Biotechnological production of deferoxamine and analogs thereof

Abstract

The present invention relates to a recombinant microbial cell for the production of at least one compound of structural formula II from a carbon source: # imgabs0 # formula IIn = 2-3R1 = H or COCH3 or CH2CH2COX wherein X = OH or O-R2 = CH3 or CH2CH2COX wherein X = OH or O-wherein said cell comprises a genetic modification to increase the activity of E4 relative to its wild type cell wherein: E4 is deferoxamine or bisukaline synthetase (EC 6.3.-) (E4i), the present invention relates to deferoxamine compounds capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid to deferoxamine B or H or at least one other linear deferoxamine or bisukaline according to formula II.

Description

Field of the Invention

[0001] The present invention relates to the biotechnological production of deferoxamine and its analogs. In particular, the present invention relates to recombinant cells capable of biotechnologically producing deferoxamine B and its analogs. Background of the Invention

[0003] Desferrioxamine B, also known as deferoxamine B, is an iron (III) chelating molecule produced by the bacterium Streptomyces pilosus (S. pilosus) and other Actinomycetes. Bacteria produce deferoxamine B to bind trace metals in the local environment. For example, iron, although a requirement for growth, is rare and / or poorly soluble in water and therefore not readily available to cells, and iron is bound by deferoxamine B.

[0004] Deferoxamine B, H and E and other N-hydroxy-N-succinylpentylamine-based siderophores such as bisucaberins are produced by a dedicated biosynthetic pathway that requires four enzymatic activities and starts from L-lysine. Deferoxamine B, H and E and other N-hydroxy-N-succinylpentylamine-based siderophores such as bisucaberins are secreted into the surrounding environment by unknown mechanisms. After metal binding, the metal-siderophore complex is bound by specific receptor proteins (e.g. DesE in Streptomyces coelicolor) and is subsequently seen to be taken up by a dedicated uptake system such as the ABC transporter FhuABCD in Erwinia amylovora. The release of metal ions from the extremely stable metal-siderophore complexes within the cell can occur via three different mechanisms: enzyme-mediated siderophore hydrolysis (e.g. DesF in Streptomyces coelicolor), proton-assisted complex dissociation and reduction of the metal center (k of Fe2+). D Only 2.85×10 -5 ).

[0005] Deferoxamine B is used clinically to treat patients with secondary iron overload, which can occur as a complication of treatment for transfusion-dependent blood disorders, including beta-thalassemia, sickle cell anemia, and myelodysplastic syndrome. Transfusion-dependent iron overload is the most common metal poisoning condition worldwide, with the highest mortality rate. Because deferoxamine B is effective in removing iron from plasma and is non-toxic, it has become a very effective treatment modality to reduce this mortality rate.

[0006] Deferoxamine B is sold by companies such as Novartis as Deferoxamine B is currently marketed and is produced almost exclusively for pharmaceutical applications, most notably for the treatment of iron poisoning (due to genetic conditions or iron overload hemochromatosis) and aluminum poisoning in dialysis patients. Deferoxamine B, which is predominantly used in these pharmaceutical applications, is produced today exclusively by fermentation using wild-type Streptomyces strains (e.g., S. tricholoma and S. parvulus). This approach has a number of disadvantages, among other problems.

[0007] That is, the current production method of deferoxamine B has low production performance characteristics, such as biomass specific productivity q P , volume productivity Q P , product yield Y on substrate X / S And product concentration. This leads to high manufacturing costs. Further, because deferoxamine B is formed using a simple fermentation process of a wild-type Streptomyces strain, there are many by-products in the fermentation broth produced. Streptomyces species are powerful producers of many secondary metabolites (including antibiotics), which need to be separated from the required deferoxamine B by laborious and expensive refining steps. In addition, due to the complex growth requirements of Streptomyces species, wild-type Streptomyces strains generally require complex and expensive fermentation medium formulations, resulting in poor reproducibility due to batch-to-batch changes in complex medium components.

[0008] While high manufacturing costs may be acceptable for pharmaceutical applications, these costs are undesirable for other applications such as cosmetics and technical (e.g., rust removal) fields. Therefore, it is highly desirable to transfer the biosynthesis of deferoxamine B, H and other analogs to well-characterized microbial production strains. Accordingly, there is a need in the art for more efficient and affordable means of biosynthesizing deferoxamine B, H and other analogs.

[0009] Description of the invention

[0010] The present invention attempts to solve the above problems by providing a biotechnological means for producing deferoxamine and its analogs using an established microbial platform. The use of an established microbial platform to produce at least one deferoxamine and its analogs not only increases the amount of deferoxamine and its analogs produced from raw materials, but also reduces the amount of by-products formed. In addition, the genetically modified cells according to any aspect of the present invention have the advantages of being non-pathogenic and easy to culture. This makes the cells safer for production and also keeps costs lower because no special safety requirements are required during the production process in the laboratory and in the use of deferoxamine and / or its analogs. Using recombinant cells according to any aspect of the present invention, the production efficiency of deferoxamine and / or its analogs is also increased. In addition, the use of a microbial platform that can integrate the overall means of converting a carbon source into at least one deferoxamine and / or its analogs makes the conversion process simpler because only a few process steps are involved in the conversion. The dependence on Streptomyces strains for producing deferoxamine and its analogs is also removed. The cells according to any aspect of the present invention have the further advantage of being able to use various carbon substrates to produce deferoxamine and its analogs according to any aspect of the present invention. For example, simple carbon such as glucose can be used as a carbon substrate.

[0011] The cells used in accordance with any aspect of the present invention may have several advantages including:

[0012] - Higher production performance characteristics, such as biomass specific productivity q P , volume productivity Q P , product yield Y on substrate X / S and product concentration, leading to lower manufacturing costs;

[0013] - Lower diversity and / or concentration or absence of by-products in the fermentation broth simplifies the refining strategy, leading to lower manufacturing costs; and

[0014] - Availability of minimal fermentation media with defined composition, leading to lower manufacturing costs and better reproducibility.

[0015] According to one aspect of the present invention, there is provided a recombinant microbial cell for producing a compound having structural formula II from a carbon source:

[0016]

[0017] in

[0018] n=1-3

[0019] R1=H or COCH3 or CH2CH2COX, where X=OH or O-

[0020] R2=CH3 or CH2CH2COX, wherein X=OH or O-, wherein the cell comprises a genetic modification to increase the activity of E4 relative to its wild-type cell, wherein:

[0021] E4 is deferoxamine or bisucalin synthase (EC 6.3.-.-)(E 4i ), which is capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid to deferoxamine B or H or any other linear deferoxamine or bisucalline according to formula II or a combination thereof.

[0022] In particular, E4 is capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid to deferoxamine B or H or a combination thereof.

[0023] In particular, n is 2 or 3.

[0024] The compound of formula II according to any aspect of the present invention may be deferoxamine and / or its analogs. In particular, the compound may be deferoxamine B having formula II:

[0025]

[0026] in

[0027] n=2, R1=H and R2=CH3.

[0028] More specifically, deferoxamine B has the following formula.

[0029]

[0030] In one example, the compound can be deferoxamine H having Formula II:

[0031]

[0032] in

[0033] n=2, R1=H and R2=CH2CH2COX, where X=OH or O-.

[0034] Analogs of deferoxamine, structural analogs, also referred to as chemical analogs, have structures falling within Formula II and are similar to deferoxamine, but differ from deferoxamine in certain components. For example, analogs of deferoxamine may differ in one or more atoms, functional groups, or substructures, which are replaced by other atoms, groups, or substructures. Structural analogs are often isoelectronic. In one example, the compound produced according to any aspect of the invention is deferoxamine B or H.

[0035] In one example, a recombinant microbial cell is provided for producing a compound having structural formula II from a simple carbon source:

[0036]

[0037] in

[0038] n=1-3

[0039] R1=H or COCH3 or CH2CH2COX, where X=OH or O-

[0040] R2=CH3 or CH2CH2COX, wherein X=OH or O-.

[0041] In particular, n is 2 or 3.

[0042] In particular, the cell according to any aspect of the present invention comprises a genetic modification to increase the activity of E4 relative to its wild-type cell, wherein:

[0043] E4 is deferoxamine or bisucalin synthase (EC 6.3.-.-)(E 4i ), which is capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid to deferoxamine B or H or any other linear deferoxamine or bisucalline according to formula II or a combination thereof.

[0044] As used herein, the term 'recombinant' refers to a molecule or a nucleic acid encoded by such a molecule, in particular a polypeptide or nucleic acid, which as such does not occur naturally but is the result of genetic modification, or to a cell comprising a recombinant molecule. For example, if a nucleic acid molecule comprises a promoter functionally linked to a sequence encoding a catalytically active polypeptide, and the promoter has been modified so that the catalytically active polypeptide is overexpressed relative to the polypeptide level in a corresponding wild-type cell comprising the original, unaltered nucleic acid molecule, then the nucleic acid molecule is a recombinant. In addition, the term "recombinant DNA" refers to a nucleic acid sequence that does not occur naturally or has been prepared by the artificial combination of two otherwise separate nucleic acid sequence segments, i.e. by joining together small pieces of DNA that are usually not continuous. "Recombinantly produced" means an artificial combination often achieved by chemical synthesis means or by artificial manipulation of isolated nucleic acid segments, for example by genetic engineering techniques using restriction enzymes, ligases and similar recombinant techniques, as described, for example, by Sambrook et al., Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, NY; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989) and DNA Cloning: A Practical Approach, Volumes I and II (Editor D. N. Glover) IREL Press, Oxford, (1985). In another example, a cell is recombinant if it has been modified, particularly a cell that has undergone genetic engineering and is not naturally occurring. The term "recombinant cell" as used herein refers to a cell that has been genetically modified to contain at least one heterologous gene encoding at least one heterologous protein, such as an enzyme. Recombinant cells can express heterologous proteins. Proteins can participate in metabolic pathways for the production of desired metabolites. In another example, 'recombinant cells' refer to cells that have expressed a specific enzyme, and the expression of the enzyme is modified using genetic engineering. In this example, endogenously expressed genes are genetically modified using methods known in the art to increase or decrease the expression of the gene. Exemplary cells include prokaryotic cells and eukaryotic cells. Exemplary prokaryotic cells include bacteria, such as Corynebacterium glutamicum (C. glutamicum), such as genetically modified Corynebacterium glutamicum.

[0045] The recombinant microbial cells used according to any aspect of the present invention can be prokaryotes or eukaryotes. These cells are isolated cells. These can be mammalian cells (e.g., cells from humans), plant cells, or microorganisms such as yeast, fungi, or bacteria, wherein microorganisms, particularly bacteria and yeast, are preferred.

[0046] Suitable bacteria, yeasts or fungi are in particular those which have been deposited as bacteria, yeasts or fungal strains at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ), Brunswick, Germany. Bacteria suitable according to the invention belong to the genera listed below:

[0047] -http: / / www.dsmz.de / species / bacteria.htm,

[0048] Suitable yeasts according to the invention belong to those genera listed below:

[0049] -http: / / www.dsmz.de / species / yeasts.htm

[0050] Also suitable fungi according to the invention are those of the genera listed below:

[0051] - http: / / www.dsmz.de / species / fungi.htm.

[0052] In particular, the cell may be selected from the group consisting of Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia, Hansenula, Kluyveromyces, romyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium, and Cupriavidus. More particularly, the cell may be selected from the group consisting of Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Burkholderia andropogonis, B. brasilensis, B. caledonica, B. caribensis, B. caryophylli, B. fungi, and B. fungorum), B. gladioli, B. glathei, B. glumae, B. graminis, B. hospita, B. kururiensis, B. phenazinium, B. phymatum, B.phytofirmans, B. plantarii, B. sacchari, B. singaporensis, B. sordidicola, B. terricola, B. tropica, B. tuberum, B. ubonensis, B. unamae, B. xenovorans, B. anthina, B. pyrrocinia, B. thailandensis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Escherichia coli, Hansenula polymorpha, Kluveromyces lactis lactis), Methylobacterium extorquens, Paracoccus versutus, Pseudomonas argentinensis, Pseudomonas borbori, Pseudomonas citronellolis, Pseudomonas flavescens, Pseudomonas mendocina, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas straminea, Pseudomonas aurantiaca, Pseudomonas flavovorans, Pseudomonas oleovor ... P.aureofaciens), P.chlororaphis, P.fragi, P.lundensis, P.taetrolens, P.antarctica, P.azotoformans, 'P.blatchfordae', P.brassicacearum, P.brenneri, P.cedrina, P.corrugata, P.fluorescens.fluorescens), Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas meridiana, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas panacis, Pseudomonas proteolytica, Pseudomonas rosenbergii, P. rhodesiae), P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. cremoricolorata, P. fulva, P. monteilii, P. mosselii, P. parafulva ), Pseudomonas putida, Pseudomonas balearica, Pseudomonas stutzeri, Pseudomonas amygdali, Pseudomonas avellanae, Pseudomonas caricapapayae, Pseudomonas cichorii, P.coronafaciens, Pseudomonas ficuserectae, 'P.helianthi', P.meliae, Pseudomonas savastanoi, Pseudomonas syringae, Pseudomonas tomato (P.tomato), Pseudomonas viridiflava (P.viridiflava), Pseudomonas abietaniphila (P.abietaniphila), P.acidophila, Pseudomonas agarici (P.agarici), Pseudomonas alcaliphila (P.alcaliphila), Pseudomonas alkanolytica (P.alkanolytica), Pseudomonas amyloderamosa (P.amyloderamosa), Pseudomonas asplenii (P.azotifigens), Pseudomonas cannabina (P.cannabina), P.coenobios, Pseudomonas icing (P.congelans), P.costantinii, P.cruciviae, P.delhiensis, P.excibis, P.extremorientalis, P.frederiksbergensis, P.fuscovaginae, P.gelidicola, P.grimontii, P.indica, P.jessenii , Pseudomonas jinjuensis, Pseudomonas kilonensis, Pseudomonas knackmussii, Pseudomonas koreensis, Pseudomonas lini, Pseudomonas lutea, Pseudomonas moravianus, Pseudomonas otitidis, Pseudomonas pachastrellae, Pseudomonas palleroniana, P. papaveris, Pseudomonas sludge .peli), P.perolens, P.poae, P.pohangensis, P.psychrophila, P.psychrotolerans, P.rathonis, P.reptilivora, P.resiniphila, P.rhizosphaerae, P.rubescens, P.salomonii, P.segitis, P.sept ica), Pseudomonas simiae, Pseudomonas suis, Pseudomonas thermotolerans, Pseudomonas aeruginosa, Pseudomonas tremae, Pseudomonas trivialis, P. turbinellae, P. tuticorinensis, Pseudomonas umsongensis, Pseudomonas vancouverensis, Pseudomonas vranovensis, Pseudomonas aureus, Pseudomonas truncat ...In some embodiments, the cell may be selected from the group consisting of bacteria, bacteria, and fungi. In some embodiments, the cell may be selected from the group consisting of bacteria, bacteria, and fungi. In some embodiments, the cell may be selected from the group consisting of bacteria, bacteria, and fungi. Even more particularly, the cell may be selected from Pseudomonas putida, Escherichia coli, Burkholderia thailandensis, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Burkholderia thailandensis, Klebsiella oxytoca, Rhizobium meliloti, Bacillus subtilis, Vibrio natriuresis and Corynebacterium glutamicum. More particularly, the cell may be Corynebacterium glutamicum or Escherichia coli. .

[0053] The terms "foreign," "exogenous," and "heterologous" are used interchangeably herein and refer to a molecule, such as a polynucleotide (e.g., a gene), a protein (e.g., an enzyme), or a metabolite, that is produced or expressed in a cell from a microorganism with a genetic modification (i.e., a recombinant cell) but not in a cell from a microorganism without any genetic modification (i.e., a wild-type cell).

[0054] As used herein in conjunction with cells or microorganisms, the phrase "wild type" can represent a cell having a genome structure in a form as naturally visible in the wild. The term may be applicable to both whole cells and various genes. Therefore, the term 'wild type' can also include cells that have been genetically modified in other aspects (i.e., with respect to one or more genes), but are unrelated to the gene of interest. Therefore, the term "wild type" does not include such cells in which the gene sequence of a specific gene of interest has been artificially changed at least in part using a recombinant method. Therefore, the wild-type cell according to any aspect of the present invention refers to a cell that has no genetic mutations about the entire genome and / or a specific gene. Therefore, in an example, a wild-type cell about enzyme E1 can refer to a cell with natural / unchanged expression of enzyme E1 in the cell. Wild-type cells about enzymes E2, E3, E4, E5, etc. can be explained in the same manner, and can refer to cells with natural / unchanged expression of enzymes E2, E3, E4, E5, etc., respectively, in the cell.

[0055] The terms "native," "natural," "endogenous," and "homologous" are used interchangeably and refer to a molecule, such as a polynucleotide (e.g., a gene), protein (e.g., an enzyme), or metabolite, produced or expressed in a cell from a microorganism without any genetic modification.

[0056] The terms "production" and "expression" are used interchangeably herein and refer to the transcription of a gene and / or the translation of an mRNA transcript into a protein by a cell.

[0057] As used herein, the term "feedstock" refers to nutrients supplied to recombinant cells in a culture medium for producing desired molecules (e.g., metabolites). For example, a carbon source such as biomass or a carbon compound derived from biomass is a feedstock for microorganisms in a fermentation process or other growth context such as a live vaccine vector or immunotherapy. The feedstock may contain nutrients as well as energy sources.

