Method for producing spermidine

Abstract

The invention provides a method for producing spermidine comprising the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4- aspartylphosphate by a kinase; b1) converting the L-ornithine of step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate of step a2) to L-aspartate-4- semialdehyde by a dehydrogenase; c1) converting the putrescine of step b1) and the L- aspartate-4-semialdehyde of step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine of step c1) to spermidine by a decarboxylase. Further, the present invention provides a host cell for producing spermidine, and the use of said host cell for producing spermidine.

Description

METHOD FOR PRODUCING SPERMIDINETECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a method for producing spermidine. Specifically, the present invention provides a method for producing spermidine, comprising the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4- aspartylphosphate by a kinase; b1) converting the L-ornithine of step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate of step a2) to L-aspartate-4- semialdehyde by a dehydrogenase; c1) converting the putrescine of step b1) and the L- aspartate-4-semialdehyde of step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine of step c1) to spermidine by a decarboxylase. The present invention further provides a host cell for producing spermidine and the use of said host cell for producing spermidine.BACKGROUND ART

[0002] Spermidine is a naturally occurring polyamine that plays a crucial role in various biological processes, such as DNA synthesis, cell growth and differentiation. The study of spermidine synthesis and its regulation is a rapidly growing area of research, with potential implications for the development of novel therapies for various diseases7. In Escherichia coli, spermidine is synthesized by the enzymatic transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The transformation is catalyzed by the enzyme spermidine synthase, SpeE. E. coli can synthesize putrescine via two metabolic routes. One route yields putrescine directly by the decarboxylation of ornithine by the ornithine decarboxylase SpeC. Alternatively, SpeA first decarboxylates L-arginine to agmatine, which is then converted to putrescine by SpeB through the release of urea. Spermidine synthesis itself is essential for the maintenance of cellular homeostasis and proper physiological function. L- glutamic acid (L-glutamate) and L-arginine are amino acid precursors of spermidine.

[0003] Besides the widespread pathway via dcSAM, an alternative spermidine biosynthesis pathway exists, which does not rely on dcSAM as a cofactor and amino group donor. It employs carboxyspermidine as an alternative donor for the third amino group of spermidine2and is widespread among other bacteria next to E. coli, e.g. Vibrio spp. or Campylobacter jejunii. E. coli naturally does not use the carboxyspermidine pathway. The carboxyspermidine pathway represents a branch of the common spermidine pathway to circumvent the dcSAM step. Insteadof the addition of a third amino group to putrescine, the two amino groups of putrescine are added to the amino acid backbone of aspartic acid: Putrescine is first condensed with L- aspartate-4-semialdehyde to carboxyspermidine by the enzyme carboxyspermidine dehydrogenase (CASDH). This reaction requires NADPH as the co-factor. In the next step, carboxyspermidine decarboxylase (CASDC) decarboxylates carboxyspermidine to form spermidine.

[0004] Several microorganisms, such as Vibrio cholerae, Vibrio alginolyticus and Campylobacter jejunii, Clostridium leptum and Lactobacillus rogosae synthesize spermidine via carboxyspermidine and thus share CASDH and CASDC genes.3Various enzymes have already been described and tested for activity.3'5For example, Liang and co-workers engineered E. coli for the synthesis of spermidine from L-homoserine and putrescine in biotransformation reactions using cell extracts and a whole cell catalyst.6L-homoserine was converted to aspartate-4- semialdehyde by the homoserine dehydrogenase from S. cerevisiae. They used recombinant CASDH from Agrobacterium fabrum str. C58 (afcasdh, accession number: MW800769) and recombinant CASDC from Butyrivibrio crossotus DSM 2876, while Nakao et al.4focussed on CASDC from Vibrio alginolyticus. Under optimized conditions, Liang and co-workers obtained roughly 29 mM spermidine from 30 mM L-homoserine and 30 mM putrescine in 24 h, which represented a molar conversion rate of 95%. However, this approach is not very well suitable for upscaling, because the precursors L-homoserine and putrescine are expensive and not readily available in food grade. Chemically synthesized spermidine costs about 8-10000 € / kg as a bulk product. Thus, there is a need to provide a method for producing spermidine from inexpensive substrates and starting materials.

[0005] Thus, so far, several methods for the chemical synthesis of spermidine have been described. However, those methods either suffer from issues of expensive substrates or are environmental unfriendly. Additionally, effective conversion of carboxyspermidine to spermidine is a further challenge.

[0006] With the present invention, the inventors provide a sustainable, cheap, food-grade biosynthesis pathway for spermidine so that it can be used as a food additive. The inventors were able by the present invention to synthesize spermidine via carboxyspermidine from L- aspartate and L-arginine, which are inexpensive nutrients and food additives.SUMMARY OF THE INVENTION

[0007] The above mentioned problems are solved by the subject-matter as defined in the claims and as defined herein.

[0008] The present invention provides a method for producing spermidine, comprising the following steps:a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4-aspartylphosphate by a kinase; b1) converting the L-ornithine of step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate of step a2) to L-aspartate-4-semialdehyde by a dehydrogenase; c1) converting the putrescine of step b1) and the L-aspartate-4-semialdehyde of step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine of step c1) to spermidine by a decarboxylase.

[0009] In some embodiments of the method of the present invention, the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are from Paracoccus denitrificans, and / or the arginase of step a1) is from Bacillus subtilis, and / or the kinase of step a2) is from Escherichia coli, and / or the decarboxylase of step b1) is from Escherichia coli, and / or the dehydrogenase of step b2) is from Escherichia coli.

[0010] In some embodiments of the method of the present invention, the arginase of step a1) is an arginase of EC-number 3.5.3.1 , preferably the arginase of step a1)(i) has the amino acid sequence shown in SEQ ID NO: 1,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 2, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 2.

[0011] In some embodiments of the method of the present invention, the kinase of step a2) is an aspartate kinase, preferably an aspartate kinase of EC-number 2.7.2.4. More preferably, the aspartate kinase of step a2)(i) has the amino acid sequence shown in SEQ ID NO: 3,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 3,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 4, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 4.

[0012] In some embodiments of the method of the present invention, the decarboxylase of step b1) is an ornithine decarboxylase, preferably an ornithine decarboxylase of EC-number 4.1.1.17. More preferably, the ornithine decarboxylase of step b1)(i) has the amino acid sequence shown in SEQ ID NO: 5,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 5,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 6, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 6.

[0013] In some embodiments of the method of the present invention, the dehydrogenase of step b2) is an aspartate-semialdehyde-dehydrogenase, preferably an aspartate-semialdehyde- dehydrogenase of EC-number 1.2.1.11. More preferably, the aspartate-semialdehyde- dehydrogenase of step b2)(i) has the amino acid sequence shown in SEQ ID NO: 7,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 7,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 8, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 8.

[0014] In some embodiments of the method of the present invention, the dehydrogenase of step c1) is a carboxyspermidine dehydrogenase, preferably a carboxyspermidine dehydrogenase of EC-number 1.5.1.43. More preferably, the carboxyspermidine dehydrogenase of step c1)(i) has the amino acid sequence shown in SEQ ID NO: 9,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 9,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 10, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 10.

[0015] In some embodiments of the method of the present invention, the decarboxylase of step c2) is a carboxyspermidine decarboxylase, preferably a carboxyspermidine decarboxylase of EC-number 4.1.1.96. More preferably, the carboxyspermidine decarboxylase(i) has the amino acid sequence shown in SEQ ID NO: 11 ,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 11,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0016] In some embodiments of the method of the present invention, the arginase of step a1), and / or the kinase of step a2), and / or the decarboxylase of step b1), and / or the dehydrogenase of step b2), and / or the dehydrogenase of step c1), and / or the decarboxylase of step c2) is / are present as purified enzyme(s) or is / are comprised in a cell extract(s).

[0017] In some embodiments of the method of the present invention, step a2) further comprises the conversion of ATP to ADP. Preferably, the regeneration of ATP from ADP is by using apolyphosphate kinase, more preferably a polyphosphate kinase of EC-number 2.7.4.1. Additionally or alternatively, step b2) may further comprise the conversion of NADPH to NADP+. Preferably, the regeneration of NADPH from NADP+is by using a glucose-1 -dehydrogenase and / or by addition of glucose, more preferably by using a glucose-1-dehydrogenase of EC- number 1.1.1.119 by conversion of glucose to gluconolactone. Additionally or alternatively, c1) may further comprise the conversion of NADPH to NADP+. Preferably, the regeneration of NADPH from NADP+is by using a glucose-1 -dehydrogenase and / or by addition of glucose, more preferably by using a glucose-1-dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone.

[0018] The present invention further provides a host cell for producing spermidine, wherein said host cell is genetically modified to overexpress at least one, preferably all, enzyme(s) selected from the group consisting of an arginase, an aspartate kinase, an ornithine decarboxylase, an aspartate-semialdehyde-dehydrogenase, a carboxyspermidine dehydrogenase and a carboxyspermidine decarboxylase.

[0019] For the host cell of the present invention it is preferred that the arginase is an arginase of EC-number 3.5.3.1, and / or that the aspartate kinase is an aspartate kinase of EC-number 2.7.2.4, and / or that the ornithine decarboxylase is an ornithine decarboxylase of EC-number 4.1.1.17, and / or that the aspartate-semialdehyde-dehydrogenase is an aspartate-semialdehyde- dehydrogenase of EC-number 1.2.1.11 , and / or that the carboxyspermidine dehydrogenase is a carboxyspermidine dehydrogenase of EC-number 1.5.1.43, and / or that the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96.

