FIELD OF THE INVENTION

[001] The present invention relates generally to genetic and metabolic engineering and biological preparation of 5-aminovalerate, and more specifically to a recombinant cell genetically engineered to produce an increased amount of 5-aminovalerate compared to its wildtype, a method for preparing 5-aminovalerate using the recombinant cell, and the use of the recombinant cell for preparing 5-aminovalerate.

BACKGROUND OF THE INVENTION

[002] Increasing concerns with regard to environmental problems and fossil resource availability have led to increased interest in the production of chemicals and materials from renewable biomass through biorefineries. Several platform bacteria such as Escherichia coli and Corynebacterium glutamicum have been metabolically engineered to produce alcohols, polymers, and platform chemicals from renewable resources.

[003] 5 -Amino valerate is a potential C5 platform chemical for synthesis of valerolactam, 5- hydroxyvalerate, glutarate, and 1,5-pentanediol. It is naturally produced from L-lysine through the 5-aminovalerate pathway, wherein L-lysine 2-monooxygenase (DavB) catalyzes L-lysine into 5-aminovaleramide and delta-aminovaleramidase (DavA) further converts 5- aminovaleramide into 5-aminovalerate. It is known that expression of the Pseudomonas putida DavB and DavA proteins in Escherichia coli entails production of 5-aminovalerate from L- lysine (Park et al. Metabolic Engineering. 16 (2013) 42-47). Purified DavA and DavB proteins can also produce 5-aminovalerate from L-lysine in a cell-free system (Liu et al. Scientific Reports. Article number: 5657 (2014)).

[004] However, there still remains a considerable need for new technologies in order for biologically derived 5-aminovalerate to become a commercially viable option.

SUMMARY OF THE INVENTION

[005] The inventors of the present invention have found that said need can be met by the provision of a recombinant cell genetically engineered to produce an increased amount of 5- aminovalerate compared to its wildtype, and a method for preparing 5-aminovalerate using said recombinant cell. [006] In one aspect, the present invention therefore relates to a recombinant cell, wherein said cell has been genetically engineered such that it produces an increased amount of 5- aminovalerate compared to its wildtype.

[007] In various embodiments, the recombinant cell is a microbial cell, preferably a microbial cell selected from the group consisting of Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida cells, and most preferably a Corynebacterium glutamicum cell.

[008] In various embodiments, the recombinant cell has, compared to its wildtype, (1) an increased activity of an enzyme El that catalyzes the amino group transfer of cadaverine; and/or (2) an increased activity of an enzyme E2 that catalyzes the dehydrogenation of 5- aminopentanal.

[009] In various embodiments, enzyme El is a putrescine aminotransferase. Enzyme El may be an EC 2.6.1.82 class enzyme, for example an PLP-dependent putrescine:2-oxoglutaric acid aminotransferase (PatA).

[010] In particularly preferred embodiments, enzyme El a) has the amino acid sequence of SEQ ID NO: l ; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).

[011] In preferred embodiments, enzyme E2 is a γ-aminobutyraldehyde dehydrogenase. Enzyme E2 may be an EC 1.2.1.19 class enzyme, for example a γ-aminobutyraldehyde dehydrogenase (PatD).

[012] In particularly preferred embodiments, enzyme E2: a) has the amino acid sequence of SEQ ID NO:2; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).

[013] In various embodiments, the recombinant cell has, compared to its wildtype, an increased activity of an enzyme E3 that catalyzes the decarboxylation of lysine. Enzyme E3 may be a lysine decarboxylase, e.g. an EC 4.1.1.18 class enzyme. In preferred embodiments, enzyme E3 is Escherichia coli lysine decarboxylase (LdcC). [014] In particularly preferred embodiments, enzyme E3: a) has the amino acid sequence of SEQ ID NO:3; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).

[015] In various preferred embodiments, the recombinant cell of the invention is a Corymb acterium glutamicum cell, preferably a Corymb acterium glutamicum GRLysl cell, wherein (a) the sugR gene as set forth in SEQ ID NO: 8 or a fragment thereof is deleted in said cell; (b) the IdhA gene as set forth in SEQ ID NO: 9 or a fragment thereof is deleted in said cell; (c) the snaA gene as set forth in SEQ ID NO: 10 or a fragment thereof is deleted in said cell; (d) the lysE gene as set forth in SEQ ID NO: 11 or a fragment thereof is deleted in said cell; and/or (e) the lysG gene as set forth in SEQ ID NO: 12 or a fragment thereof is deleted in said cell.

[016] In another aspect, the invention is directed to a method for preparing 5-aminovalerate, the method comprising culturing the recombinant cell in a growth medium under conditions allowing production of 5-aminovalerate, and recovering 5-aminovalerate from the medium.

[017] In still another aspect, the invention encompasses use of the recombinant cell for preparing 5-aminovalerate.

BRIEF DESCRIPTION OF THE DRAWINGS

[018] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[019] Figure 1 is a schematic diagram showing a pathway for 5-aminovalerate production, wherein lysine decarboxylase (LdcC) catalyzes the conversion of L-lysine into cadaverine, PatA catalyzes the conversion of cadaverine into 5-aminopentanal, and PatD catalyzes the conversion of 5-aminopentanal into 5-aminovalerate.

[020] Figure 2 is a gene map of recombinant plasmid )EKEx3-patDA encoding PatA and PatD derived from Escherichia coli.

[021] Figure 3 is a gene map of recombinant plasmid pVWExl -IdcC encoding LdcC derived from Escherichia coli.