[0058] As used herein, the term "carbon source" refers to a substance suitable for use as a source of carbon for the recombinant cell according to any aspect of the present invention to produce deferoxamine and / or its analogs. In other words, the carbon source is considered as a raw material for forming deferoxamine and / or its analogs. Carbon sources include, but are not limited to, glucose, biomass hydrolysate, starch, sucrose, cellulose, hemicellulose, xylose, lignin, and monomer components of these substrates. In a non-limiting manner, the carbon source can include various organic compounds in various forms, including polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, and peptides. Examples of these include various monosaccharides, such as glucose, dextrose (D-glucose), maltose, oligosaccharides, polysaccharides, saturated or unsaturated fatty acids, succinic acid, lactic acid, acetic acid, ethanol, rice bran, molasses, corn decomposition liquid, cellulose decomposition liquid, and aforesaid mixtures. In particular, the carbon source can be selected from glucose, sucrose, xylose, arabinose, mannose, lysine, and cadaverine. More particularly, the carbon source used according to any aspect of the present invention can be a simple carbon source. The term "simple carbon source" is understood to mean a carbon source in which at least one C-C bond in the carbon skeleton has been broken. In particular, the simple carbon source can be at least one carbohydrate, such as glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose, glycine and hemicellulose, but the carbon source can also include glycerol or very simple organic molecules, such as CO2, CO or synthesis gas.

[0059] The term "substrate" as used herein refers to a compound that is converted into another compound by the action of one or more enzymes, or is intended to be used for such conversion. The term includes not only a single type of compound, but also any combination of compounds, such as solutions, mixtures or other substances containing at least one substrate or its derivatives. In addition, the term "substrate" includes not only providing a carbon source such as sugar compounds suitable for use as raw materials derived from biomass, but also includes intermediates and final product metabolites used in pathways associated with the microorganisms of the metabolic manipulation described in this specification.

[0060] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to an organic polymer comprising two or more monomers including nucleotides, nucleosides or their analogs, and include but are not limited to single-stranded or double-stranded sense or antisense deoxyribonucleic acid (DNA) of any length, and, where appropriate, single-stranded or double-stranded sense or antisense ribonucleic acid (RNA) of any length, including siRNA.

[0061] The terms "protein" and "polypeptide" are used interchangeably herein and refer to an organic polymer composed of two or more amino acid monomers and / or analogs and linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.

[0062] The terms "amino acid" and "amino acid monomer" are used interchangeably herein and refer to natural or synthetic amino acids, such as glycine and the D- or L- optical isomers. As used herein, the term "amino acid analog" refers to an amino acid in which one or more individual atoms have been replaced by a different atom or a different functional group.

[0063] Any enzyme used according to any aspect of the present invention can be an isolated enzyme.Especially, the enzyme used according to any aspect of the present invention can be in active state, and in the presence of all cofactors, substrates, auxiliary and / or activated polypeptides or factors necessary for its activity, use.As used herein, the term "isolated" means that the enzyme of interest is enriched compared to its naturally occurring cell therein.Enzyme can be enriched by SDS polyacrylamide electrophoresis and / or activity assay.For example, as judged by visual inspection of polyacrylamide gel after staining with Coomassie brilliant blue dye, the enzyme of interest can constitute more than 5,10,20,50,75,80,85,90,95 or 99% of all polypeptides present in the preparation.The enzyme used according to any aspect of the present invention can be recombinant.

[0064] Technicians can use any method known in the art to genetically modify cells or microorganisms. According to any aspect of the present invention, genetically modified cells can be genetically modified so that within a limited time interval, within 2 hours, particularly within 8 hours or 24 hours, it forms deferoxamine and / or its analogs that are at least one or two times that of wild-type cells, especially at least 10 times, at least 100 times, at least 1000 times or at least 10000 times. The increase in product formation can be determined, for example, by: in a suitable nutrient medium, under the same conditions (same cell density, same nutrient medium, same culture conditions), each separately cultivates the time interval specified by the cell and wild-type cell according to any aspect of the present invention, and then determines the amount of the target product (deferoxamine and / or its analogs) in the nutrient medium.

[0065] Genetically modified cells or microorganisms may be genetically different from wild-type cells or microorganisms. The genetic difference between the genetically modified microorganism according to any aspect of the present invention and the wild-type microorganism may be that there are complete genes, amino acids, nucleotides, etc. in the genetically modified microorganism, which may not be present in the wild-type microorganism. In an example, the genetically modified microorganism according to any aspect of the present invention may include an enzyme that enables the microorganism to produce more deferoxamine and / or its analogs compared to wild-type cells. Relative to the genetically modified microorganism of the present invention, the wild-type microorganism may not have the activity of the enzyme that enables the genetically modified microorganism to produce deferoxamine and / or its analogs or does not have the detectable activity of the enzyme. As used herein, the term 'genetically modified microorganism' can be used interchangeably with the term 'genetically modified cell'. The genetic modification according to any aspect of the present invention is carried out on the cell of the microorganism.

[0066] The cell according to any aspect of the present invention is genetically transformed according to any method known in the art. In particular, the cell can be produced according to the method disclosed in WO2013024114.

[0067] As used herein, the phrase 'the genetically modified cell has increased activity and / or expression of an enzyme compared to its wild type' refers to an increase in the activity of the respective enzyme by at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, still more particularly at least 1000-fold, and even more particularly at least 10000-fold.

[0068] As used herein, the phrase "increased enzyme activity and / or expression" is to be understood as increased intracellular activity. Basically, the increase in enzymatic activity can be achieved by increasing the copy number of one or more gene sequences encoding the enzyme, using a strong promoter or adopting a gene or allele encoding a corresponding enzyme with increased activity, changing the codon utilization of the gene, increasing the half-life of mRNA or enzyme in various ways, modifying the regulation of gene expression and optionally by combining these measures. Using a vector containing the desired gene, the allele of the gene or a part thereof, and a vector that makes gene expression possible, for example, by transformation, transduction, conjugation or a combination of these methods to produce a genetically modified cell used according to any aspect of the present invention. Heterologous expression is achieved in particular by integrating the gene or allele into the chromosome of the cell or in a vector that replicates outside the chromosome. In particular, the activity or expression of the enzyme can be increased or enhanced in the cell by a method selected from the following:

[0069] a) introducing one or more promoters operably linked to a gene encoding an enzyme into the chromosome of said cell,

[0070] b) increasing the copy number of the gene encoding the enzyme by introducing one or more expression vectors into the cell, and

[0071] c) combinations thereof.

[0072] In particular, the cell according to any aspect of the invention comprises the following genetic modifications:

[0073] a) at least one promoter operably linked to a gene encoding any one of the enzymes in a suitable chromosome of the cell, or

[0074] b) at least one expression vector in the cell to increase the copy number of the gene encoding the enzyme, or

[0075] c) Combination of (a) and (b)

[0076] To increase the expression of enzyme activity.

[0077] In one example, the enzymes are E1, E2, E3 and E4. Even more particularly, the cell according to any aspect of the invention comprises the following genetic modifications:

[0078] a) at least one promoter operably linked to a gene encoding any one of enzymes E1, E2, E3 and E4 in a suitable chromosome of the cell, or

[0079] b) at least one expression vector in the cell to increase the copy number of the genes encoding the enzymes E1, E2, E3 and E4, or

[0080] c) a combination of (a) and (b),

[0081] To increase the expression of enzymes E1, E2, E3 and E4 in a cell according to any aspect of the invention.

[0082] In another example, the cell according to any aspect of the present invention may comprise a further genetic modification to comprise

[0083] a) at least one promoter operably linked to a suitable chromosome of the cell encoding the enzyme E6-E 14 and E 17 -E 19 Any of the genes in

[0084] b) at least one expression vector in the cell to increase the amount of the gene encoding the enzyme E6-E 14 and E 17 -E 19 The number of copies of any gene in

[0085] c) Combination of (a) and (b)

[0086] To increase the enzyme E6-E 14 and E 17 -E 19Any of the activities and / or

[0087] d) Encoding enzyme E 15 and E 16 Foreign DNA in at least one gene of

[0088] e) Deletion of enzyme E 15 and E 16 at least a portion of a gene of at least one of;

[0089] f) Encoding enzyme E 15 and E 16 at least one gene and / or at least one point mutation in the regulatory sequence of the gene, RNA interference (siRNA), antisense RNA; or

[0090] g) A combination of (d), (e) and / or (f)

[0091] To reduce the enzyme E 15 and E 16 at least one activity.

[0092] In one example, the cell according to any aspect of the invention comprises a genetic modification resulting in an increase in the activity of at least enzymes E2, E3, E4, and comprises a further genetic modification to increase the production of lysine. In this example, the increase in lysine production may be due to the cell having a further genetic modification, which increases an enzyme selected from E6-E 14 and E 17 -E 19 The activity of at least one enzyme selected from E 15 and E 16 activity of at least one enzyme.

[0093] In this context, the term 'suitable chromosome' refers to the original chromosome where the genes encoding enzymes E1, E2 and / or E3 are located. Thus, the suitable chromosome is the source chromosome from which the genes are derived.

[0094] In the same context, the phrase "enzyme E" used in relation to any aspect of the invention x "reduction of activity and / or expression" can be understood to mean that the activity is reduced to at most 0.5, in particular at most 0.1, more particularly at most 0.01, even more particularly at most 0.001 and most particularly at most 0.0001 times. The phrase "reduced activity" also includes undetectable activity ("zero activity"). The reduction of the activity of an enzyme can be achieved, for example, by selective mutation or other measures known to those skilled in the art for reducing the activity of an enzyme. In particular, those skilled in the art, for example at least in Dubeau et al. 2009. Singh & 2008., Lee et al., 2009, etc., for modifying and reducing protein expression and simultaneously reducing enzymatic activity by interrupting specific genes. The reduction of enzymatic activity in cells according to any aspect of the present invention can be achieved by modifying a gene comprising one of the nucleic acid sequences, wherein the modification is selected from the group comprising or consisting of: inserting foreign DNA into the gene, deleting at least a part of the gene, point mutation in the gene sequence, RNA interference (siRNA), antisense RNA, or modification (insertion, deletion or point mutation) of regulatory sequences such as promoters and terminators or ribosome binding sites flanking the gene. In particular, in order to reduce the enzymatic activity in the cell, the cell may comprise

[0095] d) foreign DNA in genes encoding enzymes;

[0096] e) deleting at least a portion of a gene encoding an enzyme;

[0097] f) at least one point mutation, RNA interference (siRNA), antisense RNA in the gene encoding the enzyme and / or the regulatory sequence of the gene; or

[0098] g) A combination of (d), (e) and (f)

[0099] The expression of the enzymes and genes mentioned above and all enzymes and genes mentioned below can be determined by means of one- and two-dimensional protein gel separation followed by optical identification of the protein concentration in the gel using appropriate evaluation software.

[0100] If the increase in enzyme activity is based solely on increased expression of the corresponding gene, the quantification of the increase in enzyme activity can be simply determined by comparing one-way or two-way protein separation between wild-type cells and genetically modified cells. A common method for preparing protein gels with bacteria and identifying proteins is by the procedure described by Hermann et al. (Electrophoresis, 22: 1712-23 (2001). Protein concentrations can also be analyzed by Western blot hybridization with antibodies specific for the protein to be determined (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989), followed by optical evaluation with appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647). This approach is also always an option when possible products of the reactions catalyzed by the enzyme activity to be determined may be rapidly metabolized in the microorganism, or the activity in the wild type itself is too low to adequately determine the enzyme activity to be determined based on production formation.

[0101] The cell according to any aspect of the invention is genetically modified, or may comprise a genetic modification that increases the activity of E4 relative to its wild-type cell, wherein:

[0102] E4 is a deferoxamine synthetase (EC 6.3.-.-) that converts N5-aminopentyl-N-(hydroxy)-succinamic acid to deferoxamine B or H ( 4i ), or a bisukaline synthase (EC 6.3.-.-) capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid into bisukaline B (EC 6.3.-.-) (E 4iii ).

[0103] In particular, E 4i and / or E 4iii It may also be referred to as deferoxamine E biosynthesis protein (DesD), which is generally from the siderophore synthase superfamily, group C, siderophore synthase component, ligase. 4i and / or E 4iii It may also be referred to as a lucA / lucC family siderophore biosynthesis protein. In particular, E 4i and / or E 4iiiThe DesD may be from Streptomyces coelicolor (NUV53721.1), Streptomyces species I5 (WP_199217166.1), Streptomyces species PAM3C (WP_216716553.1), Streptomyces species ZS0098 (WP_122217405.1), Streptomyces clarithromyces (MQL65620.1), Streptomyces species RK31 (WP_210906076.1), Streptomyces species E1N211 (WP_114873828.1), Streptomyces mutans (WP_189366531.1). ), Streptomyces species HNS054 (WP_048457761.1), Acidophilus actinomycetes (NEA78436.1), Streptomyces species E2N171 (WP_121720278.1), Streptomyces species Z38 (WP_156699444.1), Streptomyces species I4 (2020) (WP_199206418.1), Streptomyces species BSE7-9 (WP_199215200.1), Streptomyces species SMS_SU21 (WP_102641628.1), Streptomyces species MNU103 (WP_230228103.1), Streptomyces mattii (GGT60866.1), Streptomyces griseorhizium (GGQ93410.1), Streptomyces isothiomycin (GGQ52613.1), Acidoactinomycetes (WP_203550771.1), Streptomyces species 4F (ALV50330.1), Acidoactinomycetes (WP_163087759.1), Streptomyces griseorhizium (WP_037639707.1), Streptomyces species di50b (SCD87662.1), Streptomyces spp. (WP_163087759.1), Streptomyces griseorhizium (WP_037639707.1), Streptomyces species di50b (SCD87662.1), Streptomyces spp. ), Streptomyces species F-7 (WP_093767213.1), Streptomyces albus (GHC05289.1), Streptomyces cellulosus (GHE36661.1), Streptomyces vera (GHE82670.1), Streptomyces species DH20 (WP_228916771.1), Streptomyces vera (WP_225641487.1), Streptomyces vera (WP_225629337.1), Streptomyces donde (WP_150156578.1), Streptomyces pseudogrey (WP_225654118.1), Streptomyces species NRRL F-5527 (WP_031060221.1), Streptomyces species T12 (WP_145827641.1), Streptomyces species GESEQ-13 (WP_210633990.1), Streptomyces gancidicus (WP_006131549.1), Streptomyces species NRRL S-1314 (WP_031017402.1), Streptomyces (WP_215209976.1), Streptomyces pseudogrey group 8 WP_189406281.19, Streptomyces (WP_028959208.1), Streptomyces species S-9 (WP_203350072.1), Streptomyces species McG8 (WP_215188574.1), Streptomyces (WP_019525182.1), Streptomyces species DH-12 (WP_106414112.1), Streptomyces morocco (WP_149 548375.1), Streptomyces malachite (WP_190164277.1), Streptomyces spp. (WP_142195204.1), Streptomyces bald (WP_142232870.1), Streptomyces species SM1 (WP_103541520.1), Streptomyces fugata (WP_037931697.1), Streptomyces species CHD11 (WP_215205174.1), Streptomyces spp. (WP_030825118.1), Streptomyces species NRRL S-37 (WP_030869328.1), Streptomyces species AC558_RSS880 (WP_217127651.1), Streptomyces species DH5 (WP_228966782.1), Streptomyces pilosus (WP_189555973.1), Streptomyces pilosus (WP_189595561.1), Streptomyces griseus (WP_190095096.1), Streptomyces species NRRL WC-3626 (WP_030217188.1), Streptomyces species 13-12-16 (WP_085570643.1), Streptomyces microtubule spiralis (WP_145869101.1), Streptomyces griseus (WP_004931111.1), Streptomyces species CB02400 (WP_073931736.1), Streptomyces species SLBN-134 (WP_142170640.1), Streptomyces species (WP_125506338.1), Streptomyces species NA02536 (WP_176117313.1), and Streptomyces pyogenes (WP_004931111.1). P_121546082.1), Leeuwenhoek's Streptomyces (WP_048573206.1), Leeuwenhoek's Streptomyces (WP_029382123.1), White Hill Streptomyces (WP_184760469.1), Chlorochromogenic Streptomyces (WP_048584025.1), Chlorochromogenic Streptomyces (WP_053200122.1), Streptomyces (WP_215154693.1), Chlorochromogenic Streptomyces (WP_004001039.1), Streptomyces species ISL-14 (MBT2673677.1), Streptomyces cyaneogriseus (WP_044381715.1).1), Streptomyces griseomycini (WP_193473351.1), Streptomyces curare (WP_062155921.1), Streptomyces species uw30 (WP_147999450.1), Streptomyces caeruleatus (WP_062720821.1), Streptomyces harenosi (WP_164393243.1), Streptomyces griseostramineus (WP_184825094.1), Streptomyces bicolor (WP_031478546.1), Streptomyces regali (WP_062703122.1), Streptomyces species cf386 (WP_093908265.1), Streptomyces azureus (WP_059420174.1), Streptomyces schottenbergensis (WP_150502416.1), Streptomyces swartbergensis (WP_086603881.1), Streptomyces species CB02414 (WP_073723308.1), Streptomyces schottenbergensis (WP_176577001.1), Streptomyces species KS_5 (WP_030944363.1), Streptomyces species Tu102 (WP_214335617.1), Streptomyces species (NUT26959.1), Streptomyces cyaneochromogenes (WP_126392412.1), Streptomyces spinulosus (WP_196462534.1), Streptomyces species HGB0020 (WP_016434996.1), Streptomyces rubrum (WP_150481013.1), Streptomyces species TRM S81-3 (WP_188181662.1), Streptomyces formicus (ATL27942.1GI:1259310061), Micrococcus luteus Mu201 (SJN33943.1GI:1145375606), Nocardia species JB363 (SIO88003.1GI:1143070538), Enterobacteriaceae bacterium)bta3-1(AJR02043.1 GI:764043809), Streptomyces venezuelae(CUM41034.1GI:932870023), Anopheles elizabethii NUHP1(AIL46076.1 GI:675104783), Streptomyces sp. PAMC 26508 (AGJ55096.1 GI:478746516), Halomonas sp. TD01 (CAH1042919.1 GI:2174847834), Corynebacterium xericum (SLM95104.1 GI:1188028261), Streptomyces sp. CS113 (WP_087809199.1 GI:1205101816), Streptomyces cyanineensis anthocyanicus)(WP_189283040.1GI:1906517697), Streptomyces(WP_161377959.1GI:1801827430), Streptomyces(WP_121702379.1GI:1495472518), Streptomyces species LBUM 1480(QTU54121.1GI:2025348499), Streptomyces griseofuscus)(QNT95273.1GI:1906918764), Streptomyces(WP_106517219.1GI:1370674867), Streptomyces cyanineensis(WP_191849566.1GI:1912958734), Streptomyces tangdei(WP_189742138.1GI:1907050264), Streptomyces(WP_164410815.1GI:1817780094), Streptomyces lincolnensis(QMV07193.1GI:1886115140), Streptomyces coelicolor(QKN66532.1 GI:1851833442), Streptomyces donde (WP_164458103.1GI:1817832929), Streptomyces rubrogriseus (WP_164277235.1GI:1817618112), Streptomyces species SID10362 (WP_165285357.1GI:1819611045), Streptomyces (WP_030400073.1GI:663404431), Streptomyces (WP_011028585.1GI:499338877), Streptomyces donde (WP_159327807.1GI:1797887667), Streptomyces coelicolor A3 (2) (QFI42889.1 GI:1759037058), Streptomyces (WP_093456207.1GI:1225447207), Streptomyces species LRa12 (WP_136207792.1GI:1621656934), Streptomyces species E5N91 (WP_121709362.1GI:1495480291), Streptomyces rubra (WP_109032792.1GI:1389010967), Streptomyces species MH60 (WP_104632151.1GI:1351098821), Streptomyces diastaticus (WP_102929444.1GI:1332972289), Streptomyces species M1013 (WP_076974458.1GI:1141434617), Streptomyces diastaticus (WP_102929444.1GI:1332972289), Streptomyces species M1013 (WP_076974458.1GI:1141434617), Streptomyces rubra canus)(WP_059300524.1GI:976140927), Streptomyces rubrum (WP_030866491.1GI:664338305), Streptomyces (WP_003976015.1GI:490073840), Streptomyces ipomoeae (TQE33528.1 GI:1697190356), Streptomyces ipomoeae (TQE25637.1 GI:1697182207), Streptomyces sporangiiformans (TPQ23018.1 GI:1693045467), Streptomyces species Akac8 (THC55740.1GI:1616616223), Streptomyces species LRa12 (THA97810.1 GI: 1616076702), Streptomyces species H23 (WP_134652861.1 GI: 1604860203), Streptomyces griseoviridis (AZS87080.1 GI: 1547213271), Achromobacter xylosoxidans NBRC15126 = ATCC 27061 (AHC47453.1 GI: 566051780), Streptomyces lividans 1326 (EOY47845.1 GI: 509518532), Streptomyces alpina (UJA16738.1 GI: 2179595515), Streptomyces alpina (UJA08397.1 GI: 2179587162), Streptomyces hyderabadensis (WP_226024665.1GI:2112773893), Streptomyces species S10 (2018) (WP_127893570.1GI:1553510828), Streptomyces species NL15-2K (GCB49135.1 GI:1493708162), Streptomyces solani 87.22 (CBG72816.1 GI: 260649701), Micromonospora sp. B006 (AXO35037.1 GI: 1450456453), Streptomyces venezuelae (ALO08594.1 GI: 952470256), Brevibacterium sp. SW0106-09 (GAP78407.1 GI: 926973312), Pseudoalteromonas sp. SW0106-04 (GAP73945.1 GI: 924441201), Anopheles elizabethii (KMU63886.1 GI: 874590592), or Streptomyces venezuelae ATCC 10712 (CCA55859.1 GI: 328882620).

