WO2015132213A1 - Process for preparing terminal amino carboxylic acids and amino aldehydes by means of a recombinant microorganism - Google Patents

Abstract

The present invention relates to a process for preparing terminal amino carboxylic acids and amino aldehydes from diamines using a recombinant microorganism. More particularly, the present invention relates to a process for preparing gamma-aminobutyric acid (GABA) or gamma-aminobutyraldehyde (ABAL) using a recombinant microorganism. The present invention also relates to the use of recombinant microorganisms comprising DNA molecules in a deregulated form which improve the preparation of terminal amino carboxylic acids and/or amino aldehydes, for example gamma-aminobutyric acid or gamma-aminobutyraldehyde, and to recombinant DNA molecules and polypeptides which are used to produce the microorganism.

Description

 Process for the preparation of terminal aminocarboxylic acids and aminoaldehydes by means of a recombinant microorganism

description

The present invention relates to a process for the preparation of terminal aminocarboxylic acids and aminoaldehydes from diamines using a recombinant microorganism. More particularly, the present invention relates to a process for producing gamma-aminobutyric acid (GABA) or gamma-aminobutyraldehyde (ABAL) using a recombinant microorganism. The present invention also relates to the use of recombinant microorganisms comprising DNA molecules in a deregulated form which enhance the production of gamma-aminobutyric acid, as well as recombinant DNA molecules and polypeptides used to produce the microorganism. Moreover, the invention relates to the provision of corresponding microorganisms. The present invention provides, inter alia, a microorganism which has an increased level of a putrescine: 2-oxoglutarate aminotransferase or an increased level of a gamma-glutamyl-putrescine synthase and a gamma-glutamyl-putrescine oxidase compared to the native level, or a combination of the features described.

As far as a process for the preparation of a terminal aminocarboxylic acid is concerned Compared to the native level, an elevated level of a putrescine: 2-oxoglutarate aminotransferase and a gamma-aminobutyraldehyde dehydrogenase.

In particular, the present invention provides, in particular, a microorganism which has an increased level of a gamma-glutamyl-putrescine synthase and a gamma-glutamyl-putrescine oxidase and a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase and a level compared to a native level Gamma-glutamyl gamma-aminobutyric acid hydrolase. In addition, the present invention provides a microorganism having, compared to a native level, an enhanced level of a putrescine: 2-oxoglutarate aminotransferase and a gamma-glutamyl-putrescine oxidase.

In another aspect, the present invention provides a microorganism as described above, and a process for producing gamma-aminobutyric acid by cultivating the microorganism.

In another aspect, the present invention provides a microorganism as described above, and a process for producing gamma-aminobutyraldehyde by cultivating the microorganism.

In a further aspect, the present invention provides a microorganism having increased putrescine-forming activity. In a further aspect, the present invention provides a microorganism having any combination of the above-described embodiments of the invention and having no or a level-reduced ornithine carbamoyltransferase activity and / or none or one of native level activity decreased activity of the arginine repressor, no or a level reduced activity of the GCN5-related N-acetyltransferase and / or no or a level reduced activity of a major facilitator superfamily permease.

According to the invention, it may be provided that in order to increase or increase the level of a specific enzyme compared to the native level of the enzyme, a plasmid is introduced into a microorganism which contains at least one sequence which codes for the corresponding enzyme. In particular, it may be provided according to the invention that a plasmid comprises at least one sequence of the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID NO. 11 and SEQ ID NO. 13.

For the purposes of the invention, the term "native level" with reference to an enzyme means the level or the concentration of an enzyme in a genetically unmodified organism of the same species, whereby the native level may also be zero while the fiction, according to preferred deregulations for influencing the level of an enzyme.

A preferred way of deregulating putrescine genes: 2-oxoglutarate aminotransferase, gamma-aminobutyraldehyde dehydrogenase, gamma-aminobutyraldehyde dehydrogenase, gamma-glutamyl-putrescine synthase, gamma-glutamyl-putrescine oxidase, gamma-glutamyl-gamma-aminobutyric acid Hydrolase is a "high" mutation which increases gene activity, eg, by gene amplification using strong expression signals and / or point mutations that increase enzymatic activity. A preferred way of deregulating the genes of ornithine carbamoyltransferase, arginine repressor, GCN5-related N-acetyltransferase is a "down" mutation involving gene activity, eg by gene deletion or disruption, use of weak expression signals and / or point mutations involving the enzymatic Destroying or reducing activity lowers.

According to the invention it may be provided that in a native or genetically modified microorganism at least one sequence of the group consisting of SEQ ID. No. 15, SEQ ID. No. 17, SEQ ID. No. 19, SEQ ID. No. 21, SEQ ID. No. 23 and SEQ ID. No. 25 is deleted or disrupted.

To increase the yield of gamma-aminobutyric acid, a preferred part of the invention is the deregulation of the acetyl-CoA dependent GCN5-related N-acetyltransferase responsible for the acetylation of the prepared putrescine, especially the lowering of the activity e.g. by deletion or disruption of the gene.

It has been observed that the production capacity of a microorganism for ornithine can be improved by deregulating the activity of the arginine repressor ArgR of the gamma-aminobutyric acid producing microorganism, preferably by reducing its activity by deletion or disruption of the gene coding for the arginine repressor.

In another embodiment of the invention, the microorganism has a reduced capacity to export putrescine by deregulating the activity of the major facilitator super-family permease of the gamma-aminobutyric acid producing microorganism, preferably by reducing its activity by deletion or disruption of the major facilitator superfamily permease coding gene. To increase the yield of gamma-aminobutyric acid, in a further aspect, the present invention provides a microorganism having no or reduced activity of the 4-aminobutyrate aminotransferase, which catalyzes the conversion of the gamma-aminobutyric acid formed to succinate-semialdehyde.

In a further embodiment of the invention, the microorganism has a reduced capacity to import and / or degrade gamma-aminobutyric acid by deregulating the activity of the amino acid permease GabP or the aminotransferase GabT of the gamma-aminobutyric acid producing microorganism, preferably by reducing its activity by deletion or disruption of the gene coding for the permease or transferase.

Another important aspect of the present invention involves culturing or cultivating the recombinant microorganisms described herein to produce the desired product gamma-aminobutyric acid.