[022] Figure 4 is a schematic representation of a new metabolic route from l-lysine to 5AVA. Enzymatic reactions and the involved proteins are depicted. Dashed arrows represent more than one enzymatic reaction. Enzymes in grey boxes were added by heterologous expression of the respective genes from E. coli and are unknown in C. glutamicum. Abbreviations: 5AVA, 5- aminovalerate; LdcC, l-lysine decarboxylase; PatA, putrescine transaminase; PatD, 5- aminopentanal dehydrogenase; LysE, 1-lysine efflux permease; CgmA, putative putrescine/cadaverine efflux permease.

[023] Figure 5 shows the dependence of the growth rate of C. glutamicum on the 5AVA concentration added to glucose minimal medium.

[024] Figure 6 shows the growth (filled circles) and 5AVA concentration (empty circles) in C. glutamicum wild type. Cells were grown in CGXII medium with 4% (w/v) glucose and supplemented with about 20 mM of 5AVA after 9h of growth. Values and error bars represent the mean and the standard deviation of duplicate cultivations.

[025] Figure 7 shows genes that are differentially expressed in Corynebacterium glutamicum grown in glucose minimal medium in the presence of 5AVA as compared to sodium chloride.

[026] Figure 8 shows accumulation of 5AVA and side-products by different C. glutamicum strains.

DETAILED DESCRIPTION OF THE INVENTION

[027] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the invention. The various embodiments described herein are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments. It is thus understood that although certain embodiments are disclosed separately, all combinations of two or more of such embodiments are intended to fall within the scope of the instant invention.

[028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[029] The object of the present invention is to provide a means for biological preparation of 5- aminovalerate. To this end, 5-aminovalerate production adopting a new pathway, i.e. L-lysine to cadaverine to 5-aminopentanal to 5-aminovalerate, is enabled by recombinant overexpression of certain enzymes essential for said pathway in a cell.

[030] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element.

[031] As used herein, the term "cadaverine" refers to pentane- 1,5 -diamine, and the term "5- aminovalerate" refers to 5-aminopentanoate. [032] In one aspect, the present invention relates to a recombinant cell, wherein said cell has been genetically engineered such that it produces an increased amount of 5-aminovalerate compared to its wildtype.

[033] The term "wildtype", as used herein, is preferably understood as meaning a cell whose genome has been generated naturally by evolution. The term is used both for the cell as a whole and for individual genes. Consequently, the term "wildtype" specifically does not include those cells and genes whose gene sequences have been modified at least partially by humans by means of recombinant processes.

[034] The term "genetically engineered", as used herein, refers to the use of molecular biology methods to manipulate nucleic acid sequences and introduce nucleic acid molecules into host organisms. The term "recombinant cell", as used herein, means a cell that has been subjected to recombinant DNA manipulations, such as the introduction of exogenous nucleic acid molecule, resulting in a cell that is in a form not found originally in nature. Recombinant cells are generated, for example, by transformation, transfection, transduction, conjugation or a combination of these methods, using a vector which contains the desired gene, an allele of said gene or parts thereof and a promoter enabling the gene to be expressed. Overexpression is achieved in particular by integrating the gene or the alleles in the chromosome of the cell or in an extrachromosomally replicating vector. Methods of creating recombinant cells are well known in the art. For example, see MOLECULAR CLONING: A LABORATORY MANUAL (Sambrook et al, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989), or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., eds., John Wiley & Sons, New York, 1987).

[035] The term "increased amount", as used herein in relation to the production of 5- aminovalerate by the described cells compared to wildtype cells, means that the recombinant cell produces at least twice, particularly preferably at least 10 times, additionally preferably at least 100 times, additionally still more preferably at least 1000 times and most preferably at least 10 000 times more 5-aminovalerate than the wildtype cell within a defined time interval. The increase in product formation may be determined, for example, by culturing the recombinant and wildtype cells separately under the same conditions (same cell density, same growth medium, same culturing conditions) in a suitable growth medium for a particular time interval and then determining the amount of 5-aminovalerate in the cell supernatant.

[036] The cell of the present invention may be a prokaryote or eukaryote cell and may be a mammalian cell, a plant cell or a microbial cell such as yeast, fungus or bacterium, with particular preference being given to microbial cells. [037] In various embodiments, the recombinant cell is a microbial cell selected from the group consisting of Escherichia coli, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevibacterium flavum, Brevibacterium lactofermentum, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Methylobacterium extorquens, Ralstonia eutropha, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida cells, and most preferably is a Corynebacterium glutamicum cell.

[038] In various embodiments, the recombinant cell has, compared to its wildtype, (1) an increased activity of an enzyme El that catalyzes the amino group transfer of cadaverine; and/or (2) an increased activity of an enzyme E2 that catalyzes the dehydrogenation of 5- aminopentanal.

[039] The term "an increased activity" of an enzyme in a recombinant cell, as used herein, refers to an increase in the activity determined relative to the wildtype cell. Increased activities can be the result of, for example, increased amount of protein expressed by a recombinant cell (e.g., as the result of increased number of copies of DNA sequences encoding the protein, increased number of mRNA transcripts encoding the protein, and/or increased amount of protein translation of the protein from mRNA); changes in the structure of the protein (e.g., changes to the primary structure, such as, changes to the protein's coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (e.g., decreased degradation of the protein). In certain instances, the coding polynucleotide sequences for the proteins described herein are codon optimized for expression in a particular cell. The increased enzyme activity is, for example, increased by at least 20 %, preferably at least 50 %, more preferably at least 100 % compared to the wildtype cell and may be determined by suitable methods known to those skilled in the art.

[040] The term "enzyme El that catalyzes the amino group transfer of cadaverine", as used herein, includes any enzyme that catalyzes the exchange of an amine group of cadaverine with a keto group of another molecule, and thereby converts cadaverine into 5-aminopentanal.