[0104] In another example, E 4i and / or E 4iii It may also be referred to as chain A or B, deferoxamine E biosynthesis protein DesD. In this example, DesD may be the sequence with accession number 6XRC_B GI: 1950842083 or 6XRC_A GI: 1950842082 or 6NL2_B GI: 179970866 or 6NL2_A GI: 1799708661. DesD may also be from Actinokineospora spheciospongiae (EWC61612.1 GI:583002104), Lipobacterium simplex (IY15768.1 GI:723622292), Hydrobacterium martensii (BBF86345.1 GI:1435241517), or Salinisphaera sp. LB1 (AWN17872.1 GI:1393627878).

[0105] In another example, E 4i and / or E 4iiiIt may also be called the deferoxamine E biosynthesis protein DesC or a minor component of the siderophore synthase, an acetyltransferase. DesC may be from a plant metagenome with the following accession numbers: VGO10018.1 GI:1613564736, VFR50243.1 GI:1591769475, VFR49444.1 GI:1591759418, VFR42273.1 GI:1591739264, VFR87867.1 GI:1591734832, VFR79172.1 GI:1591731713, VFR90674.1 GI:1591727121, VFR73697.1 GI:1591713709, VFR35670.1 GI:1591707861, VFR17966.1GI:1591706236, VFR24793.1 GI:1591690017, or VFR74915.1GI:1591683123, or from Streptomyces formicae (ATL27941.1 GI:1259310060), Orrelladioscoreae (SOE50046.1GI:1253556130), Nocardiopsis sp. JB363 (SIO88002.1GI:1143070537), Orrella dioscoreae (SBT27366.1 GI:1037119874), Streptomyces venezuelae (CUM41035.1 GI:932870024), Anopheles elizabethii NUHP1 (AIL46075.1 GI:675104782), Achromobacter xylosoxidans NBRC 15126 = ATCC27061 (AHC47452.1 GI:566051779), Streptomyces lividans 1326 (EOY47844.1 GI:509518531), Streptomyces species PAMC 26508 (AGJ55095.1 GI:478746515), Lipobacterium simplex (AIY15770.1 GI:723622294), Streptomyces species NL15-2K (GCB49136.1 GI:1493708163), or Aquabacterium martensii (BBF86346.1 GI:1435241518).

[0106] In a further example, E 4i and / or E 4iiiIt may also be referred to as chain D, deferoxamine siderophore biosynthesis protein DfoC, having accession number 507O_D GI: 1351638365, or chain C, deferoxamine siderophore biosynthesis protein DfoC, having accession number 507O_C GI: 1351638364, or chain B, deferoxamine siderophore biosynthesis protein DfoC, having accession number 507O_B GI: 1351638363, or chain A, deferoxamine siderophore biosynthesis protein DfoC, having accession number 507O_A GI: 1351638362, or deferoxamine siderophore biosynthesis protein DfoC from Pantoea agglomerans. agglomerans)(AWD37890.1), Pantoea agglomerans (WP_187507962.1), Pantoea agglomerans (HAT00136.1), Pantoea agglomerans (WP_010247060.1), Pantoea agglomerans (WP_201500586.1), Pantoea agglomerans (WP_124890596.1), Pantoea agglomerans (WP_187496373.1), Pantoea agglomerans (WP_163852072.1), Pantoea agglomerans (WP_154210121.1), Pantoea agglomerans (WP_033760513.1), Pantoea agglomerans (WP _187512465.1), Pantoea agglomerans (WP_207253898.1), Pantoea agglomerans (WP_187495053.1), Pantoea agglomerans (WP_060679208.1), Pantoea species EKM10T (WP_167430078.1), Pantoea agglomerans (WP_187500286.1), Pantoea agglomerans (WP_187492242.1), Pantoea agglomerans (WP_163763414.1), Pantoea agglomerans (WP_122459787.1), Pantoea agglomerans (WP_061902164.1), Pantoea vagans (WP_061703165.1), Pantoea agglomerans (MBS7708200.1), Pantoea spp. (WP_159226815.1), Pantoea agglomerans (WP_187513802.1), Pantoea vagans (WP_050679331.1), Pantoea agglomerans (WP_187484792.1), Pantoea agglomerans (WP_163799890.1), Pantoea species OVA07A (WP_223565934.1), Pantoea agglomerans (WP_191917404.1), Pantoea agglomerans (WP_033773381.1), Pantoea agglomerans (WP_064690802.1), Pantoea agglomerans (WP_111534208.1), Pantoea agglomerans (WP_223564415.1), Pantoea agglomerans (WP_129063349.1), Pantoea agglomerans (WP_191920297.1), Pantoea agglomerans (WP_163641337.1), Pantoea agglomerans (WP_022624353.1), Pantoea agglomerans (WP_187492906.1), Pantoea agglomerans (WP_157353121.1), Pantoea species (RZK07467.1), Pantoea agglomerans (WP_191914122.1), Pantoea agglomerans (WP_045140091.1), Pantoea species EKM20T (WP_167433056.1), Pantoea agglomerans (WP_187501430.1), Pantoea genus (WP_167423161.1), Pantoea agglomerans (WP_191921680.1), Pantoea agglomerans (WP_ 163658684.1), Pantoea agglomerans (WP_172608645.1), Pantoea agglomerans (WP_192073070.1), Pantoea species CFSAN033090 (WP_052270704.1), Pantoea agglomerans (WP_187511066.1), Pantoea agglomerans (WP_158149880.1), Pantoea agglomerans (WP_132499089.1), Pantoea agglomerans (MBT8497800.1), Plantobacillus brevis (WP_122889392.1), Pantoea agglomerans (WP_192033741.1), Pantoea agglomerans (WP_033768945.1), Pantoea agglomerans (WP_187511066.1). 515506.1), Pantoea agglomerans (WP_182505410.1), Pantoea agglomerans (WP_182500640.1), Pantoea agglomerans (WP_158132336.1), Pantoea agglomerans (WP_010670384.1), Pantoea agglomerans (WP_187506786.1), Pantoea agglomerans (WP_179256483.1), Pantoea agglomerans (WP_115765004.1), Pantoea agglomerans (WP_191922816.1), Pantoea agglomerans (WP_191914536.1), Pantoea agglomerans (AWD37885.1), Pantoea agglomerans (WP_033780267.1), Pantoea agglomerans (WP_ 062758877.1), Pantoea agglomerans (WP_115388310.1), Pantoea agglomerans (WP_089414760.1), Pantoea agglomerans (WP_191918109.1), Pantoea agglomerans (WP_137228077.1), Pantoea agglomerans (WP_039389954.1), Pantoea agglomerans (WP_191924048.1), Pantoea agglomerans (WP_191920726.1), Pantoea agglomerans (WP_163845109.1), Pantoea species paga (WP_143990819.1), Pantoea agglomerans (WP_031590726.1), Pantoea agglomerans (WP_143789436.1).1), Pantoea vagans (WP_083069696.1), Pantoea agglomerans (WP_033787056.1), Pantoea agglomerans (WP_208003520.1), Pantoea agglomerans (WP_235765353.1), Pantoea pleurotus (WP_182686862.1), Pantoea agglomerans (WP_069026761.1), Pantoea agglomerans (WP_163638696.1), Pantoea agglomerans (WP_208442935.1), Pantoea agglomerans (WP_207091826.1), Pantoea (WP_064702866.1), Pantoea agglomerans (WP_086906737.1), Pantoea agglomerans (KEY40395.1), Pantoea species OV426 (WP_090965308.1), Pantoea spp. (WP_150037408.1), Pantoea eucalyptica (AWD37908.1), Pantoea spp. (WP_150011559.1), Pantoea species WMus005 (WP_179898291.1), Pantoea eucalyptica (AWD37911.1), Pantoea vagans (WP_161736340.1), Erwinia amylovora (QJQ67970.1 GI:1839296356), Erwinia amylovora (QJQ64271.1 GI:1839292579), Erwinia amylovora (QJQ60469.1 GI:1839288766), Erwinia amylovora (QJQ56770.1 GI:1839285065), Erwinia amylovora (QJQ53072.1 GI:1839281365), Erwinia amylovora (WP_004160308.1), Erwinia amylovora CFBP1430 (5O7O_A), Erwinia amylovora (QJQ53072.1), Erwinia amylovora NBRC 12687 = CFBP1232 (GAJ89919.1), Erwinia amylovora (WP_168428660.1), Erwinia amylovora (WP_099350829.1), Erwinia amylovora (WP_168384929.1), Erwinia amylovora (WP_168404295.1), Erwinia amylovora MR1 (CCP08595.1), Erwinia amylovora (WP_00 4171023.1), Erwinia amylovora (WP_168402725.1), Erwinia amylovora (WP_168394396.1), Erwinia species Ejp617 (ADP10409.1), Erwinia species Ejp617 (WP_041474304.1), Erwinia pearensis (WP_012669441.1), Erwinia nigromatis (WP_023656445.1).1), Erwinia tasmaniensis (WP_012442727.1), Xenorhabdus carinii JM26 (PHM78217.1 GI:1269095937), Xenorhabdus carinii JM26 (PHM78208.1 GI:1269095928), Xenorhabdus hominickii (PHM56127.1 GI:1269072847), Xenorhabdus mauleonii (PHM37952.1 GI:1269054189), Xenorhabdus mauleonii (PHM37944.1 GI:1269054181), Xenorhabdus budapestensis (PHM29821.1 GI:1269045843), Xenorhabdus budapestensis (PHM29812.1 GI:1269045834), Erwinia amylovora CFBP1430 (CBA23308.1 GI:291555140), Erwinia pearensis DSM 12163 (CAY75871.1 GI:283479955), Erwinia amylovora LA637 (CDK23310.1 GI:566688504), Erwinia amylovora LA636 (CDK19939.1 GI:565455063), Erwinia amylovora LA635 (CDK16572.1 GI:565423714), Erwinia nigromatis CFBP 5888 (CCG88689.1 GI:560105768), Erwinia amylovora MR1 (CCP08595.1 GI:478726734), Erwinia amylovora Ea644 (CCP04532.1 GI:478723237), Erwinia amylovora UPN527 (CCP00585.1 GI:478719346), Erwinia amylovora NBRC 12687=CFBP 1232 (CCO95265.1 GI:478715307), Erwinia amylovora 01SFR-BO (CCO91470.1 GI:478711918), Erwinia amylovora CFBP 2585 (CCO87679.1 GI:478708144), Erwinia amylovora Ea266 (CCO83917.1 GI:478703932), Erwinia amylovora Ea356 (CCO80113.1 GI:478700098), Erwinia amylovora ACW56400 (EKV52685.1 GI:426274945), or Erwinia amylovora ATCC BAA-2158 (CBX82136.1 GI:312173882).

[0107] In particular, E 4i and / or E 4iii The C-terminal domain of the protein DesC from Streptomyces coelicolor, Streptomyces rhodopsinus, Streptomyces pilosula, IucA / IucC from Bacillus cereus, DesC from Corynebacterium siccatus, or DfoC from Erwinia amylovora or Pantoea agglomerans. More particularly, E 4ii or E 4iv selected from the group consisting of Streptomyces coelicolor (NUV53721.1), Streptomyces rhodochrous, Streptomyces tricholoma (WP_189555973.1), Erwinia amylovora (WP_004160308.1), Pantoea agglomerans (AWD37890.1) and Corynebacterium xerogenes (SLM95104.1 GI: 1188028261), and even more particularly, E 4ii or E 4iv Contains relative to SEQ ID NO:6 (E 4a )、SEQ ID NO:18(E 4b )、SEQ ID NO:28(E 4c ), SEQ ID NO:34(E 4d )'s C-terminal domain, SEQ ID NO: 54 (E 4e ) or SEQ ID NO:55 (E 4f ) have at least 70% sequence identity.

[0108] The cell according to any aspect of the present invention may be further genetically modified, or may comprise a genetic modification that increases the activity of at least one enzyme selected from E1, E2 and E3 relative to its wild-type cell, wherein

[0109] E1 is a lysine decarboxylase (EC: 4.1.1.18) that can convert lysine to cadaverine;

[0110] E2 is a cadaverine N5-monooxygenase (EC 1.14.13.-) capable of converting cadaverine to N5-hydroxy-cadaverine; and

[0111] E3 is an N5-aminopentyl-N-(hydroxy)-succinamic acid synthase (EC: 2.3.-.-) capable of converting N5-hydroxy-cadaverine and succinyl-CoA into N5-aminopentyl-N-(hydroxy)-succinamic acid.

[0112] Lysine decarboxylase (E1) is able to convert lysine to cadaverine. Specifically, E1 converts lysine to cadaverine, also known as 1,5-pentanediamine. Suitable polynucleotides encoding lysine decarboxylase (E1) can be obtained, for example, from the following strains: Escherichia coli, Bacillus halodurans, Bacillus cereus, Bacillus subtilis, Bacillus thuringensis, Burkholderia ambifaria, Burkholderia vietnamensia, Burkholderia cenocepatia, Chromobacterium violaceum, Corynebacterium xerosis, Selenomonas ruminantium, Vibrio cholerae, Vibrio parahaemolyticus, Streptomyces coelicolor, Streptomyces pubescens, Streptomyces rubrum. violaceoruber), Eikenalla corrodens, Eubacterium acidaminophilum, Francisella tulariensis, Geobacillus kaustophilus, Salmonella typhi, Salmonella typhimurium, Hafnia alvei, Neisseria meningitidis, Thermoplasma acidophilum, Plasmodium falciparum, Kineococcus radiotolerans, Oceanobacillus iheyensis, Pyrococcus abyssi, Porochlorococcus marinus, Proteus vulgaris), Rhodoferaxferrireducens, Saccharophagus degradans, Streptococcus pneumoniae, Synechococcus sp. The amino acid sequences of these lysine decarboxylases have been registered in the database (GenBank).

