WO2019228937A1

WO2019228937A1 - Means and methods for the production of glutarate

Info

Publication number: WO2019228937A1
Application number: PCT/EP2019/063559
Authority: WO
Inventors: Christoph Wittmann; Judith Becker; Lars GLAESER; Christina ROHLES
Original assignee: Universitaet des Saarlandes
Current assignee: Universitaet des Saarlandes
Priority date: 2018-05-28
Filing date: 2019-05-27
Publication date: 2019-12-05
Anticipated expiration: 2020-11-28

Abstract

The present invention relates to a polynucleotide encoding NCgl0464, a host cell comprising said polynucleotide and optionally further polynucleotides. It also provides for a use of the polynucleotides, vectors or host cell for the production of glutarate and methods for producing glutarate. NCgl0464 is a 5-aminovalerate permease to be overexpressed and the further polynucleotides may encode gabT, gabA, gabB and/or gabT to be overexpressed. Also, a polynucleotide encoding lysE may be inactivated in the host cell.

Description

MEANS AND METHODS FOR PRODUCTION OF GLUTARATE

TECHNICAL FIELD OF THE INVENTION

[1] The present invention relates to a polynucleotide encoding NCgl0464, a host cell comprising said polynucleotide and optionally further polynucleotides. It also provides for a use of the polynucleotides, vectors or host cell for the production of glutarate and methods for producing glutarate.

BACKGROUND ART

[2] Today’s petrochemical industry is challenged by the ever-increasing demand for commodity chemicals, polymer materials and related compounds, which is additionally aggravated by the ever-decreasing accessibility of fossil resources as base material. Hence, substantial effort has been made for providing alternative“green” routes to produce platform chemicals through microbial fermentation processes. Compounds of interest comprise organic acids such as succinate, lactate, and itaconic acid, diamines including putrescine and cadaverine and diols, all being applicable as building blocks for bio-based plastics.

[3] In this regard, also glutarate is an attractive carbon-5 building block for the production of nylon from renewable feedstocks. By now, Escherichia coli has been in the focus of interest as platform for either de novo biosynthesis of glutarate or for biotransformation thereof from L- lysine. As glutarate is a degradation product of the proteinogenic amino acid L-lysine, the non- pathogenic Gram-positive soil bacterium Corynebacterium glutamicum, a well-established industrial L-lysine producer, has been recently been chosen as metabolic chassis for the production of this carbon-5 platform chemicals (Rohles et al. 2016).

[4] Beyond the excellent availability of genetic tools, and knowledge of its physiology and large-scale fermentation, engineered C. glutamicum has only recently been established as centerpiece for the production of L-lysine-derived cadaverine within a pipeline towards the manufacturing of the fully bio-based polyamide PA5.10. In Rohles et al, the metabolic pathway from L-lysine towards 5-aminovalerate was reconstituted by stable genome-based implementation of the Pseudomonas putida KT2440 genes davB, encoding lysine monooxygenase, and davA, encoding 5-aminovaleramide amidase (Fig. 1 ), which yielded strain AVA-1 producing L-lysine, 5-aminovalerate and glutarate. This strain was then further optimized for increased production of 5-aminovalerate and glutarate via deletion of lysE, encoding the L- lysine exporter (strain AVA-2). [5] However, there is still a need to provide improved means and methods for the production of glutarate. The technical problem therefor is to comply with this need.

SUMMARY OF THE INVENTION

[6] The inventors of the present application surprisingly found that the gene NCgl0464 encodes for a permease of 5-aminovalerate. NCgl0464 has never been related to any activity associated with 5-aminovalerate. The activity or function of NCgl0464 is useful in the bio production of glutarate as it facilitates the re-import of 5-aminovalerate, a precursor of glutarate, into the host cell. Increasing the activity of NCgl0464 in the model organism Corynebacterium glutamicum increased the amount of glutarate that can be isolated from the culture medium (see e.g. Examples 3, 4 and 5). The invention provides a polynucleotide encoding NCgl0464, a host cell comprising said polynucleotide and optionally further polynucleotides. It also provides for a use of the polynucleotides, vectors or host cell for the production of glutarate and methods for producing glutarate.

[7] Accordingly, the invention relates in a first aspect to a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 ; (b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 2; (c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has 5-aminovalerate permease activity; (d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has 5-aminovalerate permease activity; and (e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 1 and having 5-aminovalerate permease activity, wherein said polynucleotide is operably linked to an expression control sequence which allows overexpression of said polynucleotide in a host cell.

[8] Preferably, the expression control sequence is a promoter and/or the expression control sequence is heterologous to said polynucleotide. More preferably, said expression control sequence is selected from the group consisting of EFTU (SEQ ID NO: 25), SOD (SEQ ID NO: 26), Tac (SEQ ID NO: 27), ilvC (SEQ ID NO: 28), Trp (SEQ ID NO: 29), PL (SEQ ID NO: 30), PR (SEQ ID NO: 31 ), lacUV5 (SEQ ID NO: 32), T7lac (SEQ ID NO: 33), AraBD (SEQ ID NO: 34), H3s (SEQ ID NO: 35), H4 (SEQ ID NO: 36), H5 (SEQ ID NO: 37), H17 (SEQ ID NO: 38), H28 (SEQ ID NO: 39), H30 (SEQ ID NO: 40), H34 (SEQ ID NO: 41 ), H36 (SEQ ID NO: 42) and H72 (SEQ ID NO: 43). [9] The invention further relates to a vector comprising the polynucleotide of the invention.

[10] The invention further relates to a host cell genetically engineered with the vector of the invention, or comprising a polynucleotide of the invention or overexpressing a polynucleotide of any one of the first aspect items (a) to (e).

[11] Preferably, the host cell of the invention further comprises a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:3; (b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:4; (c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has glutarate-semialdehyde dehydrogenase activity; (d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has glutarate-semialdehyde dehydrogenase activity; and (e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 3 and having glutarate- semialdehyde dehydrogenase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

[12] Preferably the host cell of the invention further comprises a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 5; (b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 6; (c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has aminotransferase activity; (d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has aminotransferase activity; and (e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 5 and having aminotransferase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

[13] The invention also relates to the host cell of the invention, wherein preferably in said host cell a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:7; (b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:8; and (c) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 7 and having lysine exporter activity, is inactivated.

[14] Preferably the host cell of the invention further comprises a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:9; (b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:10; (c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has aminovaleramidase activity; (d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has aminovaleramidase activity; and (e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 9 and having aminovaleramidase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

[15] Preferably the host cell of the invention further comprises a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 1 ; (b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 12; (c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has monooxygenase activity; (d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has monooxygenase activity; and (e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 1 1 and having monooxygenase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

[16] Preferably, the host cell of the invention has at least 95 % sequence identity to any of the 16S of Corynebacterium glutamicum strain DSMZ 20300 RNA sequences set forth in SEQ ID NO: 13-18 and/or to any one of 23S RNA sequences set forth in SEQ ID NO: 19-24 of Corynebacterium glutamicum strain DSMZ 20300.

[17] Preferably, the host cell of the invention is selected from the group consisting of Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Corynebacterium crenatum, Brevibacterium tianjinese, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium efficiens and Escherichia coli. Even more preferably, the host cell of the invention is Corynebacterium glutamicum, most preferably Corynebacterium glutamicum (DSMZ 20300).

[18] The invention also relates to a use of the polynucleotides of the invention, the vector of the invention or the host cells of the invention for the production of glutarate.

[19] The invention further relates to a method for producing glutarate, comprising the steps:

(i) culturing a host cell of the invention under conditions allowing the production of glutarate; and

(ii) obtaining glutarate from the culture medium.

[20] The invention further relates to a method for producing glutarate, comprising the steps: (i) providing a host cell; (ii) increasing the activity of NCgl0464, optionally by genetically engineering the host cell with the polynucleotide of the invention or the vector of the invention; (vii) culturing the host cell in a suitable culture medium; and (viii) obtaining glutarate from the culture medium, optionally further comprising at least one of the following steps: (iii) increasing the activity of gabT, (iv) decreasing the activity of lysE, (v) increasing the activity of gabA and/or gabB, and/or (vi) increasing the activity of gabD.

BRIEF DESCRIPTION OF THE DRAWINGS

[21] Fig. 1 depicts the metabolic pathway for production of 5-aminovalerate and glutarate in Corynebacterium glutamicum. Heterologous genes davBA from P. putida were integrated into the genome via homologous recombination to reconstruct the 5-aminovalerate pathway (Rohles et al., 2016).

[22] Fig. 2 depicts the growth and production characteristics of the 5-aminovalerate and glutarate producing strains C. glutamicum AVA-2 (A) and GTA-1 (B). Both strains were cultivated in shake flasks at 30°C in a chemically defined medium. The cultivation profiles show growth, product formation and glucose consumption over time and represent mean values and corresponding standard deviations from three biological replicates.

[23] Fig. 3 depicts the growth and production characteristics of the glutarate producing C. glutamicum strain GTA-1 eftu0464 (GTA-2). Cultivations took place in shake flasks at 30°C in a chemically defined medium. The cultivation profile shows growth, product formation and glucose consumption over time and represents mean values and corresponding standard deviations from three biological replicates.