[041] The term "enzyme E2 that catalyzes the dehydrogenation of 5-aminopentanal", as used herein, includes any enzyme that catalyzes the removal of hydrogen atoms from the aldehyde group of 5-aminopentanal, and thereby converts 5-aminopentanal into 5 -amino valerate. [042] In preferred embodiments, (1) the enzyme El is a putrescine aminotransferase and/or (2) the enzyme E2 is a γ-aminobutyraldehyde dehydrogenase.

[043] Transaminases are a class of enzymes that catalyze a type of reaction between an amino acid and an a-keto acid, wherein the amine (-NH2) group on one molecule is exchanged with the keto (=0) group on the other molecule. Putrescine aminotransferases are best known in the art to catalyze the aminotransferase reaction from putrescine to 2-oxoglutarate, leading to glutamate and 4-aminobutanal, but it has been found that these enzymes can also catalyse transamination of cadaverine.

[044] Dehydrogenases are a class of enzymes that catalyze the removal of hydrogen atoms from a particular molecule. It is known in the art that γ-aminobutyraldehyde dehydrogenases from some species exhibit broad substrate specificity and have a marked preference for straight- chain aldehydes (up to 7 carbon atoms) as substrates.

[045] In preferred embodiments, enzyme El is an EC 2.6.1.82 class enzyme. The abbreviation "EC" and accompanying notations, as used herein, are references to the enzyme classification as established by the nomenclature committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMD).

[046] In further preferred embodiments, enzyme El is PLP-dependent putrescine:2- oxoglutaric acid aminotransferase (PatA). This enzyme can exchange the amine group of cadaverine with the keto group of alpha-ketoglutarate to produce 5-aminopentanal and glutamate.

[047] In particularly preferred embodiments, enzyme El : a) has the amino acid sequence of SEQ ID NO: l ; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).

[048] Preferably said enzyme El has an amino acid sequence that has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to SEQ ID NO: l .

[049] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity or similarity or homology and performs a statistical analysis of the identity or similarity or homology between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).

[050] The term "functional fragment", as used herein, refers to a fragment of the enzyme that retains the characteristic catalytic activity of said enzyme.

[051 ] In preferred embodiments, enzyme E2 is an EC 1.2.1.19 class enzyme. [052] In further preferred embodiments, enzyme E2 is γ-aminobutyraldehyde dehydrogenase (PatD). This enzyme acts on the aldehyde group of 5-aminopentanal with oxidized beta- nicotinamide adenine dinucleotide (NAD ⁺ ) as acceptor to produce 5-aminovalerate and reduced beta-nicotinamide adenine dinucleotide (NADH).

[053] In particularly preferred embodiments, enzyme E2: a) has the amino acid sequence of SEQ ID NO:2; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).

[054] Preferably said enzyme E2 has an amino acid sequence that has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to SEQ ID NO:2.

[055] In various embodiments, the recombinant cell has, compared to its wildtype, an increased activity of an enzyme E3 that catalyzes the decarboxylation of lysine.

[056] The term "enzyme E3 that catalyzes the decarboxylation of lysine", as used herein, includes any enzyme that catalyzes the conversion of L-lysine into cadaverine.

[057] In preferred embodiments, enzyme E3 is a lysine decarboxylase.

[058] Decarboxylases are a class of enzymes that catalyze the removal of a carbon dioxide from a carboxylic group. Lysine decarboxylases are enzymes that catalyze the decarboxylation of L-lysine generating cadaverine.

[059] In further preferred embodiments, enzyme E3 is an EC 4.1.1.18 class enzyme.

[060] In particularly preferred embodiments, enzyme E3 is Escherichia coli lysine decarboxylase (ldcC).

[061] In various embodiments, enzyme E3: a) has the amino acid sequence of SEQ ID NO:3; or b) has at least 80% sequence identity to the amino acid sequence as defined in a); or c) is a functional fragment of a) or b).

[062] Preferably said enzyme E3 has an amino acid sequence that has at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% sequence identity to SEQ ID NO:3.

[063] It is also contemplated that in certain embodiments, a hybrid enzyme having the enzymatic activities of enzymes El and E2 and/or enzyme E3 and having the desired substrate specificity for 5-aminovalerate synthesis may be used, with such an enzyme being generated by recombinant DNA technology. Alternatively, an enzyme which does initially not have the specific properties as defined herein can be modified, for example by altering the amino acid sequence thereof, in order to generate an enzyme having the enzymatic activities of enzymes El and E2 and/or enzyme E3 and having the desired substrate specificity. [064] In certain embodiments, the recombinant cell is further genetically engineered to increase the production of 5-aminovalerate, for example, by promoting the uptake or synthesis of a precursor thereof such as L-lysine or cadaverine, enhancing a positive feedback or reducing a negative feedback regulating biosynthesis thereof, reducing the catabolism or degradation thereof, or increasing the tolerance of the cell towards 5-aminovalerate and precursors thereof.

[065] In various preferred embodiments, the recombinant cell of the invention is a Corynebacterium glutamicum cell, preferably a Corynebacterium glutamicum GRLysl cell, wherein (a) the sugR gene as set forth in SEQ ID NO: 8 or a fragment thereof is deleted in said cell; (b) the ldhA gene as set forth in SEQ ID NO: 9 or a fragment thereof is deleted in said cell; (c) the snaA gene as set forth in SEQ ID NO: 10 or a fragment thereof is deleted in said cell; (d) the lysE gene as set forth in SEQ ID NO: l 1 or a fragment thereof is deleted in said cell; and/or (e) the lysG gene as set forth in SEQ ID NO: 12 or a fragment thereof is deleted in said cell.

[066] The deleted fragments of the nucleotide sequences set forth in SEQ ID Nos: 8-12 comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 80, at least 100, at least 130, at least 150, at least 180, at least 200 or at least 250 nucleotides.

[067] The Corynebacterium glutamicum GRLysl cell is well-known in art by Unthan et al. (Unthan et al., Biotechnol. J. 2015, 10, 290-301) and also characterized in Table 1 (B) of this document.