Thus, one embodiment of the invention comprises a gamma-aminobutyric acid production system comprising a microorganism comprising a deregulated ornithine decarboxylase and a deregulated putrescine: 2-oxoglutarate aminotransferase and a deregulated gamma-aminobutyraldehyde dehydrogenase and / or a deregulated gamma-glutamyl-putrescine Synthase, a deregulated gamma-glutamyl-putrescine oxidase, a deregulated gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase and a deregulated gamma-glutamate GABA hydrolase, a fermentation medium suitable for cultivating this microorganism, and technical systems to aid production of gamma-aminobutyric acid ,

The production of GABA from glutamate using the activity of a glutamate decarboxylase (GAD, EC 4.1.1.15) has already been described in the literature. Genes for Glutamate decarboxylases have been introduced into Escherichia coli, various Lactobacillus species (De Biase et al., 1996, Hiraga et al., 2008, Komatsuzaki et al., 2008) and enterococci (Tamura et al., 2010). In lactic acid bacteria, the formation of GABA was observed with the addition of glutamate to the fermentation medium (Hanya et al., 2007; Bo et al., 2013; Gao and Jiang, 2014). Depending on the strain used, the optimal conditions for GABA fermentation vary. The most important factors affecting GABA production, such as carbon source, glutamate concentration, temperature, pyridoxal-5-phosphate, and pH, have already been characterized (Ran et al., 2007; Li et al., 2010, Lu et al. Komatsuzaki et al., 2005; Yang et al., 2008). Another approach describes the conversion of glutamate to GABA using immobilized E. coli glutamate decarboxylase (S. Lee et al., 2013).

Fig. 1 shows a schematic representation of the GABA biosynthesis in a recombinant Corynebacterium glutamicum.

As a more cost-effective alternative, the production of GABA directly from carbohydrates has been established by expression of a glutamate decarboxylase (Takahashi et al., 2012, Shi et al., 2013, Shi and Li, 2011, Li et al., 2013, Lee et al., 2012; Liao and Cho, 2011). In order to achieve effective production of GABA in C. glutamicum, the E. coli gadB gene was overexpressed in the glutamate-producing C. glutamicum ATCC 13032. This strain yielded 8.07 + 1.53 g / L extracellular GABA at 96 hours in GABA production medium with 50 g / L glucose and without glutamate addition. Addition of 0.1 mM pyridoxal-5-phosphate increased GABA production to a yield of 12.37 + 0.88 g / L after 72 h, which corresponds to a productivity of 0.172 g / L / h (Takahashi et al , 2012). In a study to further optimize GABA synthesis, recombinant C. glutamicum strains were used to improve the intracellular glutamate concentration. In order to increase the flow of 2-oxoglutarate to glutamate, the serine / threonine Protein kinase G encoding gene pknG deleted. This enzyme participates in a regulatory mechanism that controls the activity of 2-oxoglutarate dehydrogenase (Kimura, 2002, Schultz et al., 2009, Niebisch et al., 2006). Previous work has already shown that reduction of the 2-oxoglutarate dehydrogenase complex, which catalyzes the conversion of 2-oxoglutarate to succinyl-CoA as part of the TCA cycle, is an important factor in glutamate production with C. glutamicum ( Schultz et al., 2007; Boulahya et al., 2010). By overexpression of E. coli GadB in a C. glutamicum strain with a pkn deletion in medium containing 100 g / L glucose and 0.1 mM pyridoxal-5-phosphate, GABA production could be reduced by one compared to precursor with intact PnkG Factor from 2.3 to 31.1 + 0.41 g / L (0.26 g / L / h) after 120 hours. The yield of GABA from glucose reached 0.89 mol / mol (Okai et al., 2014).

In addition to the overexpression of glutamate decarboxylase gadB from E. coli, the glutamate decarboxylases gadBl and gadB2 from LactobaciUus brevis Lb85 were also overexpressed in C. glutamicum (Shi and Li, 2011, Shi et al., 2013). In this case, each gene encoding glutamate decarboxylase was plasmid-based both individually and also gadB2 in combination with the upstream L-glutamate / GABA antiporter gadC (gadCgadB2) and gadC-gadB2 with the upstream regulatory gene gadR (gadR-gadC-gadB2 ) overexpressed. Shake flask cultivations of these four recombinant C. glutamicum strains showed the highest GABA titer for the strain after 72 hours with the gadR gadC gadB2 overexpression (2.15 gL ^-1 ), compared to 1.25 gL ^-1 for gadC - gadB2 and 0.88 gL ^"1 for gadB2 (Shi and Li, 2011) A further approach to optimize GABA production was the co-overexpression of the two glutamate decarboxylases from LactobaciUus brevis Lb85 GADB1 and GADB2. Compared to GABA Production of the strains expressing gadBl or gadB2 alone was more than twice as high in the gadBl and gadB2 coexpression strain, and by optimizing the urea addition, the GABA titer could be increased from 4.02 + 0.95 to 18 , 66 + 2,11 gL ^{-1 increased} after 84 hours. In a fed-batch fermentation, the titer was 26.32 gL ^-1 after 60 hours (Shi et al., 2013).

In addition to the use of the glutamate decarboxylases of E. coli and Lactobacillus brevis, there are other approaches that involve the use of plant or chimeric glutamate decarboxylases (Zelder et al., 2010).

As an alternative to the previously reported routes for the biotechnological production of GABA, this invention describes the synthesis of GABA from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase. The heterologous expression of gamma-aminobutyraldehyde dehydrogenase (PatD) and putrescine: 2-oxoglutarate aminotransferase (PatA), preferably from E. coli, ensures the subsequent conversion of putrescine into GABA.