[24] Fig. 4 depicts growth and production characteristics of glutarate producing C. glutamicum strains. The strains AVA-2 (A, B), GTA-1 (C, D), and GTA-4 (E, F) were cultivated in shake flasks at 30 °C in a chemically defined glucose medium. The cultivation profiles show the growth, the product formation and the glucose consumption over time (A, C, E) and the yields (B, D, F). Error bars represent standard deviations from three biological replicates.

[25] Fig. 5A depicts the growth and production characteristics of the glutarate producing C. glutamicum strain GTA-4 under fed-batch conditions. The cultivation profile shows growth, product formation and glucose consumption over time and represents mean values and corresponding standard deviations from three biological replicates

[26] Fig. 5B depicts the amount of glutarate produced by glutarate-producing C. glutamicum strain GTA-4 per added amount of sugar.

[27] Fig. 6 depicts a labeling study of C. glutamicum for elucidation of 5- aminovalerate/glutarate metabolism. (A) Carbon transition during formation of glutarate from glucose and 5-aminovalerate. Non-labeled carbon atoms are given in white, labeled carbon atoms in grey. (B) TIC spectrum showing supernatant sample of AVA-2, grown on naturally labeled glucose. (C) Mass isotopomer distribution of glutarate contained in supernatant samples of C. glutamicum AVA-2 (1^st bar) and GTA-3 (2^nd bar) grown on U-¹³C glucose without (left) and with (right) addition of naturally labeled 5-aminovalerate. In addition the mass isotopomer distributions of glutarate from a standard solution (3rd bar) and of a supernatant sample of AVA- 2, grown on naturally labeled glucose (4th bar) are given. The GC-MS raw data were corrected for natural isotopes.

DETAILED DESCRIPTION OF THE INVENTION

[28] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.

[29] The present invention provides a polynucleotide encoding NCgl0464, a host cell comprising said polynucleotide and optionally further polynucleotides. It also provides for a use of the polynucleotides, vectors or host cell for the production of glutarate and methods for producing glutarate.

[30] The inventors surprisingly discovered that NCIg0464 is not only a GABA-specific transporter (Zhao et al. 2012) but also functions as an permease of 5-aminovalerate. This surprising function can be used to increase the production of glutarate by eliminating the loss of an important precursor in glutarate production. [31] Accordingly, the invention relates to polynucleotides, herein also referred to as NCgl0464-polynucleotides, comprising a nucleotide sequence encoding NCgl0464 or a fragment thereof, wherein said polynucleotide is operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell. In a first aspect, the polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1. In a second aspect, tie polynucleotide has or consists of the nucleotide sequence as shown in SEQ ID NO: 2. In a third aspect, the polynucleotide has a nucleotide sequence encoding a fragment of a polypeptide encoded by the first or second aspect, wherein said fragment has 5-aminovalerate permease activity. In a fourth aspect, the polynucleotide has a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of the first to the third aspect, wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has 5-aminovalerate permease activity. In a fifth aspect, the polynucleotide has a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to the amino acid sequence as shown in SEQ ID NO: 1 and has 5-aminovalerate permease activity. The invention may also relate to polynucleotides of the 1^st to 5^th aspect of NCgl0464-polynucleotide, which are not operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell.

[32] “Fragment” as used herein describes a part of a certain nucleotide sequence, gene, amino acid sequence or a protein. Such a fragment may be shortened by at least one amino acid at N-terminal or C-terminal of a protein or at least one nucleotide at the 5’- or 3’-end of a polynucleotide. A fragment of a polypeptide may lack 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 amino acids at the N-terminus and/or C-terminus. A fragment of a polynucleotide may lack 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nucleotides at the 5’ end and/or the 3’ end. An important feature of the fragments is that they retain their ability to carry out the activity of the source sequence. When compared to the source sequence, the activity of the fragment may be at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 %. The activity of a fragment may also be higher than the activity of the source sequence.

[33] A variety of sequence based alignment methodologies, which are well known to those skilled in the art, can be used to determine identity among sequences. These include, but are not limited to, the local identity/homology algorithm of Smith, F. and Waterman, M. S. (1981 ) Adv. Appl. Math. 2: 482-89, homology alignment algorithm of Peason, W. R. and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85: 2444-48, Basic Local Alignment Search Tool (BLAST) described by Altschul, S. F. et al. (1990) J. Mol. Biol. 215: 403-10, or the Best Fit program described by Devereau, J. et al. (1984) Nucleic Acids. Res. 12: 387-95, and the FastA and TFASTA alignment programs, preferably using default settings or by inspection. Alternatively, an alignment may be done manually/visually for amino acids sequences as follows: the percent identity between an amino acid sequence in question and the amino acid sequence of NCgl0464, GabD, GabT, LysE, GabA, GabB, 16S RNA or 23S RNA as defined herein is determined by pairwise alignment in such a way that the maximum identity is obtained between both amino acid sequences. The identical amino acid residues between both amino acid sequences are counted and divided by the total number of residues of the amino acid sequence of NCgl0464, GabD, GabT, LysE, GabA, GabB, 16S RNA or 23S RNA (including positions that do not contain amino acid residues, e.g. one or more gaps) yielding the percentage of identity. A similar method applies to nucleotide sequences: An alignment may be done manually/visually for nucleotide sequences as follows: the percent identity between an nucleotide sequence in question and the nucleotide sequence of NCgl0464, GabD, GabT, LysE, GabA, GabB, 16S RNA or 23S RNA as defined herein is determined by pairwise alignment in such a way that the maximum identity is obtained between both nucleotide sequences. The identical nucleotides between both nucleotide sequences are counted and divided by the total number of nucleotides of the nucleotide sequence of NCgl0464, GabD, GabT, LysE, GabA, GabB, 16S RNA or 23S RNA (including positions that do not contain nucleotides, e.g. one or more gaps) yielding the percentage of identity.

[34] “5 -aminovalerate permease activity” as used herein is the ability to allow the transport of 5-aminovalerate through the cell membrane of a host cell. This ability can be quantified by means known to a person skilled in the art. For example, the amount of 5-aminovalerate can be determined by quantitative mass spectrometry, chromatographic analysis like HPLC, RP-HPLC or as described in Example 1. To determine whether a fragment still has 5-aminovalerate permease activity, the fragment may be incorporated into a liposome or microsome filled with a liquid comprising a given concentration of 5-aminovalerate. The liposome comprising NCgl0464 will then be transferred into an environment comprising no 5-aminovalerate and the concentration of 5-aminovalerate will be measured after a given time. If 5-aminovalerate is measured at a level of at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % of the level transported outside by the full-length NCgl0464 in the environment, the fragment has 5- aminovalerate permease activity.

[35] The term“derivative” as used herein relates to derivatives or variants of a protein or peptide that include modifications of the amino acid sequence, for example by substitution, deletion, insertion or chemical modification. Such variants include proteins, wherein one or more amino acids have been replaced by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline, or wherein one or more amino acid residues are conservatively substituted compared to said polypeptide. A“conservative substitution” as used herein is an amino acid substitution that changes an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size). Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.

[36] All polynucleotides of the invention, especially NCgl0464-polynucleotides, may be operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell. An expression control sequence is sometimes also called a “regulatory sequence”. This provides an increased activity of the protein encoded by the polynucleotide. Such an expression control sequence may be a promotor and/or may be heterologous to said polynucleotide. Preferably, the promotor is selected from the group consisting of EFTU (SEQ ID NO: 25), SOD (SEQ ID NO: 26), Tac (SEQ ID NO: 27), ilvC (SEQ ID NO: 28), Trp (SEQ ID NO: 29), PL (SEQ ID NO: 30), PR (SEQ ID NO: 31 ), lacUV5 (SEQ ID NO: 32), T7lac (SEQ ID NO: 33), AraBD (SEQ ID NO: 34) H3 (SEQ ID NO: 35), H4 (SEQ ID NO: 36), H5 (SEQ ID NO: 37), H17 (SEQ ID NO: 38), H28 (SEQ ID NO: 39), H30 (SEQ ID NO: 40), H34 (SEQ ID NO: 41 ), H36 (SEQ ID NO: 42) and H72 (SEQ ID NO: 43) Further, the expression control sequence may be controllable by the addition or removal of a chemical agent, e.g. lactose, IPTG (Isopropyl b-D-l-thiogalactopyranoside) or else.

[37] The invention also relates to a vector comprising the polynucleotides of the invention. This vector may additionally comprise a promotor to allow the overexpression of the polynucleotide in a host cell, a selection marker, to enrich host cells comprising the vector, or comprise further polynucleotides mentioned within this application. Examples for a vector include pClik_int_sacB or pClik_5A_MCS.