[0020] Further, the host cell of the present invention is preferably genetically modified to overexpress a carboxyspermidine decarboxylase. More preferably, the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96. Even more preferably, the carboxyspermidine decarboxylase has the amino acid sequence shown in SEQ ID NO: 11 and / or is encoded by a nucleotide sequence shown in SEQ ID NO: 12.

[0021] Preferably, the host cell of the present invention is a bacterial host cell, a yeast host cell, a plant host cell or an insect host cell. Preferably, the bacterial host cell is a probiotic bacterial host cell. More preferably, the bacterial host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida. Preferably, the host cell of the present invention may also be a yeast host cell. The yeast host cell may be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia pastoris and Kluyveromyces lactis.

[0022] In a further aspect, the present invention provides the use of the host cell according to the present invention for producing spermidine, preferably in a mammal.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows the biosynthesis of spermidine in E. coli and the integration of the recombinant carboxyspermidine pathway. E. coli enzymes are indicated, non-E. coli enzymes of the carboxyspermidine pathway are indicated in bold. Metabolites relevant for this study are highlighted in light grey.

[0024] Figure 2 shows the reconstituted biosynthesis cascade of spermidine via carboxyspermidine starting from L-arginine and L-aspartate as inexpensive precursors. Recombinant enzymes and their EC numbers are indicated.

[0025] Figure 3 shows that PdCASDC swiftly converted carboxyspermidine to spermidine.

[0026] Figure 4 shows that exclusively putrescine was produced from L-arginine and L- aspartate-4-semialdehyde. Putrescine did not further react to carboxyspermidine or spermidine.

[0027] Figure 5 shows that ARG1 and SpeC converted L-arginine efficiently to putrescine.

[0028] Figure 6 shows that L-aspartate-4-semialdehyde appears not to be inhibiting when it is constantly converted to carboxyspermidine and spermidine. After 2 h, no conversion to spermidine was visible.

[0029] Figure 7 shows that the complete biocascade converted L-aspartate and L-arginine to spermidine. After 2 h, no conversion to spermidine was visible.

[0030] Figure 8 shows maps of the vectors used in this study. Figure 8A shows pET21- argl_speC; Figure 8B shows pET21-thrA_asd; Figure 8C shows pET29-PdCASDC; Figure 8D shows pET29-VaCASDC; Figure 8E shows pET29-PdCASDH. The used abbreviations have the following meaning: AmpR, ampicillin resistance marker; bom, basis of mobility region; H6, hexahistidine-tag; KanR, kanamycin resistance marker; lacl, lac repressor; lacO, lac operator; ori, origin of replication; Placl, lacl promoter; PlacUV5, lacUV5 promoter; Ptac, tac promoter; RBS, ribosome binding site; rom, encodes repressor of primer protein; ‘rrnB T2’, partial sequence of the rrnB T2 terminator; T7p, T7 promoter; T7t, T7 terminator; TEV, cleavage site for tobacco etch virus (TEV) protease.

[0031] Figure 9 shows the conversion of carboxyspermidine to spermidine by PdCASDC and VaCASDC after different storage conditions. The mean of three technical triplicates is shown.

[0032] Figure 10 shows the conversion of carboxyspermidine to spermidine by immobilized recombinant PdCASDC and VaCASDC. A single measurement is shown.DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will now be described hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalogue of all the different ways in which theinvention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0034] Unless otherwise defined herein, scientific and technical terms used herein will have the meanings that are commonly understood by those of ordinary skill in the art.

[0035] Generally, nomenclatures used in connection with techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization, described herein, are those well-known and commonly used in the art.

[0036] In one aspect, the present invention provides a method for producing spermidine, comprising the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4-aspartylphosphate by a kinase; b1) converting the L-ornithine of step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate of step a2) to L-aspartate-4-semialdehyde by a dehydrogenase; c1) converting the putrescine of step b1) and the L-aspartate-4-semialdehyde of step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine of step c1) to spermidine by a decarboxylase.

[0037] In some embodiments of the method for producing spermidine according to the present invention, the method comprises the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4-aspartylphosphate by a kinase; b1) converting the L-ornithine produced in step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate produced in step a2) to L-aspartate-4-semialdehyde by a dehydrogenase; c1) converting the putrescine produced in step b1) and the L-aspartate-4-semialdehyde produced in step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine produced in step c1) to spermidine by a decarboxylase.

[0038] In some embodiments of the method for producing spermidine according to the present invention, the method comprises the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4-aspartylphosphate by a kinase; b1) converting the L-ornithine obtained in step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate obtained in step a2) to L-aspartate-4-semialdehyde by a dehydrogenase; c1) converting the putrescine obtained in step b1) and the L-aspartate-4-semialdehyde obtained in step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine obtained in step c1) to spermidine by a decarboxylase.

[0039] As used herein, the term “arginase” refers to any enzyme that is able to hydrolyse arginine into ornithine and urea.

[0040] Further, the term “kinase” as used herein means an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates, specifically, in the case of an aspartate kinase, as described herein and in the context of the present invention, the transfer of (a) phosphate group(s) from aspartate to aspartylphosphate. This process is known as phosphorylation, where the high-energy ATP molecule donates a phosphate group to the substrate molecule. This transesterification produces a phosphorylated substrate and ADP.

[0041] As used herein, the expression “decarboxylase” refers to an enzyme that catalyses the removal of carbon dioxide from a compound.

[0042] Further, the term “dehydrogenase” as used herein means any enzyme that accelerates the removal of hydrogen from metabolites and its transfer to other substances.

[0043] As used herein, L-arginine is presented by the following chemical structure:

[0044] Further, L-aspartate as used in the present invention is characterized by the following chemical structure:

[0045] As used herein, L-ornithine is characterized by the following chemical structure:

[0046] Further, L-4-aspartylphosphate as used in the present invention is characterized by the following chemical structure:

[0047] As used herein, putrescine is characterized by the following chemical structure:

[0048] Further, L-aspartate-4-semialdehyde as used in the present invention is characterized by the following chemical structure:

[0049] As used herein, carboxyspermidine is characterized by the following chemical structure:

[0050] Further, spermidine as used in the present invention is characterized by the following chemical structure:

[0051] Also, as used herein, the terms "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" refer to RNA or DNA, including cDNA, a DNA fragment or portion, genomic DNA, synthetic DNA, plasmid DNA, mRNA, and antisense RNA, any of which can be single-stranded or double-stranded, linear or branched, or a hybrid thereof. Nucleic acid molecules and / or nucleotide sequences provided herein are presented herein in the 5'- to 3'-direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth e.g. in the World Intellectual Property Organization (WIPO) Standard ST.25.

[0052] As used herein, the terms “peptide”, “polypeptide”, and “protein” are used interchangeably to indicate a polymer of amino acid residues.

[0053] Nucleic acid molecules and / or nucleotide sequences and / or protein sequences provided herein may be exogenous or heterologous, preferably heterologous. As used herein, the terms "exogenous" or "heterologous" when used with respect to a nucleic acid (RNA or DNA), protein or gene refer to a nucleic acid, protein or gene which occurs non-naturally as part of the cell, organism, genome, RNA or DNA sequence into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Such an exogenous gene could be a gene from another species or strain, a modified, mutated or evolved version of a gene naturally occurring in the host cell or a chimeric version of a gene naturally occurring in the host cell or fusion genes. In these former cases, the modification, mutation or evolution causes a change in the nucleotide sequence of the gene to thereby obtain a modified, mutated or evolved gene with another nucleotide sequence as compared to the gene naturally occurring in the host cell. Evolved gene refers to genes encoding evolved genes and obtained by genetic modification, such as mutation or exposure to an evolutionary pressure, to derive a new gene with a different nucleotide sequence as compared to the wild type or native gene. A chimeric gene is formed through the combination of portions of one or more coding sequences to produce a new gene. These modifications are distinct from a fusion gene, which merges whole gene sequences into a single reading frame and often retain their original functions.

[0054] As used herein, the term “enzyme” is defined as a protein which catalyses a chemical or a biochemical reaction in a cell.

[0055] In some embodiments of the method of the present invention, the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are from Paracoccus denitrificans, and / or the arginase of step a1) is from Bacillus subtilis, and / or the kinase of step a2) is from Escherichia coli, and / or the decarboxylase of step b1) is from Escherichia coli, and / or the dehydrogenase of step b2) is from Escherichia coli. In some preferred embodiments of the method of the present invention, the dehydrogenase of step c1) and the decarboxylase of step c2) are from Paracoccus denitrificans, and the arginase of step a1) is from Bacillus subtilis, and the kinase of step a2) is from Escherichia coli, and the decarboxylase of step b1) is from Escherichia coli, and the dehydrogenase of step b2) is from Escherichia coli.

[0056] It is preferred for the method of the present invention that the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are from Paracoccus denitrificans. It is preferred for the method of the present invention that the dehydrogenase used in step c1) and / or the decarboxylase used in step c2) is / are from Paracoccus denitrificans. It is also preferred for the method of the present invention that the dehydrogenase of step c1) or the decarboxylase of step c2) is / are from Paracoccus denitrificans. It is also preferred for the method of the present invention that the dehydrogenase used in step c1) or the decarboxylase used in step c2) is / are from Paracoccus denitrificans. It is further preferred for the method of the present invention that the dehydrogenase of step c1) and the decarboxylase of step c2) are from Paracoccus denitrificans. It is further preferred for the method of the present invention that the dehydrogenase used in step c1) and the decarboxylase used in step c2) are from Paracoccus denitrificans.