As a further alternative to the routes to biotechnological production of GABA shown so far, this invention describes the synthesis of GABA from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase. Heterologous expression of a gamma-glutamyl-putrescine synthase (PuuA), a gamma-glutamyl-putrescine oxidase (PuuB), a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) and a gamma-glutamayl-GABA hydrolase (PuuD) , preferably from E. coli, ensures the subsequent conversion of putrescine to GABA. As a further alternative to the routes to biotechnological production of GABA shown so far, this invention describes the synthesis of GABA from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase. By heterologous expression of putrescine: 2-oxoglutarate Aminotransferase (PatA) and a gamma-glutamyl gamma-aminobutyraldehyde dehydrogenase (PuuC), preferably from E. coli, ensures the subsequent conversion of putrescine to GABA. As an alternative to the routes to biotechnological production of ABAL (gamma-aminobutyraldehyde) shown so far, this invention describes the synthesis of ABAL from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase. The heterologous expression of aminotransferase (PatA), preferably from E. coli, ensures the subsequent conversion of putrescine into ABAL.

As a further alternative to the routes to biotechnological production of ABAL shown so far, this invention describes the synthesis of ABAL from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase. The heterologous expression of a gamma-glutamyl-putrescine synthase (PuuA), a gamma-glutamyl-putrescine oxidase (PuuB), preferably from E. coli, ensures the subsequent conversion of putrescine into ABAL.

Microorganisms and plasmids preferably used in this invention are listed in Table 1 and Table 2. The ornithine-producing strain C. glutamicum ORN1 (ATCC 13032 with argF and argR deletion) may preferably serve as a starting strain for introducing further modifications. As a strain for the cloning of PCR products, the E. coli DH5a can be preferably used. The plasmids pVWxl and pEKEx3, which replicate in C. glutamicum, fiction, according to the plasmid-based overexpression of genes were used. The episomal expression vectors preferably contain a multiple cloning site, an origin of replication for E. coli and C. glutamicum and a kanamycin or a spectinomycin resistance as a selection marker. The non-replicating in C. glutamicum, integrative plasmids pK18mobsacB and pK19mobsacB with a multiple cloning site, an origin of replication for E. coli as well as a kanamycin resistance and the Bacillus subtilis levansucrase gene sacB as selection markers served preferentially as vectors for the genomic integration of gene deletions. In this case, the corresponding plasmids with the truncated gene sequence can preferably be used to replace the wild-type gene chromosomally by the mutated gene.

Table 1: Strains used in this study.

Table 2: Plasmids used in this study.

 media

For the culture of E. coli strains, LB medium with 10 gL ^-1 tryptone, 5 gL ^-1 yeast extract and 10 gL ^-1 sodium chloride can be routinely used with preference. The precultures for cultivations of C. glutamicum can preferably be carried out in BHI complex medium with 37 gL ^-1 BHI. CGXII minimal medium with the following concentrations (per liter) may preferably be used for the main culture: 20 g (NH ₄ ) ₂ SO ₄ , 5 g urea, 1 g KH ₂ PO ₄ , 1 g K ₂ HPO ₄ , 42 g MOPS, 0 , 25 g MgSO ₄ ● 7H ₂ O, 10 mg CaCl ₂ , 20 mg biotin, 1 mg FeSO ₄ ● 7H ₂ O, 1 mg MnS0 ₄ ● H ₂ O, 0.1 mg ZnSO ₄ ● 7H ₂ O , 20 μg CuS0 ₄ , 2 μg NiCl ₂ ● 6H ₂ O, 30 μg protocatechuic acid. To optimize the production of GABA, preference may be given to using the modified minimal medium CGXII1 / 2, in which the ammonium and urea concentrations have been halved in comparison to the starting medium; all other media components can remain unchanged. The media for cultivating strains with episomally replicating plasmids may advantageously additionally contain 25 μg mL ¹ kanamycin or 100 μg mL ¹ spectinomycin and 1 mM isopropyl-β-Dl-thiogalactopyranoside (IPTG).

cultivations

Single colonies of C. glutamicum and E. coli can preferably be used as inoculum for the preculture and at 30 ° C. or 37 ° C. in 500 ml of chicane flasks with 50 ml of BHI or LB medium on a rotary shaker (120 rpm). incubate overnight. CGXII minimal medium can be used for the main cultures of C. glutamicum cultivations [1]. For example, in each case 50 ml of CGXII medium were inoculated with an OD of 1 and incubated at 30 and 120 rpm. The growth was monitored by measuring the optical density at 600 nm. At this wavelength, an optical density of 1 corresponds to a cell dry weight (CDW) of 0.25 g / L. Gamma-aminobutyric acid (GABA) tolerance assays were performed in a BioLector (m2p Labs, Baesweiter, Germany), while growth behavior and GABA production were studied in baffled flasks. In this case, 50 ml LB medium were inoculated with a single colony from an agar plate and cultured overnight. The cells were harvested by centrifugation (4000 xg, 10 min) and washed twice with CGXII minimal medium without carbon source. Thereafter, 50 ml of CGXII medium with a corresponding glucose concentration and the corresponding media additives were added and inoculated with a starting OD of 1.0.

GABA tolerance test

 To study the tolerance of C. glutamicum to GABA, growth of C. glutamicum wild-type was monitored in CGXII minimal medium with addition of various concentrations of GABA. For this purpose, 5 ml of LB medium were inoculated from an agar plate and incubated overnight. Subsequently, 50 mL LB were inoculated with the overnight culture and cultured until the exponential phase. Then the cells were harvested by centrifugation (4000 x g, 10 min) and washed twice with CGXII minimal medium without carbon source. Finally, 1 mL each of CGXII minimal medium with carbon source and the corresponding media additives as well as 50 mM, 100 mM, 200 mM or 500 mM GABA (Merck KGaA, Darmstadt, Germany) were inoculated with an optical density of 1.0. The test was performed in the BioLector (m2p Labs, Baesweiler, Germany) in a FlowerPlate (48 well MTP, flower, m2p Labs, Baesweiler, Germany).