[068] In another aspect, the invention is directed to a method for preparing 5-aminovalerate, the method comprising culturing the recombinant cell in a growth medium under conditions allowing production of 5-aminovalerate, and recovering 5-aminovalerate from the medium.

[069] In some embodiments, exogenous L-lysine is introduced to the culture in a suitable manner, for example added at the beginning or at a certain stage during culturing or fed continuously, to promote the synthesis of 5-aminovalerate by the recombinant cell. In other embodiments, it is also possible that cadaverine is directly introduced into the culture as the substrate.

[070] The recombinant cell according to the invention may be cultured in growth medium in a continuous process or in a batch process (batch culture) or in a fed-batch process or repeated fed-batch process for the purpose of producing 5-aminovalerate. A semi-continuous process, as described in GB-A-1009370 for example, is also conceivable. A review of other known culturing methods is described in the textbook by Chmiel ("Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik" [Bioprocessing 1. Introduction to bioprocessing] (Gustav Fischer Verlag, Stuttgart, Germany, 1991)) or in the textbook by Storhas ("Bioreaktoren and periphere Einrichtungen" [Bioreactors and peripheral equipment], Vieweg Verlag, Brunswick/Wiesbaden, Germany, 1994).

[071] The growth medium to be used must be suited to the requirements of the particular cell. Culture media for various microorganisms are described in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C, USA, 1981).

[072] Carbon sources which may be used are carbohydrates [such as, for example, monosaccharides (e.g. glucose, fructose, galactose, arabinose, xylose), oligosaccharides (e.g. maltose, saccharose, lactose), and polysacharides (e.g. starch, hydrolysed starch, cellulose, hydrolysed cellulose, hemicellulose, hydrolysed hemicellulose)], amino sugars (e.g. glucosamine, N-acetylglucosamine), and reaction products thereof such as, for example, sugar alcohols and polyhydroxy acids; carbon dioxide; organic mono-, di- and tricarboxylic acids optionally carrying 1 or more, e.g. 1, 2, 3 or 4, hydroxyl groups, e.g. acetic acid, tartaric acid, itaconic acid, succinic acid, propionic acid, lactic acid, 3-hydroxypropionic acid, fumaric acid, maleic acid, 2,5-furandicarboxylic acid, glutaric acid, laevulinic acid, gluconic acid, aconitic acid, succinic acid and diaminopimelic acid, citric acid; lipids; oils or fats such as, for example, rapeseed oil, soya oil, palm oil, sunflower oil, groundnut oil and coconut oil; saturated and unsaturated fatty acids, preferably with from 10 to 22 carbons, for example γ-linolenic acid, dihomo-y-linolenic acid, arachidonic acid, palmitic acid, stearic acid, linoleic acid, eicosapentaenoic acid and docosahexaenoic acid; hydrocarbons such as methane; alcohols, for example with from 1 to 22 carbons, e.g. butanol, methanol, ethanol; diols, preferably with from 3 to 8 carbons, e.g. propanediol and butanediol; polyhydric (also referred to as higher) alcohols with 3 or more, for example 3, 4, 5 or 6, OH groups, e.g. glycerol, sorbitol, mannitol, xylitol and arabinitol; ketones, preferably with from 3 to 10 carbons and, where appropriate, 1 or more hydroxyl groups, e.g. acetone and acetoin; lactones, e.g. γ-butyrolactone, cyclodextrins, biopolymers, e.g. polyhydroxyacetate, polyesters, e.g. polylactide, polysaccharides, polyisoprenoids, polyamides; aromatic compounds, e.g. aromatic amines, vanillin and indigo; proteins, for example enzymes such as amylases, pectinases, acidic, hybrid or neutral cellulases, esterases such aslipases, pancreases, proteases, xylanases and oxidoreductases such as laccase, catalase and peroxidase, glucanases, phytases; carotenoids, e.g. lycopene, (3-carotene, astaxanthin, zeaxanthin and canthaxanthin; proteinogenic and non-proteinogenic amino acids, e.g. lysine, glutamate, methionine, phenylalanine, aspartic acid, tryptophan and threonine; purine and pyrimidine bases; nucleosides and nucleotides, e.g. nicotinamide-adenine dinucleotide (NAD) and adenosine 5'-monophosphate (AMP); and also precursors and derivatives, for example salts of the acids mentioned, of the compounds mentioned above. [073] These substances may be used individually or as mixture. Particular preference is given to using carbohydrates, in particular glucose.

[074] Nitrogen sources which may be used are organic compounds containing nitrogen, such as glucosamine, N-acetylglucosamine, peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as mixture.

[075] Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium salts.

[076] The culture medium should furthermore contain metal salts such as, for example, magnesium sulphate or iron sulphate, which are required for growth.

[077] Essential growth substances such as amino acids and vitamins may be used in addition to the substances mentioned above.

[078] The pH of the culture is controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulphuric acid in a suitable manner. Foaming can be controlled by using antifoams such as fatty acid polyglycol esters, for example.

[079] Aerobic conditions may be maintained by introducing into the culture oxygen or oxygen-containing gas mixtures such as air, for example. The culture temperature is usually from 20 °C to 45 °C and preferably from 25 °C to 40 °C.

[080] The purification of 5 -amino valerate from the growth medium is preferably carried out continuously, it being furthermore preferred in this context also to produce 5-aminovalerate by fermentation in a continuous manner, so that the entire process from production of 5- aminovalerate up to its purification from the fermentation broth can be carried out continuously. For continuous purification of the preparation of 5-aminovalerate from the growth medium, the latter is continuously passed through a device for removing the cells employed during fermentation, preferably through a filter with a cut-off in the range from 20 to 200 kDa, where solid/liquid separation takes place. It is also feasible to employ a centrifuge, a suitable sedimentation device or a combination of these devices, it being especially preferred to first separate at least part of the cells by sedimentation and subsequently to feed the fermentation broth, which has been partly relieved of the cells, to ultrafiltration or to a centrifugation device.