[0113] Suitable lysine decarboxylases that can be used in the method according to any aspect of the invention are understood to be enzymes capable of decarboxylating lysine, in particular L-lysine, and alleles or mutants thereof.

[0114] In particular, the lysine decarboxylase (E1) is selected from Escherichia coli, Corynebacterium xerogenes, Streptomyces coelicolor, Streptomyces rhodochrous and Streptomyces pilosus, whose safety has been confirmed. More particularly, E1 may be selected from Streptomyces rhodochrous, Streptomyces pilosus and Escherichia coli. Even more particularly, E1 comprises a residue relative to SEQ ID NO: 15 (E 1a )、SEQ ID NO:25(E 1b ) or SEQ ID NO:38 (E 1c ) at least 70% sequence identity. These sequences are freely available in internationally accessible databases, such as the databases of the National Library of Medicine and the National Institute of Health (NIH). The same sequence is also freely available on the colibri web server of the Institute Pasteur (France) under the gene name cadA. The same sequence is also freely available through the web server ExPasy maintained by the Swiss Institute of Bioinformatics under the gene name cadA.

[0115] E2 is a cadaverine N5-monooxygenase capable of converting cadaverine to N5-hydroxycadaverine. In particular, E2 converts cadaverine to N5-hydroxycadaverine. E2 may also be referred to as DesB, or a lysine N(6)-hydroxylase / L-ornithine N(5)-oxygenase family protein or L-lysine 6-monooxygenase (dfoA). More specifically, E2 can be from Streptomyces leeuwenhoekii (CQR63915.1 GI: 822880125), Streptomyces venezuelae ATCC 10712 (CCA55857.1 GI: 328882618), Streptomyces fulvissimus DSM 40593 (AGK77332.1 GI: 485094149), Streptomyces cess cabiei 87.22 (CBG72818.1 GI: 260649703), Streptomyces species PAMC 26508 (AGJ55094.1 GI: 478746514), Streptomyces coelicolor A3 (2) (CAB87220.1 GI:7544047), Streptomyces coelicolor CAI-140, Streptomyces ambofaciens ATCC 23877 (AKZ55867.1 GI:917649198), Streptomyces ambofaciens (CAL18383.1 GI:115501977), Streptomyces species (QTK16919.1 GI:2021361636), Streptomyces species OM5714 (KAF2779951.1 GI:1812043702), Streptomyces species CBMAI 2042 (RLV66814.1 GI:1495247117), Nocardiopsis sp. JB363 (SIO88001.1 GI:1143070536), Streptomyces venezuelae (ALO08592.1 GI:952470254), Streptomyces venezuelae (CUM41036.1 GI:932870025), Streptomyces formicae (ATL27940.1 GI:1259310059), Streptomyces rhodochrous, Streptomyces hairy, Orrella dioscoreae (SOE50134.1 GI:1253556185), Orrella dioscoreae (SBT27311.1 GI:1037119819), Streptomyces species NL15-2K (GCB49137.1GI:1493708164), Micromonospora sp. B006 (AXO35035.1 GI:1450456451), Streptomyces bottropensis ATCC 25435 (EMF50428.1 GI:456384850), Actinobacteria bacterium OV450 (KPI33963.1 GI:930403533), Micromonospora saelicesensis (RAN96846.1 GI:1408858225), or plant metagenomes with the following accession numbers: VGO10077.1 GI:1613564795, VFR50302.1 GI:1591769534、VFR49623.1GI:1591759477、VFR70778.1 GI:1591736834、VFR85324.1GI:1591730130、VFR90816.1 GI:1591727176、VFR74009.1GI:1591713764、VFR35744.1 GI:1591707917、VFR18051.1GI:1591706292、VFR25000.1 GI:1591690076, or VFR75029.1GI:1591683182. .

[0116] E2 may also be referred to as an iron carrier biosynthesis protein, a monooxygenase, and may also be selected from Brachybacterium truncatum (SLM95113.1), Brachybacterium tyrofermentans (WP_193116041.1), Brachybacterium ginsengisoli (WP_096799042.1), Brachybacterium sp. HMSC06H03 (WP_070499498.1), Actinomycetales bacterium (NMA75681.1), candidate species Brachybacterium intestinipullorum (HJC70675.1), Brachybacterium species AG952 (WP_133677657.1), Brachybacterium paracoagulans (WP_096799042.1), Brachybacterium sp. HMSC06H03 (WP_070499498.1), Actinomycetales bacterium (NMA75681.1), candidate species Brachybacterium intestinipullorum (HJC70675.1), Brachybacterium species AG952 (WP_133677657.1), Brachybacterium paraconglomeratum)(WP_010552292.1), Brachybacterium species SW0106-09(WP_053916852.1), Brachybacterium species Sponge(WP_062950357.1), Brachybacterium species SGAir0954(WP_137770520.1), Brachybacterium phenoliresistens(WP_038369982.1), Brachybacterium subflavum(WP_152352729.1), Brachybacteriumendophyticum(WP_109276394.1), Brachybacterium sacelli(WP_209904120.1), Brachybacterium halotolerans)(WP_200500938.1), Brevibacterium species CBA3105(WP_228358231.1), Brevibacterium species P6-10-X1(WP_083713362.1), Brevibacterium species P6-10-X1(APX34711.1), Brevibacterium species FME24(WP_193105844.1), Brevibacterium species YJGR34(WP_114855299.1), Isoptericola Cucumis(WP_188524545.1), Microbacterium marinilacus(WP_221859754.1).1), Agreia bicolorata (WP_044440800.1), Isoptericola variabilis (WP_144881770.1), Isoptericola variabilis (WP_013837804.1), Xylanimonas oleitrophica (WP_111251928.1), Brevibacterium (WP_113902445.1), Antribacter species KLBMP9083 (WP_236088632.1), Isoptericola (WP_106267363.1), Xylanimonas cellulosilytica (WP_012878564.1), Isoptericola chiayiensis (WP_172152929.1), Promicromonospora citrifolia (WP_013837804.1), Xylanimonas oleitrophica (WP_111251928.1), Brevibacterium (WP_113902445.1), Antribacter species KLBMP9083 (WP_236088632.1), Isoptericola (WP_106267363.1), Xylanimonas cellulosilytica (WP_012878564.1), Promicromonospora citrea)(WP_171102616.1), Isoptericola jiangsuensis(WP_098462726.1), Brevibacterium renqingii(WP_209323545.1), Brevibacterium renqingii(WP_209371577.1), Brevibacterium species W7.2(WP_211978854.1), Brevibacterium casei(WP_198499439.1), Brevibacteriumsandarakinum(WP_092106697.1), Brevibacterium linens(WP_052239998.1), Brevibacterium species RIT 803 (WP_204232889.1), Brevibacterium species VCM10 (WP_025776857.1), Brevibacterium iodinum (WP_101543359.1), Brevibacterium spp. (WP_137827005.1), Brevibacterium species CFH 10365 (WP_152347365.1), Brevibacterium eriumpigmentatum (WP_167198402.1), Brevibacterium siliguriense (WP_092011613.1), Brevibacterium limosum (WP_166822890.1).1), Brevibacterium sediminis (WP_181272454.1), Brevibacterium aurantiacum (WP_096178869.1), Brevibacterium aurantiacum (WP_114384950.1), Brevibacterium sp. UCMA11754 (WP_235346449.1), Brevibacterium aurantiacum (WP_143924264.1), Brevibacterium antiquum (WP_198396798.1), Brevibacterium sp. CCUG 69071 (WP_230744765.1), Brevibacterium marinum (WP_167950433.1), Brevibacterium sp. HY170 (WP_231442298.1), Brevibacterium sp. S22 (WP_135810285.1), Brevibacterium aurantiacum (WP_193102124.1), Brevibacterium aurantiacum (WP_193086562.1), Brevibacterium aurantiacum (WP_101639104.1), Brevibacterium aurantiacum (WP_125178290.1), Brevibacterium aurantiacum (WP_135446719.1), Brevibacterium epidermidis (WP_098731558.1), Brevibacterium linens (WP_062862175.1), Brevibacterium epidermidis (WP_062243347.1), Brevibacterium linens (WP_139908726.1), Brevibacterium epidermidis (HJE77366.1), Brevibacterium aurantiacum (WP_096157398.1), Brevibacterium aurantiacum (WP_193084517.1), Brevibacterium aurantiacum (WP_101584429.1), Brevibacterium aurantiacum (WP_096167625.1), Brevibacterium aurantiacum (WP_096145959.1), Brevibacterium aurantiacum (WP_096160423.1), Brevibacterium aurantiacum (WP_069600710.1), Brevibacterium aurantiacum (WP_009883551.1), Brevibacterium species UCMA11752 (WP_235350997.1), Brevibacterium species MG-1 (WP_139468396.1), candidate species Brevibacteriumintestinavium (HJA61508.1), Brevibacterium species SMBL_HHYL_HB1 (WP_212129584.1), Brevibacterium atlanticum (WP_166973261.1), Brevibacteriumaurantiacum (WP_193078848.1), Brevibacterium aurantiacum (WP_096163051.1), Brevibacterium species CT2-23B (WP_172170764.1), Brevibacterium expansum (HJF75769.1), Brevibacterium aurantiacum (WP_101598305.1), Brevibacterium species 'Marine' (WP_169252964.1), Brevibacterium species S111 (WP_135540314.1), Brevibacterium oceani)(WP_210602990.1), Brevibacterium oceanicum (WP_181273084.1), Brevibacterium archaea (WP_101620895.1), Brevibacterium species 239c (WP_101574167.1), Brevibacterium species FME17 (WP_193096434.1), Brevibacterium species FME37 (WP_193073001.1), Brevibacterium (WP_009376218.1), Brevibacterium permense)(WP_173151746.1), Brevibacterium casei (WP_095376411.1), Brevibacterium casei (WP_119296730.1), Brevibacterium casei (WP_063249380.1), or Brevibacterium casei (WP_144588713.1). .

[0117] In another example, E2 may also be referred to as a lysine N(6)-hydroxylase / L-ornithine N(5)-oxygenase family protein or L-lysine 6-monooxygenase (dfoA), and may be from Actinospica acidiphila (NEA95037.1), Streptomyces species I5 (WP_199217164.1), Streptomyces species ZS0098 (WP_122217404.1), Streptomyces species RK31 (WP_122217404.1), Streptomyces species RK32 (WP_122217404.1), Streptomyces species RK33 (WP_122217404.1), Streptomyces species RK34 (WP_122217404.1), Streptomyces species RK35 (WP_122217404.1), Streptomyces species RK36 (WP_122217404.1), Streptomyces species RK37 (WP_122217404.1), Streptomyces species RK38 (WP_122217404.1), Streptomyces species RK39 (WP_122217404.1), Streptomyces species RK40 (WP_122217404.1), Streptomyces species RK41 (WP_122217404.1), Streptomyces species RK42 (WP_122217404.1), Streptomyces species RK43 (WP_122217404.1), Streptomyces species RK44 (WP_122217404.1), Streptomyces species RK45 (WP_122217404.1), Streptom P_210906078.1), Streptomyces species BSE7-9 (WP_128126623.1), Streptomyces species I4 (2020) (WP_199206420.1), Streptomyces species PAM3C (WP_216716554.1), Streptomyces species E2N171 (WP_121720277.1), Streptomyces species E1N211 (WP_114873827.1), Streptomyces vinaceus)(QL65622.1), acidophilic actinomycetes(NEA78438.1), Streptomyces species Z38(WP_156699446.1), Streptomyces species SMS_SU21(WP_102641626.1), Streptomyces variabilis(WP_189366530.1), Streptomyces species HNS054(WP_048457763.1), Streptomyces species 4F(ALV50328.1), Streptomyces matensis(GGT60880.1), acidophilic actinomycetes(WP_203550773.1), Streptomyces cellulosae(GHE36673.1), Streptomyces variabilis(WP_189366530.1), Streptomyces species HNS054(WP_048457763.1), Streptomyces species 4F(ALV50328.1), Streptomyces matensis(GGT60880.1), acidophilic actinomycetes(WP_203550773.1), Streptomyces cellulosae(GHE36673.1), Streptomyces griseorubens)(WP_037639705.1), Streptomyces griseorubens (GGQ93424.1), Streptomyces werraensis (WP_190000755.1), Streptomyces species di50b (SCD87686.1), Streptomyces species MNU103 (WP_230228098.1), Streptomyces werraensis (WP_225629336.1), Actinoacidophilus actinomycetes (WP_163087763.1), Streptomyces species GESEQ-13 (WP_210633992.1), Streptomyces isothiazolini (WP_163087763.1), Streptomyces species GESEQ-14 (WP_210633992.1), Streptomyces spp. althioticus)(GGQ52599.1), Streptomyces species XHT-2(WP_161107407.1), Streptomyces spp.(WP_028959209.1), Streptomyces albogriseolus (GHC05278.1), Streptomyces species McG8 (MBT2905834.1), Streptomyces (WP_086020175.1), Streptomyces species DH-12 (WP_106414114.1), Streptomyces (WP_093767215.1), Streptomyces species DH20 (WP_228916768.1), Streptomyces tendae (WP_150156576.1), Streptomyces species T12 (WP_145827643.1), Streptomyces (WP_215209974.1), Streptomyces (WP_006131547.1), Streptomyces species NRRL F-5527 (WP_031060218.1), Streptomyces pseudogriseolus (WP_225654116.1), Streptomyces hair (WP_189555971.1), Streptomyces malachitofuscus (WP_190164278.1), Streptomyces griseoloalbus (GGV56407.1), Streptomyces (WP_125506339.1), Streptomyces albaduncus (WP_184760471.1), Streptomyces (WP_161176537.1), Streptomyces bald (WP_161176537.1), Streptomyces malachitofuscus (WP_190164278.1), Streptomyces griseoloalbus (GGV56407.1), Streptomyces (WP_125506339.1), Streptomyces albaduncus (WP_184760471.1), Streptomyces (WP_161176537.1), Streptomyces calvus)(WP_142232871.1), Streptomyces bald (WP_142195206.1), Streptomyces viridosporus (WP_081236127.1), Streptomyces marokkonensis (WP_149548377.1), Streptomyces spp. (WP_040907108.1), Streptomyces species NWU49 (WP_109540000.1), Streptomyces viridosporus (WP_004987747.1), Streptomyces spp. fungicidicus)(WP_121546080.1), Streptomyces species DH5(WP_228966784.1), Streptomyces chlorosporus(WP_016826156.1), Streptomyces species NA02536(WP_176117311.1), Streptomyces species AC558_RSS880(WP_217127653.1), Streptomyces species 13-12-16 (WP_085570641.1), Streptomyces species Tu 3180 (WP_159533060.1), Streptomyces species CB02400 (WP_073931738.1), Streptomyces griseoflavus (WP_190095098.1), Streptomyces capillispiralis (WP_145869103.1), Streptomyces (WP_184825089.1), Streptomyces species CHD11 (WP_215205176.1), Streptomyces species NRRL S-37 (WP_030869334.1), Streptomyces species SM1 (WP_103541522.1), Streptomyces toyocaensis (WP_037931691.1), Streptomyces poonensis (WP_189862605.1), Streptomyces species NRRL WC-3626 (WP_030217203.1), Streptomyces rubradiris (WP_189995711.1), Streptomyces spp. (WP_086696892.1), Streptomyces rubradiris (WP_189995711.1). corchorusii)(WP_059261504.1), Streptomyces species EAS-AB2608(WP_059249696.1), Streptomyces species FBKL.4005(WP_094373911.1), Streptomyces hygroscopicus(WP_014672887.1), Streptomyces populi(WP_103552166.1), Streptomyces spongiae (WP_152774423.1), Streptomyces aurantiogriseus (WP_189941416.1), Streptomyces (WP_030825112.1), Streptomyces cyanogenus (WP_208032394.1), Streptomyces hygroscopicus (WP_058080637.1), Streptomyces glaucum Tu4000 (EFL41232.1), Streptomyces (WP_030782431.1), Streptomyces species Ru62 (WP_103810221.1), Streptomyces ferrugineus (WP_194045781.1).1), Streptomyces species M2CJ-2 (WP_202277307.1), Streptomyces hirsutus (WP_055628641.1), Streptomyces triticiradicis (WP_151470815.1), Streptomyces (WP_189754126.1), Streptomyces species NRRL WC-3725 (WP_031028778.1), Streptomyces phyllanthi (WP_152789148.1), Streptomyces phylanthi (WP_055595050.1), Streptomyces achromogenes (WP_030618669.1), Streptomyces species NRRL B-3648 (WP_053710495.1), Streptomyces (WP_031094998.1), Streptomyces species IMTB2501 (WP_076088759.1), Pantoea (WP_010247061.1), Pantoea agglomerans (WP_158132335.1), Pantoea agglomerans (WP_163641339.1), Pantoea (WP_03 9389956.1), Pantoea agglomerans (WP_191924047.1), Pantoea agglomerans (WP_111534207.1), Pantoea agglomerans (WP_172608646.1), Pantoea agglomerans (WP_060679206.1), Pantoea agglomerans (WP_201500585.1), Pantoea agglomerans (WP_187500285.1), Pantoea agglomerans (WP_033787054.1), Pantoea spp. (WP_124890595.1), 1), Pantoea agglomerans (WP_163852070.1), Pantoea genus (WP_154210120.1), Pantoea agglomerans (WP_187506787.1), Pantoea agglomerans (WP_010670383.1), Pantoea agglomerans (WP_143789438.1), Pantoea species (RZK07466.1), Pantoea agglomerans (MBS7708199.1), Pantoea agglomerans (WP_089414761 ... 2500641.1), Pantoea agglomerans (WP_193585707.1), Pantoea agglomerans (WP_191921679.1), Pantoea agglomerans (WP_163794175.1), Pantoea agglomerans (WP_208003523.1), Pantoea agglomerans (WP_064690801.1), Pantoea agglomerans (WP_069026760.1), Pantoea agglomerans (WP_033768947.1), Pantoea agglomerans (WP_086906736.1), Pantoea agglomerans (WP_062758876.1), Pantoea agglomerans (WP_191938763.1), Pantoea agglomerans (WP_163658683.1), Pantoea agglomerans (WP_163845107.1), Pantoea agglomerans (WP_163638697.1), Pantoea agglomerans (WP_115765005.1), Pantoea species WMus005 (WP_179898292.1), Pantoea genus (WP_039661441.1), Pantoea eucalypti (AWD37912.1), Pantoea genus (WP_105099754.1), Pantoea vagans (WP_095707818.1), Pantoea (WP_008924643.1), Pantoea vagans (WP_135910763.1), Pantoea species S62 (WP_201251339.1), Pantoea vagans (WP_107320322.1), Pantoea eucalyptus (WP_187513086.1), Pantoea agglomerans (PEI06058.1), Pantoea species PNT01 (WP_192377898.1), Pantoea eucalyptus (AWD37907.1), Pantoea eucalyptus (AWD37904.1), Pantoea jilinensis (QXG56954.1), Pantoea species OV426 (WP_090965310.1), Pantoea vagans (WP_161736341.1), Pantoea vagans (WP_061060758.1), Pantoea vagans (WP_033735770.1), Pantoea vagans (WP_013196309.1), Pantoea (WP_150011557.1), Pantoea agglomerans (WP_222925733.1), Pantoea (WP_046102688.1), Siphoviridae sp. (DAL43156.1), Plautia stali) F-type symbiont (WP_058958199.1), Pantoea (WP_009092930.1), Pantoea species EKM101V (WP_167428608.1), Pantoeadeleyi (WP_140917098.1), Pantoea species ARC607 (WP_111138385.1), Timema californicum (CAD7569022.1), Enterobacter soli (WP_014063925.1), Pantoea agglomerans (WP_233988771.1), Erwinia sp. 198 (WP_125287552.1), Erwinia sp. (HBV39119.1), Pantoea allii (WP_218994922.1), Pantoea sp. 3_1284 (WP_113655326.1), Pantoea sp. ICBG1758 (WP_104085642.1), Pantoea sp. (WP_063878329.1), Pantoea sp. PSNIH1 (WP_145339666.1), Pantoea sp. Ae16 (WP_071668352.1), Pantoea sp. (WP_040113515.1), Pantoea sp. RIT 413 (WP_108569844.1), Pantoea spp. eucrina)(WP_065647647.1), Pantoea species Acro-807(WP_166714195.1), Pantoea sp.(WP_094111611.1), Pantoea allii(WP_096011383.1), Erwinia species 9145(WP_034916568.1), Erwinia species ErVv1(WP_067700592.1), Erwinia spp.(WP_034947936.1), Serratia sp. M24T3(WP_009636530.1), Pantoea stewartia(WP_039338790.1), Rouxiella badensis(WP_165429960.1), Rouxiella badensis (WP_084912800.1), Pantoea (WP_110267872.1), Pantoea stuartii (WP_054634525.1), Rouxiella badensis (WP_227989822.1), Pantoea (WP_033740201.1), Pantoea stuartii (WP_058702088.1), Pantoea stuartii (WP_185199936.1), Erwiniatasmaniensis (WP_012442726.1), Erwinia piriflorinigrans (WP_023656444.1), Pantoea pineappleensis (WP_012442726.1), Erwinia tasmanien ... ananatis)(WP_019106389.1), Pantoea ananatis(WP_03024531.1), Pantoea ananatis(WP_194761606.1), Pantoea ananatis(WP_064352874.1), Erwinia amylovora(WP_004160307.1), Erwinia amylovora (WP_099350830.1), Erwinia amylovora CFBP1430 (5O8P_A), Erwinia amylovora (WP_168394395.1), Erwinia amylovora (WP_168415207.1), Erwinia amylovora (WP_004171022.1), Erwinia pyrifoliae DSM 12163 (CAY75870.1), Erwinia pyrifoliae (WP_012669440.1), Erwinia species Ejp617 (WP_014542490.1), Erwinia pyrifoliae typographi)(WP_034899164.1), Erwinia spp.(WP_013202517.1), Erwinia billingiae(WP_053142707.1), Pantoea ananas(WP_210512032.1), Pantoea ananas(WP_161611345.1), Pantoea ananas(AWD37900.1), Pantoea spp.(WP_014606631.1), Pantoea species BAV 3049(WP_158784840.1), Pantoea ananas(WP_029568444.1), Pantoea ananas(WP_110956821.1), Pantoea ananas(WP_105077110.1), Mixta mediterraneensis (WP_193405262.1), Pantoea ananas (WP_210451676.1), Pantoea ananas (WP_028724620.1), Erwinia psidii (WP_124232197.1), Pantoea ananas (WP_058705541.1), Erwinia iniecta (WP_052902396.1), Pantoea species IMH (WP_024968324.1), Erwinia spp. (WP_099707479.1), or Pantoea agglomerans (WP_111534207.1).