Recombinant DNA techniques

For PCRs and the purification of PCR fragments as well as for the construction, purification and analysis of plasmid DNA and the transformation of E. coli, standard protocols can preferably be used according to the invention. The extraction of chromosomal DNA from E. coli and C. glutamicum takes place, for example, by means of phenol Chloroform-isoamyl alcohol. The oligonucleotides (primers) preferably used for the vector construction are listed in Table 3. Standard methods such as PCR, restriction and ligation can be performed as described elsewhere (Sambrook & Russell, 2001). All plasmids can preferably be cloned and propagated in E. coli DH5a. For the amplification of speC and patDA, genomic DNA of E. coli MG 1655 can preferably be used as template. The PCR can be carried out, for example, using the KOD Hot Start DNA Polymerase (Novagen, Darmstadt, Germany). The amplification product patD can preferably be cloned into pEKEx3 via the restriction sites PstI / BamHI, which resulted in the plasmid pEKEx3 patD. Thereafter, the plasmid can be purified with the QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany) and the patA amplification product can be cloned into pEKEx3 patD via BamHI / SacI. The start codon of the gene patA can preferably be changed from TTG to ATG. For the deletion of the gabTDP operon (cg0566-0568), the integrative vector pK19mobsacB can preferably be used (Schäfer et al., 1994). The flanking genomic regions of gabT and gabP necessary for integration via homologous recombination could be amplified from genomic DNA of the wild-type C. glutamicum using the primer pairs TDDP_A / gabTDP_B and gabTDP_C / gabTDP_D (Table 3). After purification, the two PCR products can be linked by crossover PCR using the primer pair gabTDP_A / gabTDP_D. The resulting product is preferably cloned into pK19mobsacB via the SmaI site, yielding the deletion plasmid pK19mobsacBDgabTDP (Table 2). This plasmid can preferably be used for directional deletion of the gabTDP operon by two-step homologous recombination. After the first recombination, integration of the vector can be selected via the kanamycin resistance cassette. Integration of the vector into the genome may preferably result in sucrose sensitivity due to the sacB gene product levansucrase. The selection of the clones that have excised the deletion vector in a second recombination event s can be detected by a resistance to sucrose. The deletion of the gabTDP operon can be done by PCR analysis using the primer pair gave TDP_A / gabTDP _D verification.

E. coli cells can preferably be transformed by heat shock (Sambrook & Russell, 2001) and C. glutamicum cells can be transformed by electroporation at 2.5 kV, 200 Ω and 25 μF (van der Rest, Lange, & Molenaar, 1999 ). All cloned DNA fragments can preferably be checked by sequencing.

Table 3: Primers suitable for cloning.

6 shows a plasmid map of pEKEx3 suitable for producing a microorganism according to the invention, the DNA sequence of which is listed as SEQ ID NO: 27: E. coli / C. glutamicum shuttle vector for IPTG-inducible gene expression (Ptac, laclq, pBLI oriVCg, specR); 7 shows a plasmid map of pEKEx3patDA suitable for producing a microorganism according to the invention whose DNA sequence is listed as SEQ ID NO: 28: pEKEx3 derivative for IPTG-inducible gene expression of patD and patA from E. coli MG1655;

8 shows a plasmid map of pK18mobsacB_de \ cgl722 which is suitable for the preparation of a microorganism according to the invention and whose DNA sequence is listed as SEQ ID No. 29: pK18mobsacB with cgl722 deletion construct;

9 shows a plasmid map of pK19mobsacB_delgabTDP which is suitable for producing a microorganism according to the invention and whose DNA sequence is listed as SEQ ID No. 30: pK19mobsacB with gabTDP deletion construct; 10 shows a plasmid map of pK19mobsacB_de \ cgmA suitable for producing a microorganism according to the invention whose DNA sequence is listed as SEQ ID NO: 31 31: pK19mobsacB with cgmA deletion construct;

11 shows a plasmid map of pK19mobsacB_delcgmR suitable for producing a microorganism according to the invention, the DNA sequence of which is listed as SEQ ID No. 32: pK19mobsacB with cgmR deletion construct;

FIG. 12 shows a plasmid map of pK19mobsacB_delargFR suitable for producing a microorganism according to the invention, the DNA sequence of which is listed as SEQ ID NO: 33: pK19mobsacB with argFR deletion construct. 13 shows a plasmid map of pEKEx3patA which is suitable for the preparation of a microorganism according to the invention and whose DNA sequence is listed as SEQ ID NO: 34: pEKEx3 derivative for IPTG-inducible gene expression of patA from E. coli MG 1655 FIG. 14 shows one for the production a plasmid according to the invention of pEKEx3puuAB whose DNA sequence is listed as SEQ ID NO: 35: pEKEx3 derivative for IPTG-inducible gene expression of puuA and puuB from E. coli MG1655 Fig. 15 shows a plasmid suitable for the preparation of a microorganism according to the Invention of pEKEx3patApuuC whose DNA sequence is listed as SEQ ID No. 36: pEKEx3 derivative for IPTG-inducible gene expression of patA and puuC from E. coli MG1655 FIG. 16 shows a plasmid map of pEKEx3puuADCB suitable for producing a microorganism according to the invention SEQ ID NO: 38: pEKEx3 derivative for IPTG-inducible gene expression of p uuA, puuB, puuC and puuD from E. coli MG 1655 Fig. 17 shows a plasmid map of pVWExl suitable for producing a microorganism according to the invention whose DNA sequence is listed as SEQ ID NO: 37: E. coli / C. glutamicum shuttle vector for IPTG-inducible gene expression (Ptac, laclq, pBLI oriVCg, kanR) FIG. 18 shows a plasmid map of pVWExlspeC suitable for producing a microorganism according to the invention whose DNA sequence is listed as SEQ ID No. 39: pVWExl derivative for IPTG inducible gene expression of speC from E. coli MG1655 Quantification of GABA, putrescine, N-acetyl-putrescine and amino acids

In one embodiment of the invention, samples were taken for the quantification of GABA during culture and the cells were removed by centrifugation (13,000 x g, 10 min). The supernatant was transferred to a new reaction tube, diluted with L-asparagine as an internal standard, and purified by HPLC (1200 series, Agilent Technologies Deutschland GmbH, Boeblingen, Germany) with ortho-phthalaldehyde (OPA) precolumn derivatization and fluorescence chromatography. Detection (FLD G1321A, Series 1200, Agilent Technologies). The system used was equipped with a precolumn (LiChrospher 100 RP 18 × C5μ (40 × 4 mm), CS-Chromatography Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP 18 × 5 μm (125 × 4 mm), CS- Chromatography Service GmbH, Langerwehe, Germany). For the gradient required for the elution, 0.25% Na acetate (pH 6) and methanol were used as eluents at a flow rate of 0.7 mL min-1. The injection volume was 5 μL ×.