[0057] It is preferred for the method of the present invention that the arginase of step a1) is from Bacillus subtilis. It is also preferred for the method of the present invention that the arginase used in step a1) is from Bacillus subtilis.

[0058] It is further preferred for the method of the present invention that the kinase of step a2) is from Escherichia coli. It is also preferred for the method of the present invention that the kinase used in step a2) is from Escherichia coli.

[0059] It is preferred for the method of the present invention that the decarboxylase of step b1) is from Escherichia coli. It is also preferred for the method of the present invention that the decarboxylase used in step b1) is from Escherichia coli.

[0060] It is preferred for the method of the present invention that the dehydrogenase of step b2) is from Escherichia coli. It is also preferred for the method of the present invention that the dehydrogenase used in step b2) is from Escherichia coli.

[0061] In some embodiments of the method of the present invention, the arginase of step a1) is an arginase of EC-number 3.5.3.1.

[0062] It is preferred that said arginase of step a1)(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 ,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1 ,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 2, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 2.

[0063] It is also preferred that said arginase of step a1)(i) has the amino acid sequence shown in SEQ ID NO: 1 ,(ii) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 2, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 2.

[0064] It is preferred that said arginase of step a1) or used in step a1) comprises the amino acid sequence as shown in SEQ ID NO: 1. It is also preferred that said arginase of step a1) has the amino acid sequence shown in SEQ ID NO: 1. It is also preferred that said arginase of step a1) consists of the amino acid sequence shown in SEQ ID NO: 1. It is also preferred that said arginase of step a1) or used in step a1) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1. It is also preferred that said arginase of step a1) or used in step a1) is encoded by a nucleotide sequence shown in SEQ ID NO: 2. It is also preferred that said arginase of step a1) or used in step a1) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 2.

[0065] In some embodiments of the method of the present invention, the kinase of step a2) is an aspartate kinase.

[0066] It is preferred that the kinase of step a2) is an aspartate kinase of EC-number 2.7.2.4.

[0067] It is further preferred that the aspartate kinase of step a2)(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 3,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 4, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 4.

[0068] It is further preferred that the aspartate kinase of step a2)(i) has the amino acid sequence shown in SEQ ID NO: 3,(ii) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 4, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 4.

[0069] It is further preferred that the aspartate kinase of step a2) comprises the amino acid sequence shown in SEQ ID NO: 3. It is also preferred that the aspartate kinase of step a2) has the amino acid sequence shown in SEQ ID NO: 3. It is further preferred that the aspartate kinase of step a2) consists of the amino acid sequence shown in SEQ ID NO: 3. It is also preferred that the aspartate kinase of step a2) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3. It is further preferred that the aspartate kinase of step a2) is encoded by a nucleotide sequence shown in SEQ ID NO: 4. It is also preferred that the aspartate kinase of step a2) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 4.

[0070] In some embodiments of the method of the present invention, the decarboxylase of step b1) is an ornithine decarboxylase.

[0071] It is preferred that the decarboxylase of step b1) is an ornithine decarboxylase of EC- number 4.1.1.17.

[0072] It is further preferred that the ornithine decarboxylase of step b1)(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 5,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 6, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 6.

[0073] It is further preferred that the ornithine decarboxylase of step b1)(i) has the amino acid sequence shown in SEQ ID NO: 5,(ii) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 6, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 6.

[0074] It is further preferred that the ornithine decarboxylase of step b1) or used in step b1) comprises the amino acid sequence shown in SEQ ID NO: 5. It is also preferred that the ornithine decarboxylase of step b1) has the amino acid sequence shown in SEQ ID NO: 5. It is further preferred that the ornithine decarboxylase of step b1) consists of the amino acid sequence shown in SEQ ID NO: 5. It is also preferred that the ornithine decarboxylase of step b1) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQID NO: 5. It is also preferred that the ornithine decarboxylase of step b1) is encoded by a nucleotide sequence shown in SEQ ID NO: 6. It is further preferred that the ornithine decarboxylase of step b1) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 6.

[0075] In some embodiments of the method of the present invention, the dehydrogenase of step b2) is an aspartate-semialdehyde-dehydrogenase.

[0076] It is preferred that the dehydrogenase of step b2) is an aspartate-semialdehyde- dehydrogenase of EC-number 1.2.1.11.

[0077] It is further preferred that the aspartate-semialdehyde-dehydrogenase of step b2)(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 7,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 7,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 8, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 8.

[0078] It is also preferred that the aspartate-semialdehyde-dehydrogenase of step b2)(i) has the amino acid sequence shown in SEQ ID NO: 7,(ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 7,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 8, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 8.

[0079] It is further preferred that the aspartate-semialdehyde-dehydrogenase of step b2) comprises the amino acid sequence shown in SEQ ID NO: 7. It is also preferred that the aspartate-semialdehyde-dehydrogenase of step b2) has the amino acid sequence shown in SEQ ID NO: 7. It is further preferred that the aspartate-semialdehyde-dehydrogenase of stepb2) consists of the amino acid sequence shown in SEQ ID NO: 7. It is also preferred that the aspartate-semialdehyde-dehydrogenase of step b2) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 7. It is further preferred that the aspartate-semialdehyde-dehydrogenase of step b2) is encoded by a nucleotide sequence shown in SEQ ID NO: 8. It is also preferred that the aspartate-semialdehyde-dehydrogenase of step b2) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 8.

[0080] In some embodiments of the method of the present invention, the dehydrogenase of step c1) is a carboxyspermidine dehydrogenase.

[0081] It is preferred that the dehydrogenase of step c1) is a carboxyspermidine dehydrogenase of EC-number 1.5.1.43.

[0082] It is further preferred that the carboxyspermidine dehydrogenase of step c1)(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 9,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 9,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 10, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 10.

[0083] It is also preferred that the carboxyspermidine dehydrogenase of step c1)(i) has the amino acid sequence shown in SEQ ID NO: 9,(ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 9,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 10, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 10.

[0084] It is further preferred that the carboxyspermidine dehydrogenase of step c1) comprises the amino acid sequence shown in SEQ ID NO: 9. It is also preferred that the carboxyspermidine dehydrogenase of step c1) has the amino acid sequence shown in SEQ ID NO: 9. It is further preferred that the carboxyspermidine dehydrogenase of step c1) consists of the amino acid sequence shown in SEQ ID NO: 9. It is also preferred that the carboxyspermidine dehydrogenase of step c1) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 9. It is further preferred that the carboxyspermidine dehydrogenase of step c1) is encoded by a nucleotide sequence shown in SEQ ID NO: 10. It is also preferred that the carboxyspermidine dehydrogenase of step c1) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 10.

[0085] In some embodiments of the method of the present invention, the decarboxylase of step c2) is a carboxyspermidine decarboxylase.

[0086] It is preferred that the decarboxylase of step c2) is a carboxyspermidine decarboxylase of EC-number 4.1.1.96.

[0087] It is further preferred that the carboxyspermidine decarboxylase of step c2)(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 11 ,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11 ,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0088] It is also preferred that the carboxyspermidine decarboxylase of step c2)(i) has the amino acid sequence shown in SEQ ID NO: 11 ,(ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0089] It is further preferred that the carboxyspermidine decarboxylase of step c2) comprises the amino acid sequence shown in SEQ ID NO: 11. It is also preferred that the carboxyspermidine decarboxylase of step c2) has the amino acid sequence shown in SEQ ID NO: 11. It is further preferred that the carboxyspermidine decarboxylase of step c2) consists of the amino acid sequence shown in SEQ ID NO: 11. It is also preferred that the carboxyspermidine decarboxylase of step c2) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11. It is further preferred that the carboxyspermidine decarboxylase of step c2) is encoded by a nucleotide sequence shown in SEQ ID NO: 12. It is also preferred that the carboxyspermidine decarboxylase of step c2) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0090] It is also preferred for the method of the present invention that the arginase of step a1), and / or the kinase of step a2), and / or the decarboxylase of step b1), and / or the dehydrogenase of step b2), and / or dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are present as purified enzyme(s) or is / are comprised in a cell extract(s).

[0091] It is further preferred for the method of the present invention that the arginase of step a1) is present as a purified enzyme. It is also preferred for the method of the present invention that the arginase of step a1) is comprised in a cell extract.

[0092] It is further preferred for the method of the present invention that the kinase of step a2) is present as a purified enzyme. It is also preferred for the method of the present invention that the kinase of step a2) is comprised in a cell extract.

[0093] It is further preferred for the method of the present invention that the decarboxylase of step b1) is present as a purified enzyme. It is also preferred for the method of the present invention that the decarboxylase of step b1) is comprised in a cell extract.

[0094] It is further preferred for the method of the present invention that the dehydrogenase of step b2) is present as a purified enzyme. It is also preferred for the method of the present invention that the dehydrogenase of step b2) is comprised in a cell extract.

[0095] It is further preferred for the method of the present invention that the dehydrogenase of step c1) is present as a purified enzyme. It is also preferred for the method of the present invention that the dehydrogenase of step c1) is comprised in a cell extract.

[0096] It is further preferred for the method of the present invention that the decarboxylase of step c2) is present as a purified enzyme. It is also preferred for the method of the present invention that the decarboxylase of step c2) is comprised in a cell extract.