[0118] In particular, E2 can be from Streptomyces coelicolor, Streptomyces rubrocephala, Streptomyces tricholoma, Erwinia amylovora, Pantoea agglomerans or Corynebacterium driesii. More particularly, E2 can be selected from Streptomyces coelicolor (NUV53723.1), Streptomyces rubrocephala, Streptomyces tricholoma (WP_189555971.1), Erwinia amylovora (WP_004160307.1), Pantoea agglomerans (WP_010247061.1) or Corynebacterium driesii (SLM95113.1). Even more particularly, E2 can comprise relative to SEQ ID NO:4 (E 2a )、SEQ ID NO:16(E2b )、SEQ ID NO:26(E 2c )、SEQ ID NO:33(E 2d )、SEQ ID NO:56(E 2e ) or SEQ ID NO:57 (E 2f ) have at least 70% sequence identity.

[0119] E3 is an N5-aminopentyl-N-(hydroxy)-succinamic acid synthase (EC: 2.3.-.-), which is capable of converting N5-hydroxy-cadaverine and succinyl-CoA to N5-aminopentyl-N-(hydroxy)-succinamic acid. In particular, enzyme E3 converts N5-hydroxy-cadaverine and succinyl-CoA to N5-aminopentyl-N-(hydroxy)-succinamic acid. E3 can be an acetyltransferase or GNAT family N-acetyltransferase, or also known as deferoxamine E biosynthesis protein DesD, or siderophore synthase superfamily group C, or siderophore synthase component ligase. More specifically, E3 can be from Streptomyces (WP_048457762.1), Streptomyces species I4 (2020) (WP_199206419.1), Streptomyces species I5 (WP_199217165.1), Streptomyces species Z38 (WP_156699445.1), Streptomyces species RK31 (WP_210906077.1), Acidophilus actinomycetes (WP_163087761.1), Streptomyces (WP_102641627.1), Streptomyces species di50b (SCD87678.1), Streptomyces vera (WP_190000756.1), Streptomyces griseus (GGQ93417.1), Streptomyces species MNU103 (WP_23022 8100.1), Streptomyces mattii (GGT60873.1), Streptomyces griseo-red (WP_033274713.1), Streptomyces (WP_136238514.1), Acidophilic actinomycetes (WP_203550772.1), Streptomyces (WP_215209975.1), Streptomyces cellulosus (GHE36667.1), Streptomyces (WP_019525183.1), Streptomyces donde (WP_150156577.1), Streptomyces isothiomycin (GGQ52607.1), Streptomyces species GESEQ-13 (WP_210633991.1), Streptomyces species F-7 (WP_093767214.1), Streptomyces flavo rubiginosus)(WP_189480783.1), Streptomyces species T12(WP_145827642.1), Streptomyces species DH20(WP_228916770.1), Streptomyces viridodiastaticus(WP_189909874.1), Streptomyces species(WP_006131548.1), Streptomyces species 2BBP-J2(WP_167741843.1), Streptomyces pseudogrey(WP_225654117.1), Streptomyces species DH-12(WP_106414113.1), Streptomyces tricholoma(WP_189555972.1).1), Streptomyces morocco (WP_149548376.1), Streptomyces species CB02400 (WP_073931737.1), Streptomyces (WP_164333385.1), Streptomyces species SM1 (WP_103541521.1), Streptomyces (WP_192229625.1), Streptomyces species CHD11 (WP_215205175.1), Streptomyces species AC558_RSS880 (WP_217127652.1), Streptomyces (WP_171113987.1), Streptomyces spinoverrucosus)(WP_141313135.1), Streptomyces fugata (WP_037931694.1), Streptomyces species 13-12-16 (WP_085570642.1), Streptomyces species DH5 (WP_228966783.1), Streptomyces griseoflavus (WP_004931114.1), Streptomyces species NA02536 (WP_176117312.1), Streptomyces (WP_142195205.1), Streptomyces spinoverrucosus (WP_196462535.1), Streptomyces species SLBN-134 (WP_142170642.1), Streptomyces lomondii lomondensis)(WP_190048216.1), Streptomyces species JCM17656(QWA22516.1), Streptomyces microtubule spiral (WP_145869102.1), Streptomyces spp.(WP_215154691.1), Streptomyces viridochromogenes(WP_004001040.1), Streptomyces spp.(WP_125510363.1), Streptomyces chromofuscus(WP_189699031.1), Streptomyces griseus(WP_190095097.1), Streptomyces spp.(WP_126900012.1), Streptomyces species NRRL WC-3626(WP_030217191.1), Streptomyces curare curacoi)(WP_062155922.1), Streptomyces malachite brown (GGX09391.1), Streptomyces malachite brown (WP_190164476.1), Streptomyces afghaniensis 772 (EPJ38539.1), Streptomyces fungicide (WP_121546081.1), Streptomyces chartreusis (WP_107905561.1).1), Streptomyces species WAC 05379 (WP_125529404.1), Streptomyces wuyuanensis (WP_093654151.1), Streptomyces cesswartbergensis (WP_086603880.1), Streptomyces edulis (WP_176577002.1), Streptomyces bellus (WP_193505879.1), Streptomyces coeruleorubidus (WP_150481012.1), Streptomyces dysideae (WP_067021295.1), Streptomyces africanus (WP_086560822.1), Streptomyces cyanobacteria ...603880.1), Streptomyces wuyuanensis (WP_093654151.1), Streptomyces wuyuanensis (WP_093654151.1), Streptomyces wuyuanensis (WP_086603880.1), Streptomyces edulis (WP_176577002.1), Streptomyces bellus (WP_193505879.1), Streptomyces coeruleorubidus (WP_150481012.1), Streptomyces dysideae (WP_067021295.1), Streptomyces africanus (WP iakyrus)(WP_033308050.1), Streptomyces djakartensis(WP_190197820.1), Streptomyces species XY152(WP_053636056.1), Streptomyces(WP_030825115.1), Streptomyces species SID5643(WP_161369364.1), Streptomyces caelestis(WP_184984045.1), Streptomyces qaidamensis(WP_062927006.1), Streptomyces regalis(WP_062703120.1), Streptomyces umbrinus)(WP_190227783.1), Streptomyces species NRRL S-146(WP_031102640.1), Streptomyces ochraceus(WP_189843349.1), Streptomyces collinus(MBB5811277.1), Streptomyces species WAC04114(WP_221757552.1), Streptomyces indica(WP_234847835.1), Streptomyces massasporeus(WP_189588732.1), Streptomyces spp.(WP_104779799.1), Streptomyces violaceochromogenes(WP_191845917.1), Streptomyces montanus(WP_138046192.1), Streptomyces species A244 (WP_107455124.1), Streptomyces (WP_031130500.1), Streptomyces paradoxus (WP_184558435.1), Streptomyces tuirus (WP_190899118.1), Streptomyces species NRRLS-475 (WP_030838074.1), Streptomyces hawaiiensis (WP_175432717.1), Streptomyces purpurascens (WP_189723836.1), Streptomyces violet (WP_189723836.1), Streptomyces janthinus) (WP_193478232.1), Streptomyces sp. AK010 (WP_185013999.1), Streptomyces luteogriseus (WP_184910807.1), or Streptomyces tricholoma (WP_189555972.1).

[0120] In one example, E3 can be an acetyltransferase from Streptomyces sp. (QTK16920.1 GI: 2021361637), Desulfosarcina cetonica (VTR67244.1 GI: 2039686980), Streptomyces leewenhoek (CQR63914.1 GI: 822880124), Streptomyces sp. PAMC 26508 (AGJ55095.1 GI: 478746515), Streptomyces solani 87.22 (CBG72817.1 GI: 260649702), Streptomyces coelicolor A3 (2) (CAB87221.1 GI: 7544048), Streptomyces rhodochrous, Pseudarthrobacter siccitolerans (CCQ47788.1 GI:635350230), Streptomyces dioscoreae (CAL18384.1 GI:115501978), Arthrobacter sp. 9AX (VXB93179.1 GI:17893988879), Streptomyces dioscoreae ATCC 23877 (AKZ55868.1 GI:917649199), Streptomyces formicus (ATL27941.1 GI:1259310060), Orrelladioscoreae (SOE50046.1 GI:1253556130), Pseudomonas sp. JB363 (SIO88002.1 GI:1143070537), Orrella dioscoreae (SBT27366.1 GI:1037119874), Streptomyces venezuelae (CUM41035.1GI:932870024), Elizabethkingia anophelis NUHP1 (AIL46075.1 GI:675104782), Achromobacter xylosoxidans NBRC15126=ATCC 27061 (AHC47452.1 GI:566051779), Desulfosarcina cetonica (VTR65358.1GI:2039688767), Desulfosarcina cetonica (VTR65683.1 GI:2039688264), Desulfosarcina cetonica (VTR64064.1 GI:2039690162), Desulfosarcina cetonica(VTR64557.1GI:2039689733), Desulfosarcina cetonica(VTR64494.1 GI:2039689672), Desulfosarcina cetonica(VTR65495.1 GI:2039688584), Desulfosarcina cetonica(VTR65880.1 GI:2039688461), Desulfosarcina cetonica(VTR65802.1 GI:2039688383), Desulfosarcina cetonica(VTR67337.1GI:2039686714), Desulfosarcina cetonica(VTR67722.1 GI:2039686548), Desulfosarcina cetonica (VTR67458.1 GI:2039686284), Desulfosarcina cetonica(TR67453.1 GI:2039686279), Desulfosarcina cetonica(VTR68479.1 GI:2039685800), Desulfosarcina cetonica(VTR68453.1GI:2039685774), Desulfosarcina cetonica(VTR69949.1 GI:2039683894), Desulfosarcina cetonica(VTR70394.1 GI:2039683642), Desulfosarcina cetonica(VTR70461.1 GI:2039683453), Desulfosarcina cetonica(VTR70443.1 GI:2039683435), Pimelobactersimplex (AIY15770.1 GI:723622294), Streptomyces species NL15-2K (CB49136.1 GI:1493708163), Aquitalea magnusonii (BBF86346.1 GI:1435241518), Micromonospora species B006 (AXO35036.1 GI:1450456452), Venezuelan Streptomyces (ALO08593.1 GI:952470255), Streptomyces pojo ATCC 25435 (EMF50429.1 GI:456384851), Venezuelan Streptomyces ATCC10712 (CCA55858.1 GI:328882619), Drosophila melanogaster (CAA17683.1 GI:2924547), Desulfosarcina cetonica (VTR64151.1 GI:2039689961), Desulfosarcina cetonica (VTR64706.1 GI:2039689502), Desulfosarcina cetonica (VTR65910.1 GI:2039688142), Desulfosarcina cetonica (VTR65903.1 GI:2039688135), Desulfosarcina cetonica (VTR68069.1 GI:2039685916), Desulfosarcina cetonica (VTR69458.1 GI:2039684725), Desulfosarcina cetonica (VTR63766.1 GI:2039690355), Desulfosarcina cetonica (VTR64871.1 GI:2039689205), Desulfosarcina cetonica (VTR64990.1 GI:2039688983), Desulfosarcina cetonica (VTR65346.1 GI:2039688755), Desulfosarcina cetonica (VTR65983.1 GI:2039688006), Desulfosarcina cetonica (VTR66132.1 GI:2039687832), Desulfosarcina cetonica (VTR67865.1 GI:2039686223), Desulfosarcina cetonica (VTR67945.1 GI:2039686120), Desulfosarcina cetonica (VTR68557.1 GI:2039685499), Desulfosarcina cetonica (VTR69047.1 GI:2039685372), Desulfosarcina cetonica (VTR68952.1 GI:2039685277), Desulfosarcina cetonica (VTR69234.1 GI:2039684917), Desulfosarcina cetonica (VTR69164.1 GI:2039684847), Desulfosarcina cetonica (VTR69124.1 GI:2039684807), Desulfosarcina cetonica (VTR69451.1GI:2039684718), Desulfosarcina cetonica (VTR69548.1 GI: 2039684504), Desulfosarcina cetonica (VTR70126.1 GI: 2039684068), Desulfosarcina cetonica (VTR70315.1 GI: 2039683696), Desulfosarcina cetonica (VTR70566.1 GI:2039683558), Streptomyces malaysiensis (ATL84885.1 GI:1266921847), Streptomyces fusca DSM 40593 (AGK77333.1 GI:485094150), Brevibacterium oceanicum (WP_181273085.1), Brevibacterium species HY170 (WP_231442296.1), Brevibacterium species FME17 (WP_193096435.1), Brevibacterium lactis (WP_009376215.1), Brevibacterium euchromatum (WP_167198399.1), Brevibacterium atlanticum (WP_166973259.1), Brevibacterium (WP_137827008.1), Brevibacterium species S22 (TGD29889.1), Brevibacterium epidermidis (WP_098731557.1), Brevibacterium expansum (HJF75768.1), Brevibacterium species LS14 (WP_131248027.1), Brevibacterium species VCM10 (WP_025776856.1), Brevibacterium species UCMA 11752 (WP_235350998.1), Brevibacterium permianense (WP_173151748.1), Brevibacterium aurantiacum (WP_125240720.1), Brevibacterium aurantiacum (WP_193078849.1), Brevibacterium aurantiacum (WP_096145960.1), Brevibacterium aurantiacum (WP_114384949.1), Brevibacterium siliguri (WP_092011616.1), Brevibacterium aurantiacum(WP_096160424.1), Brevibacterium aurantiacum(WP_193086563.1), Brevibacterium species 'Marine'(WP_169252963.1), Brevibacterium aurantiacum(WP_135446718.1), Brevibacterium aurantiacum (WP_193084516.1), Brevibacterium marine (WP_167950434.1), Brevibacterium aurantiacum (WP_101639105.1), Brevibacteriumlimosum (WP_166822893.1), Brevibacterium aurantiacum(WP_096167624.1), Brevibacterium aurantiacum(WP_143924263.1), Brevibacterium aurantiacum(WP_096178868.1), Brevibacterium aurantiacum(WP_096157397.1), Brevibacterium aurantiacum(WP_096163052.1), Brevibacterium aurantiacum (WP_009883550.1), Brevibacterium aurantiacum (WP_069600709.1), Brevibacterium aurantiacum (WP_101557621.1), Brevibacterium casei (WP_119296729.1), Brevibacterium sp. CFH 10365(WP_152347364.1)、Brevibacterium aurantiacum (WP_101598306.1), Brevibacterium casei (WP_082834881.1), Brevibacterium casei (WP_144588711.1), Agreia bicolorata (WP_078715081.1), Brevibacterium species SMBL_HHYL_HB1 (WP_212129586.1), Brevibacterium casei (KZE22294.1), Brevibacterium casei (WP_232623671.1), Brevibacterium casei (QQB16215.1), Agreia bicolorata (WP_044440798.1), Mycobacteroides abscessus subsp.abscessus) (SIH96297.1), Brevibacterium lactis (WP_101624625.1), Brevibacterium lactis (WP_190247010.1), Brevibacterium sp. S111 (WP_135540312.1), Brevibacterium sp. W7.2 (WP_211979250.1), Corynebacterium xericum (SLM95104.1 GI:1188028261), or from plant metagenomes with the following accession numbers: GO10018.1 GI:1613564736, VFR50243.1 GI:1591769475, VFR49444.1 GI:1591759418. GI:1591739264、VFR87867.1GI:1591734832、VFR79172.1 GI:1591731713、VFR90674.1GI:1591727121、VFR73697.1 GI:1591713709, VFR35670.1GI:1591707861, VFR17966.1GI:1591706236, VFR24793.1GI:1591690017, or VFR74915.1GI:1591683123. .