[081] After the cells have been removed, the fermentation product which is enriched with regard to its 5-aminovalerate content, is fed to a separation system, preferably a multistep separation system. This separation system provides for a plurality of separation steps which are connected in series, from which steps in each case return lines lead away and back to the fermentation tank. Furthermore, exit pipes lead out of the respective separation steps. The individual separation steps may operate by electrodialysis, reverse osmosis, ultrafiltration or nanofiltration. As a rule, the individual separation steps comprise membrane separation devices. The individual separation steps are selected due to the nature and the extent of the fermentation by-products and residual substrates.

[082] Besides being removed by means of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration, where the end product obtained is an aqueous 5 -amino valerate solution, 5- aminovalerate may also be removed by extractive methods from the fermentation solution which has been relieved of cells, in which case pure 5 -amino valerate may ultimately be obtained.

[083] The above described purification of 5-aminovalerate can also be carried out non- continuously and the respective methods are also encompassed by the present invention.

[084] The 5-aminovalerate prepared by the method according to the invention may be neutralized before, during or after purification, for which purpose for example bases such as alkali metal or alkaline earth metal hydroxides, e.g. calcium hydroxide or sodium hydroxide or else N¾ or NH4OH for example, can be employed.

[085] In certain embodiments, an in vitro cell-free system is established and utilized for the production of 5-aminovalerate. For example, in the presence of all the substances necessary for the enzymatic reactions, cadaverine may be contacted with purified enzymes El and E2, or L- lysine may be contacted with purified enzymes El, E2 and E3 to produce 5-aminovalerate.

[086] In still another aspect, the invention encompasses use of the recombinant cell for preparing 5-aminovalerate.

[087] The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments, but that its scope is defined by the appended claims.

EXAMPLES

Materials and Methods

A. Bacterial strains and plasmids

The bacteria and plasmids used in the experiments were shown in Table 1 (A) and (B).

Table 1 (A) and (B). Bacterial strains and plasmids used in the experiments

(A) cgl524), CGP2 (cgl746-cgl752) and CGP3 (cgl 890- cg2071); also named DM1933 ACGP123

GRLysl derivative with the expression plasmids

5AVA5

pVWExl -IdcC and pEKEx3-patDA

(Perez- GRLysl AsugR In-frame deletion of sugR (cg2115) in GRLysl Garcia et al. 2016)

GRLys \ AsugR derivative with the expression plasmids

5AVA3

pVWExl -IdcC and pEKEx3-patDA

(Perez-

In- frame deletion of sugR (cg2115) and IdhA (cg3219)

GRLys 1 AsugRAldhA Garcia et in GRLysl

al. 2016)

GRLys \ AsugRAldhA derivative with the expression

5AVA4

plasmids pVWExl -IdcC and pEKEx3-patDA

GRLys 1 AsugRAldhAAlys In- frame deletion of sugR (cg2115), IdhA (cg3219),

EG lysE (cgl424) and lysG (cgl425) in GRLysl

GRLysl AsugRAldhA AlysEG derivative with the

5AVA6 expression plasmids pVWExl -IdcC and pEKEx3- patDA

In- frame deletion of sugR (cg2115), IdhA (cg3219),

GRLys 1 AsugRAldhAAlys

lysE (cgl424), lysG (cgl425) and snaA (cgl722)

EGAsnaA

in GRLysl

GRLysl AsugRAldhA AlysEG AsnaA derivative with the

5AVA7 expression plasmids pVWExl -IdcC and pEKEx3- patDA

Plasmids

Km ^R ; E. colilC. glutamicum shuttle vector for

(Schafer et pKl SmobsacB construction of insertion and deletion mutants in C.

al. 1994) glutamicum (pK18 oriVEc sacB lacZa)

Km ^R ; E. colilC. glutamicum shuttle vector for

(Schafer et pK19mobsacB construction of insertion and deletion mutants in C.

al. 1994) glutamicum (pK18 oriVEc sacB lacZa)

(Engels and pKl 9mobsacB- AsugR pKl 9mobsacB with a sugR (cg2115) deletion construct Wendisch

2007)

B. Growth conditions of bacteria

[088] Escherichia coli DH5a cells were used for gene cloning. Corynebacterium glutamicum and Escherichia coli cells were routinely grown in lysogeny broth (LB) (10 g/L of tryptone, 5 g/L yeast extract and 10 g/L sodium chloride) in 500 mL baffled flasks on a rotary shaker (120 rpm) at 30 °C or 37 °C, respectively. CGXII minimal medium was also used for general growth of Corynebacterium glutamicum. Growth was followed by measuring optical density at 600 nm. An optical density of 1 at this wavelength corresponds to 0.25 g/L cell dry weight. If necessary, the growth medium was supplemented with kanamycin (25 μg/mL), spectinomycin (100 μg/mL) and isopropyl β-D-l-thiogalactopyranoside (IPTG) (1 mM).