[0097] "Extract", as used in the context of "cell extract" in this invention, means a product of any type of cell, as defined above, obtained by chemical or mechanical action, such as pressure, distillation, evaporation, etc. The extracts may include all or any individually selected component or combination of cell components, including concentrated products of the active components. Such extract components include, but are not limited to, RNA, DNA, lipids, all amino acid-based structures, including peptides and proteins, carbohydrates, or combinations thereof. Extracts contemplated by the present invention include, but are not limited to, extracts from the organisms Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida or Saccharomyces cerevisiae.

[0098] In some embodiments of the method of the present invention, step a2) further comprises the conversion of ATP to ADP. Preferably, the regeneration of ATP from ADP is by using a polyphosphate kinase, more preferably a polyphosphate kinase of EC-number 2.7.4.1.

[0099] In some embodiments of the method of the present invention, step b2) further comprises the conversion of NADPH to NADP+. Preferably, the regeneration of NADPH from NADP+is by using a glucose- 1 -dehydrogenase and / or by addition of glucose, more preferably by using a glucose-1-dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone.

[0100] In some embodiments of the method of the present invention, step c1) further comprises the conversion of NADPH to NADP+. Preferably, the regeneration of NADPH from NADP+is by using a glucose- 1 -dehydrogenase and / or by addition of glucose, more preferably by using a glucose-1-dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone.

[0101] For example, the inventors of the present invention provide a cascade for producing spermidine by recombinantly expressing for example the following genes in e.g. E. coli, which are depicted as an exemplary embodiment in Figure 2 shown herein:- L-arginine branch, arg1 (arginase, EC 3.5.3.1) from Bacillus subtilis and speC (ornithine decarboxylase, EC 4.1.1.17) from E. coir,- L-aspartate branch, thrA (aspartate kinase, EC 2.7.2.4) and asd (aspartate semialdehyde dehydrogenase, EC 1.2.1.11) from E. coli. ThrA is a bifunctional enzyme that catalyzes the first step in the biosynthesis of lysine and homoserine, and indirectly methionine and threonine, as well as the final step in homoserine biosynthesis. ASD catalyzes an intermediary step in homoserine biosynthesis where it generates L-aspartate-4-semialdehyde.- Carboxyspermidine branch, CASDH (carboxyspermidine dehydrogenase, EC 1.5.1.43) and CASDC (carboxyspermidine decarboxylase, EC 4.1.1.96) from Paracoccus denitrificans (PdCASDH).

[0102] NADPH is preferably recycled using commercially available, food-grade GDH (glucose 1 -dehydrogenase (NADP+), EC 1.1.1.119). Soluble protein fractions containing the recombinant enzymes may be prepared as described herein, mixed and the reaction may be started by the addition of L-arginine, L-aspartate and glucose for the entire biocascade or the corresponding intermediate to follow the individual reactions.

[0103] Enzyme Commission (EC) numbers, referred to throughout this description, are according to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) in its resource “Enzyme Nomenclature” (1992, including Supplements 6-17) available, for example, as “Enzyme nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes”, Webb, E. C. (1992), San Diego: Published for the International Union of Biochemistry and Molecular Biology by Academic Press (ISBN 0-12-227164-5). This is a numerical classification scheme based on the chemical reactions catalyzed by each enzyme class.

[0104] The terms "sequence identity", "identity", "identical" or being "identical" as used in the present invention means the percentage of pair-wise identical residues, following homology alignment of a sequence of a polypeptide of the present invention with a sequence in question, with respect to the number of residues in the longer of these two sequences. The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). The percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10'3) including the respective sequences. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0105] In a further aspect, the present invention is directed to a method for producing spermidine from carboxyspermidine comprising the step of converting carboxyspermidine to spermidine by a carboxyspermidine decarboxylase.

[0106] It is preferred for that method of the present invention that the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96.

[0107] It is further preferred for that method of the present invention that the carboxyspermidine decarboxylase(i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 11 ,(ii) comprises or consists of an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11 ,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0108] It is also preferred for that method of the present invention that the carboxyspermidine decarboxylase(i) has the amino acid sequence shown in SEQ ID NO: 11 ,(ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11 ,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0109] It is further preferred for that method of the present invention that the carboxyspermidine decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 11. It is also preferred that said carboxyspermidine decarboxylase has the amino acid sequence shown in SEQ ID NO: 11. It is further preferred that said carboxyspermidine decarboxylase consists of the amino acid sequence shown in SEQ ID NO: 11. It is also preferred that said carboxyspermidine decarboxylase has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence shown in SEQ ID NO: 11. It is further preferred that said carboxyspermidine decarboxylase is encoded by a nucleotide sequence shown in SEQ ID NO: 12. It is also preferred that said carboxyspermidine decarboxylase is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0110] In a further aspect, the present invention provides a host cell for producing spermidine, wherein said host cell is genetically modified to overexpress at least one, preferably all, enzyme(s) selected from the group consisting of an arginase, an aspartate kinase, an ornithine decarboxylase, an aspartate-semialdehyde-dehydrogenase, a carboxyspermidine dehydrogenase and a carboxyspermidine decarboxylase.

[0111] It is preferred for the host cell for producing spermidine of the present invention that said host cell is genetically modified to overexpress an arginase, an aspartate kinase, an ornithine decarboxylase, an aspartate-semialdehyde-dehydrogenase, a carboxyspermidine dehydrogenase and a carboxyspermidine decarboxylase.

[0112] “Cell", as used herein, means the smallest structural unit of living matter capable of functioning autonomously, consisting of one or more nuclei, cytoplasm and various organelles, all together surrounded by a semipermeable membrane.

[0113] The term "overexpress", "overexpresses" or "overexpression" as used herein refers to higher levels of activity of a gene, e.g. transcription of the gene; higher levels of translation of mRNA into protein; and / or higher levels of production of a gene product, e.g. polypeptide, than would be in the cell in its native or control, e.g. not transformed with the particular heterologous or recombinant polypeptides being overexpressed, state. A typical example of an overexpressed gene is a gene under transcription control of another promoter as compared to the native promoter of the gene. Also, or alternatively, other changes in the control elements of a gene, such as enhancers, could be used to overexpress the particular gene. Furthermore, modifications that affect, i.e. increase, the translation of the mRNA transcribed from the gene could, alternatively or in addition, be used to achieve an overexpressed gene as used herein. These terms can also refer to an increase in the number of copies of a gene and / or an increase in the amount of mRNA and / or gene product in the cell. Overexpression can result in levels that are 25%, 50%, 100%, 200%, 500%, 1000%, 2000% or higher in the cell, or any range therein, as compared to control levels.

[0114] As used herein, the term “modified”, when it is used with respect to an organism, refers to a host organism that has been modified to increase production of spermidine, as compared with an otherwise identical host organism that has not been so modified. In principle, such “modification” in accordance with the present disclosure may comprise any physiological, genetic, chemical, or other modification that appropriately alters production of spermidine in ahost organism as compared with such production in an otherwise identical organism which is not subject to the said modification. In most of the embodiments, however, the modification will comprise a genetic modification. In certain embodiments, as described herein, the modification comprises introducing genes into a host cell. In some embodiments, a modification comprises at least one physiological, chemical, genetic, or other modification; in other embodiments, a modification comprises more than one chemical, genetic, physiological, or other modification. In certain aspects where more than one modification is made use of, such modifications can include any combinations of physiological, genetic, chemical, or other modification (e.g., one or more genetic, chemical and / or physiological modification(s)). Genetic modifications which boost the activity of a polypeptide include, but are not limited to: introducing one or more copies of a gene encoding the polypeptide (which may distinguish from any gene already present in the host cell encoding a polypeptide having the same activity); altering a gene present in the cell to increase transcription or translation of the gene (e.g., altering, adding additional sequence to, replacement of one or more nucleotides, deleting sequence from, or swapping, for example, regulatory, a promoter or other sequence); and altering the sequence (e.g. non-coding or coding) of a gene encoding the polypeptide to boost activity (e.g., by increasing enzyme activity, decrease feedback inhibition, targeting a specific subcellular location, boost mRNA stability, boost protein stability). Genetic modifications that reduce activity of a polypeptide include, but are not limited to: deleting a portion or all of a gene encoding the polypeptide; inserting a nucleic acid sequence which disrupts a gene encoding the polypeptide; changing a gene present in the cell to reduce transcription or translation of the gene or stability of the mRNA or polypeptide encoded by the gene (for example, by adding additional sequence to, altering, deleting sequence from, replacement of one or more nucleotides, or swapping, for example, replacement of one or more nucleotides, a promoter, regulatory or other sequence).

[0115] It is preferred for the host cell of the present invention that the arginase is an arginase of EC-number 3.5.3.1, and / or that the aspartate kinase is an aspartate kinase of EC- number 2.7.2.4, and / or that the ornithine decarboxylase is an ornithine decarboxylase of EC- number 4.1.1.17, and / or that the aspartate-semialdehyde-dehydrogenase is an aspartate- semialdehyde-dehydrogenase of EC-number 1.2.1.11, and / or that the carboxyspermidine dehydrogenase is a carboxyspermidine dehydrogenase of EC-number 1.5.1.43, and / or that the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96.

[0116] It is further preferred for the host cell of the present invention that the host cell is genetically modified to overexpress an arginase of EC-number 3.5.3.1 , wherein the arginase of EC-number 3.5.3.1 (i) has the amino acid sequence shown in SEQ ID NO: 1, (ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 1 , (iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 2, or (iv) is encoded by a nucleotide sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 2.