[38] The invention also provides a host cell. Such a host cell provides the machinery for the production of glutarate. This host cell may be genetically engineered with the vector of the invention, comprise a polynucleotide of the invention or overexpress the NCgl0464- polynucleotide not linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell. Such a host cell is especially useful for the production of glutarate as it allows the re-import of 5-aminovalerate into the host cell by using the activity of NCgl0464. Thus, the important precursor 5-aminovalerate is not lost. Preferred host cells are selected from the group consisting of Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Corynebacterium crenatum, Brevibacterium tianjinese, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium efficiens and Escherichia coli. More preferably, the host cell is Corynebacterium glutamicum, even more preferably Corynebacterium glutamicum (DSMZ 20300). Another preferred host cell is characterized by its similarity to the 16S and/or 23S RNA of Corynebacterium glutamicum strain DSMZ 20300: In particular, the preferred host cell has at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to any of the 16S of Corynebacterium glutamicum strain DSMZ 20300 RNA sequences set forth in SEQ ID NO: 13-18 and/or to any one of 23S RNA sequences set forth in SEQ ID NO: 19-24 of Corynebacterium glutamicum strain DSMZ 20300.

[39] The host cell of the invention may further comprise polynucleotides relating to GabD, GabT, GabA or GabB. These are further suitable to increase the production of glutarate. A polynucleotide relating to LysE may be inactivated in the host cell. Inactivation of LysE prevents the export of lysine, which is a starting material of the biosynthesis of glutarate, thereby optimizing the biosynthetic pathway.

[40] GabD, also known as NCgl0463 or DavD, is a glutarate-semialdehyde dehydrogenase. A preferred GabD is shown in the amino acid sequence of SEQ ID NO: 3 or the nucleotide sequence of SEQ ID NO: 4.“Glutarate-semialdehyde dehydrogenase activity” as used herein describes the catalysis of glutarate-semialdehyde and NAD(P)H to glutarate and NAD(P) (see Fig. 1 ). The activity may be determined by an increase in the optical density at 340 nm as a result of reduced coenzyme [NAD(P)H] The reduced NAD(P)H shows an increased absorption at 340 nm. An overexpression or increased activity of gabD may result in an increased production of glutarate by rerouting the metabolism to an increased production of glutarate. A variant or fragment of GabD that has glutarate-semialdehyde dehydrogenase activity has at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % activity compared to a polypeptide with the amino acid sequence of SEQ ID NO: 3.

[41] Accordingly, the present invention relates to the host cell of the invention further comprising a polynucleotide, herein also referred to as the gabD-polynucleotide, comprising a nucleotide sequence encoding gabD or a fragment thereof, wherein said polynucleotide is operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell or amplified in said host cell. In a first aspect, the polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SE Q ID NO: 3. In a second aspect, the polynucleotide has or consists of the nucleotide sequence as shown in SEQ ID NO: 4. In a third aspect, the polynucleotide has a nucleotide sequence encoding a fragment of a polypeptide encoded by the first or second aspect, wherein said fragment has glutarate-semialdehyde dehydrogenase activity. In a fourth aspect, the polynucleotide has a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of the first to the third aspect, wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has glutarate-semialdehyde dehydrogenase activity. In a fifth aspect, the polynucleotide has a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to the amino acid sequence as shown in SEQ ID NO: 3 and has glutarate-semialdehyde dehydrogenase activity.

[42] GabT, also known as NCgl0462 or DavT, is an aminotransferase or also known as transaminase. As shown in Example 2, gabT increases the production of glutarate. A preferred gabT is shown in the amino acid sequence of SEQ ID NO: 5 or the nucleotide sequence of SEQ ID NO: 6.“aminotransferase activity” as used herein describes the catalysis of the reaction from 5-aminovalerate to glutarate semialdehyde, in which the 5-amino residue is transferred to an o ketoglutarate. The activity can be measured by the following assay: 0.25 ml of crude cell extract are mixed with 2.5 ml buffer solution (100 mM TRIS/HCI, 100 mM KOI, 10 mM MgCI2, pH 8.5), 0.1 ml 5-aminovalerate (100 mM), 0.1 ml oketoglutarate (100 mM), 0.05 ml pyridoxal-5- phosphate (10 mM) and 2 ml water, split up in 200 pi aliquots in 1.5 ml. eppendorf tubes. The eppendorf tubes are incubated at 30 °C in a thermomixer. At different time points, the tubes are boiled in a second thermomixer at 100 °C for five minutes in order to stop the enzymatic reaction. After cooling on ice, the tubes are centrifuged for 20 min at 21 130 g and 4 °C. The supernatants are diluted 1 :1 with 222.07 mM DL-oamine-n-butyrate solution (ABU) and analyzed by HPLC to determine the amount of 5-aminovalerate. Another exemplary method could be the observation of the decrease of the educts 5-aminovalerate and/or oketoglutarate or the increase of the products glutarate semialdehyde and/or glutamate. There are various methods known to a person skilled in the art to determine the concentration of aminovalerate o ketoglutarate, glutarate semialdehyde and/or glutamate. Examples of such techniques include quantitative mass spectrometry, chromatography like e.g. RP-HPLC or size exclusion chromatography or also gas chromatography. A variant or fragment of GabT that has glutarate- semialdehyde dehydrogenase activity has at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % activity compared to a polypeptide with the amino acid sequence of SEQ ID NO: 5.

[43] Accordingly, the present invention relates to the host cell of the invention further comprising a polynucleotide, herein also referred to as gabT-polynucleotide, comprising a nucleotide sequence encoding gabT or a fragment thereof, wherein said polynucleotide is operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell or amplified in said host cell. In a first aspect, the gabT- polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SE Q ID NO: 5. In a second aspect, the gabT-polynucleotide has or consists of the nucleotide sequence as shown in SEQ ID NO: 6. In a third aspect, the gabT- polynucleotide has a nucleotide sequence encoding a fragment of a polypeptide encoded by the first or second aspect, wherein said fragment has aminotransferase activity. In a fourth aspect, the gabT-polynucleotide has a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of the first to the third aspect, wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has aminotransferase activity. In a fifth aspect, the gabT- polynucleotide has a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to the amino acid sequence as shown in SEQ ID NO: 5 and has aminotransferase activity.

[44] GabA, also known as DavA, is a 5-aminovaleramide amidase. A preferred GabA is shown in the amino acid sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10.“aminovaleramidase activity” as used herein describes the catalysis of the reaction from aminovaleramide to 5-aminovalerate (see also Fig. 1 ). The activity of gabA can be measured by the decrease of the educt aminovaleramide or the increase of the product aminovalerate concentration. There are various methods known to a person skilled in the art to determine the concentration of aminovaleramide or aminovalerate. Examples of such techniques include quantitative mass spectrometry, chromatography like e.g. RP-HPLC or size exclusion chromatography or also gas chromatography. A variant or fragment of gabA that has aminovaleramidase activity has at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % activity compared to a polypeptide with the amino acid sequence of SEQ ID NO: 9.

[45] Accordingly, the present invention relates to the host cell of the invention further comprising a polynucleotide, herein also referred to as gabA-polynucleotide, comprising a nucleotide sequence encoding gabA or a fragment thereof, wherein said polynucleotide is operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell or amplified in said host cell. In a first aspect, the gabA- polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amho acid sequence as shown in SEQ ID NO: 9. In a second aspect, the gabA-polynucleotide has or consists of the nucleotide sequence as shown in SEQ ID NO: 10. In a third aspect, the gabA- polynucleotide has a nucleotide sequence encoding a fragment of a polypeptide encoded by the first or second aspect, wherein said fragment has 5-aminovaleramide amidase activity. In a fourth aspect, the gabA-polynucleotide has a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of the first to the third aspect, wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has 5-aminovaleramide amidase activity. In a fifth aspect, the gabA-polynucleotide has a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to the amino acid sequence as shown in SEQ ID NO: 9 and has 5-aminovaleramide amidase activity.

[46] GabB, also known as DavB, is a lysine monooxygenase. A preferred GabB is shown in the amino acid sequence of SEQ ID NO: 11 or the nucleotide sequence of SEQ ID NO: 12. “Lysine monooxygenase” activity as used herein describes the catal^is of the reaction from L- lysine to aminovaleramide (see also Fig. 1 ). The activity can be measured by the consumption rate of oxygen as e.g. described in Liu et al. 2014, Scientific Reports, 4, 5657. A variant or fragment of GabB that has Lysine monooxygenase activity has at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % activity compared to a polypeptide with the amino acid sequence of SEQ ID NO: 1 1.

[47] Accordingly, the present invention relates to the host cell of the invention further comprising a polynucleotide, herein also referred to as gabB-polynucleotide, comprising a nucleotide sequence encoding gabB or a fragment thereof, wherein said polynucleotide is operably linked to an expression control sequence, which allows overexpression of said polynucleotide in a host cell or amplified in said host cell. In a first aspect, the gabB- polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 1. In a second aspect, the gabB-polynucleotide has or consists of the nucleotide sequence as shown in SEQ ID NO: 12. In a third aspect, the gabB polynucleotide has a nucleotide sequence encoding a fragment of a polypeptide encoded by the first or second aspect, wherein said fragment has lysine monooxygenase activity. In a fourth aspect, the gabB-polynucleotide has a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of the first to the third aspect, wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has lysine monooxygenase activity. In a fifth aspect, the gabB-polynucleotide has a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to the amino acid sequence as shown in SEQ ID NO: 1 1 and has monooxygenase activity.