Transcriptome

In a preferred embodiment of the invention, in order to carry out comparative transcriptome analyzes of a GABA producer with a strain which can not produce GABA, the C. glutamicum strains GAB A4 and PUT21 (pEKEx3patA) were cultured in modified minimal medium until the middle exponential phase was reached Centrifugation harvested and stored at -80 ° C until RNA isolation. The isolation of the RNA and its labeling were as described by Wendisch (2003). To determine the relative levels of expression by hybridization to DNA microarrays of the entire genome of C. glutamicum (Wendisch, 2003), three microarrays were performed with DNA from three independent cultivations. The hybridization was carried out as described by Hüser et al. (2003). The raw data was evaluated with the program ImaGene Premium. Genes with a significantly altered mRNA level (P <0.05) by a factor of at least 2.0 were considered to be differentially expressed. Measurement of enzyme activities

 In a preferred embodiment of the invention, enzyme activities were measured in crude extracts prepared from late exponential phase cells grown in LB medium (with 1 mM IPTG). The cells were harvested (4000 x g, 4 ° C, 10 min), washed twice with 150 mM NaCl and frozen at -80 ° C. The cell pellets were resuspended in 1 ml of resuspension buffer (0.1 M potassium phosphate, pH 7.5, 1 mM DTT) and sonicated (sonoplus Sonoplus GM 200, sonotrode M72, Bandelin Electronic GmbH & Co. KG, Berlin, Germany) for 6 min (Cycle 0.5, amplitude 55) digested and then centrifuged for 90 min at 4 ° C and 13200 rpm. After centrifugation, the cell debris-free supernatant was used in the enzymatic reactions. Protein concentrations were determined by the method of Bradford with bovine serum albumin as the standard (Bradford, 1976). For the indicated enzyme activities, one unit corresponds to the amount of enzyme which catalyzes the formation of 1 μm product in 1 minute.

The activity of putrescine transaminase was determined according to a protocol of Albrecht and Vogel (1964), with the following modifications: a 0.5 mL batch contained 0.1 M Tris-HCl buffer pH 8.0, 15 mM a-ketoglutarate, 25 mM putrescine, 1mM pyridoxal-5'-phosphate and 10mM EDTA. One batch was incubated for 30 min at 37 ° C. The reaction was started by the addition of putrescine and the change in absorbance was measured with the Shimadzu Spectrophotometer UV1700 at 440 nm.

The activity of γ-aminobutyraldehyde dehydrogenase was determined as previously described (Schneider and Reitzer, 2012). The assay solution contained 50 mM glycine buffer, pH 9.5, 0.28 mM NAD and 0.5 mM γ-aminobutyraldehyde (fresh prepared and finally added). The reaction was carried out at 37 ° C and the change in absorbance at 340 nm (ε340nm = 6.22 mM ^-1 cm ^-1 ) was measured. GABA tolerance test

 The effects of increased educt and product concentrations on the production strain are of great importance in biotechnological processes with whole-cell biocatalysts. So far no studies on the effect of GABA on the growth of C. glutamicum are known. To study the effect of GABA on the growth of C. glutamicum, the wild type was cultured in BioLector in minimal medium with addition of various concentrations of GABA (0 mM, 50 mM, 100 mM, 200 mM, 500 mM and 1 M) (Fig 2).

Fig. 2 shows the study of the GABA tolerance of C. glutamicum wild type when cultivated in CGXII minimal medium at 30 ° C with the addition of various GABA concentrations. The data represent averages of duplicate determinations with the associated standard deviations.

In the presence of concentrations up to 200 mM GABA, the growth of C. glutamicum WT was comparable to that of the control without GABA. From an addition of 500 mM GABA, a reduction in the growth rate of about 25% and a slight reduction in biomass formation (7%) could be observed. In addition, the lag phase was extended by about 2 h with the addition of 500 mM GABA and the growth rate was reduced by almost 30% (FIG. 3). From the values shown in Fig. 3, the GABA concentration Ki at which the growth rate is reduced by half could be calculated to be 1050 mM.

FIG. 3 shows the examination of the tolerance of C. glutamicum wild-type ATCC13032 to GABA.

GABA production in C. glutamicum by overexpression of speC and patDA Since GABA is not a natural product of the metabolism of C. glutamicum, the expression of heterologous genes is the key modification for the production of GABA, both glutamate and putrescine. Heterologous overexpression of E. coli glutamate decarboxylase turned into a strain designed for the production of GABA from glutamate (Takahashi et al., 2012). A preferred embodiment of the invention provides the synthesis of GABA via putrescine. Since putrescine is not a natural metabolite of C. glutamicum, this route requires heterologous expression of ornithine decarboxylase, which catalyzes the conversion of ornithine to putrescine. There are two distinct routes to GABA synthesis from putrescine: the glutamylated putrescine pathway, which consists of four enzymes, and the transaminase pathway, which catalyzes the conversion of putrescine into GABA via aminobutyraldehyde in two steps.

As a starting strategy for GABA production according to the invention, the genes of the transaminase pathway patDA were plasmid-overexpressed under the IPTG-inducible promoter Ptac in the putrescine producer C. glutamicum PUT21 (AargRF pVWExl-speC-5 '2i-argF) , The resulting strain C. glutamicum GABA1 (AargRF pVWExl-speC-5 '2i-argF pEKEx3patDA) was cultured in CGXII minimal medium with 4% glucose and 1 mM IPTG and the growth and production properties compared with those of PUT21 pEKEx3 (Table 4). (Figures 4 and 5).