[0117] It is further preferred for the host cell of the present invention that the host cell is genetically modified to overexpress an aspartate kinase of EC-number 2.7.2.4, wherein the aspartate kinase of EC-number 2.7.2.4 (i) has the amino acid sequence shown in SEQ ID NO: 3, (ii) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 3, (iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 4, or (iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 4.

[0118] It is further preferred for the host cell of the present invention that the host cell is genetically modified to overexpress an ornithine decarboxylase of EC-number 4.1.1.17, wherein the ornithine decarboxylase of EC-number 4.1.1.17 (i) has the amino acid sequence shown in SEQ ID NO: 5, (ii) has an amino acid sequence which is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 5, (iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 6, or (iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 6.

[0119] It is further preferred for the host cell of the present invention that the host cell is genetically modified to overexpress an aspartate-semialdehyde-dehydrogenase of EC-number 1.2.1.11 , wherein the aspartate-semialdehyde-dehydrogenase of EC-number 1.2.1.11 (i) has the amino acid sequence shown in SEQ ID NO: 7, (ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 7, (iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 8, or (iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 8.

[0120] It is further preferred for the host cell of the present invention that the host cell is genetically modified to overexpress a carboxyspermidine dehydrogenase of EC-number 1.5.1.43, wherein the carboxyspermidine dehydrogenase of EC-number 1.5.1.43 (i) has the amino acid sequence shown in SEQ ID NO: 9, (ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 9, (iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 10, or (iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 10.

[0121] It is also preferred for the host cell of the present invention that the host cell is genetically modified to overexpress a carboxyspermidine decarboxylase, wherein the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96. It is further preferred for the host cell of the present invention that the carboxyspermidine decarboxylase (i) has the amino acid sequence shown in SEQ ID NO: 11, (ii) has an amino acid sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 11, (iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or (iv) is encoded by a nucleotide sequence which is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 12.

[0122] It is preferred for the host cell of the present invention that the host cell is a bacterial host cell, a yeast host cell, a plant host cell or an insect host cell. For example, the host cell of the present invention may be a bacterial host cell. Further, the host cell of the present invention may be a probiotic bacterial host cell. The term “probiotic” as used in the present invention may mean a live microorganism or live microorganisms that are intended to have health benefits when consumed or applied to the body. They can be found in yogurt and other fermented foods, dietary supplements and beauty products. Probiotics may contain a variety of microorganisms. The most common are bacteria that belong to groups called Lactobacillus and Bifidobacterium. Other bacteria may also be used as probiotics, and so may yeasts such as Saccharomyces boulardii. For example, the bacterial host cell may be selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonasputida. Further, the host cell of the present invention may be a yeast host cell. For example, the yeast host cell may be selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia pastoris and Kluyveromyces lactis. Further, the host cell of the present invention may be a plant host cell. For example, the plant host cell may be selected from the group consisting of Arabidopsis spp., preferably Arabidopsis thaliana, Nicotiana spp., preferably Nicotiana tabacum, Triticum spp., preferably Triticum aestivum, Zea mays and Glycine max. Further, the host cell of the present invention may be an insect host cell. For example, the insect host cell may be selected from the group consisting of Spodoptera frugiperda, Trichoplusia ni and Drosophila melanogaster.

[0123] In a further aspect, the present invention provides the use of the host cell according to the invention and as described herein for producing spermidine, preferably in a mammal. In a further aspect, the present invention provides the non-medical use of the host cell according to the present invention and as described herein for producing spermidine, preferably in a mammal.

[0124] The following sequences are used herein and in the context of the present invention:

[0125] Table 1 : Amino acid sequences used herein and in the context of the present invention.

[0126] Table 2: Nucleotide sequences used herein and in the context of the present invention.

[0127] The present invention is further characterized by the following items:1. A method for producing spermidine, comprising the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4-aspartylphosphate by a kinase; b1) converting the L-ornithine of step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate of step a2) to L-aspartate-4-semialdehyde by a dehydrogenase; c1) converting the putrescine of step b1) and the L-aspartate-4-semialdehyde of step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine of step c1) to spermidine by a decarboxylase.2. The method of item 1, wherein the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are from Paracoccus denitrificans, and / or wherein the arginase of step a1) is from Bacillus subtilis, and / or wherein the kinase of step a2) is from Escherichia coli, and / or wherein the decarboxylase of step b1) is from Escherichia coli, and / or wherein the dehydrogenase of step b2) is from Escherichia coli.3. The method of item 1 or 2, wherein the arginase of step a1) is an arginase of EC-number 3.5.3.1, preferably wherein the arginase of step a1)(i) has the amino acid sequence shown in SEQ ID NO: 1 ,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 2, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 2.4. The method of any one of the preceding items, wherein the kinase of step a2) is an aspartate kinase, preferably an aspartate kinase of EC-number 2.7.2.4, more preferably wherein the aspartate kinase of step a2)(i) has the amino acid sequence shown in SEQ ID NO: 3,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 3,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 4, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 4.5. The method of any one of the preceding items, wherein the decarboxylase of step b1) is an ornithine decarboxylase, preferably an ornithine decarboxylase of EC-number 4.1.1.17, more preferably wherein the ornithine decarboxylase of step b1)(i) has the amino acid sequence shown in SEQ ID NO: 5,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 5,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 6, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 6.6. The method of any one of the preceding items, wherein the dehydrogenase of step b2) is an aspartate-semialdehyde-dehydrogenase, preferably an aspartate-semialdehyde-dehydrogenase of EC-number 1.2.1.11 , more preferably wherein the aspartate-semialdehyde-dehydrogenase of step b2)(i) has the amino acid sequence shown in SEQ ID NO: 7,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 7,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 8, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 8.7. The method of any one of the preceding items, wherein the dehydrogenase of step c1) is a carboxyspermidine dehydrogenase, preferably a carboxyspermidine dehydrogenase of EC- number 1.5.1.43, more preferably wherein the carboxyspermidine dehydrogenase of step c1)(i) has the amino acid sequence shown in SEQ ID NO: 9,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 9,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 10, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 10.8. The method of any one of the preceding items, wherein the decarboxylase of step c2) is a carboxyspermidine decarboxylase, preferably a carboxyspermidine decarboxylase of EC- number 4.1.1.96, more preferably wherein the carboxyspermidine decarboxylase(i) has the amino acid sequence shown in SEQ ID NO: 11 ,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 11,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 12.9. Method of any one of the preceding items, wherein the arginase of step a1), and / or the kinase of step a2), and / or the decarboxylase of step b1), and / or the dehydrogenase of step b2), and / or the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are present as purified enzyme(s) or is / are comprised in a cell extract(s).10. Method of any one of the preceding items, wherein step a2) further comprises the conversion of ATP to ADP, preferably wherein the regeneration of ATP from ADP is by using a polyphosphate kinase, more preferably a polyphosphate kinase of EC-number 2.7.4.1, and / or wherein step b2) further comprises the conversion of NADPH to NADP+, preferably wherein the regeneration of NADPH from NADP+is by using a glucose-1-dehydrogenase and / or by addition of glucose, more preferably by using a glucose- 1 -dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone, and / or wherein step c1) further comprises the conversion of NADPH to NADP+, preferably wherein the regeneration of NADPH from NADP+is by using a glucose-1-dehydrogenase and / or by addition of glucose, more preferably by using a glucose- 1 -dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone.11. Host cell for producing spermidine, wherein said host cell is genetically modified to overexpress at least one, preferably all, enzyme(s) selected from the group consisting of an arginase, an aspartate kinase, an ornithine decarboxylase, an aspartate-semialdehyde-dehydrogenase, a carboxyspermidine dehydrogenase and a carboxyspermidine decarboxylase.12. The host cell of item 11 , wherein the arginase is an arginase of EC-number 3.5.3.1, and / or wherein the aspartate kinase is an aspartate kinase of EC-number 2.7.2.4, and / or wherein the ornithine decarboxylase is an ornithine decarboxylase of EC-number 4.1.1.17, and / or wherein the aspartate-semialdehyde-dehydrogenase is an aspartate-semialdehyde- dehydrogenase of EC-number 1.2.1.11, and / or wherein the carboxyspermidine dehydrogenase is a carboxyspermidine dehydrogenase of EC- number 1.5.1.43, and / or wherein the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC- number 4.1.1.96.13. The host cell of item 11 or 12, wherein the host cell is genetically modified to overexpress a carboxyspermidine decarboxylase, wherein the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96, preferably wherein the carboxyspermidine decarboxylase has the amino acid sequence shown in SEQ ID NO: 11 and / or is encoded by a nucleotide sequence shown in SEQ ID NO: 12.14. The host cell of any one of items 11 to 13, wherein the host cell is a bacterial host cell, a yeast host cell, a plant host cell or an insect host cell, preferably wherein the bacterial host cell is a probiotic bacterial host cell, more preferably wherein the bacterial host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida, or preferably wherein the yeast host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia pastoris and Kluyveromyces lactis, or preferably wherein the plant host cell is selected from the group consisting of Arabidopsis spp., more preferably Arabidopsis thaliana, Nicotiana spp., more preferably Nicotiana tabacum, Triticum spp., more preferably Triticum aestivum, Zea mays and Glycine max, or preferably wherein the insect host cell is selected from the group consisting of Spodoptera frugiperda, Trichoplusia ni and Drosophila melanogaster.15. Use of the host cell according to any one of items 11 to 14 for producing spermidine, preferably in a mammal.* * * * *

[0128] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method”includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0129] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0130] The term "and / or", wherever used herein, includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0131] When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 20 %, up to 10 % or up to 5 % of the given value, including the respective value.