[48] As outlined above, lysine is an important starting product in the biosynthesis of glutarate that is exported by lysE and therefore removed from this pathway. By inactivating a polynucleotide encoding lysE in a host cell, lysine cannot be diverted from the glutarate biosynthesis pathway by lysE. [49] Accordingly, the present invention relates to the host cell of the invention, wherein in said host cell a polypeptide, herein also referred to as lysE-polynucleotide, comprising a nucleotide sequence encoding lysE or a fragment thereof, wherein said polynucleotide is inactivated in the host cell. In a first aspect, the polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 7. In a second aspect, the polynucleotide has or consists of the nucleotide sequence as shown in SEQ ID NO: 8. In a third aspect, the polynucleotide has a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50%, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 98 % or at least 99 % identical to the amino acid sequence as shown in SEQ ID NO: 7 and has lysine exporter activity.

[50] “Inactivating a polynucleotide” as used herein relates to reducing the number of the polynucleotide in a host cell or to mutate the polynucleotide comprised in a host cell to prevent its translation or expression and/or to prevent its activity. Said inactivation can be achieved by destroying the genetic information encoding the polynucleotide in the genome or plasmid ofthe host cell by molecular and/or genetic engineering. Molecular and/or genetic engineering methods for inactivation of the polynucleotide may be e.g. the use of restriction enzymes, CRISPR or TALEN.

[51] The polynucleotides of the invention, the vector of the invention or the host cells of the invention may be used for the production of glutarate. Accordingly, the present invention relates to the use the polynucleotides of the invention, the vector of the invention or the host cells of the invention may be used for the production of glutarate.

[52] The invention further relates to a method for producing glutarate. In one embodiment, the method for producing glutarate comprises the steps of culturing a host cell according to the invention under conditions allowing the production of glutarate and obtaining glutarate from the culture medium.

[53] “Conditions allowing the production of glutarate” describes an environment, in which the host cell, e.g. a bacterium, is able to obtain enough nutrients to be able to produce any protein needed for the production of glutarate and is provided sufficient amounts of resources that can be transformed to the desired product glutarate. Such an environment preferably is a culture medium. Examples for suitable culture media are known to a person skilled in the art and include a chemically defined minimal medium (CDM): CDM has glucose as sole carbon source. All solutions are autoclaved prior to usage, except for the vitamin solution, trace elements and 3,4-dihydrobenzoic acid, which are sterile-filtered. The buffer solution is prepared by adding buffer P2 to buffer P1 till a pH of 7.8 is reached. Table 1 : Stock solutions for CDM

Solution A 1 L

NaCI 20 g

CaCI2 1.1 g

MgSQ4^* H20 _ 4 g

Solution B 1 L

(NH4)2S04 150 g

Set pH to 7 with NaOH _

Fe Solution 1 L

FeS04^* 7H20 2 g

Set pH to 1 with HCI

Buffer P1 _ 1 L

K2HP04 348.2 g

Buffer P2 1 L

KH2PQ4 _ 272.1 g

Substrate Solution _ 1 L

Glucose Monohydrate _ 1 10 g

Trace elements solution 1 L

FeCI3^*6H20 200 mg

MnS04^*H20 200 mg

ZnS04^*H20 50 mg

CuCI2^*2H20 20 mg

Na2B4O7^*10H2O 20 mg

(NH4)6Mo7024^*4H20 10 mg

Set pH to 1 with HCI _

Vitamin solution _ 1 L

Biotin 25 mg

Thiamine/HCI 50 mg

Ca-Panthotenate _ 50 mg

3.4-Dihydroxybenzoic acid (DHB) solution 1 L

3.4-DHB 30 g

Add to 1 L H20, set pH to 12 with NaOH

Table 2: Chemically defined minimal medium (CDM)

Minimal Medium 1 L

Solution A 0.5 L

Solution B 0.1 L

Buffer Solution 0.1 L

Substrate Solution 0.1 L

Vitamin Solution 0.02 L

Trace Elements 0.01 L

Fe Solution 0.01 L

DHB Solution 0.001 L

H20 0.159 L

[54] The obtaining of glutarate from the culture medium can be carried out by separating the host cells from the medium, e.g. by centrifugation, and then isolating the glutarate out of the culture medium, e.g. by a chromatographic method, like e.g. HPLC. Alternatively or additionally, the host cells may be lysed prior to the isolation of glutarate out of the culture medium. [55] The invention also relates to a second embodiment of a method of producing glutarate. In this second embodiment of the invention the method comprises the provision of a host cell, increasing the activity of NCgl0464, culturing the host cell in a suitable culture medium and obtaining glutarate from the culture medium.

[56] Preferred host cells of the second embodiment of the method of producing glutarate may be selected from the group consisting of Corynebacterium glutamicum, Brevi bacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Corynebacterium crenatum, Brevibacterium tianjinese, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium efficiens and Escherichia coli. More preferably, the host cell is Corynebacterium glutamicum, even more preferably Corynebacterium glutamicum (DSMZ 20300). Another preferred host cell is characterized by its similarity to the 16S and/or 23S RNA of Corynebacterium glutamicum strain DSMZ 20300: In particular, the preferred host cell has at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % sequence identity to any of the 16S of Corynebacterium glutamicum strain DSMZ 20300 RNA sequences set forth in SEQ ID NO: 13-18 and/or to any one of 23S RNA sequences set forth in SEQ ID NO: 19-24 of Corynebacterium glutamicum strain DSMZ 20300.

[57] “Increasing the activity of NCgl0464” as used herein is defined as increasing the catalytic activity of a host cell in total and/or increasing the activity of NCgl0464 itself. Increasing the activity includes overexpression of NCgl0464 as set forth in SEQ ID NO: 1 or a fragment or variant thereof. In one embodiment, increasing the activity of NCgl0464 includes genetically engineering the host cell with an NCgl0464-polynucleotide or vector of the invention. By this, the expression of the encoded protein is increased and as a consequence, the activity of the encoded protein is increased. In another embodiment, the activity is increased by increasing the copy number of an NCgl0464-encoding gene within the host cell (see e.g. Examples 3 or 4).

[58] The method of the invention for producing glutarate may also further comprise increasing the activity of gabT, gabA, gabB and/or gabD. “Increasing the activity” as used within this context is defined as increasing the catalytic activity of a host cell in total anchor increasing the activity of gabT, gabA, gabB and/or gabD itself. I.e., if the expression of gabT, gabA, gabB and/or gabD is increased, the activity is increased. It is irrelevant how this increased expression is achieved. In one embodiment, increasing the activity of gabT, gabA, gabB and/or gabD includes genetically engineering the host cell with the gabT-, gabA-, gabB- and/or gabD- polynucleotide of the invention. By this, the expression of the encoded protein is increased and as a consequence, the activity of the encoded protein is increased. In another embodiment, the activity is increased by increasing the copy number of the respective encoding gene within the host cell. [59] The second embodiment of a method of the invention of producing glutarate may also further comprise decreasing the activity of lysE, optionally by inactivating the polynucleotide encoding lysE in the host cell.“Decreasing the activity of lysE” includes the reduction of the catalytic activity performed by lysE in the host cell by any manner. Examples of such manners are the inactivation of the polypeptide encoding lysE. Said inactivation can be achieved by destroying the genetic information encoding lysE in the genome or plasmid of the host cell by molecular and/or genetic engineering, and/or providing an inhibitor of lysE. Molecular and/or genetic engineering methods for inactivation of lysE may be e.g. the use of restriction enzymes, CRISPR or TALEN. Another possible way to decrease the activity of lysE would be the use of an inhibitor specific for lysE.

_****

[60] 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.

[61] 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.

[62] 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".

[63] The term“less than” or in turn“more than” does not include the concrete number.

[64] 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 %.

[65] 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. [66] The term“including” means“including but not limited to”.“Including” and“including but not limited to” are used interchangeably.

[67] 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.

[68] 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.

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

[70] 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.

[71] The invention is also characterized by the following items:

[72] Item 1 : A polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 ;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 2;

(c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has 5-aminovalerate permease activity;

(d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has 5-aminovalerate permease activity; and

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 1 and having 5-aminovalerate permease activity,

wherein said polynucleotide is operably linked to an expression control sequence which allows overexpression of said polynucleotide in a host cell. [73] Item 2: The polynucleotide of item 1 , wherein said expression control sequence is a promoter.

[74] Item 3: The polynucleotide of item 1 or 2, wherein said expression control sequence is heterologous to said polynucleotide.

[75] Item 4: The polynucleotide of any one of items 1 to 3, wherein said expression control sequence is selected from the group consisting of EFTU (SEQ ID NO: 25), SOD (SEQ ID NO: 26), Tac (SEQ ID NO: 27), ilvC (SEQ ID NO: 28), Trp (SEQ ID NO: 29), PL (SEQ ID NO: 30), PR (SEQ ID NO: 31 ), lacUV5 (SEQ ID NO: 32), T7lac (SEQ ID NO: 33), AraBD (SEQ ID NO: 34), H3 (SEQ ID NO: 35), H4 (SEQ ID NO: 36), H5 (SEQ ID NO: 37), H17 (SEQ ID NO: 38), H28 (SEQ ID NO: 39), H30 (SEQ ID NO: 40), H34 (SEQ ID NO: 41 ), H36 (SEQ ID NO: 42) and H72 (SEQ ID NO: 43).

