounds of Formula II to XVI:

9. Compound according to claim 7, selected from the following compounds of Formula XVII to XX:

where Ri, R ₂ , R ₃ and R ₄ are, each and independently, hydrogen or a protecting group; wherein the protecting group comprises an alkyl group, an alkyl cyclo group, a heterocyclic cycloalkyl group, a hydroxyalkyl hydro group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, an alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or a combination of them; for the preparation of a pharmaceutical composition for the treatment of cancer.

1 1. Use according to claim 10, wherein the type of cancer is selected from: breast cancer, lung cancer and colon cancer.

12. Use of a compound according to claims 7 to 9 for the preparation of a pharmaceutical composition for the treatment of neurodegenerative diseases. 13. Use of a compound of Formula (I):

where Ri, R ₂ , R ₃ and R4 are, each and independently, hydrogen or a protecting group; wherein the protecting group comprises an alkyl group, an alkyl cyclo group, a heterocyclic cycloalkyl group, a hydroxyalkyl hydro group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, an alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or a combination of them; for the preparation of a pharmaceutical composition for the treatment of infectious diseases.

14. Pharmaceutical composition comprising at least one of the compounds of claims 7 to 9 and at least one pharmaceutically acceptable excipient.

15. A method for the treatment of cancer comprising the administration to the patient of a therapeutically effective amount of a compound of Formula (I):

where Ri, R ₂ , R ₃ and R4 are, each and independently, hydrogen or a protecting group; wherein the protecting group comprises an alkyl group, an alkyl cyclo group, a heterocyclic cycloalkyl group, a hydroxyalkyl hydro group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, an alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or a combination of them. 16. Method according to claim 14, wherein the type of cancer is selected from: breast cancer, lung cancer and colon cancer.

17. Method for the treatment of neurodegenerative diseases comprising the administration to the patient of a therapeutically effective amount of a compound according to claims 7 to 9 or of a pharmaceutical composition according to claim 14.

18. Method for the treatment of infectious diseases comprising the administration to the patient of a therapeutically effective amount of a compound of Formula (I):

where Ri, R ₂ , R ₃ and R ₄ are, each and independently, hydrogen or a protecting group; where the protecting group may consist of an alkyl group, a cycloalkyl group, a heterocyclic cycloalkyl group, a hydroxyalkyl hydro group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group , an alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or A combination of them.

(l) where Ri, R ₂ , R 3 and R ₄ are, each and independently, hydrogen or a protecting group; wherein the protecting group comprises an alkyl group, a cycloalkyl group, a heterocyclic cycloalkyl group, a hydroxyalkyl group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group, a group alkylaryl, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or a combination of they; to be used in the treatment of cancer.

20. A compound according to claim 19 for use in the treatment of a type of cancer selected from: breast cancer, lung cancer and colon cancer.

21. Compound according to claims 7 to 9 for use in the treatment of neurodegenerative diseases.

22. Compound of Formula (I):

(I) where Ri, R ₂ , R 3 and R 4 are, each and independently, hydrogen or a protecting group; where the protecting group may consist of an alkyl group, a cycloalkyl group, a heterocyclic cycloalkyl group, a hydroxyalkyl hydro group, a halogenated alkyl group, an alkoxyalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heterocyclic aryl group , an alkylaryl group, an ester group, a ketone group, a carbonate group, a carboxylic acid group, an aldehyde group, a ketone group, an oxime group, a nitrile group, a urethane group, a silyl group, a sulfoxy group or a combination of them; to be used in the treatment of infectious diseases.

23. Method for obtaining acylated derivatives of colismicin A and / or colismicin C, which comprises reacting colismicin A and / or colismicin C with an acylating agent in the presence of a hydrolase enzyme.