[0132] The term “less than” or in turn “more than” does not include the concrete number. For example, “less than 20” means less than the number indicated. Similarly, “more than” or “greater than” means more than or greater than the indicated number, e.g. “more than 80 %” means more than or greater than the indicated number of 80 %.

[0133] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes, when used herein, with the term “having”. When used herein, “consisting of" excludes any element, step, or ingredient not specified.

[0134] The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0135] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0136] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0137] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0138] A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.EXAMPLES

[0139] MATERIALS AND METHODS:

[0140] Chemicals, strains and enzymes

[0141] All chemicals were purchased from Sigma Aldrich (St. Louis, MO) or Carl Roth GmbH (Karlsruhe, Germany) unless indicated otherwise. Glucose dehydrogenase from Pseudomonas sp. was from Sigma-Aldrich (> 200 U / mg; product number 19359; CAS 9028-53- 9). E. coli gene expression strain BL21 (E. coli B F“ ompT hsdSB(rB~ mB”) dcm+gal) was from Merck KGaA (Darmstadt, Germany). The inventors used E. coli NEB5-alpha (E. coli fhuA2A(argF-lacZ)U169 phoA glnV44 $80A(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) from New England Biolabs (Ipswich, MA) for cloning.

[0142] Construction of expression vectors

[0143] The inventors used the following sequences to generate the expression constructs of the enzymes: E. coli aspartate kinase l / homoserine dehydrogenase I (SEQ ID NO: 3), ThrA, NCBI NP_414543.1; E. coli aspartate semialdehyde dehydrogenase (SEQ ID NO: 7), ASD, NCBI NP_417891.1; arginase from Bacillus subtilis (SEQ ID NO: 1), ARG1 ,7NCBI NP_391912.1; E. coli ornithine decarboxylase (SEQ ID NO: 5), SpeC, NCBI NP_417440.4; carboxyspermidine dehydrogenase (CASDH) (SEQ ID NO: 9) from Paracoccus denitrificans PD1222, NCBI ABL71039.1; carboxyspermidine decarboxylase (CASDC) (SEQ ID NO: 11) from Paracoccus denitrificans strain 1222, NCBI A1B696 and from Vibrio alginolyticus, NCBI A0A2I3C6S9. The amino acid sequences of non-E. coli enzymes were reverse-translated using the integrated software from Twist Bioscience and codon optimized for E. coli. ThrA / ASD and ARG1 / SpeC coexpression vectors were ordered as synthetic DNA from Twist Bioscience (San Francisco, CA) yielding constructs pET21-thrA_ASD and pET21-ARG_speC. pET29-PdCASDH, pET29-PdCASDC, and pET29-\ / aCASDC were constructed with an additional tac promoter and rrnB1 terminator to allow protein expression without the T7 polymerase. All constructs were sequence verified prior delivery. The maps of the constructs are shown in Figure 8 as given herein.

[0144] Preparation of cells and lysates for the biotransformations

[0145] E. coli strain BL21 was transformed with the vectors pET21-ARG_speC, pET21- thrA_ASD, pET29-PdCASDH, pET29-PdCASDC and pET29-\ / aCASDC. The resulting expression strains were inoculated to a starting attenuance at 600 nm (D6oo) of 0.1 in 400 mL 2xTY 5052 medium (16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCI)8in a 2 L baffled flask.

[0146] The medium contained 40 pg / mL kanamycin or 100 pg / mL ampicillin for the maintenance of the pET29 or pET21 constructs, respectively. Recombinant gene expression was performed by autoinduction with 2xTY 5052 medium according to Studier et al.8,9The culture was incubated at 37 °C and 120 rpm shaking for 4 h, then the temperature was switched to 20 °C and incubation was continued with shaking for 24 h. Cells were harvested by centrifugation at 3220 ref at 4 °C for 20 min in 50 ml conical polypropylene tubes (Greiner Bio- One GmbH, Kremsmunster, Austria). The resulting pellets were frozen and stored at -20 °C until lysate preparation.

[0147] The lysates were prepared by sonication and subsequent removal of cell debris by centrifugation. To prepare the lysates, the frozen cells corresponding to 200 mL culture were filled up to 20 mL with buffer (50 mM KPi, pH 7.4; 1 mM dithiothreitol), thawed on ice and resuspended. The cells in buffer were subsequently sonicated with a Branson Sonifier 25 for 6 min with a 30 s pause after 3 min. The sonication was performed in an aluminum cap on ice to allow proper cooling. The disrupted cells were centrifuged at 48384 ref at 4 °C for 45 min. The resulting cleared lysate was then used for biotransformation on the same day.

[0148] Biotransformation conditions

[0149] All biotransformations were performed in the same buffer under identical assay conditions. The reaction buffer contained 50 mM Kpi pH 7.6; 20 mM DTT; 0.1 mM pyridoxal phosphate; 25 mM ATP; 0.5 mM NADP; 0.01 mg / ml GDH (>200 ll / rng); and 55.5 mM glucose. The substrates were added to the buffer at a concentration of 20 mM. For reactions with two substrates, e.g., L-aspartate and L-arginine, both substrates were present at 20 mM. The reactions were performed in 2 mL polypropylene tubes in a final volume of 1.5 mL. The samples were incubated at 30 °C and with 500 rpm shaking in an Eppendorf Thermomix C. Aliquots were sampled at 0 h, 2 h and after 24 h.

[0150] Comparison of PofCASDC and VaCASDC

[0151] For the isolation and purification of PdCASDC and VaCASDC, the correspondingE. coli expression strains were cultivated as described above for the cell lysates. Cell pellets were resuspended in 25 mL KPi buffer (50 mM, pH 7.4) and disrupted by sonification on ice (three cycles of 30 s sonification and 100 s cooling break each). To prepare the soluble protein fraction, the cell homogenate was centrifuged at 40000 x g at 4 °C for 15 min to pellet the cell debris. For enzyme purification, 2 mL Ni-Sepharose (His-Select Nickel affinity gel, Merck) were dispensed into PD10 columns and were rinsed with 10 column volumes (CVs) of H2O followed by washing with 10 CVs of wash buffer (KPi buffer, pH 7.4; 20 mM imidazole; 0.5 M NaCI). The soluble protein fraction was diluted 1:1 (v / v) with sample dilution buffer (KPi buffer, pH 7.4; 40 mM imidazole; 1 M NaCI), 10 mL were loaded on the column and incubated for 10 min at RT.Subsequently, the sample was drained and collected. Both steps were repeated until the entire soluble protein fraction had been passed over the column. Then the column was washed three times with wash buffer. Bound proteins were eluted by adding three times 1 CV of elution buffer (KPi buffer, pH 7.4; 500 mM imidazole, 0.5 M NaCI). The eluate was desalted using 5 mL HiTrap desalting columns (Cytiva Europe GmbH, Vienna, Austria) and rebuffered into storage buffer (KPi buffer, pH 7.4; 1 mM DTT; 0.1 mM pyridoxal phosphate).

[0152] CASDC activity was analyzed in reaction buffer containing 50 mM KPi pH 7.4;1 mM DTT; 0.1 mM pyridoxal phosphate and 20 mM carboxyspermidine. The purified enzymes were added to a final concentration of 0.1 mg / mL and the reaction proceeded at 30 °C for 120 min unless indicated otherwise. For the heat shock treatment, the standard reaction mixture was incubated at 60 °C for five minutes and afterwards, the samples were incubated at 30 °C for 90 min. All samples were run in triplicate including standard reactions without heat shock. To assess the pH stability of the CASDC enzymes, reaction buffers adjusted to pH 6.5, 7.4 and 8.5 were employed. To test the impact of different storage conditions on the enzyme activity, the enzymes were stored at 4 °C and -20 °C, as well as shock-frozen in liquid nitrogen and thawed on ice trice. Enzymes that had been stored under different conditions were then used in a standard activity assay as described above. 1 mg of PdCASDC and VaCASDC in storage buffer were immobilized on 1 mL HisTrap FF Ni-sepharose columns (Cytiva) for 5 min at RT and unbound protein was washed off with storage buffer. The immobilized enzymes were incubated with 1 mL reaction buffer containing 20 mM carboxyspermidine for 5 min at 30 °C and afterwards, the reaction mixture was harvested using a syringe. The incubation with reaction buffer was repeated 4 times.

[0153] HPLC analytics of polyamines

[0154] The method was adapted from Yufei et al. (2017).70Polyamines were analyzed on an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with an Agilent Advance BIO AA column (4.6 x 100 mm, 2.7 micron, P.N. 655950-802; Agilent Technologies), a binary pump and variable wavelength detector (VWD) running the proprietary software Chemstation B04.03 SP3 (Agilent Technologies). The mobile phases were 10 mM Na2PO4, 10 mM B4Na2O7, pH 8.2, as solvent A and acetonitrile / methanol / H2O (45 / 45 / 10; v / v / v) as solvent B. All chemicals were purchased at HPLC grade.