[76] Item 5: A vector comprising the polynucleotide of any one of items 1 to 4.

[77] Item 6: A host cell genetically engineered with the vector of item 5, or comprising a polynucleotide as defined in any one of item 1 to 4 or overexpressing a polynucleotide of any one of items 1 (a) to (e).

[78] Item 7: The host cell of item 6, further comprising a polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:3;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:4;

(c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has glutarate-semialdehyde dehydrogenase activity;

(d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has glutarate-semialdehyde dehydrogenase activity; and

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 3 and having glutarate-semialdehyde dehydrogenase activity,

wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

[79] Item 8: The host cell of item 6 or 7, further comprising a polynucleotide selected from the group consisting of (a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:5;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:6;

(c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has aminotransferase activity;

(d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has aminotransferase activity; and

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 5 and having aminotransferase activity,

[80] Item 9: The host cell of any one of items 6 to 8, wherein in said host cell a polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:7;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:8; and

(c) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 7 and having lysine exporter activity,

is inactivated.

[81] Item 10: The host cell of any one of items 6 to 9, further comprising a polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:9;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:10;

(c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has aminovaleramidase activity;

(d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has aminovaleramidase activity; and (e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 9 and having aminovaleramidase activity,

[82] Item 1 1 : The host cell of any one of items 6 to 10, further comprising a polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 1 1 ;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 12;

(c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has monooxygenase activity;

(d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has monooxygenase activity; and

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 11 and having monooxygenase activity,

[83] Item 12: The host cell of any one of items 6 to 1 1 , wherein the host cell has at least 95% sequence identity to any of the 16S of Corynebacterium glutamicum strain DSMZ 20300 RNA sequences set forth in SEQ ID NO: 13-18 and/or to any one of 23S RNA sequences set forth in SEQ ID NO: 19-24 of Corynebacterium glutamicum strain DSMZ 20300.

[84] Item 13: The host cell of any one of items 6 to 1 1 , wherein the host cell is selected from the group consisting of Corynebacterium glutamicum, Brevi bacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Corynebacterium crenatum, Brevibacterium tianjinese, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium efficiens and Escherichia coli.

[85] Item 14: The host cell of any one of items 6 to 11 , wherein the host cell is Corynebacterium glutamicum, preferably Corynebacterium glutamicum (DSMZ 20300).

[86] Item 15: Use of the polynucleotide of any one of items 1 to 4, the vector of item 5 or the host cell of any one of items 6 to 14 for the production of glutarate. [87] Item 16: A method for producing glutarate, comprising the steps:

(i) culturing a host cell according to any one of items 6 to 13 under conditions allowing the production of glutarate; and

(ii) obtaining glutarate from the culture medium.

[88] Item 17: A method for producing glutarate, comprising the steps:

(i) providing a host cell;

(ii) increasing the activity of NCgl0464, optionally by genetically engineering the host cell with the polynucleotide of any one of items 1 to 4 or the vector of item 5;

(vii) culturing the host cell in a suitable culture medium; and

(viii) obtaining glutarate from the culture medium.

[89] Item 18: The method of item 17, further comprising

(iii) increasing the activity of gabT, optionally by genetically engineering the host cell with the polynucleotide of item 8.

[90] Item 19: The method of items 17 and 18, further comprising

(iv) decreasing the activity of lysE, optionally by inactivating the polynucleotide of item 9 in the host cell.

[91] Item 20: The method of items 17 to 19, further comprising

(v) increasing the activity of gabA and/or gabB, optionally by genetically engineering the host cell with the polynucleotide of items 10 and/or 1 1.

[92] Item 21 : The method of items 17 to 20, further comprising

(vi) increasing the activity of gabD, optionally by genetically engineering the host cell with the polynucleotide of items 7.

EXAMPLES

Example 1 : Materials and Methods

Complex media

[93] Cultivations of bacterial strains took place in different complex media. All solutions used were autoclaved (HICLAVE HG-80, Hirayama, Manufacturing Corporation, Saitama, Japan, 121 °C for 45 min) or sterile-filtered (0.2 pm Ultrafree-MC, Millipore, Billerica, Massachusetts, USA) prior to usage. Preparation of agar required addition of 20 g/l of granulated agar, before autoclaving the media. The antibiotics kanamycin (Carl Roth, Karlsruhe, Germany) and tetracycline (Sigma Aldrich, St. Louis) were used in concentrations of 50 mg/L and 12.5 mg/L respectively and were added to the autoclaved medium before use, when required. For preparation of antibiotic-containing agar plates, kanamycin was added after autoclaving to the media at a temperature below 50°C, whereas tetracycline was plated directly onto the agar plates at room temperature.

Table 3: Composition of complex media.

Brain heart infusion (BHI)-Medium 1 L

BHI (Bacto Laboratories, New Jersey, USA) 37 g

H20 Ad 1000 mL

BHIS-Medium 1 L

BHI (Bacto Laboratories, New Jersey, USA) 37 g

H20 Ad 750 mL

Added after autoclaving

Sorbitol (2 M) 250 mL

CM-Sucrose agar plates 1 L

Peptone (Bacto Laboratories, New Jersey, USA) 10 g

Beef extract (Bacto Laboratories, New Jersey, USA) 25 g

Yeast extract (Fluka, Buchs, Austria) 5 g

NaCI 2.5 g

Sucrose (50%) 200 mL

Agar (Bacto Laboratories, New Jersey, USA) 25 g

H20 Ad 725 ml

Added after autoclaving

Glucose (40%) 25 mL

Urea (40 g/L) 50 mL

Cultivation techniques

[94] Cultivations were performed in a rotary shaker at 230 rpm (Infors Multitron, Basel, Switzerland) with a shaking diameter of five centimeters. To ensure sufficient mixing and oxygen supply, the cultivations occurred in baffled shake flasks filled to 10% of the total volume. C. glutamicum cells were cultivated at 30°C.

Cultivation of Corynebacterium qlutamicum

[95] The inoculation of the first pre-culture in BHI-medium was performed by picking single colonies from BHI-agar plates and cultivating them over-night. After determination of the optical density Oϋbbo, the overnight pre-culture was harvested by centrifugation at 8500 x g (Biofuge Stratos, Heraeus, Hanau, Germany), the supernatant discarded and the cells resuspended in fresh minimal medium to inoculate the second pre-culture with an OD between 0.2 and 1. The main culture was inoculated with exponentially growing cells from the second pre culture. All cultivations were performed in biological triplicates. Strains with the episomal plasmid pClik_5A_MCS were cultivated with 50 mg/ml_ kanamycin added to the medium.

Fed-batch cultivation in stirred tank bioreactors

[96] The production performance of the glutarate-producer C. glutamicum GTA-4 was evaluated in a fed-batch process. However, this method is also applicable to other strains as well. Fermentation was carried out in a glucose molasses medium, containing per liter: 72.4 g L ¹ sugar cane molasses, 50 g L ¹ glucose, 35 g L ¹ yeast extract, 20 g L ¹ (NH₄)₂S0₄, 100 mg L ¹ MgS0 , 60 mg L ¹ Ca-pantothenate, 18 mg L ¹ nicotinamide, 15 mg L ¹ thiamine-HCI, 1 1 mg L ¹ FeS0 -7H₂0, 10 mg L ¹ citrate, 9 mg L ¹ biotin, 250 mI_ H₃P0 (85 %) and 5 ml. Antifoam 204 (Sigma-Aldrich). The initial batch process in a working volume of 300 mL in 1 L DASGIP bioreactors (Eppendorf, Julich, Germany) was inoculated with exponentially growing cells from pre-cultures on BHI medium. Dissolved oxygen-based feeding (500 g L-¹ glucose, 162.5 g L ¹ sugar cane molasses, 40 g L ¹ (NH₄)₂S0 , 15 g L ¹ yeast extract, 2 mL antifoam) was initiated when the sugar concentration was depleted. Feed shots of 5 mL were automatically added, when the 0₂ level rose above 45%. Cultivation temperature and aeration rate were kept constant at 30°C and 1 vvm, respectively. The pH and the pC^ level were monitored online with a pH electrode (Mettler Toledo, ΰίbbbh, Germany) and a p(¾ electrode (Hamilton, Hochst, Germany). The pH was kept constant at 7.0 ± 0.05 by automated addition of 10 M NaOH. The dissolved oxygen level was maintained at saturation above 30 % by variation of stirrer speed and oxygen content in the gas inflow. Data acquisition and process operations were controlled by DASGIP control software 4.0 (Eppendorf, Julich, Germany).

Sample collection

[97] During cultivations, 1 mL samples were taken at different time points in an aseptically manner. The optical density (OD660) was measured and documented. The samples were centrifuged at 21 130 x g and 4°C (Eppendorf Centrifuge 5424, Rotor 52508 5424R, Eppendorf, Germany) for several minutes and the supernatant was transferred to a fresh eppendorf tube. Until further analysis, the supernatants were stored at -20°C.