Der Stamm GABA1 akkumulierte bei Kultivierung in CGXII Minimalmedium mit 4 % (w/v) Glucose und 1 mM IPTG 5,3 gL^-1 GABA. Es konnte kein Putrescin im Überstand nachgewiesen werden, was auf eine vollständige Umsetzung des zur Verfügung stehenden Putrescins zu GABA schließen lässt. Somit konnte gezeigt werden, dass durch heterologe Überexpression der Gene patDA des Transaminase- Weges aus E. coli in einem Putrescin- Produzenten die Produktion von GABA in C. glutamicum ermöglicht wurde. Unter der Annahme, dass die Glucose zum Ende der Kultivierung komplett verbraucht war, lässt sich daraus eine Ausbeute von 0,13 g g^"1 berechnen. Die maximale Produktivität von 0,18 g L^"1 h^{" 1} nach 30 h ist der höchste Wert, der bisher für die biotechnologische GABA-Produktion auf Glucose als Haupt- Kohlenstoffquelle erreicht wurde. Erwartungsgemäß konnte im Überstand von PUT21 (pEKEx3) kein GABA sondern nur Putrescin detektiert werden. Table 4: Accumulation of GABA and putrescine upon cultivation of C. glutamicum GABA1 and its precursor PUT21 (pEKEx3) in CGXII minimal medium with 4% (w / v) glucose and 1 mM IPTG.

The strain GABA1 when cultivated in CGXII minimal medium with 4% (w / v) glucose and 1 mM IPTG accumulated 5.3 gL ^-1 GABA. No putrescine could be detected in the supernatant, which indicates a complete conversion of the available putrescine to GABA. Thus, it could be shown that the production of GABA in C. glutamicum was made possible by heterologous overexpression of the genes patDA of the transaminase pathway from E. coli in a putrescine producer. Assuming that the glucose was completely consumed at the end of the cultivation, it can be calculated a yield of 0.13 gg ^"1. The maximum productivity of 0.18 g L ^{" 1} h ^{" 1} after 30 h is the highest value As expected, it was not possible to detect GABA in the supernatant of PUT21 (pEKEx3) but only putrescine in the supernatant of PUT21 (pEKEx3).

4 shows the growth profile of the parent strain C. glutamicum PUT21 pEKEx3 and of the GABA producers GABA1 (AargRF pVWExl-speC-5 '2i-argF pEKEx3patDA) generated therefrom, GABA2 (AargRFAcgmA pVWExl-speC-5' ₂ i-argF pEKEx3patDA) and GAB A3 (AargRFAcgmR pVWExl-speC-5 ' ₂ i-argF pEKEx3patDA) in CGXII minimal medium with 4% (w / v) glucose and 1mM IPTG.

PUT21 (pEKEx3) showed a slightly lower growth rate than GABA1 (0.19 h ^-1 ) with μ = 0.17 h ^-1 . The final biomass concentration after 26 hours was 20 gL ^-1 for GABA1, while that for the putrescine producer PUT21 (pEKEx3) was significantly lower (FIG. 5).

Figure 5 shows the GABA titer of the various C. glutamicum GABA production strains after 30 hours. All strains were cultured in chicane flasks in CGXII minimal medium with 4% glucose (w / v) and 1 mM IPTG. The data represent averages of duplicate determinations with the associated standard deviations. The values given in FIG. 5 represent the endpoints of the cultivations after 30 hours. As already shown above, no GABA could be detected in the culture supernatant of PUT21 (pEKEx3), which served as a negative control. In contrast, in the culture supernatant of GABA1, a steady increase in GABA concentration was observed to a final value of 5.3 g L ^-1 GABA; Putrescine as a by-product could not be detected. The byproduct N-acetylputrescine (Nguyen et al., 2014) described for putrescine production with C. glutamicum, which reached a final concentration of 3.2 gL ^-1 in PUT21 (pEKEx3) cultivation, could also be found in the supernatant GABA 1 cultivation are detected. However, the final concentration of 1.2 gL ^-1 was significantly lower than that of the parent strain.

Elimination of putrescine export by deletion of cgmA

In order to prevent the export of putrescine, the precursor of GABA, and thus to prevent the outflow of carbon and the formation of undesired by-products, the gene cgmA, which encodes the permease of the major facilitator superfamily, which is mainly involved in putrescine export, was encoded (Nguyen Schneider et al., 2014), deleted in GABA 1. Since deletion of cgmA drastically reduces the concentration of putrescine in the culture supernatant, it should make available more putrescine intracellularly for conversion to GABA, the production characteristics of the resulting strain GABA2 was also studied in minimal medium with glucose as the sole carbon source, but the additional modification did not improve the production properties compared to the precursor strain GABA1 and after 30 h a titer of 5.1 gL ^-1 GABA was measured Adoption of a complete glucose verb a yield of 0.13 g (GABA) g ^-1 (glucose) corresponds (Fig. 5). The final concentration of the by-product N-acetyl-putrescine was also comparable to that of the precursor strain GABA1 at 1.2 gL ^-1 . Thus, no positive effect on GABA production by inhibiting putrescine export could be demonstrated in the investigated strain background GABA1 become. Since no putrescine could be detected in the supernatant during shake flask cultivation of GABA1 in minimal medium, this observation is not surprising. However, by further stem optimization or under modified culture conditions in other medium, the formation of putrescine as a by-product may occur and thus the deletion of CgmA may play an important role.

However, the slight decrease in GABA titers in GABA2 compared to GABA1 with intact permease may also indicate that CgmA is also responsible for the export of GABA to C. glutamicum.

Export of GABA to C. glutamicum

 In the prior art, so far no protein has been identified in C. glutamicum which is responsible for the export of GABA. To identify a putative GABA export protein, a comparative DNA microarray was performed with the GABA producer GABA4 and the non-producing strain PUT21 (pEKEx3-patA). Table 5 shows all genes with a different expression (P <0.05 and at least two-fold up or down regulation (M> 1 equals 2-fold)) in both strains, which could be detected in at least two out of three replicates. Table 5: Comparative transcriptome analysis of the GABA-producing strain C. lutamicum GAB A4 and the non-GABA producer PUT21 (EKEx3-atA).

One of the highest upregulations in the GABA producer GABA4 compared to a strain unable to produce GABA revealed the cgmR gene encoding a transcriptional regulator of the TetR family (cg2984). Upregulation of this gene has already been demonstrated by comparative transcriptome analyzes for putrescine and cadaverine-producing C. glutamicum strains (Kind et al., 2011, Nguyen et al., 2014). According to the invention, cgmR was now deleted in PUT21 pEKEx3-patDA, which resulted in C. glutamicum GABA3. The production properties of this strain were also examined in CGXII minimal medium and compared with those of its starting strain GABA1 (Table 6).