[0155] The method included a dilution step during pre-column derivatization with diluent C consisting of 100 mL solvent A mixed with 1.2 mL phosphoric acid. For derivatization the inventors used the following injector program: Step 1 : draw 4 pL borate buffer at max. speed; step 2: draw 4 pL from sample at max. speed; step 3: mix 8 pL in seat at max. speed for 5 times; step 4: wait 0.4 min; step 5: draw 6 pL OPA at max. speed; step 6: mix 14 pL in seat at max. speed for 10 times; step 7: draw 20 pL diluent C at max. speed; step 8: mix 20 pL in seatat max. speed for 8 times; step 9: inject; step 10: wait 0.1 min; step 11: valve by-pass; step 12: wash needle 3 times in acetonitrile.

[0156] The separation of the analytes was performed in an 18 min run with a flow rate of 1.5 mL / min with the following gradient settings: 2% B from 0 to 0.35 min followed by a gradient to 57% B until 13.4 min and to 100% B until 13.5 min. 100% B was maintained until 15.7 min and lowered to 2% B until 15.8 min. 2% B was maintained until 18 min. The column was operated at 40 °C and the signals were detected with an Agilent 1100 VWD at 338 nm. Pure substances were used to prepare calibration curves and spiking of the samples with pure substances served for peak identification.

[0157] RESULTS AND DISCUSSION

[0158] Set-up of the biotransformation reactions

[0159] The biotransformations were set up as described in the Materials and Methods- section. Table 3 as given herein below gives an overview of the individual reactions, the relevant proteins, substrates and expected products.

[0160] Table 3: Biotransformation reactions. 20 mM of each substrate were used. Abbreviations: Arg, L-arginine; ARG1, arginase; Asa, L-aspartate-4-semialdehyde; ASD, aspartate-semialdehyde dehydrogenase; Asp, L-aspartate; Cs, carboxyspermidine; PdCASDC, carboxyspermidine decarboxylase from Paracoccus denitrificans', PdCASDH, carboxyspermidine dehydrogenase from Paracoccus denitrificans’, Put, putrescine; Spe, spermidine; SpeC, ornithine decarboxylase; ThrA, aspartate kinase; n.a., not applicable; n.d., not detectable.

[0161] PofCASDC rapidly converted carboxyspermidine to spermidine

[0162] The decarboxylation of carboxyspermidine to spermidine by PdCASDC (Table 3, reaction #1) occurred rapidly. Already after 2 h, most carboxyspermidine had been converted and after 24 h the spermidine concentration reached 13.8 mM with no carboxyspermidine left.

[0163] High concentrations of L-aspartate-4-semialdehyde inhibit the carboxyspermidine branch of the biocascade

[0164] L-aspartate-4-semialdehyde and putrescine were not converted to carboxyspermidine by PdCASDH (Table 3, reaction #2). The substrate concentrations remained constant within 24 h, which indicates that PdCASDH was inactive. Neither were these two substrates converted to spermidine by a combination of PdCASDH and PdCASDC (Table 3, reaction #5).

[0165] When L-aspartate-4-semialdehyde was present at high concentrations together with L-arginine, putrescine was efficiently produced but did not react further to carboxyspermidine or spermidine (Figure 3). After 24 h, this reaction (Table 3, reaction #7) produced exclusively 10 mM putrescine. Obviously, high concentrations of L-aspartate-4- semialdehyde inhibit the carboxyspermidine branch of the biocascade limiting or inactivating the CASDH.4The inventors were also unable to detect the conversion of L-aspartate to L-aspartate- 4-semialdehyde by ThrA and ASD (Table 3, reaction #3). Potentially, L-aspartate-4- semialdehyde is over-reduced to L-homoserine, whose analysis the inventors had not included. Since there was no downstream reaction included that would consume L-aspartate-4- semialdehyde, the reverse oxidation of L-homoserine to L-aspartate-4-semialdehyde could have been inefficient.

[0166] The L-arginine branch of the biocascade was highly active

[0167] ARG1 and SpeC produced 12.4 mM putrescine from 20 mM arginine within 24 h (Table 3, reaction #4).

[0168] Spermidine was produced through a functional L-aspartate branch and putrescine

[0169] After 24 h, the biocascade that had been programmed with 20 mM L-aspartate and putrescine each (Table 3, reaction #6) yielded 3 mM spermidine product. Obviously, L-aspartate-4-semialdehyde did not accumulate such that the flux through the biocascade was inhibited by the inactivation, e.g. of PdCASDH.

[0170] The biocascade converted L-aspartate and L-arginine to spermidine

[0171] The inventors detected 4.8 mM spermidine and 9.2 mM putrescine after 24 h of conversion of 20 mM L-arginine and L-aspartate each (Table 3, reaction #8). The inventors were unable to detect L-aspartate-4-semialdehyde. This indicates that the entire biocascade is functional in a mixture of lysate preparations.

[0172] Comparison of PofCASDC and VaCASDC

[0173] The inventors compared the temperature stability, resilience to heat shock, pH stability, stability towards freeze-thaw cycles and activity after immobilization of the CASDCs from Paracoccus denitrificans (PdCASDC) and Vibrio alginolyticus (VaCASDC). For these experiments, both enzymes were purified via their N-terminal hexahistidine-tag and the conversion of carboxyspermidine to spermidine was analyzed under the indicated conditions.

[0174] The thermostability of the enzymes was assessed at 30 °C, 45 °C and 60 °C for 120 min. Both enzymes were most active at 30 °C, where quantitative conversion occurred, with a rapid decline of the activity at the higher temperatures. When the enzymes were briefly heat shocked to 60 °C before the reaction could proceed at 30 °C, the activity also dropped. Thus, PdCASDC and VaCASDC are both equally heat sensitive.

[0175] Next, the inventors assessed the pH stability of the CASDCs at pH 6.5, 7.4 and 8.5. Again, the reaction was allowed to proceed at 30 °C for 120 min. Both enzymes quantitatively converted carboxyspermidine to spermidine under all pH conditions. Their activity does not seem to be pH sensitive in this pH range.

[0176] PdCASDC and VaCASDC were shock-frozen in liquid nitrogen and thawed on ice three times (freeze-thaw cycle). Their reactivity with carboxyspermidine was compared with that of enzymes that had been stored at 4 °C and -20 °C. Spermidine formation occurred at 30 °C for 60 min. The activity of both enzymes was independent of the storage conditions, i.e. refrigeration or freezing versus freeze-thaw cycles. However, VaCASDC’s activity was slightly reduced (80-90% conversion) while PdCASDC showed again quantitative conversion. It appears that PdCASDC tolerates low temperatures as well as temperature fluctuation slightly better than VaCASDC.

[0177] Finally, the inventors analyzed the reactivity of PdCASDC and VaCASDC after they had been immobilized on Ni-sepharose matrix via their N-terminal hexahistidine tag. The immobilized enzymes were repeatedly incubated with carboxyspermidine (see the Materials and Methods-section for details), and the spermidine content of the reaction mixtures collected at 0, 5, 10, 15 and 20 min was determined. Immobilized PdCASDC was more reactive than\ / aCASDC, because the inventors observed 60% conversion of carboxyspermidine already in the eluate collected after 5 min and it reached its maximum (90%) already after 10 min. VaCASDC reached 40% conversion after 5 min, which increased to 60% after 10 and 15 min and reached a maximum of 70% in the eluate collected after 20 min.

[0178] CONCLUSION

[0179] Recombinant PdCASDH and PdCASDC are excellent enzymes for the biosynthesis of spermidine via carboxyspermidine. While PdCASDC and \ / aCASDC performed comparably with respect to heat sensitivity, resilience to heat shock, or pH stability, the first outperformed the latter with regard to storage stability and activity in immobilized form.

[0180] The inventors were unable to observe the conversion of L-aspartate to L- aspartate-4-semialdehyde (Table 3, reaction #3), most probably due to an insufficiently low limit-of-detection combined with a putative overreduction to the alcohol L-homoserine.

[0181] The synthesis of putrescine worked very well, a yield of 12.4 mM (62% molar conversion) was reached after 24 h. The single reactions where L-aspartate-4-semialdehyde was used as a substrate did not yield any desired product (Table 3, reactions #2, 5 and 7). This may be expected if PdCASDH was inactivated permanently by high L-aspartate-4- semialdehyde concentrations as described previously.6,11

[0182] Spermidine was produced at 3 mM (15% conversion rate) by a functional L- aspartate branch together with putrescine (Table 3, reaction #6), most probably because L- aspartate-4-semialdehyde was produced and consumed continuously and as such did not accumulate to inhibit PdCASDH. PdCASDC is an avid producer, it efficiently converted carboxyspermidine into spermidine (13.8 mM, 69% molar conversion; Table 3, reaction #1).