[089] If not mentioned otherwise, C. glutamicum strains were cultured in 500 mL baffled flasks. For 5AVA production experiments BHI seed culture was inoculated from an agar plate and grown overnight. The cells were harvested by centrifugation (4000 x g, 10 min) and washed once with CGXII medium lacking the carbon source. Subsequently, CGXII medium containing a given concentration of carbon along with the necessary supplements was inoculated to an optical density between 0.5-1.0. For the growth experiment with 5AVA as sole carbon or nitrogen source, C. glutamicum was inoculated to an optical density of 0.5 and grown in baffled flasks using CGXII medium with 30 mM of 5AVA as sole carbon source or in free-nitrogen CGXII medium supplemented with 90 mM of 5AVA as sole nitrogen source. To verify if GABA was converted in other products, cells were grown in CGXII medium until optical density of about 20 and then 20 mM of 5AVA was added. For the 5AVA tolerance assays, a second pre -culture was grown until exponential phase and used to inoculate the main culture. CGXII medium was supplemented with different 5AVA concentrations and grown in 48-well flower plate using the Biolector microfermentation system (m2p labs GmbH, Aachen, Germany).

C. Quantification of amino acids and polyamines

[090] For quantification of extracellular metabolites, aliquots of the culture were harvested and the cells were removed by centrifugation (13000 x g, 10 min). The supernatant was transferred to a new tube and analyzed using a high-pressure liquid chromatography (HPLC) system (1200 series, Angilent Technologies Deutschland GmbH, Boblingen, Germany). For quantification of amino acids and polyamines, samples were derivatised with ortho- phthaldialdehyde (OPA) and separated on a system consisting of a pre -column (LiChrospher 100 PvP8 EC-δμ, 40 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP8 Ε^5μ, 125 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and detected with a fluorescence detector (FLD G1321A, 1200 series, Agilent Technologies). L-asparagine was used as internal standard. Elution buffer gradient consisted of 0.25% Na-acetate (pH 6), as the polar phase and methanol as the nonpolar phase were used at a flow rate of 1.0 mL min ^"1 and the injection volume was 5 μΐ. The product yield was calculated by dividing the 5AVA titer by the amount of glucose used.

D. Transcriptome analysis using DNA microarrays

[091] To identify gene expression changes due to the addition of 5AVA to the growth medium, C. glutamicum wild type was grown in CGXII minimal medium with 4% glucose and either 200 mM 5AVA or 200 mM sodium chloride. Cells were harvested in exponential phase of growth by centrifugation (4000 x g, 10 min, 4 °C) and kept at -80 °C.

[092] RNA isolation was performed as described (Wendisch 2003) and the RNA was kept at - 80 °C until further use. DNA microarray analysis, synthesis of fluorescently labelled cDNA from total RNA, DNA microarray hybridization, and gene expression analysis were performed as described previously (Netzer et al. 2004; Polen et al. 2007). The data are available as Gene Expression Omnibus GSE83413 data set at http://www.ncbi.nlm.nih.gov/geo/.

Example 1: Construction of an expression vector encoding PatA and PatD [093] PCR primers listed in table 2 were obtained from Metabion GmbH (Martinsried, Germany). Escherichia coli DH5a cells were used for the preparation of plasmids. Genes were PCR amplified with KOD Hot Start DNA Polymerase (NOVAGEN, Darmstadt, Germany) using genomic DNA of Escherichia coli MG1655 cells as the template. The plasmids were isolated using the QIAprep spin miniprep kit (QIAGEN, Hilden, Germany), and the PCR products were purified using PCR purification kit or MinElute PCR purification kit (QIAGEN, Hilden, Germany). Escherichia coli cells were transformed by heat shock and Corynebacterium glutamicum cells were transformed by electroporation at 2.5 kV, 200 Ω and 25 uF. All cloned DNA fragments were verified by sequencing.

Table 2. Primers used for the cloning of patA and patD

Start and stop codons are in bold, the ribosome binding sites are in italics, and the restriction sites are underlined.

[094] In order to clone patA and patD into an expression vector, the genes were PCR amplified using the respective primer pairs listed in Table 2. The patD gene was first cloned into the PstVBamHl restriction sites in pEKEx3 to generate plasmid ρΕΚΕχ3-£>αίΖλ Subsequently, the patA gene was cloned into the BamHVSacl restriction sites in pEKEx3-patD to generate the plasmid \)EKEx3-patDA as shown in Figure 2. The start codon of the patA gene was changed from ttg to atg.

Example 2: Generation of recombinant Corynebacterium glutamicum cells overexpressing enzymes PatA, PatD and LdcC

Corynebacterium glutamicum DM1933 cells were transformed with the expression vector pVWExl -IdcC encoding lysine decarboxylase (LdcC) from Escherichia coli to generate Cadi cells, which are capable of converting L-lysine into cadaverine. These cells were further transformed with the expression plasmid pEKEx3 _patDA encoding PatA and PatD to generate 5AVA1 cells. Example 3: Fermentative production of 5-aminovalerate using recombinant Corynebacterium glutamicum cells overexpressing enzymes PatA, PatD and LdcC

The production of 5-aminovalerate by recombinant 5AVA1 cells was performed in baffled flasks. Briefly, 5AVA1 cells were cultured in 50 mL of LB medium overnight. Then, the cells were harvested by centrifugation (4000 x g, 10 min) and washed twice with CGXII minimal medium without any carbon source. Finally, the cells were further inoculated into 50 mL of CGXII medium containing 4% (w/v) of glucose and the necessary supplements to grow to an optical density of 1.0.