Transformation of C. qlutamicum

[98] Transformation of C. glutamicum cells was performed, using a combination of electroporation and heat shock (Van der Rest, Lange, & Molenaar, 1999). Electrocompetent cells were generated by cultivating cells in BHI-medium at 18 °C, until an Oϋqbo oί 0.6 to 2 was reached. Cells were then harvested by centrifugation (8500 x g, 5 min, 4°C) (Biofuge Stratos, Heraeus, Hanau, Germany) and washed twice with cold glycerol (10%). The pellet was resuspended in 8 mL glycerol (10%) per gram cells and split into aliquots (200 pL) in fresh eppendorf tubes. The aliquots were used for subsequent transformation or stored at -80°C for several days.

[99] At the beginning of the transformation, electrocompetent cells were thawed on ice. After adding 5 pg of plasmid DNA, the suspension was transferred into a pre-cooled electroporation cuvette (Gene Pulser 0.2 cm gap, Bio-Rad, Hercules, California, USA) and incubated for five minutes on ice. After adding 400 mI cold glycerol (10%) to the cuvettes, the electroporation was performed in an electroporation pulser (PenePulser XCell, Bio-Rad, Hercules, California, USA) using 2.5 kV, 25 mF and 400 W. Immediately after transformation, the electroporated cells were transferred into 4 mL pre-warmed (46°C) BHIS-medium. After heat shock for six minutes at 46°C, cells were incubated for three hours at 30°C and 230 rpm. The cells were harvested by centrifugation (8500 x g, 2 minutes, RT) and the supernatant was decanted, before the cells were resuspended in the residual medium, plated on BHISKan agar plates and incubated at 30°C for at least two days, representing the 1st recombination event. Positive colonies were screened using colony-PCR and incubated in BHI medium overnight before they have been plated on CM-Sac plates for the 2nd recombination event. Single colonies were successively plated on CM-Sac and BHkan plates. Positive cells should grow on CM-Sac but not on BHkan plates due to their loss of the kanamycin resistance.

Optical density (OP) and cell dry weight

[100] The optical density of C. glutamicum was measured using a photometer (UV-1600PC Spectrometer, VWR, Radnor, Pennsylvania, USA) at a wavelength of 660 nm (ODteo) with water as reference. In the measurement scale from 0.1 to 0.5, the optical density fits to the linear range. If a measured sample was above this range, a dilution with water was prepared. Every dilution was prepared on a balance in duplicates (Secura®, Sartorius, Gottingen, Germany). The cell dry weight (CDW) (g/L) was determined using the following equation (Rohles et al. 2016):

CDW (g/L) = 0.32 * ODeeo

Determination of organic acids

[101] Measurement of glucose and glutarate was performed by reverse phase chromatography using a HPLC Agilent 1260 Infinity Series system (Agilent Technologies, Waldbronn, Germany). Using an isocratic flow of 3.5 mM H2SO4, samples were separated in an Aminex HPX-87H column (Bio-Rad, Hercules, California, USA), heated to 55°C. Before the separation column, a Microgard Cation₊ H₊ 30x4.6 column (Bio-Rad, California, USA) was installed. The detection of glucose and glutarate was performed via the refractive index wifi a Rl-detector (Agilent 1260 RID G1362A, Agilent Technologies, Waldbronn, Germany).

Determination of amino acids and their derivatives

[102] Determination of amino acids and their derivatives, like 5-aminovalerate, was performed by a HPLC Agilent 1260 Infinity Series system (Agilent Technologies, Waldbronn, Germany). For this purpose, the samples had to be derivatized with o-phthaldialdehyde (OPA) before injection (Kromer et al. 2005).

[103] Separation took place in a 5 pm C18 1 10 A150x4.6 Column (Gemini, Phenomex; Aschaffenburg, Germany) at 40°C using two different eluents (eluent A: 40 mM NaFLPO^ 0.5 g/L sodium azide, set to pH 7.8 and eluent B: acetonitrile:methanol:H20, 45:45:10 [V:V:V]). A G1321 A fluorescence detector at RT was used (Agilent Technologies, Waldbronn, Germany) for detection of the amino acids. [104] Table 4 and Table 5 represent the eluent profiles for the methods, used either for determination of cultivation samples or samples taken from enzyme assay.

Table 4: Eluent profile for eluent A and B for the separation of samples taken from cultivations with a flow rate of 1 ml/min.

Time [min] Eluent A [%] Eluent B [%]

0 100 0

0 70 30

14 56 44

14.5 0 100

16.5 0 100

17 70 30

19 70 30

Table 5: Eluent profile for eluent A and B for the separation of samples taken from enzyme assay with a flowrate of 1.0 mL/min.

Time [min] Eluent A [%] Eluent B [%]

7 93 7

23 61 39

23.5 0 100

26.5 0 100

27 100 0

30 100 0

Cell extraction

[105] The cells for the production of the cell extract were taken from the respective main culture in chemically defined minimal medium. Culture broth was harvested during the exponential growth phase and centrifuged for five minutes at 8000 x g and 4°C (Biofuge Stratos, Heraeus, Hanau, Germany). After a washing step with 4 ml. of buffer solution (Table 9), the cell pellet was resuspended in 4 ml. buffer solution. The cell suspension was then transferred in 1 ml. aliquots in 2 ml. tubes, filled with 0.1 mm silica spheres (MP Biomedicals, Santa Ana, California, USA). Cell lysis was performed twice in a rotation mill at 5500 rpm for 30 seconds (Precellys 24, PEQLAB Biotechnology GmbH, Erlangen, Germany). In order to avoid protein denaturation by high temperature, tubes were cooled on ice for two minutes between each lysis step. After lysis, the tubes were centrifuged for 20 minutes at 21.130 x g and 4°C (Eppendorf Centrifuge 5424, Rotor 52508 5424R, Eppendorf, Germany) and the supernatant corresponding to the crude cell extract was stored on ice, until further use.

Determination of protein concentration

[106] The protein concentration in the crude cell extract was determined using the Pierce BCA Protein Assay Kit (Thermo Fischer Scientific, Rockford, Illinois, USA). The crude cell extract was diluted 1 :10 on a balance in triplicates. The preparation of the samples was performed according to the manufacturer’s protocol.

5-aminotransamidase assay

[107] 0.25 ml of crude cell extract were mixed with 2.5 ml buffer solution (100 mM TRIS/HCI, 100 mM KCI, 10 mM MgCI2, pH 8.5), 0.1 ml 5-aminovalerate (100 mM), 0.1 ml oketoglutarate (100 mM), 0.05 ml pyridoxal-5-phosphate (10 mM) and 2 ml water, split up in 200 mI aliquots in 1.5 ml. eppendorf tubes. The eppendorf tubes are incubated at 30 °C in a thermomixer. At different time points, the tubes are boiled in a second thermomixer at 100 °C for five minutes in order to stop the enzymatic reaction. After cooling on ice, the tubes are centrifuged for 20 min at 21130 g and 4 °C. The supernatants are diluted 1 :1 with 222.07 mM DL-oamine-n-butyrate solution (ABU) and analyzed by HPLC to determine the amount of 5-aminovalerate.

GC-MS analysis of metabolites in the culture supernatant.

[108] The labeling pattern of metabolites in culture supernatant was analyzed after derivatization into the t-butyl-dimethylsilyl derivates (Becker et al. 2015). For this purpose, 10 mI_ culture supernatant was dried under a nitrogen stream, followed by incubation with 50 mI_ dimethylformamide (0.1 % pyrimidine) and 50 mI_ methyl-t-butyldimethylsilyl-trifluoroacetamide (Macherey and Nagel, Duren, Germany) for 30 minutes at 80 °C. The obtained derivatives were analyzed by gas chromatography-mass spectrometry (GC/MS 7890A, 5975C quadrupole detector, Agilent Technologies, Santa Clara, CA, USA) (Lange et al. 2017). In addition, pure glutarate was analyzed as reference standard.

Example 2: Overexpression of gabT in C. glutamicum AVA-2 for enhanced formation of glutarate

[109] C. glutamicum AVA-2, a 5-aminovalerate producer strain was chosen as microbial chassis to design a glutarate overproducing mutant (Rohles et al. 2016). As first target, the gene gabT, encoding 5-aminovalerate-transaminase, should be overexpressed. The enzyme may be mainly responsible for conversion of 5-aminovalerate into glutarate-semialdehyde, the precursor of glutarate. For this purpose, the native promotor was replaced by the strong constitutive promotor Pettu. The increased expression should result in a higher flux towards glutarate.

[110] In order to validate the effect of an overexpressed gabT gene on glutarate production, the novel strain GTA-1 was cultivated on glucose and compared to its parent strain C. glutamicum AVA-2 (Figure 2). The novel strain accumulated about 15 mM glutarate and only minor amounts of 5-aminovalerate as by-product (1 mM) (Figure 2B). Within about 22 h, the substrate glucose was completely consumed and converted into the two products and into biomass, whereby cells grew exponentially. For comparison, the parent strain produced glutarate to a significantly weaker extent and still secreted high levels of 5-aminovalerate: final titers were 8 mM glutarate and 9 mM 5-aminovalerate. Obviously, the genetic modification was highly beneficial for glutarate production (Figure 2A). This successful integration is evident not only by the cultivation profiles, but also by the parameters listed in Table 6.