[0183] The flux through the entire biocascade was even better with L-aspartate and L- arginine as the substrates (Table 3, reaction #8). A total of 4.7 mM spermidine was produced, in addition 10.8 mM of putrescine were formed. Obviously, the continuous production of L- aspartate-4-semialdehyde and putrescin balances the reaction. When L-aspartate-4- semialdehyde is constantly consumed either its over- reduction to L-homoserine can be avoided or L-homoserine can be re-oxidized to re-enter the cascade.6

[0184] The presented biocascade is shown as herein above active in a mixture of lysate preparations.REFERENCE(1) Madeo, F., Eisenberg, T., Pietrocola, F., and Kroemer, G. (2018) Spermidine in health and disease. Science 359 (6374), eaan2788. https: / / doi.org / 10.1126 / science.aan2788(2) Lee, J., Sperandio, V., Frantz, D. E., Longgood, J., Camilli, A., Phillips, M. A., and Michael, A. J. (2009) An alternative polyamine biosynthetic pathway is widespread in bacteria and essential for biofilm formation in Vibrio cholerae J. Biol. Chem. 284 (15), 9899-9907. https: / / doi.org / 10.1074 / jbc.M900110200(3) Hanfrey, C. C., Pearson, B. M., Hazeldine, S., Lee, J., Gaskin, D. J., Woster, P. M., Phillips, M. A., and Michael, A. J. (2011) Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota J. Biol. Chem. 286 (50), 43301-43312. https: / / doi.org / 10.1074 / jbc.M111.307835(4) Nakao, H., Shinoda, S., and Yamamoto, S. (1991) Purification and some properties of carboxynorspermidine synthase participating in a novel biosynthetic pathway for norspermidine in Vibrio alginolyticus. J. Gen. Microbiol. 137 (7), 1737-1742. https: / / doi.org / 10.1099 / 00221287- 137-7-1737(5) Sakanaka, M., Sugiyama, Y., Kitakata, A., Katayama, T., and Kurihara, S. (2016) Carboxyspermidine decarboxylase of the prominent intestinal microbiota species Bacteroides thetaiotaomicron is required for spermidine biosynthesis and contributes to normal growth. Amino Acids 48 (10), 2443-2451. https: / / doi.org / 10.1007 / s00726-016-2233-0(6) Liang, X., Deng, H., Bai, Y., Fan, T.-P., Zheng, X., and Cai, Y. (2022) Highly efficient biosynthesis of spermidine from L-homoserine and putrescine using an engineered Escherichia coli with NADPH self-sufficient system. Appl. Microbiol. Biotechnol. 106 (17), 5479-5493. https : / / doi . org / 10.1007 / s00253-022- 12110-x(7) Yu, J. -J., Park, K.-B., Kim, S.-G., and Oh, S.-H. (2013) Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168. J. Microbiol. 51 (2), 222-228. https: / / doi.org / 10.1007 / s12275-013-2669-9(8) Studier, F. W. (2014) Stable expression clones and auto-induction for protein production in E. coli. Methods Mol. Biol. 1091, 17-32. https: / / doi.org / 10.1007 / 978-1-62703-691-7_2(9) Studier, F. W. (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 41 (1), 207-234. https: / / doi.Org / 10.1016 / j.pep.2005.01.016(10) Yufei, L. (2017) Application Note: Analysis of Amino Acids Derived Online Using an Agilent AdvanceBio AAA Column, p 6, Agilent Technologies, Inc.(11) Nakao, H., Shinoda, S., and Yamamoto, S. (1990) Purification and properties of carboxynorspermidine decarboxylase, a novel enzyme involved in norspermidine biosynthesis, from Vibrio alginolyticus. J. Gen. Microbiol. 136 (9), 1699-1704. https : / / doi . org / 10.1099 / 00221287-136-9-1699

Claims

CLAIMS:

1. A method for producing spermidine, comprising the following steps: a1) converting L-arginine to L-ornithine by an arginase; a2) converting L-aspartate to L-4-aspartylphosphate by a kinase; b1) converting the L-ornithine of step a1) to putrescine by a decarboxylase; b2) converting the L-4-aspartylphosphate of step a2) to L-aspartate-4-semialdehyde by a dehydrogenase; c1) converting the putrescine of step b1) and the L-aspartate-4-semialdehyde of step b2) to carboxyspermidine by a dehydrogenase; and c2) converting the carboxyspermidine of step c1) to spermidine by a decarboxylase.

2. The method of claim 1, wherein the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are from Paracoccus denitrificans, and / or wherein the arginase of step a1) is from Bacillus subtilis, and / or wherein the kinase of step a2) is from Escherichia coli, and / or wherein the decarboxylase of step b1) is from Escherichia coli, and / or wherein the dehydrogenase of step b2) is from Escherichia coli.

3. The method of claim 1 or 2, wherein the arginase of step a1) is an arginase of EC-number 3.5.3.1, preferably wherein the arginase of step a1)(i) has the amino acid sequence shown in SEQ ID NO: 1 ,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 1,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 2, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 2.

4. The method of any one of the preceding claims, wherein the kinase of step a2) is an aspartate kinase, preferably an aspartate kinase of EC-number 2.7.2.4, more preferably wherein the aspartate kinase of step a2)(i) has the amino acid sequence shown in SEQ ID NO: 3,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 3,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 4, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 4.

5. The method of any one of the preceding claims, wherein the decarboxylase of step b1) is an ornithine decarboxylase, preferably an ornithine decarboxylase of EC-number 4.1.1.17, more preferably wherein the ornithine decarboxylase of step b1)(i) has the amino acid sequence shown in SEQ ID NO: 5,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 5,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 6, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 6.

6. The method of any one of the preceding claims, wherein the dehydrogenase of step b2) is an aspartate-semialdehyde-dehydrogenase, preferably an aspartate-semialdehyde-dehydrogenase of EC-number 1.2.1.11 , more preferably wherein the aspartate-semialdehyde-dehydrogenase of step b2)(i) has the amino acid sequence shown in SEQ ID NO: 7,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 7,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 8, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 8.

7. The method of any one of the preceding claims, wherein the dehydrogenase of step c1) is a carboxyspermidine dehydrogenase, preferably a carboxyspermidine dehydrogenase of EC- number 1.5.1.43, more preferably wherein the carboxyspermidine dehydrogenase of step c1)(i) has the amino acid sequence shown in SEQ ID NO: 9,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 9,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 10, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 10.

8. The method of any one of the preceding claims, wherein the decarboxylase of step c2) is a carboxyspermidine decarboxylase, preferably a carboxyspermidine decarboxylase of EC- number 4.1.1.96, more preferably wherein the carboxyspermidine decarboxylase(i) has the amino acid sequence shown in SEQ ID NO: 11 ,(ii) has an amino acid sequence which is at least 70% identical to the amino acid sequence shown in SEQ ID NO: 11,(iii) is encoded by a nucleotide sequence shown in SEQ ID NO: 12, or(iv) is encoded by a nucleotide sequence which is at least 70% identical to the nucleotide sequence shown in SEQ ID NO: 12.

9. Method of any one of the preceding claims, wherein the arginase of step a1), and / or the kinase of step a2), and / or the decarboxylase of step b1), and / or the dehydrogenase of step b2), and / or the dehydrogenase of step c1) and / or the decarboxylase of step c2) is / are present as purified enzyme(s) or is / are comprised in a cell extract(s).

10. Method of any one of the preceding claims, wherein step a2) further comprises the conversion of ATP to ADP, preferably wherein the regeneration of ATP from ADP is by using a polyphosphate kinase, more preferably a polyphosphate kinase of EC-number 2.7.4.1 , and / or wherein step b2) further comprises the conversion of NADPH to NADP+, preferably wherein the regeneration of NADPH from NADP+is by using a glucose-1-dehydrogenase and / or by addition of glucose, more preferably by using a glucose- 1 -dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone, and / or wherein step c1) further comprises the conversion of NADPH to NADP+, preferably wherein the regeneration of NADPH from NADP+is by using a glucose-1-dehydrogenase and / or by addition of glucose, more preferably by using a glucose- 1 -dehydrogenase of EC-number 1.1.1.119 by conversion of glucose to gluconolactone.11 . Host cell for producing spermidine, wherein said host cell is genetically modified to overexpress at least one, preferably all, enzyme(s) selected from the group consisting of an arginase, an aspartate kinase, an ornithine decarboxylase, an aspartate-semialdehyde-dehydrogenase, a carboxyspermidine dehydrogenase and a carboxyspermidine decarboxylase.

12. The host cell of claim 11 , wherein the arginase is an arginase of EC-number 3.5.3.1, and / or wherein the aspartate kinase is an aspartate kinase of EC-number 2.7.2.4, and / or wherein the ornithine decarboxylase is an ornithine decarboxylase of EC-number 4.1.1.17, and / or wherein the aspartate-semialdehyde-dehydrogenase is an aspartate-semialdehyde- dehydrogenase of EC-number 1.2.1.11, and / or wherein the carboxyspermidine dehydrogenase is a carboxyspermidine dehydrogenase of EC- number 1.5.1.43, and / orwherein the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC- number 4.1.1.96.

13. The host cell of claim 11 or 12, wherein the host cell is genetically modified to overexpress a carboxyspermidine decarboxylase, wherein the carboxyspermidine decarboxylase is a carboxyspermidine decarboxylase of EC-number 4.1.1.96, preferably wherein the carboxyspermidine decarboxylase has the amino acid sequence shown in SEQ ID NO: 11 and / or is encoded by a nucleotide sequence shown in SEQ ID NO: 12.

14. The host cell of any one of claims 11 to 13, wherein the host cell is a bacterial host cell, a yeast host cell, a plant host cell or an insect host cell, preferably wherein the bacterial host cell is a probiotic bacterial host cell, more preferably wherein the bacterial host cell is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida, or preferably wherein the yeast host cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Pichia pastoris and Kluyveromyces lactis, or preferably wherein the plant host cell is selected from the group consisting of Arabidopsis spp., more preferably Arabidopsis thaliana, Nicotiana spp., more preferably Nicotiana tabacum, Triticum spp., more preferably Triticum aestivum, Zea mays and Glycine max, or preferably wherein the insect host cell is selected from the group consisting of Spodoptera frugiperda, Trichoplusia ni and Drosophila melanogaster.

15. Use of the host cell according to any one of claims 11 to 14 for producing spermidine, preferably in a mammal.

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