Example 4: Quantification of 5-aminovalerate fermentatively produced using recombinant Corynebacterium glutamicum cells overexpressing enzymes PatA, PatD and LdcC

[095] For quantification of extracellular 5-aminovalerate, aliquots of the culture were harvested and the cells were removed by centrifugation (13000 x g, 10 min) and the supernatant was transferred to a new tube and analyzed using a high-performance liquid chromatography (HPLC) system (1200 series, Angilent Technologies Deutschland GmbH, Boblingen, Germany). For the detection of 5-aminovalerate, samples were derivatized with ortho- phthaldialdehyde (OPA) and separated on a system consisting of a pre -column (LiChrospher 100 RP18 EC-δμ, 40 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP18 Ε^5μ, 125 x 4 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and detected with a fluorescence detector (FLD G1321A, 1200 series, Agilent Technologies). L-asparagine was used as internal standard. Elution buffer gradient consisted of 0.25% Na-acetate (pH 6), as the polar phase and methanol as the nonpolar phase were used at a flow rate of 0.7 mL min-1 and the injection volume was 5 μΐ. For the detection of cadaverine, samples were derivatised with OPA and separated on an system consisting of a pre -column (LiChrospher 100 RP8 Ε^5μ, 40 x 4.6 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany) and the main column (LiChrospher 100 RP8 Ε^5μ, 125 x 4.6 mm, CS-Chromatographie Service GmbH, Langerwehe, Germany). 1,7- diaminoheptane was used as internal standard. Elution buffer gradient consisted of 0.25% Na- acetate (pH 6), as the polar phase and methanol as the nonpolar phase were used at a flow rate of 1.0 mL min-1 and the injection volume was 5 μΐ.

Example 5: Comparison of recombinant Corynebacterium glutamicum Cadi and 5A VA1 cells in the production of 5-aminovalerate

[096] Cadi and 5AVA1 cells were used for the production of 5-aminovalerate, and 5- amino valerate produced by these cells were subjected to HPLC quantification. As shown in Table 3, Cadi cells expressing LdcC did not produce 5 -amino valerate to a detectable level. To the contrary, 5AVA1 cells expressing LdcC, PatA and PatD produced 5-aminovalerate at a concentration of 21.0 ± 0.2 mM.

Table 3. Concentration of 5-aminovalerate produced by the recombinant Corynebacterium glutamicum cells

[097] Example 6: 5A VA tolerance assay, 5A VA uptake and 5A VA conversion

The toxicity of the product to the cells is of utmost importance in biotechnical processes, since product inhibition often limits productivity. To our knowledge the response of C. glutamicum to extracellular 5AVA has not yet been studied. In order to determine the influence of 5AVA on growth, different concentrations of 5AVA (0 mM; 10 mM; 20 mM; 40 mM; 80 mM; 160 mM; 320 mM; 640 mM and 1280 mM) were added to CGXII minimal medium with 4 % (w/v) glucose and the growth rate of C. glutamicum wild type was calculated. For concentrations of 5AVA up to 160 mM the growth rate was mildly affected, while growth was slower for higher concentrations of 5AVA (Fig. 5). Approximately, 1.1 M of 5AVA reduced the maximum growth rate to half (Fig. 5). Thus, C. glutamicum is an adequate host for the production of 5AVA to high concentrations.

[098] To evaluate if metabolic engineering strategies would be necessary to apply to avoid 5AVA re-uptake, C. glutamicum wild type was grown in CGXII minimal medium with 30 mM of 5AVA as sole carbon source or in free-nitrogen CGXII minimal medium with 90 mM 5AVA as sole nitrogen source. For both cases, the cells did not grow indicating that C. glutamicum is not able to use 5AVA as sole carbon or nitrogen source (data not shown) and 5AVA re-uptake will not be a problem in 5AVA strain engineering.

[099] To verify if product degradation is a problem to design high 5AVA productions strains, C. glutamicum wild type was grown in CGXII minimal medium with 4 % (w/v) glucose up to an OD6oonm of 20. Then 20 mM of 5AVA was supplemented to the medium and samples were harvested at this time point, after 15h, 25h and 48h to quantify 5AVA by HPLC. 5AVA degradation was not observed (Fig. 6), therefore, no genetic manipulation will be necessary to avoid product degradation. [0100] C. glutamicum cells growing exponentially in glucose minimal medium supplemented with 200 mM either 5AVA or sodium chloride were harvested and RNA was prepared. The transcriptome analysis showed that several genes were differentially expressed (Fig. 7). The results revealed increased expression of several genes of carbon metabolism such as cdaS (cyclomaltodextrinase), glgC (glucose- 1 -phosphate adenylyltransferase), cgl373 (putative glyoxalase), pfkA (6-phosphofructokinase), nagD (putative phosphate involved in N- acetylglucosamine metabolism), mdh (malate dehydrogenase), and butA (1-2,3 -butanediol dehydrogenase/acetoin reductase). Also several membrane proteins had increased mRNA levels, as well as, sucE (succinate exporter). On other hand, two genes involved in amino acid biosynthesis (himP and cysK) showed decreased expression, as well as, one gene involved in thiamine biosynthesis (nadS). Gene expression response indicated that product inhibition is not limiting 5AVA production.

Example 8: Heterologous expression of IdcC, patA and patD from E. coli enabled 5A VA production from glucose fermentation in Corynebacterium glutamicum wild type

[0101] C. glutamicum wild type is able to synthesize the amino acid 1-lysine, however, it is unable to use this amino acid for catabolic purposes (Bellmann et al. 2001), therefore, cannot produce naturally 5AVA. In fact, C. glutamicum needs to be metabolic engineered by expressing heterologous genes to produce the 1-lysine-derived compounds cadaverine (Kind et al. 2010a; Mimitsuka et al. 2007) and 1-pipecolic acid (Perez-Garcia et al. 2016). 5AVA production can be obtained by the decarboxylation of 1-lysine into the diamine cadaverine, which in turn can be transaminated and further dehydrogenated into the non-protein amino acid 5AVA (patent application, 10 2015 114 785.8). Therefore, the gene encoding the constitutive 1- lysine decarboxylase (LdcC) from E. coli was cloned into the IPTG-inducible expression plasmid pVWExl (Peters- Wendisch et al. 2001) and the genes encoding the putrescine transaminase (PatA) and the γ-aminobutyraldehyde dehydrogenase (PatD) from E. coli were cloned in the IPTG-inducible expression plasmid pEKEx3 (Stansen et al. 2005). Transformation of C. glutamicum wild type with the plasmids pVWExl -IdcC and pEKEx3-patDA (Table 1 (B)) yielded C. glutamicum strain 5AVA2 (Table 1 (B)). When cultured in shake flasks using CGXII minimal medium with 4 % (w/v) glucose and 1 mM IPTG, the parental WT strain (pVWExl )(pEKEx3) did not produce any 5AVA (Fig. 8), while 5AVA2 produced 7.5 mM of 5AVA (Fig. 8), proving for the first time that is possible to produce 5AVA by metabolic engineering C. glutamicum. The production of 1-lysine, cadaverine and N-acetylcadaverine as side-products could also be detected (Fig. 8). Example 9: 5 A VA production using strains engineered for high l-lysine titers