Table 6: Growth and production performance of the C. glutamicum strains AVA-2 and GTA-1 during batch cultivations in shake flasks using chemically defined minimal medium with glucose as sole carbon source. Data shown represent mean values and corresponding standard deviations from three biological replicates. Shown are the specific growth rate (m), the specific substrate uptake rate (q_s), the specific production rates for 5-aminovalerate (q_Ava) and glutarate (q_Git), the biomass yield (Uc_/s) and yield for 5-aminovalerate (YAva_/s) and glutarate (YGH/S)·

[111] Both strains revealed similar growth, indicating that the overexpression of gabT had no influence on the overall fitness of the strain. The novel strain GTA-1 showed increased substrate uptake rate q_s. In addition, also the specific glutarate production rate (qcit) was enhanced. Moreover, the strain exhibited a twofold increased glutarate yield and an eightfold lower 5-aminovalerate yield. In summary, the overexpression of gabT obviously resulted in a faster conversion from 5-aminovalerate to glutarate-semialdehyde (GSA), which was then converted into glutarate by the subsequent reaction. The increased flux towards glutarate was due to a higher activity of 5-aminovalerate transaminase and glutarate-semialdehyde dehydrogenase.

[112] The constant production characteristic of the strains however showed that once glutarate is secreted, no re-import was observable during glutarate uptake studies. Here, Corynebacterium glutamicum Lys-12 cells (non-glutarate producing cells) were cultivated using minimal medium, supplemented with the indicated amounts auf glutarate and the intracellular concentration of glutarate was determined at the beginning of the stimulation and after 24.5 h. The following table shows the results

[113] As can be seen, there was no change of intracellular glutarate concentration after external application of glutarate, showing there is no re-import of glutarate supporting the usefulness of the method of the invention.

[114] The improved expression of gabT via integration of the strong promotor Pee led to higher amounts of 5-aminovalerate transaminase, the key enzyme to convert 5-aminovalerate into glutarate-semialdehyde, the precursor of glutarate, resulting in an overall higher flux towards glutarate. With a production titer of 2 g/L, C. glutamicum GTA-1 shows the highest de novo synthesis of glutarate reported and surpasses recent attempts with feed of the precursors L- lysine and a-ketoglutarate to recombinant E. coli (1.7 g/L) (Park et al. 2013) and extending the a-keto acid carbon chain and decarboxylation pathway in £. coli (0.42 g/L) (Wang et al. 2017). De novo biosynthesis of glutarate in C. glutamicum from renewable feedstocks was successively realized, which makes feeding of expensive external substrates, e.g. L-lysine and a-ketoglutarate obsolete.

Example 3: Overexpression of NCgl0464 in GTA-1 under control of P_eftu promoter

[115] C. glutamicum NCgl0464 is annotated as amino-acid permease and was previously described as g-aminobutyric acid (GABA) transporter (GabPc_g) (Zhao et al. 2012). Deletion of this transporter in C. glutamicum AVA-2 surprisingly resulted in decreased secretion of glutarate. This indicates an important function in glutarate production.

[116] The effect of NCgl0464 has been tested integratively by a second gene copy under the control of Peftu. For this purpose, the plasmid pClik_int_sacS eftu0464 was designed, which contained the sequence of NCgl0464 with the upstream located promotor Peftu. The cultivation of the strain was performed with a start optical density of ODB6O = 1 .

[117] As shown in Figure 3, the new strain C. glutamicum GTA-2 showed growth associated production of glutarate up to a maximum level of 15 mM within 1 1 h in a shake flask cultivation. At the same time, 5-aminovalerate accumulation was almost diminished. The novel strain revealed the same specific growth rate as its parent strain GTA-1. The glutarate yield increased to 268 mmol/mol.

Table 7: Growth and production performance of the C. glutamicum strains GTA-1 and GTA-1 eftu0464 (GTA-2). Batch cultivations were performed in shake flasks using chemically defined minimal medium with glucose as sole carbon source. Data shown represent mean values and corresponding standard deviations from three biological replicates. Shown are the specific growth rate (m), the specific substrate uptake rate (q_s), the specific production rates for 5- aminovalerate (q_Ava) and glutarate (q_Git), the biomass yield (Uc_/s) and yield for 5-aminovalerate (YA_VB/S) and glutarate (Y_Git/s)·

[118] Overexpression of NCgl0464 successfully increased production of glutarate, whereas 5- aminovalerate secretion was almost fully eliminated. The strain C. glutamicum GTA-2, overproducing this protein, thereby showed a production of glutarate in C. glutamicum at 2 g/L.

Example 4: Overexpression of NCgl0464 in GTA-1 under control of the Tuf-promoter

[119] The overexpression of the permease NCgl0464 was in an alternative approach tackled by inserting a second gene copy under control of the tuf promoter into the genome of the GTA-1 strain. Positive clones, which carried the desired second gene copy, were identified on basis of a 2.4 kb PCR fragment, which contained the inserted sequence in contrast to the wild type (1.1 kb). The successful integration of the construct was further confirmed by sequencing. The novel strain GTA-1 P_tUfNCgl0464 was designated GTA-4. When tested in shake flask in batch culture, the GTA-4 strain exhibited improved performance (Fig. 4). It accumulated higher amounts of glutarate and formed the product at increased yield (271 mmol mol ¹) and productivity (0.9 mmol g ¹ h ¹) (Table 8). The secretion of 5-aminovalerate was further reduced by a factor of more than three, as compared to the GTA-1 strain, to a level of only about 1 % of the glutarate formed (Table 8). Table 8: Growth and production performance of glutarate producing C. glutamicum strains during batch cultivation in shake flasks using a mineral salt medium with glucose as sole carbon source. The data comprise the specific rates for growth (m), glucose uptake (q^_c), glutarate (q_Git), and 5-aminovelarate formation (q_Ava). Additionally, the yields for biomass (Uc_/oic), glutarate (Y _Git_/Gic) _s and 5-aminovalerate (YAva_/oic) are given. Error bars represent standard deviations from three biological replicates.

[120] In this example, a different method for the extraction of glutarate was used: After cell separation via centrifugation (5 min, 8.000 xg, 4°C), the broth was vacuum-filtered (Whatman filter paper, Grade 3, Sigma-Aldrich). In order to decolorize the product solution, the obtained filtrate was mixed 12.5% (w/vol) with activated carbon. The mixture was then stirred for 1 hour. After removal of the activated carbon by filtration, glutarate was precipitated by acidification to pH 1.5 with 37% HCI. The obtained crystals were washed with deionized water and lyophilized (Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany), which finally yielded a white crystalline powder.

Example 5: Culturing GTA-4 under fed-batch conditions leads to an extremely high production of glutarate

[121] To assess the performance of the created producer under industrially relevant conditions, the GTA-4 strain was benchmarked in a fed-batch process on a glucose-molasses medium (Fig. 5). During the initial batch phase of the fermentation, an exponential growth of the strain (Fig. 5A) along with glutarate production at a yield of 0.290 mol mol ¹, i.e. 0.215 g g ¹ (Fig. 5B) was observed.

[122] After a process time of 15 h, the initially supplied sugar (75 g L ¹) was depleted and the feed phase was started. Feed pulses were automatically applied to maintain the sugar concentration at about 10 g L ¹, triggered by the signal of the dissolved oxygen (DO) probe. The glutarate level continuously increased from 15g L ¹ at the end of the batch phase to a final titer of more than 90 g L ¹. Surprisingly, the cells produced glutarate almost exclusively. The formation of 5-aminovalerate was negligible. A small amount accumulating at the end of the batch phase was completely taken up again in later process phases (Fig. 5A). Also trehalose, a typical by-product of C. glutamicum, was not formed in significant amounts.

[123] When evaluating the efficiency of the GTA-4 strain throughout the process phases (Fig. 5B), the glutarate yield increased by 2.4-fold in the feed phase to an impressive value of 0.52 g g ¹, based on the consumed sugar. This equals a molar yield of 0.71 mol mol ¹, close to the theoretical optimum of 0.75 mol mol ¹ of the pathway, and can be taken as an indication for the enormous synthetic power of the created cell factory. The space-time yield for glutarate reached a maximal value of 1.8 g L ¹ h ¹ after 24 hours. Averaged over the full process time, the production occurred at 75 % of the maximum rate (1.4 g L ¹ h ¹). In sum, the data show that the present invention enables the production of very high amounts of glutarate (up to 90 g/L).

[124] In this example, a different method for the extraction of glutarate was used: After cell separation via centrifugation (5 min, 8.000 xg, 4°C), the broth was vacuum-filtered (Whatman filter paper, Grade 3, Sigma-Aldrich). In order to decolorize the product solution, the obtained filtrate was mixed 12.5% (w/vol) with activated carbon. The mixture was then stirred for 1 hour. After removal of the activated carbon by filtration, glutarate was precipitated by acidification to pH 1.5 with 37% HCI. The obtained crystals were washed with deionized water and lyophilized (Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany), which finally yielded a white crystalline powder.