TABLE 6 Accumulation of GABA, putrescine, N-acetyl-putrescine, glutamate and ornithine of GABA-producing C. glutamicum GABA1 and GAB A3 when cultivated in CGXII minimal medium.

The deletion of cgmR in the GABA producer GABA1 did not improve the production properties. On the contrary, the final GABA titer was 3.3 gL ^"1 when cultivated in CGXII minimal medium and thus 40% lower than the precursor strain GABAl. While the concentrations of the other by-products remained unchanged, a marked increase in extracellular putrescine - Concentration can be observed. Elimination of the formation of N-acetyl-putrescine

Studies of putrescine-producing C. glutamicum strains have shown that much of the putrescine produced is acetylated prior to export. This by-product undesirable for putrescine production is also detrimental to GABA production since the N-acetyl-putrescine formed is no longer available for conversion to GABA. For the formation of N-acetyl-putrescine, as well as for the acetylation of cadaverine (Kind et al., 2012), the cgl722-encoded GCN5-like N-acetyltransferase is responsible (Nguyen et al., 2014). The deletion of the N-acetyltransferase in putrescine producer PUT21Acgl722 led to an increase of more than 50% in putrescine production with complete suppression of N-acetyl-putrescine production. Also in the cultivations of GABA1 and GAB A3, the product spectrum was investigated with respect to the formation of N-acetyl-putrescine. The results listed in Table 7 show that even in the tested GABA producers, a considerable proportion of about 20% of the products formed on N-acetyl-putrescine. The deletion of cgl722 thus surprisingly represents a target for the production of GABA in C. glutamicum. The deletion of cgl722 in GABA1 resulted in C. glutamicum GABA4, whose production properties were compared with those of its precursor. TABLE 7 Accumulation of GABA and putrescine of C. glutamicum GAB A4 and its precursor GABA1 when cultivated in CGXII minimal medium with 4% (w / v) glucose and 1 mM IPTG.

The comparison of the achieved titers of the two strains makes it clear that the deletion of the N-acetyltransferase cgl722 is advantageous for GABA production Modification is; with a value of 5.6 gL ^-1 , the titer of GABA4 was almost 10% higher than that of GABA1.

Analysis of the enzyme activities of PatD and PatA

To verify the functional expression of patD and patA, the specific activities of gamma-aminobutyraldehyde dehydrogenase (PatD) and of putrescine: 2-oxoglutarate aminotransferase (PatA) were determined. For this purpose, GABA producer GABA4 and its precursor strain PUT21Acgl722 (pEKEx3) were cultured in LB medium with 1 mM IPTG and the cells were harvested in the late exponential phase. After cell disruption by ultrasound, the cell debris was centrifuged off and the supernatant was used as crude protein extract for determining the enzyme activity. The protein concentration in the crude extract was determined by the method of Bradford with bovine serum albumin as standard. Table 8: Specific in vitro activity of PatD (gamma-aminobutyraldehyde dehydrogenase) and PatA (putrescine: 2-oxoglutarate aminotransferase) in GABA producer C. glutamicum GAB A4 and its precursor strain, the putrescine producer C. glutamicum PUT21Acgl722 (pEKEx3).

For both enzymes, surprisingly, enzyme activity could be detected in the crude extract of GABA4, whereas the control strain PUT21Acgl722 (pEKEx3) showed neither PatD nor PatA activity. Thus, a successful expression of the two genes essential for GABA synthesis in C. glutamicum could be demonstrated by the enzyme assay. Overexpression of puuC in combination with patDA

 Since the activity of gamma-aminobutyraldehyde dehydrogenase PatD is lower compared to putrescine: 2-oxoglutarate aminotransferase PatA, this reaction is a limiting step for GABA production in C. glutamicum. To increase gamma-aminobutyraldehyde dehydrogenase activity, puuC was overexpressed with patDA. Studies by (Schneider & Reitzer, 2012) have shown that in E. coli PatD and PuuC are responsible for 55 and 30% of the gamma-aminobutyraldehyde dehydrogenase activity, respectively. The residual activity of 15% is attributed to one or more previously unknown enzymes. In order to enable a common overexpression, according to the invention the vector pEKEx3-puuCpatDA was constructed and various C. glutamicum were transformed therewith. The production properties were compared with those of strains with overexpression of patDA alone.

Elimination of GABA import and mining

GabP (Cg0568) was reported by Zhao et al. (2012) was identified as a GABA importer in C. glutamicum and it was shown that a deletion of the coding gene prevents growth of C. glutamicum on GABA as the sole source of C or N, while increasing the production of GABA from glutamate (Zhao et al., 2012). The deletion of the genes responsible for the degradation is a strategy according to the invention for improving the production properties of a strain for the desired product. In the case of GABA production with C. glutamicum, the enzymes γ-aminobutyrate aminotransferase (GabT, Cg0566) and succinate semialdehyde dehydrogenase (gabD, Cg0567) encoded in the genome of C. glutamicum are described in the literature as part of the putrescine transaminase In E. coli responsible for the degradation of GABA to succinate (Schneider & Reitzer, 2012; Schneider et al., 2013). Since in C. glutamicum all three genes (gabT, gabD (succinate-semialdehyde dehydrogenase) and gabP) were in an operon, the complete operon was deleted according to the invention in the GABA producers GABA1 and GAB A4, which resulted in the formation of GABA5 (FIG. PUT21 AgabTDP (pEKEx3-patDA)) and GABA6 (PUT21Δcgl722AgabTDP (pEKEx3-patDA)). The successful deletion was detected by PCR.

To investigate the effects of the introduced modification on GABA production, C. glutamicum GABA5 and GABA6 were cultured in the shake flask in CGXII minimal medium and the production characteristics were compared with those of their precursors (Table 9). By deleting the operon gabTDP, the GABA titer in GABA6 could be increased by 10% to 7.7 gL ^-1 compared to the precursor strain GABA4. The maximum productivity was this cultivation at 0.31 g L ^{_1 _1} h to 26 h. In addition to productivity and titre, the yield could also be increased to a value of 0.20 μg ^"1 glucose.