[0121] In another example, E3 can be the deferoxamine siderophore biosynthesis protein DfoC from Pantoea agglomerans (AWD37890.1), Pantoea agglomerans (WP_187507962.1), Pantoea agglomerans (HAT00136.1), Pantoea agglomerans (WP_010247060.1), Pantoea agglomerans (WP_201500586.1), Pantoea agglomerans (WP_124890596.1), Pantoea agglomerans (WP_187496373.1), Pantoea agglomerans (WP_163852072.1), Pantoea agglomerans (WP_154210121.1), Pantoea agglomerans (WP_03376001). 513.1), Pantoea agglomerans (WP_187512465.1), Pantoea agglomerans (WP_207253898.1), Pantoea agglomerans (WP_187495053.1), Pantoea agglomerans (WP_060679208.1), Pantoea species EKM10T (WP_167430078.1), Pantoea agglomerans (WP_187500286.1), Pantoea agglomerans (WP_187492242.1), Pantoea agglomerans (WP_163763414.1), Pantoea agglomerans (WP_122459787.1), Pantoea agglomerans (WP_061902164.1), Pantoea vagans (WP_061703165.1), Pantoea agglomerans (MBS7708200.1), Pantoea spp. (WP_159226815.1), Pantoea agglomerans (WP_187513802.1), Pantoeavagans (WP_050679331.1), Pantoea agglomerans (WP_187484792.1), Pantoea agglomerans (WP_163799890.1), Pantoea species OVA07A (WP_223565934.1), Pantoea agglomerans (WP_191917404.1), Pantoea agglomerans (WP_033773381.1), Pantoea agglomerans (WP_064690802.1), Pantoeavagans (WP_111534 208.1), Pantoea agglomerans (WP_223564415.1), Pantoea agglomerans (WP_129063349.1), Pantoea agglomerans (WP_191920297.1), Pantoea agglomerans (WP_163641337.1), Pantoea agglomerans (WP_022624353.1), Pantoea agglomerans (WP_187492906.1), Pantoea agglomerans (WP_157353121.1), Pantoea species (RZK07467.1), Pantoea agglomerans (WP_191914122.1), Pantoea agglomerans (WP_045140091.1), Pantoea species EKM20T (WP_167433056.1), Pantoea agglomerans (WP_187501430.1), Pantoea (WP_167423161.1), Pantoea agglomerans (WP_191921680.1), Pantoea agglomerans (WP_163658684.1), Pantoea agglomerans (WP_172608645.1), Pantoea agglomerans (WP_192073070.1), Pantoea species CFSAN033090 (WP_052270704.1), Pantoea agglomerans (WP_187511066.1), Pantoea agglomerans (WP_158149880.1), Pantoea agglomerans (WP_132499089.1), Pantoea agglomerans (MBT8497800.1), Curtobacterium plantarum)(WP_122889392.1), Pantoea agglomerans(WP_192033741.1), Pantoea agglomerans(WP_033768945.1), Pantoea agglomerans(WP_187515506.1), Pantoea agglomerans(WP_182505410.1), Pantoea agglomerans(WP_182500640.1), Pantoea agglomerans(WP_158132336.1), Pantoea agglomerans(WP_01067038 4.1), Pantoea agglomerans (WP_187506786.1), Pantoea agglomerans (WP_179256483.1), Pantoea agglomerans (WP_115765004.1), Pantoea agglomerans (WP_191922816.1), Pantoea agglomerans (WP_191914536.1), Pantoea agglomerans (AWD37885.1), Pantoea agglomerans (WP_033780267.1), Pantoea agglomerans (WP_062758877.1), Pantoea agglomerans (WP_115388310.1), Pantoea agglomerans (WP_089414760.1), Pantoea agglomerans (WP_191918109.1), Pantoea agglomerans (WP_137228077.1), Pantoea agglomerans (WP_039389954.1), Pantoea agglomerans (WP_191924048.1), Pantoea agglomerans (WP_191920726.1), Pantoea agglomerans (WP_163845109.1), Pantoea spp. Species paga (WP_143990819.1), Pantoea agglomerans (WP_031590726.1), Pantoea agglomerans (WP_143789436.1), Pantoeavagans (WP_083069696.1), Pantoea agglomerans (WP_033787056.1), Pantoea agglomerans (WP_208003520.1), Pantoea agglomerans (WP_235765353.1), Pantoea pleuroti (WP_182686862.1), Pantoea agglomerans (WP_069026761.1), Pantoea agglomerans (WP_163638696.1).1), Pantoea agglomerans (WP_208442935.1), Pantoea agglomerans (WP_207091826.1), Pantoea agglomerans (WP_064702866.1), Pantoea agglomerans (WP_086906737.1), Pantoea agglomerans (KEY40395.1), Pantoea species OV426 (WP_090965308.1), Pantoea spp. (WP_150037408.1), Pantoea eucalyptae (AWD37908.1), Pantoea spp. (WP_150011559.1), Pantoea species WMus005 (WP_179898291.1), Pantoea eucalyptae (AWD37911.1), Pantoea vagans (WP_161736340.1), Erwinia amylovora (WP_004160308.1), Erwinia amylovora (QJQ67970.1 GI:1839296356), Erwinia amylovora (QJQ64271.1 GI:1839292579), Erwinia amylovora (QJQ60469.1 GI:1839288766), Erwinia amylovora (QJQ56770.1 GI:1839285065), Erwinia amylovora (QJQ53072.1 GI:1839281365), Xenorhabdus cabanillasii JM26 (PHM78217.1 GI:1269095937), Xenorhabdus mauleonii (PHM78208.1 GI:1269095928), Xenorhabdus hominickii (PHM56127.1GI:1269072847), GI:1269054189), Xenorhabdus mauleonii(PHM37944.1 GI:1269054181), GI:1269045834), Erwinia amylovora CFBP1430 (CBA23308.1 GI:291555140), Erwinia pearensis DSM 12163 (CAY75871.1 GI:283479955), Erwinia amylovora LA637 (CDK23310.1 GI:566688504), Erwinia amylovora LA636 (CDK19939.1 GI:565455063), Erwinia amylovora LA635 (CDK16572.1 GI:565423714), Erwinia amylovora CFBP 5888 (CCG88689.1 GI:560105768), Erwinia amylovora MR1 (CCP08595.1 GI:478726734), Erwinia amylovora Ea644 (CCP04532.1 GI:478723237), Erwinia amylovora UPN527 (CCP00585.1 GI:478719346), Erwinia amylovora NBRC 12687=CFBP 1232 (CCO95265.1 GI:478715307), Erwinia amylovora 01SFR-BO (CCO91470.1 GI:478711918), Erwinia amylovora CFBP2585 (CCO87679.1 GI:478708144), Erwinia amylovora Ea266 (CCO83917.1 GI:478703932), Erwinia amylovora Ea356 (CCO80113.1 GI:478700098), Erwinia amylovora ACW56400 (EKV52685.1 GI:426274945), or Erwinia amylovora ATCC BAA-2158 (CBX82136.1 GI:312173882).

[0122] In particular, E3 is selected from DesC from Streptomyces coelicolor, DesC from Streptomyces pilosula, Streptomyces rhodopsinus, the N-terminal domain of DesC protein from Corynebacterium sicca, Pantoea agglomerans or Erwinia amylovora. More particularly, E3 is selected from Streptomyces coelicolor, Streptomyces rhodopsinus, Streptomyces pilosula (WP_189555972.1), Corynebacterium sicca (SLM95104.1GI: 1188028261), Pantoea agglomerans (AWD37890.1) and Erwinia amylovora (WP_004160308.1). Even more particularly, E3 comprises the N-terminal domain of DesC protein from Corynebacterium sicca, Pantoea agglomerans or Erwinia amylovora relative to SEQ ID NO: 5 (E 3a )、SEQ ID NO:17(E 3b )、SEQ ID NO:27(E 3c ), SEQ ID NO:55(E 3d ), or the N-terminal domain of SEQ ID NO:54 (E 3e ) or SEQ ID NO:34 (E 3f ) have at least 70% sequence identity.

[0123] The accession numbers mentioned throughout this specification and stated in connection with the present invention correspond to the NCBI ProteinBank database entry dated 07.02.2022; typically, the version number of an entry is identified here by a "number" such as ".1".

[0124] Lysine decarboxylase (E1), cadaverine N5-monooxygenase (E2), N5-aminopentyl-N-(hydroxy)-succinamic acid synthase (E3), deferoxamine synthetase (E4) and any other enzyme examples mentioned according to any aspect of the present invention, also include proteins having the same amino acid sequence as those mentioned above except for substitution, deletion, insertion and / or addition of one or several amino acids (as long as its function is maintained). The term "several" generally means about 1 to 7, particularly about 1 to 5, more particularly about 1 to 2 in this article. Lysine decarboxylase (E1), cadaverine N5-monooxygenase (E2), N5-aminopentyl-N-(hydroxy)-succinamic acid synthase (E3), deferoxamine synthetase (E4) and any other enzyme mentioned according to any aspect of the present invention can each be a protein having an amino acid sequence that is generally not less than 70, 75, 80, 85%, particularly not less than 90%, more particularly not less than 95% identical to the original amino acid sequence, as long as its function is maintained.

[0125] The substitution, deletion, insertion and / or addition in the above amino acid sequence is particularly a conservative substitution. Examples of conservative substitution of an original amino acid by another amino acid include substitution of Ala by Ser or Thr; substitution of Arg by Gln, His or Lys; substitution of Asn by Glu, Gln, Lys, His or Asp; substitution of Asp by Asn, Glu or Gln; substitution of Cys by Ser or Ala; substitution of Gln by Asn, Glu, Lys, His, Asp or Arg; substitution of Glu by Asn, Gln, Lys or Asp; substitution of Gly by Pro; substitution of His by Asn, Lys, Gln, Arg or Tyr; Ile is replaced by Leu, Met, Val or Phe; Leu is replaced by Ile, Met, Val or Phe; Lys is replaced by Asn, Glu, Gln, His or Arg; Met is replaced by Ile, Leu, Val or Phe; Phe is replaced by Trp, Tyr, Met, Ile or Leu; Ser is replaced by Thr or Ala; Thr is replaced by Ser or Ala; Trp is replaced by Phe or Tyr; Tyr is replaced by His, Phe or Trp; and Val is replaced by Met, Ile or Leu.

[0126] In particular, the cell according to any aspect of the invention may be genetically modified or may comprise a genetic modification that increases the activity of E4 and at least two other enzymes selected from E1, E2 and E3 relative to its wild-type cell. In one example, the cell has increased expression of E4, E1 and E2, or E4, E1 and E3, or E4, E2 and E3.

[0127] Even more particularly, the cell according to any aspect of the invention is genetically modified or may comprise a genetic modification that increases all four enzymes E1, E2, E3 and E4 relative to its wild-type cell.

[0128] The cell according to any aspect of the present invention is further genetically modified or may comprise a genetic modification that increases activity to increase the production of L-lysine and / or cadaverine. In particular, the cell has an increased intracellular production of L-lysine and / or cadaverine. More particularly, the further genetic modification in the cell according to any aspect of the present invention comprises an increase in the activity of the following enzymes E6-E 19 More particularly, the further genetic modification according to any aspect of the present invention comprises an increase in the expression of at least one of the following enzymes:

[0129] - pyruvate carboxylase (EC 6.4.1.1) (E6),

[0130] -Aspartate aminotransferase (EC 2.6.1.1) (E7)

[0131] - aspartate kinases, in particular feedback-resistant aspartate kinases (EC 2.7.2.4) (E8),

[0132] - aspartate semialdehyde dehydrogenase (EC 1.2.1.11) (E9),

[0133] -Dihydrodipicolinate synthase (EC 4.3.3.7) (E 10 ),

[0134] -Dihydropicolinate reductase (EC 30 1.17.1.8) (E 11 ),

[0135] -Diaminopimelate dehydrogenase (EC 1.4.1.16) (E 12 ),

[0136] -Diaminopimelate epimerase (EC 5.1.1.7) (E 13 ),

[0137] -Diaminopimelate decarboxylase (EC 4.1.1.20) (E 14 ),

[0138] -N-succinyl-aminoketopimelate aminotransferase (EC 2.6.1.17) (E 17 ),

[0139] -2,3,4,5-tetrahydropyridine-2,6-dicarboxylic acid N-succinyltransferase (EC 2.3.1.117) (E 18 ), and / or

[0140] -Succinyldiaminopimelate desuccinylase (EC 3.5.1.18) (E 19 );

[0141] and / or reduced expression of at least one of the following enzymes:

[0142] -Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (E 15 ), and / or

[0143] -Homoserine dehydrogenase (EC 1.1.1.3) (E 16 ).

[0144] In one example, E6 can be encoded by the gene pyc P458S of Corynebacterium glutamicum disclosed at least in WO1999018228 or EP2107128. In one example, E7 can be encoded by the gene aspB of Corynebacterium glutamicum disclosed at least in EP0219027 or WO2008033001. In one example, E8 can be variant T311I and can be encoded by the gene lysC of Corynebacterium glutamicum disclosed at least in US6893848. In one example, E9 can be encoded by the gene asd of Corynebacterium glutamicum disclosed at least in EP0387527 or WO2008033001. In one example, E 10 It may be encoded by the gene dapA of Corynebacterium glutamicum disclosed at least in EP0197335. In one example, E 11 It may be encoded by the gene dapB of Corynebacterium glutamicum disclosed at least in US8637295 or EP0841395. In one example, E 12 Can be encoded by the gene ddh of Corynebacterium glutamicum described at least in EP0811682. In one example, E 13 It may be encoded by the gene dapF of Corynebacterium glutamicum described at least in US6670156. In one example, E 14 It may be encoded by the gene lysA of Corynebacterium glutamicum described at least in EP0811682. In one example, E 15 It can be encoded by the gene pck of Corynebacterium glutamicum. In one example, E16 It can be encoded by the gene homV59A of Corynebacterium glutamicum. In one example, E 17 It can be encoded by the gene dapC of Corynebacterium glutamicum. In one example, E 18 It can be encoded by the gene dapD of Corynebacterium glutamicum. In one example, E 19 It may be encoded by the gene dapE of Corynebacterium glutamicum.

[0145] In another example, the cell according to any aspect of the present invention may be genetically modified to increase a gene selected from E6 to E 19 or 14 enzymes.