[125] Example 6: Re-uptake of the intermediate 5-aminovalerate by NCgl0464 contributes to glutarate formationAn isotope experiment was designed to study the 5-aminovalerate/glutarate metabolism in C. glutamicum in more detail. Briefly, C. glutamicum AVA-2 was grown on minimal medium with ¹³C enriched [¹³C₆] glucose, additionally supplemented with naturally labeled 5-aminovalerate. An incubation without added 5-aminovalerate served as a control. The analysis of the ¹³C enrichment of the produced glutarate by GC-MS provided a direct readout, to which extent the external pathway intermediate was still available for the metabolic conversion. When 5-aminovalerate was present in the medium, the ¹³C enrichment of glutarate was substantially reduced, as compared to the control (Fig. 6, Table 2). This demonstrated that about 40% of the glutarate are derived from extracellular 5-aminovalerate. This is a beneficial feature, as transiently secreted 5-aminovalerate is not lost, but can be recycled for glutarate production.

[126] To assess the role of NCgl0464 ( cgl0841 ), the deletion of the NCgl0464 gene was realized in the parent AVA-2 strain. Such a clone was studied for its production characteristics. The ANCgl0464 mutant, designated GTA-3, revealed a substantially reduced re-cycling of external 5-aminovalerate (Fig. 6, Table 2). Only about 25% of glutarate stemmed from re-uptake of the intermediate, while 75% was synthesized from glucose. In addition, the overall glutarate production was reduced, while the 5-aminovalerate level was substantially increased (Table 9). This indicated that the protein NCgl0464 has 5-aminovalerate permease activity and explains the huge impact of NCgl0464 overexpression on glutarate production.

Table 9: Growth and production performance of glutarate producing C. glutamicum strains during batch cultivation in shake flasks using a mineral salt medium with glucose as sole carbon source. The data comprise the specific rates for growth (m), glucose uptake (qGIc), glutarate (qGIt), and 5-aminovelarate formation (qAva). Additionally, the yields for biomass (YX/GIc), glutarate (YGIt/GIc), and 5-aminovalerate (YAva/GIc) are given. Error bars represent standard deviations from three biological replicates

REFERENCES

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[129] Kind, S., Kreye, S., & Wittmann, C. (201 1 ). Metabolic engineering of cellular transport for overproduction of the platform chemical 1 ,5-diaminopentane in Corynebacterium glutamicum. Metabolic Engineering, 13(5), 617-627.

[130] Kromer, J. O., Fritz, M., Heinzle, E., & Wittmann, C. (2005). In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. Analytical Biochemistry, 340( 1 ), 171-173.

[131] Park, S. J., Kim, E. Y„ Noh, W„ Park, H. M„ Oh, Y. H„ Lee, S. H„ ... Lee, S. Y. (2013). Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals. Metabolic Engineering, 16(1 ), 42-47.

[132] Rohles, C. M., G^elmann, G., Kohlstedt, M., Wittmann, C., & Becker, J. (2016). Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon - 5 platform chemicals 5-aminovalerate and glutarate. Microbial Cell Factories, 1-13.

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[134] Wang, J., Wu, Y., Sun, X., Yuan, Q., & Yan, Y. (2017). De novo Biosynthesis of Glutarate via 58 a-Keto Acid Carbon Chain Extension and Decarboxylation Pathway in Escherichia coli. ACS Synthetic Biology, acssynbio.7b00136.

[135] Zhang, C., Chen, X., Stephanopoulos, G., & Too, H. P. (2016). Efflux transporter engineering markedly improves amorphadiene production in Escherichia coli. Biotechnology and Bioengineering, 173(8), 1755-1763.

[136] Zhao, Z., Ding, J. Y., Ma, W. hua, Zhou, N. Y., & Liu, S. J. (2012). Identification and characterization of g-Aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum. Applied and Environmental Microbiology, 78(8), 2596-2601.

Claims

1. A polynucleotide selected from the group consisting of

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 2;

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 1 and having 5-aminovalerate permease activity, wherein said polynucleotide is operably linked to an expression control sequence which allows overexpression of said polynucleotide in a host cell.

2. The polynucleotide of claim 1 , wherein said expression control sequence is a promoter.

3. The polynucleotide of claim 1 or 2, wherein said expression control sequence is heterologous to said polynucleotide.

4. The polynucleotide of any one of claims 1 to 3, wherein said expression control sequence is selected from the group consisting of EFTU (SEQ ID NO: 25), SOD (SEQ ID NO: 26), Tac (SEQ ID NO: 27), ilvC (SEQ ID NO: 28), Trp (SEQ ID NO: 29), PL (SEQ ID NO: 30), PR (SEQ ID NO: 31 ), lacUV5 (SEQ ID NO: 32), T7lac (SEQ ID NO: 33), AraBD (SEQ ID NO: 34), H3 (SEQ ID NO: 35), H4 (SEQ ID NO: 36), H5 (SEQ ID NO: 37), H17 (SEQ ID NO: 38), H28 (SEQ ID NO: 39), H30 (SEQ ID NO: 40), H34 (SEQ ID NO: 41 ), H36 (SEQ ID NO: 42) and H72 (SEQ ID NO: 43).

5. A vector comprising the polynucleotide of any one of claims 1 to 4.

6. A host cell genetically engineered with the vector of claim 5, or comprising a polynucleotide as defined in any one of claim 1 to 4 or overexpressing a polynucleotide of any one of claims 1 (a) to (e).

7. The host cell of claim 6, further comprising a polynucleotide selected from the group consisting of

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:4;

8. The host cell of claim 6 or 7, further comprising a polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO:5;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:6; (c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has aminotransferase activity;

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 5 and having aminotransferase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

9. The host cell of any one of claims 6 to 8, wherein in said host cell a polynucleotide selected from the group consisting of

is inactivated.

10. The host cell of any one of claims 6 to 9, further comprising a polynucleotide selected from the group consisting of

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO:10;

(c) a polynucleotide having a nucleotide sequence encoding a fragment of a polypeptide encoded by the polynucleotide of (a) or (b), wherein said fragment has aminovaleramidase activity; (d) a polynucleotide having a nucleotide sequence encoding a derivative of a polypeptide encoded by the polynucleotide of any one of (a) to (c), wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said derivative has aminovaleramidase activity; and

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 9 and having aminovaleramidase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

1 1. The host cell of any one of claims 6 to 10, further comprising a polynucleotide selected from the group consisting of

(a) a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the amino acid sequence as shown in SEQ ID NO: 11 ;

(b) a polynucleotide having the nucleotide sequence as shown in SEQ ID NO: 12;

(e) a polynucleotide having a nucleotide sequence encoding a polypeptide having an amino acid sequence which is at least 50% identical to the amino acid sequence as shown in SEQ ID NO: 11 and having monooxygenase activity, wherein said polynucleotide is (i) operably linked to an expression control sequence which allows overexpression of said polynucleotide in said host cell or (ii) amplified in said host cell.

12. The host cell of any one of claims 6 to 1 1 , wherein the host cell has at least 95 % sequence identity to any of the 16S of Corynebacterium glutamicum strain DSMZ 20300 RNA sequences set forth in SEQ ID NO: 13-18 and/or to any one of 23S RNA sequences set forth in SEQ ID NO: 19-24 of Corynebacterium glutamicum strain DSMZ 20300.

13. The host cell of any one of claims 6 to 1 1 , wherein the host cell is selected from the group consisting of Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Corynebacterium crenatum, Brevibacterium tianjinese, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium efficiens and Escherichia coli.

14. The host cell of any one of claims 6 to 1 1 , wherein the host cell is Corynebacterium glutamicum, preferably Corynebacterium glutamicum (DSMZ 20300).

15. Use of the polynucleotide of any one of claims 1 to 4, the vector of claim 5 or the host cell of any one of claims 6 to 14 for the production of glutarate.

16. A method for producing glutarate, comprising the steps:

(i) culturing a host cell according to any one of claims 6 to 13 under conditions allowing the production of glutarate; and

(ii) obtaining glutarate from the culture medium.

17. A method for producing glutarate, comprising the steps:

(i) providing a host cell;

(ii) increasing the activity of NCgl0464, optionally by genetically engineering the host cell with the polynucleotide of any one of claims 1 to 4 or the vector of claim 5;

(vii) culturing the host cell in a suitable culture medium; and

(viii) obtaining glutarate from the culture medium.

18. The method of claim 17, further comprising

(iii) increasing the activity of gabT, optionally by genetically engineering the host cell with the polynucleotide of claim 8.

19. The method of claims 17 and 18, further comprising

(iv) decreasing the activity of lysE, optionally by inactivating the polynucleotide of claim 9 in the host cell.

20. The method of claims 17 to 19, further comprising

(v) increasing the activity of gabA and/or gabB, optionally by genetically engineering the host cell with the polynucleotide of claims 10 and/or 11.

21 . The method of claims 17 to 20, further comprising

(vi) increasing the activity of gabD, optionally by genetically engineering the host cell with the polynucleotide of claims 7.