Table 9: Comparison of GABA titer and GABA productivity of C. glutamicum GAB A4 and GABA6 when cultivated in CGXII minimal medium with 4% (w / v) glucose and 1 mM IPTG.

media optimization

The minimal CGXII medium optimized for the production of L-lysine contains an excess of nitrogen (Eggeling and Bott, 2005). Since GABA possesses only one amino group in contrast to L lysine and its precursor putrescine and the conversion of putrescine into GABA is accompanied by the formation of glutamate from 2-oxoglutarate, the influence of the high nitrogen concentration in the medium on GABA production was investigated. To study the influence of nitrogen concentration on GABA production, the ammonium concentration in the CGXII minimal medium was reduced by 20 to 10 gL ^-1 and the urea concentration was reduced by half from 5 gL ^-1 to 2.5 gL ^-1 ; all other media components remained unchanged. GABA producers GABA1, GAB A4, GABA5 and GABA6 were cultured comparatively in CGXII and in the modified CGXIIl / 2 minimal medium (Table 10). Under the conditions tested, a significant increase in GABA titre and GABA productivity in the minimal medium with reduced ammonium concentration was observed. For C. glutamicum GABA1 and GAB A4, the increase in titer and productivity was approximately 20 and 15%, respectively. An even clearer optimization of production properties was achieved in the cultivations of GABA5. Here, both titre and productivity were increased by almost 40%. For GABA6 were titers (9.9 gL ^"1) and productivity (0.38 g ^L" V) in CGXIIl / 2 minimal medium about 20% higher than in the original medium. Table 10: Comparison of GABA concentrations and productivities of various C. glutamicum strains when cultivated in CGXII and CGXIIl / 2 Minimal medium at 4%

(w / v) glucose and 1 mM IPTG after 26 h.

Moreover, the present invention relates to a process for the preparation of terminal amino aldehydes from diamines using recombinant microorganisms. Synthesis of γ-aminobutyraldehyde (ABAL)

 For the synthesis of the C4-aminobutyraldehyde γ-aminobutyraldehyde (ABAL), the patA gene from E. coli, which codes for a putrescine: 2-oxoglutarate aminotransferase, was transformed into a C. glutamicum strain, which was expressed by expression of the speC gene from E coli for the synthesis of putrescine (C4-diamine) is overexpressed.

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Claims

Claims: 1. Genetically modified microorganism, wherein the microorganism in the  Compared to a native level an increased level  A: a putrescine: 2-oxoglutarate aminotransferase; and or  B: a gamma-glutamyl-putrescine synthase and a gamma-glutamyl Putrescine oxidase has. 2. Genetically modified microorganism according to claim 1, wherein said in the  Compared to a native level an increased level  C: a gamma-aminobutyraldehyde dehydrogenase; and or  D: has a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase. 3. Genetically modified microorganism according to claim 1, wherein said in  Compared to a native level an increased level  E: a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase and a gamma-glutamyl-gamma-aminobutyric acid hydrolase. 4. Genetically modified microorganism according to one of the preceding  Claims, wherein this has an increased level of an ornithine decarboxylase compared to a native level. 5. Genetically modified microorganism according to one of the preceding Claims wherein it has no or, compared to a native level, reduced activity of at least one enzyme of the group consisting of ornithine carbamoyltransferase, arginine repressors, GCN5-related N-acetyltransferase and major facilitator superfamily permease. A genetically engineered microorganism according to any one of the preceding claims, which has no or, compared to a native level, a reduced activity of at least one enzyme of the group consisting of 4-aminobutyrate aminotransferase (GabT) and amino acid permease (GabP). 7. Genetically modified microorganism according to one of the preceding  Claims, in comparison with a native level, of an increased level of a putrescine: 2-oxoglutarate aminotransferase and a gamma-aminobutyraldehyde dehydrogenase and an ornithine decarboxylase and a reduced or no activity of the enzymes ornithine carbamoyltransferase, arginine repressors, GCN5- has related N-acetyltransferase and permease major facilitator superfamily. 8. Genetically modified microorganism according to one of the preceding  Claims, wherein said at least one plasmid genetically engineered into the microorganism having at least one sequence of the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID NO: 11 and SEQ ID NO: 13. 9. A genetically engineered microorganism according to claim 8, wherein in this at least one sequence of the group consisting of SEQ ID. No. 15, SEQ ID. No. 17, SEQ ID. No. 19, SEQ ID. No. 21, SEQ ID. No. 23 and SEQ ID. No. 25 is genetically deleted or disrupted. 10. Genetically modified microorganism according to one of the preceding Claims, which belongs to the genus Corynebacterium, preferably to the species Corynebacterium glutamicum. 11. A process for preparing a terminal aminocarboxylic acid and / or a terminal aminoaldehyde, characterized in that a genetically modified microorganism according to any one of claims 1 to 10 is cultured in a suitable medium. 12. The method according to claim 11, characterized in that the medium between <20 gL ^-1 and> 5 gL ^-1 (NH4) ₂ S0 ₄ and / or a urea concentration between <5 gL ^-1 and> 1.5 gL ^-1 having. 13. The method according to any one of claims 11 and 12, comprising the steps: Providing a microorganism, preferably of the genus Corynebacterium; Decreasing the activity of at least one enzyme of the group consisting of ornithine carbamoyltransferase, arginine repressors, GCN5-related N-acetyltransferase, and major facilitator superfamily permease versus native activity by genetic engineering;  Increase the level of a putrescine: 2-oxoglutarate aminotransferase (PatA) and / or a gamma-glutamyl-putrescine synthase (PuuA) and a gamma-glutamyl-putrescine oxidase (PuuB) over a native level by genetic engineering; and  Culturing the recombinant microorganism thus obtained. 14. The method according to claim 13, additionally comprising the step:  Increasing the level of gamma-aminobutyraldehyde dehydrogenase (PatD); and / or a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC); and / or a gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) and a gamma-glutamyl-gamma-aminobutyric acid hydrolase (PuuD) versus a native level by genetic engineering Method. 15. The method according to claim 14, additionally comprising the step:  decreased activity of at least one enzyme of the group consisting of 4-aminobutyrate aminotransferase (GabT) and amino acid permease (GabP) versus native activity by genetic engineering.