[0102] The strain 5AVA2 produced 5AVA in C. glutamicum for the first time, however, the product titers were low meaning that the overexpression of three E. coli genes can only be the first step towards a competitive industrial strain. As 5AVA is derived from l-lysine, previous metabolic engineering strategies of C. glutamicum l-lysine production strains (Unthan et. al 2015) seem to be relevant and helpful. Thus, the l-lysine overproducer strain GRLysl (Unthan et. al 2015) was transformed with the plasmids pVWExl-WcC and pEKEx3-patDA, yielding the strain 5AVA5 (Table 1 (B)). The 5AVA5 strain produced almost 5 times more 5AVA than the strain 5AVA2 (Fig. 8).

[0103] SugR is DeoR-type transcriptional regulator that plays a key role in genetic control of a wide range of genes involved in sugar metabolism of C. glutamicum. Genes of the main glucose uptake system of C. glutamicum, the phosphoenolpyruvate phosphotransferase system (PTS), are repressed by SugR, as well as, other genes of glycolysis, namely the 1-lactate dehydrogenase encoded by IdhA that leads to 1-lactate formation (Engels et al. 2008; Engels and Wendisch 2007; Teramoto et al. 2011). More recently, deletion of sugR showed to increase l-lysine titers and volumetric productivities as well as the side -product 1-lactate (Perez-Garcia et al. 2016).

[0104] Strain 5AVA3 (5 A A5 AsugR, Table 1 (B)) was constructed under the assumption that an increase observed for the precursor l-lysine could result in the production of higher amounts of 5AVA. However, 5AVA titers were only slightly increased to 37.8 mM (Fig. 8), which might be due to the accumulation of 1-lactate as a side product.

Example 10: Medium optimization for 5A VA production

[0105] CGXII minimal medium was used in 5AVA production experiments. This medium was originally optimized for l-lysine production (Kelle et al. 2005). l-lysine is an amino acid with two amino groups while 5AVA only contains one amino group. Reduction of the nitrogen amount in the CGXII medium has been successfully applied for the production of amino acids with only one amino group, like GAB A (Jorge et al. 2016) or proline (Jensen and Wendisch 2013), therefore, it was evaluated if the reduction of nitrogen amount of the medium affected 5AVA production. Experiments using CGXII medium with half concentration of nitrogen (CGXIIm, i.e. 2.5 g L ¹ instead of 5 g L ¹ urea and 10 g L ¹ instead of 20 g L ¹ ammonium sulfate) were performed with the strain 5AVA3. The reduction of the nitrogen amount in CGXII medium led to a production of 35.8 mM of 5AVA which represents about 6% less as compared with the same strain grown in normal CGXII medium (Fig. 8), thus, in this case the reduction of the nitrogen concentration in the medium was not helpful. Example 11: Avoid production of the by-product l-lactate to boost 5A VA production

[0106] Perez-Garcia et al. revealed that the deletion of sugR in GRLysl led to the accumulation of the undesirable end-product 1-lactate and that IdhA deletion showed to help the production of the 1-lysine derive amino acid, 1-pipecolic acid (Perez-Garcia et al. 2016). Therefore, IdhA was deleted in the strain 5AVA3, yielding the strain 5AVA4 (Table 1 (B)). The AldhA strain showed an increase in the production of 5AVA to 44.9 mM which represents an increase of 19% as compared to 5AVA3 (Fig. 8) and corresponds to a yield of 202 mmol mol ^"1 glucose, indicating that avoid the formation of the by-product 1-lactate is an helpful strategy to increase 5AVA production.

Example 12: Avoid l-lysine export and the formation of N-acetylcadaverine

[0107] GRLysl strain contains two copies of the 1-lysine exporter gene lysE (Unthan et. al 2015). Deletion of one copy of lysE was done under the assumption that when only one copy of the lysE gene is present in the genome of C. glutamicum less 1-lysine would be exported and more 1-lysine would be available intracellularly to be converted into 5AVA. However, it had the contrary effect because when one copy of lysE was deleted, more 1-lysine and less 5AVA (36.8 mM) were observed extracellularly (see 5AVA6 strain, Fig. 8). The little amounts of 1-lysine detected were also detected for the WT strain that only contains a copy of the lysE gene, therefore, only by deleting both copies of this gene the loss of 1-lysine could be completely avoided.

[0108] N-acetylcadaverine is a well-known side product of cadaverine production (Kind et al. 2010a) and snaA (cgl722) was identified as the gene responsible for the acetylation of cadaverine and its deletion led to strains unable to produce N-acetylcadaverine (Kind et al. 2010b). N-acetylcadaverine was also observed as a side product of all the 5AVA production strains (Fig. 8), thus, the deletion of snaA was performed in the strain 5AVA6, yielding 5AVA7 (Table 1 (B)), which, as expected, did not produce anymore N-acetylcadaverine (Fig. 8). The 5AVA production increased to 39.2 mM as compared to the parental strain 5AVA6 (Fig. 8), however, the final 5AVA titer was lower as compared to the strain 5AVA4 (Fig. 8).