[0146] In another example, the cell according to any aspect of the invention may be genetically modified to increase the expression of at least pyruvate carboxylase (EC 6.4.1.1) (E6) and aspartate kinase (EC 2.7.2.4) (E8) as disclosed at least in JP6219481B2 to increase the production of lysine.

[0147] In a further example, the cell according to any aspect of the invention may be genetically modified to increase at least dihydrodipicolinate synthase (EC 4.3.3.7) (EC 10 ), aspartate kinase (EC 2.7.2.4) (E8), dihydropicolinate reductase (EC 30 1.17.1.8) (E 11 ), phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (E 15 ) and aspartate semialdehyde dehydrogenase (EC 1.2.1.11) (E9) as disclosed in US8062869B2.

[0148] In one example, the cell according to any aspect of the invention may be genetically modified to increase at least aspartate semialdehyde dehydrogenase (EC 1.2.1.11) (E9), dihydrodipicolinate synthase (EC 4.3.3.7) (E 10 ) and dihydropicolinate reductase (EC 30 1.17.1.8) (E 11 ) expression, as disclosed at least in JP5486029B2. The cell may be further genetically modified to increase the expression of diaminopimelate dehydrogenase (EC 1.4.1.16) (E 12 ) and / or diaminopimelate decarboxylase (EC 4.1.1.20) (E 14 ) expression.

[0149] In a further example, the cell according to any aspect of the invention may be genetically modified to increase the expression of pyruvate carboxylase (EC 6.4.1.1) (E6), aspartate kinase (EC 2.7.2.4) (E8), aspartate semialdehyde dehydrogenase (EC 1.2.1.11) (E9), dihydrodipicolinate synthase (EC 4.3.3.7) (E10), pyruvate carboxylase (EC 6.4.1.1) (E11), pyruvate carboxylase (EC 6.4.1.1) (E12), pyruvate carboxylase (EC 6.4.1.1) (E13), pyruvate carboxylase (EC 6.4.1.1) (E14), pyruvate carboxylase (EC 6.4.1.1) (E15), pyruvate carboxylase (EC 10 ), dihydropicolinate reductase (EC301.17.1.8) (E 11 ), diaminopimelate dehydrogenase (EC 1.4.1.16) (E 12 ) and / or diaminopimelate decarboxylase (EC 4.1.1.20) (E 14 ) expression and reduced phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (E 15 ) and / or homoserine dehydrogenase (EC 1.1.1.3) (E 16 ) expression. At least in US8637295, US2011039313, EP1725672 and EP1320593, it is shown that E6-E 19 Examples of sequences and means to increase and decrease expression of related enzymes.

[0150] Further, the examples use strain Corynebacterium glutamicum DM1933, and its construction is disclosed at least in Blomberg et al., 2009 (doi: 10.1128 / AEM.01844-08). This method can then be used to produce any cell that may have increased lysine production compared to wild-type cells.

[0151] According to a further aspect of the present invention, there is provided a method for producing at least one compound having structural formula II from at least one carbon source:

[0152]

[0153] n=1-3

[0154] R1=H or COCH3 or CH2CH2COX, where X=OH or O-

[0155] R2=CH3 or CH2CH2COX, where X=OH or O-

[0156] The method includes:

[0157] (a) contacting a cell according to any aspect of the invention with at least one carbon source.

[0158] In another aspect of the present invention, there is provided the use of a cell according to any aspect of the present invention for producing at least one compound of formula II from at least one carbon source:

[0159]

[0160] n=1-3

[0161] R1=H or COCH3 or CH2CH2COX, where X=OH or O-

[0162] R2=CH3 or CH2CH2COX, wherein X=OH or O-. BRIEF DESCRIPTION OF THE DRAWINGS

[0163] Figure 1 It is a multiple reaction monitoring (MRM) chromatogram of DesH (7.9 minutes) detected in a sample from Corynebacterium glutamicum DM1933int.NCGl0013 / 0014::{Ptuf}[ldcC_Ec(coCg)]pXMJ19{Ptac}{RBSopt}[des BCD_Sco(co_Cg)]{T} (plasmid of Example 2).

[0164] Figure 2 It is a multiple reaction monitoring (MRM) chromatogram of DesH (7.9 minutes) detected in a sample from Corynebacterium glutamicum DM1933int.NCG10013 / 0014::{Ptuf}[ldcC_Ec(coCg)]pXMJ19{Ptac}[desABCD_Svi] (plasmid of Example 3).

[0165] Figure 3 This is a multiple reaction monitoring (MRM) chromatogram of DesH (7.9 minutes) detected in a sample from Corynebacterium glutamicum DM1933int.NCG10013 / 0014::{Ptuf}[ldcC_Ec(coCg)]pXMJ19{Ptac}[desABCD_Sp i)] (plasmid of Example 4).

[0166] Figure 4 is a multiple reaction monitoring (MRM) chromatogram of DesH (6.8 minutes) detected in a sample from E. coli W3110 pXMJ19{Ptac}[desBCD_Sco]{T} (plasmid of Example 1).

[0167] Figure 5is a multiple reaction monitoring (MRM) chromatogram of DesH (7.7 minutes) detected in a sample from E. coli W3110 pXMJ19{Ptac}[dfoAC_Eam]{T} (plasmid of Example 5).

[0168] Figure 6 is a multiple reaction monitoring (MRM) chromatogram of DesB (5.7 minutes) detected in a sample from E. coli W3110 pXMJ19{Ptac}[dfoAC_Eam]{T} (plasmid of Example 5). Example

[0169] The foregoing describes a preferred embodiment, and as will be appreciated by those skilled in the art, the embodiment may be subject to changes or modifications in design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be covered by the scope of the claims.

[0170] Example 1

[0171] Construction of Corynebacterium glutamicum Expression Vector of Desferrioxamine Biosynthesis Gene desBCD_Sco from Streptomyces coelicolor

[0172] For heterologous expression of the desB gene (SEQ ID NO: 1), desC gene (SEQ ID NO: 2) and desD gene (SEQ ID NO: 3) from Streptomyces coelicolor CAI-140, plasmid pXMJ19{Ptac}[desBCD_Sco]{T} was constructed. The synthetic operons consisting of the following were cloned into the E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 under the IPTG-inducible promoter P tacThe operon was under the control of: desB_Sco encoding a lysine N(6)-hydroxylase / L-ornithine N(5)-oxygenase family protein (DesB, EC 1.14.13.-, SEQ ID NO: 4), desC_Sco encoding an acetyltransferase (DesC, EC 2.3-.-, SEQ ID NO: 5), and desD_Sco encoding a lucA / lucC family siderophore biosynthesis protein (DesD, EC 6.3.-.-, SEQ ID NO: 6). The terminator sequence was located downstream of the synthetic operon. The complete synthetic operon (3732 bp, SEQ ID NO: 7) including the terminator sequence was ordered from Eurofins Genomics Germany GmbH (Ebersberg, Germany) for gene synthesis. Escherichia coli / Corynebacterium glutamicum shuttle vector pXMJ19 carries the pUC replication origin for Escherichia coli and the pBL1 replication origin for the replication in Corynebacterium glutamicum ATCC 13032. For cloning, the synthetic operon restriction enzyme HindIII is cut, and is connected in the pXMJ19 that cuts with the same enzyme. The connection product is transformed into 10-beta electrocompetent Escherichia coli cells (New England BioLabs Inc., Ipswich, USA, catalog number (Cat. No.) C3020K). Clone and transformation procedure are carried out according to the manual of manufacturers. Check the correct insertion of target gene by restriction analysis, and verify the authenticity of the introduced DNA fragmentation by dna sequencing. Resulting expression vector named after pXMJ19{Ptac}[desBCD_Sco]{T}(SEQ ID NO:8, referring to table 2 below).

[0173] Example 2

[0174] Construction of a Corynebacterium glutamicum expression vector for the Streptomyces coelicolor deferoxamine biosynthesis gene desBCD_Sco codon-optimized for expression in Corynebacterium glutamicum

[0175] For heterologous expression of the desB gene (SEQ ID NO: 1), desC gene (SEQ ID NO: 2) and desD gene (SEQ ID NO: 3) from Streptomyces coelicolor CAI-140, plasmid pXMJ19{Ptac}{RBSopt}[desBCD_Sco(co_Cg)]{T} was constructed. The synthetic operons consisting of the following were cloned into the E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 under the IPTG-inducible promoter P tacUnder the control of: desB_Sco encoding lysine N (6) -hydroxylase / L- ornithine N (5) -oxygenase family protein (DesB, EC 1.14.13.-, SEQ ID NO: 4), desC_Sco encoding acetyltransferase (DesC, EC2.3-.-, SEQ ID NO: 5) and desD_Sco encoding lucA / lucC family siderophore biosynthesis protein (DesD, EC6.3.-.-, SEQ ID NO: 6). An optimized ribosome binding site (RBS) for Corynebacterium glutamicum was added upstream of the operon, and a terminator sequence was positioned downstream of the synthetic operon. Ordered the complete synthetic operon (3779bp, SEQ ID NO:9) that comprises RBS and terminator sequence from Eurofins GenomicsGermany GmbH (Ebersberg, Germany) for gene synthesis, and the DNA sequence dna of gene fragment is codon optimized for the expression in Corynebacterium glutamicum ATCC 13032.Escherichia coli / Corynebacterium glutamicum shuttle vector pXMJ19 carries the pUC replication origin that is used for intestinal bacteria and the pBL1 replication origin that is used for the replication in Corynebacterium glutamicum ATCC 13032.For cloning, the synthetic operon is cut with restriction enzyme HindIII, and is connected in the pXMJ19 that cuts with the same enzyme.The connection product is transformed in 10-beta electrocompetent Escherichia coli cells (New England BioLabs Inc., Ipswich, USA, catalog number (Cat. No.) C3020K).Clone and transformation procedure are carried out according to the manual of manufacturers.Check the correct insertion of target gene by restriction analysis, and verify the authenticity of the introduced DNA fragmentation by DNA sequencing. The resulting expression vector was named pXMJ19{Ptac}{RBSopt}[desBCD_Sco(co_Cg)]{T} (SEQ ID NO: 10, see Table 2 below).

[0176] Example 3

[0177] Construction of Corynebacterium glutamicum expression vector of the deferoxamine biosynthesis gene desABCD_Svi from Streptomyces rhodochrous

[0178] For heterologous expression of the desA gene (SEQ ID NO: 11), desB gene (SEQ ID NO: 12), desC gene (SEQ ID NO: 13) and desD gene (SEQ ID NO: 14) from Streptomyces rhodochrous A3 (2), the plasmid pXMJ19 {Ptac} [desABCD_Svi] was constructed. The complete operon consisting of the following, respectively, was cloned into the E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 under the IPTG-inducible promoter P tac The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). The primers were MW22_001fw / MW22_002rv (SEQ ID NO: 44, SEQ ID NO: 45). NO:45, see Table 1 above) and genomic DNA from Streptomyces rhodochrous A3 (2) as templates, and the entire operon was amplified via PCR. The E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 carries the pUC replication origin for E. coli and the pBL1 replication origin for replication in Corynebacterium glutamicum ATCC 13032. Restriction sites HindIII and 5-mercaptoethanol from New England BioLabs Inc., Ipswich, USA, catalog number E5520 were used. HiFi DNA Assembly Cloning Kit, PCR product (5120bp, SEQ ID NO: 19) was cloned into vector pXMJ19. The ligation product was transformed into 10-beta electrocompetent Escherichia coli cells (New England BioLabs Inc., Ipswich, USA, catalog number C3020K). The cloning and transformation procedures were performed according to the manufacturer's manual. The correct insertion of the target gene was checked by restriction analysis, and the authenticity of the introduced DNA fragment was verified by DNA sequencing. The resulting expression vector was named pXMJ19{Ptac}[desABCD_Svi](SEQ ID NO: 20, see Table 2 below).

[0179]

[0180] Table 1: Primer list

[0181] Example 4

[0182] Construction of Corynebacterium glutamicum expression vector of the deferoxamine biosynthesis gene desABCD_Spi from Streptomyces pilosus

[0183] For heterologous expression of desA gene (SEQ ID NO: 21), desB gene (SEQ ID NO: 22), desC gene (SEQ ID NO: 23) and desD gene (SEQ ID NO: 24) from Streptomyces trichomes ATCC19797, plasmid pXMJ19{Ptac}[desABCD_Spi] was constructed. The complete operon consisting of the following respectively was cloned into the E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 under the IPTG-inducible promoter P tac The primers were MW22_004fw / MW22_005rv (SEQ ID NO: 46, SEQ ID NO: 47). The primers were MW22_004fw / MW22_005rv (SEQ ID NO: 46, SEQ ID NO: 47). NO:47, see Table 1 above) and genomic DNA from Streptomyces hairyus ATCC19797 as templates, via PCR amplification of the complete operon. The E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 carries the pUC replication origin for E. coli and the pBL1 replication origin for replication in Corynebacterium glutamicum ATCC 13032. Restriction sites HindIII and 5-mercaptoethanol from New England BioLabs Inc., Ipswich, USA, catalog number E5520 were used. HiFi DNA Assembly CloningKit, PCR product (5104bp, SEQ ID NO:29) is cloned into vector pXMJ19. The ligation product is transformed into 10-beta electrocompetent Escherichia coli cells (New England BioLabs Inc., Ipswich, USA, catalog number C3020K). The cloning and transformation procedures are carried out according to the manufacturer's manual. The correct insertion of the target gene is checked by restriction analysis, and the authenticity of the introduced DNA fragment is verified by DNA sequencing. The resulting expression vector is named pXMJ19{Ptac}[desABCD_Spi](SEQ ID NO:30, see Table 2 below).

[0184] Example 5

[0185] Construction of Corynebacterium glutamicum expression vector of Erwinia amylovora deferoxamine biosynthesis gene dfoAC_Eam

[0186] For heterologous expression of the dfoA gene (SEQ ID NO: 31) and dfoC gene (SEQ ID NO: 32) from Erwinia amylovora CFBP1430, plasmid pXMJ19 {Ptac} [dfoAC_Eam] {T} was constructed. The operon consisting of the following was cloned into the E. coli / Corynebacterium glutamicum shuttle vector pXMJ19 under the IPTG-inducible promoter P tacThe dfoA_Eam of coding lysine N (6)-hydroxylase / L-ornithine N (5)-oxygenase family protein (DfoA, EC 1.14.13.-, SEQ ID NO:33) and the dfoC_Eam of coding GNAT family N-acetyltransferase (DfoC, EC 2.3.-.-, SEQ ID NO:34) are under the control of.Terminator sequence is positioned at the downstream of synthetic operon.Complete synthetic operon (3830bp, SEQ ID NO:35) including terminator sequence was ordered from Eurofins Genomics Germany GmbH (Ebersberg, Germany) for gene synthesis.Escherichia coli / Corynebacterium glutamicum shuttle vector pXMJ19 carries the pUC replication origin for Escherichia coli and the pBL1 replication origin for the replication in Corynebacterium glutamicum ATCC 13032.For cloning, synthetic operon is cut with restriction enzyme HindIII, and is connected in the pXMJ19 cut with the same enzyme. The ligation product is transformed into 10-beta electrocompetent Escherichia coli cells (New England BioLabs Inc., Ipswich, USA, catalog number (Cat. No.) C3020K). Cloning and transformation procedures are carried out according to the manufacturer's manual. Correct insertion of the target gene is checked by restriction analysis, and the authenticity of the introduced DNA fragment is verified by DNA sequencing. The resulting expression vector is named pXMJ19{Ptac}[dfoAC_Eam]{T}(SEQ ID NO:36, see table 2 below).

[0187] Plasmids Cloned genes pXMJ1 9{Ptac}[desBCD_Sco]{T} desBCD_Sco pXMJ1 9{Ptac}{RBSopt}[desBCD_Sco(co_Cg)]{T} desBCD_Sco(co_Cg) pXMJ 19{Ptac}[desABCD_Svi] desABCD_Svi pXMJ1 9{Ptac}[desABCD_Spi] desABCD_Spi pXMJ1 9{Ptac}[dfoAC_Eam]{T} dfoAC_Eam

[0188] Table 2: List of expression plasmids for Corynebacterium glutamicum / Escherichia coli

[0189] Example 6

[0190] Construction of a cadaverine producer based on Corynebacterium glutamicum by introducing L-lysine decarboxylase ldcC from Escherichia coli into the L-lysine producer strain Corynebacterium glutamicum DM1933

[0191] In order to construct the cadaverine producer based on Corynebacterium glutamicum, the intestinal bacteria ldcC gene (SEQ ID NO:37) of coding L-lysine decarboxylase (LdcC, EC4.1.1.18, SEQ ID NO:38) is integrated in the genome of lysine producer Corynebacterium glutamicum DM1933.The detailed construction of DM1933 ​​is described in Blomberg et al., 2009 (doi:10.1128 / AEM.01844-08).For the integration of ldcC gene, construct plasmid pK18mobsacB KI{Ptuf}[ldcC_Ec(co_Cg)]., the dcC gene is integrated in the intergenic region between ORF NCg10013 and ORF NCg10014, and is cloned into constitutive Corynebacterium glutamicum promoter P tuf The {Ptuf}[ldcC_Ec(co_Cg)] fusion product (SEQ ID NO: 39) was ordered from Eurofins Genomics Germany GmbH (Ebersberg, Germany) for gene synthesis.

[0192] In the first cloning step, two flanking regions of the chromosomal noncoding region between NCgl0013 and NCgl0014 were amplified by PCR using primer pairs MW_21_80 / MW_21_81 (SEQ ID NO: 48, SEQ ID NO: 49) and MW_21_82 / MW_21_83 (SEQ ID NO: 50, SEQ ID NO: 51, see Table 1), generating fragments HomA (1046 bp, SEQ ID NO: 40) and HomB (1028 bp, SEQ ID NO: 41). The restriction site EcoRI and PCR products from New England BioLabs Inc., Ipswich, USA, catalog number E5520 were used. HiFi DNA Assembly Cloning Kit, clone the two fragments into the vector pK18mobsacB ( et al., 1994, DOI: 10.1016 / 0378-1119(94)90324-7). In addition, an AscI restriction site was introduced between HomA and HomB via primers MW_21_81 / MW_21_82. The assembled product was transformed into 10-beta electrocompetent E. coli cells (New England BioLabs Inc., Ipswich, USA, catalog number C3020K). The cloning and transformation procedures were performed according to the manufacturer's manual. The correct insertion of the target gene was checked by restriction analysis, and the authenticity of the introduced DNA fragment was verified by DNA sequencing. The resulting expression vector was named pK18mobsacB[KI NCgl0013 locus] (SEQ ID NO: 42).

[0193] In the second cloning step, the {Ptuf}[ldcC_Ec(co_Cg)] fusion product (SEQ ID NO:39, 2433 bp) was amplified by PCR using primer pair MW_21_93 / MW_21_94 (SEQ ID NO:52, SEQ ID NO:53) and restriction sites AscI and PCR products from New England BioLabs Inc., Ipswich, USA, catalog number E5520 were used. HiFi DNA Assembly Cloning Kit, cloned into vector pK18mobsacB [KI NCgl0013 locus] (SEQ ID NO: 42). The assembled product is transformed into 10-beta electrocompetent Escherichia coli cells (NewEngland BioLabs Inc., Ipswich, USA, catalog number C3020K). The cloning and transformation procedures are carried out according to the manufacturer's manual. The correct insertion of the target gene is checked by restriction analysis, and the authenticity of the introduced DNA fragment is verified by DNA sequencing. The resulting knock-in plasmid is named pK18mobsacB KI {Ptuf} [ldcC_Ec (co_Cg)] (SEQ ID NO: 43).

[0194] This plasmid was transformed into Corynebacterium glutamicum DM1933 ​​via electroporation. et al. 1994 (DOI: 10.1016 / 0378-1119(94)90324-7), a double crossover was performed to insert a gene into the promoter P tufThe gene ldc_Ec(co_Cg) under the control of was integrated into the chromosome of Corynebacterium glutamicum DM1933 ​​to produce Corynebacterium glutamicum DM1933 ​​int.NCGl0013 / 0014::{Ptuf}[ldcC_Ec(coCg)].

[0195] Example 7

[0196] Construction of a deferoxamine producer based on Corynebacterium glutamicum

[0197] For the construction of the deferoxamine producer based on Corynebacterium glutamicum, the plasmid described in Example 2-4 and listed in the upper table 2 is transformed into Corynebacterium glutamicum DM1933 ​​int.NCG10013 / 0014::{Ptuf}[ldcC_Ec(coCg)] by means of electroporation. The cell is plated on the LB agar plate supplemented with chloramphenicol (7.5mg / L). The transformant is checked for the presence of the correct plasmid by plasmid preparation and analytical restriction analysis. The resulting bacterial strain is listed in Table 3.

[0198]

[0199] Table 3: List of deferoxamine producer strains based on Corynebacterium glutamicum

[0200] Example 8

[0201] Construction of a deferoxamine producer based on Escherichia coli

[0202] For the construction of the E. coli based deferoxamine producer, the plasmids described in Examples 1 and 5 and listed in Table 2 were transformed into E. coli W3110 by means of electroporation. The cells were plated on LB agar plates supplemented with chloramphenicol (20 mg / L). The transformants were checked for the presence of the correct plasmid by plasmid preparation and analytical restriction analysis. The resulting strains were grown in

[0203] Listed in Table 4.

[0204]

[0205] Table 4: List of deferoxamine producer strains based on E. coli

[0206] Example 9

[0207] Linear deferoxamine production using Corynebacterium glutamicum derivatives

[0208] For the production of linear deferoxamine derivatives (DesB / DesH), 10 ml of BHI medium (GranuCult™ BHI (Brain Heart Infusion) broth, Merck, Darmstadt, Germany, catalog number: 1.10493.0500) supplemented with chloramphenicol (7.5 mg / L) in a 100 ml baffled shake flask was inoculated with 0.1 ml of the stock culture and incubated at 30° C. and 200 rpm for 16 hours. The preculture was harvested by centrifugation (10 min, 4000 g, 4° C.) and the pellet was washed twice with 10 ml of 0.9% (w / v) NaCl. For the main culture, the preculture was used to inoculate a FlowerPlate with pH and dissolved oxygen optodes (48-well MTP, flower, Beckman Coulter Life Sciences, Baesweiler, Germany, catalog number: M2P-MTP-48-BOH1), which contained in each well 0.7 ml of CGXII medium (15 g / L glucose, 20 g / L (NH4)2SO4, 5 g / L urea, 1 g / L K2HPO4, 1 g / L KH2PO4, 0.25 g / L MgSO4 x7H2O, 42 g / L MOPS, 13.2 mg / L CaCl2, 0.2 mg / L biotin, 30 mg / L protocatechuic acid, trace element solution: 10 g / L FeSO4 x 7H2O, 10 g / L MnSO4 x H2O, 1 g / L ZnSO4 x 7H2O, 0.2g / L CuSO4, 20mg / L NiCl2 x6H2O, pH 7) to reach the initial OD 600 0.5. The main culture was incubated at 30°C and 1400 rpm and relative humidity (85%) for 24 hours in a BioLector I system (Beckman Coulter Life Sciences, Baesweiler, Germany). At the beginning of the exponential phase, the expression of the target gene was induced with 0.5 mM IPTG. At the end of the culture, the cells were harvested and the supernatant was sterile filtered with a 0.2 μm PVDF filter and stored at -20°C before analysis. The deferoxamine concentration of all strains was analyzed by LC-UV-MS (see Example 11). Deferoxamine H was detectable in the supernatant of all strains, as shown in Figure 1-3 Visible in.

[0209] Example 10

[0210] Linear deferoxamine production using Escherichia coli derivatives

[0211] For the production of linear deferoxamine derivatives (DesB / DesH), 10 ml LB medium (Carl Roth, Karlsruhe, Germany, catalog number: X968.1) supplemented with chloramphenicol (20 mg / L) in a 100 ml baffled shake flask was inoculated with 0.1 ml of the stock culture and incubated at 37° C. and 200 rpm for 16 hours. For the main culture, the preculture was used to inoculate a FlowerPlate (48-well MTP, flower, Beckman Coulter Life Sciences, Baesweiler, Germany, catalog number: M2P-MTP-48-BOH1) with pH and dissolved oxygen optodes, which contained 0.7 ml LB medium (Carl Roth, Karlsruhe, Germany, catalog number: X968.1) buffered with 100 mM MOPS, pH 7.2 and supplemented with chloramphenicol (20 mg / L) in each well to reach a starting OD 600 0.1. The main culture was incubated in a BioLector I system (Beckman Coulter LifeSciences, Baesweiler, Germany) at 37°C and 1400 rpm and relative humidity (85%) for 24 hours. At the beginning of the exponential phase, the expression of the target gene was induced with 0.5 mM IPTG. At the end of the culture, the cells were harvested and the supernatant was sterile filtered with a 0.2 μm PVDF filter and stored at -20°C before analysis. The deferoxamine concentration of all strains was analyzed by LC-UV-MS (see Example 11). Deferoxamine H (e.g., 1:1) was detected in the supernatant of all strains. Figure 4-5 In the supernatant of strain E. coli W3110pXMJ19{Ptac}[dfoAC_Eam]{T}, we can also detect deferoxamine B, as shown in Figure 6 As shown in .

[0212] Embodiment 11

[0213] HPLC-based quantification of deferoxamine B / H

[0214] Quantification of linear deferoxamine derivatives was performed by means of HPLC. Prior to analysis, the sample was centrifuged at 16100 g for 5 minutes and filtered using a 0.22 μm PVDF filter. 20 μl of the filtered supernatant was mixed with 80 μl of ammonium ferric sulfate solution and placed in an HPLC vial. The sample was stored at -20°C prior to measurement.

[0215] For the detection and quantification of linear deferoxamine derivatives, a DAD detector (198 and 430 nm) was used. 4.6x100mm, 2.6μm, Phenomenex Kinetex) was measured. The injection volume was 5μl, and the running time was 25 minutes at a flow rate of 0.8ml / min. Mobile phase A: 1L pure water, 1ml formic acid, mobile phase B: 1L acetonitrile, 1ml formic acid. The column temperature was 40°C. As reference materials, deferoxamine B mesylate (Merck KGaA, Darmstadt, Germany, catalog number D9533) and ferrioxamine E (Merck KGaA, Darmstadt, Germany, catalog number 38266) (both are iron complexes) from Streptomyces antibioticus were used.

[0216] gradient:

[0217] t[minutes] [％]A [％]B Flow rate [ml / min] Maximum pressure [bar] 0.0 95 5 0.8 400 5.0 95 5 0.8 400 13.0 70 30 0.8 400 15.0 2 98 0.8 400 20.0 2 98 0.8 400 20.01 95 5 0.8 400 25.0 95 5 0.8 400

[0218] For analytes with concentrations below the limit of quantification (LOQ), identification was performed with the aid of HPLC / ESI-MS-MS. For these measurements the multiple reaction monitoring mode (MRM) of a Triple Quadrupol Mass Spectrometer (Agilent 6410B, Santa Clara, Calif., USA) was used.

Claims

1. A recombinant microbial cell for producing at least one compound of formula II from a carbon source: n＝2-3 R1=H or COCH3 or CH2CH2COX, where X=OH or O- R2=CH3 or CH2CH2COX, wherein X=OH or O-, wherein the cell comprises a genetic modification to increase the activity of E4 relative to its wild-type cell, wherein: E4 is deferoxamine or bisucalin synthase (EC 6.3.-.-)(E 4i ), which is capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid to deferoxamine B or H or at least one other linear deferoxamine or bisucalline according to formula II.

2. The cell according to claim 1, wherein the cell comprises a further genetic modification to increase the activity of at least one enzyme selected from E1, E2 and E3 relative to its wild-type cell, in E1 is a lysine decarboxylase (EC: 4.1.1.18)； E2 is a cadaverine N5-monooxygenase (EC 1.14.13.-) capable of converting cadaverine to N5-hydroxy-cadaverine; and E3 is an N5-aminopentyl-N-(hydroxy)-succinamic acid synthase (EC: 2.3.-.-)。 3. The cell according to claim 2, wherein the cell comprises a genetic modification to increase the activity of E4 and at least two other enzymes selected from E1, E2 and E3 relative to its wild-type cell.

4. The cell according to claim 2 or 3, wherein the cell comprises a genetic modification to increase the activity of all enzymes E2, E3 and E4 relative to its wild-type cell.

5. The cell according to any one of claims 2 to 4, wherein the genetic modification is (a) at least one promoter operably linked to a gene encoding enzyme E1, E2, E3 and / or E4 introduced into a suitable chromosome of the cell, or (b) at least one expression vector to increase the copy number of the gene encoding enzyme E1, E2, E3 and / or E4 in the cell, or (c) Combination of (a) and (b) To increase the expression of enzymes E1, E2, E3 and / or E4.

6. The cell according to any one of claims 2 to 5, wherein -E1 comprises the same sequence as SEQ ID NO: 15 (E 1a )、SEQ ID NO:25(E 1b ) or SEQ ID NO:38 (E 1c ) at least 70% sequence identity; -E2 comprises the same sequence as SEQ ID NO:4 (E 2a )、SEQ ID NO:16(E 2b )、SEQ ID NO:26(E 2c )、SEQ ID NO:33(E 2d )、SEQ ID NO:56(E 2e ) or SEQ ID NO:57 (E 2f ) at least 70% sequence identity; -E3 comprises the same sequence as SEQ ID NO:5 (E 3a )、SEQ ID NO:17(E 3b )、SEQ ID NO:27(E 3c ), SEQ ID NO:55(E 3d ), or the N-terminal domain of SEQ ID NO:54 (E 3e ) or SEQ ID NO:34 (E 3f ) at least 70% sequence identity; and -E4 comprises the same sequence as SEQ ID NO: 6 (E 4a )、SEQ ID NO:18(E 4b )、SEQ ID NO:28(E 4c ), SEQ ID NO:34(E 4d ), or the C-terminal domain of SEQ ID NO:54 (E 4e ) or SEQ ID NO:55 (E 4f ) have at least 70% sequence identity.

7. The cell according to any one of the preceding claims, wherein the compound is deferoxamine B or H.

8. The cell of any of the preceding claims, wherein the cell is selected from Aspergillus species, Corynebacterium species, Brevibacterium species, Bacillus species, Acinetobacter species, Alcaligenes species, Lactobacillus species, Paracoccus species, Lactococcus species, Candida species, Pichia species, Hansenula species, Kluyveromyces species, Saccharomyces species, Escherichia species, Zymomonas species, Yarrowia species, Methylobacterium species, Ralstonia species, Pseudomonas species, Rhodospirilla species, Rhodobacter species, Burkholderia species, Clostridium species, and Cuprilobacter species.

9. The cell of any one of the preceding claims, wherein the carbon source is selected from the group consisting of glucose, sucrose, xylose, arabinose, mannose, glycerol, lysine and cadaverine.

10. The cell according to any one of the preceding claims, wherein the cell comprises a further genetic modification to produce L-lysine.

11. The cell of claim 10, wherein the further genetic modification in the cell comprises an increased activity of at least one of the following enzymes relative to a wild-type cell: - pyruvate carboxylase (EC 6.4.1.1) (E6), -Aspartate aminotransferase (EC 2.6.1.1) (E7) - aspartate kinases, in particular feedback-resistant aspartate kinases (EC 2.7.2.4) (E8), - aspartate semialdehyde dehydrogenase (EC 1.2.1.11) (E9), -Dihydrodipicolinate synthase (EC 4.3.3.7) (E 10 ), -Dihydropicolinate reductase (EC 30 1.17.1.8) (E 11 ), -Diaminopimelate dehydrogenase (EC 1.4.1.16) (E 12 ), -Diaminopimelate epimerase (EC 5.1.1.7) (E 13 ), -Diaminopimelate decarboxylase (EC 4.1.1.20) (E 14 ), -N-succinyl-aminoketopimelate aminotransferase (EC 2.6.1.17) (E 17 ), -2,3,4,5-tetrahydropyridine-2,6-dicarboxylic acid N-succinyltransferase (EC 2.3.1.117) (E 18 ), and / or -Succinyldiaminopimelate desuccinylase (EC 3.5.1.18) (E 19 ); and / or the activity of at least one of the following enzymes is reduced relative to wild-type cells: -Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (E 15 ), and / or -Homoserine dehydrogenase (EC 1.1.1.3) (E 16 ).

12. The cell of claim 10 or 11, wherein the further genetic modification in the cell comprises an increased activity of at least one of the following relative to a wild-type cell: - pyruvate carboxylase (EC 6.4.1.1) (E6), - aspartate kinase (EC 2.7.2.4) (E8), in particular feedback-resistant aspartate kinase (EC 2.7.2.4) (E8), - aspartate semialdehyde dehydrogenase (EC 1.2.1.11) (E9), -Dihydrodipicolinate synthase (EC 4.3.3.7) (E 10 ), -Dihydropicolinate reductase (EC 30 1.17.1.8) (E 11 ), -Diaminopimelate dehydrogenase (EC 1.4.1.16) (E 12 ), and / or -Diaminopimelate decarboxylase (EC 4.1.1.20) (E 14 ), and / or Relative to wild-type cells, at least one of the following activities is reduced -Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) (E 15 ), and / or -Homoserine dehydrogenase (EC 1.1.1.3) (E 16 ).

13. The cell according to any one of claims 10 to 12, wherein the further genetic modification is: a) at least one promoter operably linked to a suitable chromosome of the cell encoding the enzyme E6-E 14 and E 17 -E 19 Any of the genes in b) at least one expression vector in the cell to increase the amount of the gene encoding the enzyme E6-E 14 and E 17 -E 19 The number of copies of any gene in c) Combination of (a) and (b) To increase the enzyme E6-E 14 and E 17 -E 19 Any of the activities and / or d) Encoding enzyme E 15 and E 16 Foreign DNA in at least one gene of e) Deletion of enzyme E 15 and E 16 at least a portion of a gene of at least one of; f) Encoding enzyme E 15 and E 16 at least one gene and / or at least one point mutation in the regulatory sequence of the gene, RNA interference (siRNA), antisense RNA; or g) A combination of (d), (e) and / or (f) To reduce the enzyme E 15 and E 16 at least one activity.

14. A method of producing at least one compound of formula II from at least one carbon source: n＝2-3 R1=H or COCH3 or CH2CH2COX, where X=OH or O- R2=CH3 or CH2CH2COX, where X=OH or O- The method comprises: (a) contacting the cell according to any one of claims 1 to 13 with at least one carbon source.

15. The method according to claim 14, wherein the activity of the enzyme is increased in the cell by a method selected from the group consisting of: a) introducing at least one promoter operably linked to a gene encoding an enzyme into the chromosome of said cell, b) increasing the copy number of the gene encoding the enzyme by introducing at least one expression vector into the cell, and c) combinations thereof.

16. Use of a cell according to any one of claims 1 to 13 for producing at least one compound of formula II from at least one carbon source: n＝2-3 R1=H or COCH3 or CH2CH2COX, where X=OH or O- R2=CH3 or CH2CH2COX, wherein X=OH or O-.

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