WO2015132213A1 - Procédé de préparation d'acides aminocarboxyliques et d'aminoaldéhydes terminaux au moyen d'un micro-organisme recombinant - Google Patents

Procédé de préparation d'acides aminocarboxyliques et d'aminoaldéhydes terminaux au moyen d'un micro-organisme recombinant Download PDF

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WO2015132213A1
WO2015132213A1 PCT/EP2015/054330 EP2015054330W WO2015132213A1 WO 2015132213 A1 WO2015132213 A1 WO 2015132213A1 EP 2015054330 W EP2015054330 W EP 2015054330W WO 2015132213 A1 WO2015132213 A1 WO 2015132213A1
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gamma
putrescine
seq
gaba
glutamyl
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Stefanie KIND
Joao Jorge
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Evocatal Gmbh
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/0301—Putrescine oxidase (1.4.3.10)
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • C12Y206/01029—Diamine transaminase (2.6.1.29)
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    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01011—Glutamate--putrescine ligase (6.3.1.11)

Definitions

  • 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.
  • GABA gamma-aminobutyric acid
  • ABAL gamma-aminobutyraldehyde
  • 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.
  • the present 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.
  • 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.
  • 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.
  • the present invention provides a microorganism as described above, and a process for producing gamma-aminobutyric acid by cultivating the microorganism.
  • the present invention provides a microorganism as described above, and a process for producing gamma-aminobutyraldehyde by cultivating the microorganism.
  • the present invention provides a microorganism having increased putrescine-forming activity.
  • 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.
  • a plasmid 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ,
  • GABA glutamate decarboxylase
  • GABA fermentation 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.
  • 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).
  • 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).
  • E. coli GadB By overexpression of E. coli GadB in a C.
  • 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).
  • 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.
  • 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).
  • this invention describes the synthesis of GABA from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase.
  • putrescine gamma-aminobutyraldehyde dehydrogenase (PatD) and putrescine: 2-oxoglutarate aminotransferase (PatA), preferably from E. coli, ensures the subsequent conversion of putrescine into 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.
  • 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.
  • PatA 2-oxoglutarate Aminotransferase
  • PuuC gamma-glutamyl gamma-aminobutyraldehyde dehydrogenase
  • 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.
  • this invention describes the synthesis of ABAL from putrescine, which is formed in C. glutamicum from ornithine by decarboxylation by a heterologous ornithine decarboxylase.
  • 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.
  • the E. coli DH5a can be preferably used as a strain for the cloning of PCR products.
  • 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.
  • 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.
  • the corresponding plasmids with the truncated gene sequence can preferably be used to replace the wild-type gene chromosomally by the mutated gene.
  • 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 ⁇ g CuS0 4 , 2 ⁇ g NiCl 2 ⁇ 6H 2 O, 30 ⁇ g protocatechuic acid.
  • the media for cultivating strains with episomally replicating plasmids may advantageously additionally contain 25 ⁇ g mL 1 kanamycin or 100 ⁇ g mL 1 spectinomycin and 1 mM isopropyl- ⁇ -Dl-thiogalactopyranoside (IPTG).
  • 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.
  • an optical density of 1 corresponds to a cell dry weight (CDW) of 0.25 g / L.
  • GABA Gamma-aminobutyric acid
  • 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.
  • C. glutamicum wild-type was monitored in CGXII minimal medium with addition of various concentrations of GABA.
  • 5 ml of LB medium were inoculated from an agar plate and incubated overnight.
  • 50 mL LB were inoculated with the overnight culture and cultured until the exponential phase.
  • the cells were harvested by centrifugation (4000 x g, 10 min) and washed twice with CGXII minimal medium without carbon source.
  • 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.
  • speC and patDA genomic DNA of E.
  • the 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.
  • 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 ⁇ and 25 ⁇ F (van der Rest, Lange, & Molenaar, 1999 ). All cloned DNA fragments can preferably be checked by sequencing.
  • FIG. 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;
  • FIG. 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;
  • FIG. 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;
  • FIG. 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. 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
  • 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
  • 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 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 ⁇ C5 ⁇ (40 ⁇ 4 mm), CS-Chromatography Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP 18 ⁇ 5 ⁇ m (125 ⁇ 4 mm), CS- Chromatography Service GmbH, Langerwehe, Germany).
  • a precolumn LiChrospher 100 RP 18 ⁇ C5 ⁇ (40 ⁇ 4 mm)
  • CS-Chromatography Service GmbH Langerwehe, Germany
  • a main column LiChrospher 100 RP 18 ⁇ 5 ⁇ m (125 ⁇ 4 mm)
  • CS- Chromatography Service GmbH Langerwehe, Germany
  • 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 ⁇ L ⁇ .
  • RNA isolation 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
  • 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.
  • putrescine transaminase 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 ⁇ -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 ⁇ -aminobutyraldehyde (fresh prepared and finally added).
  • GABA tolerance test
  • 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.
  • 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.
  • Ornithine decarboxylase catalyzes the conversion of ornithine to putrescine.
  • GABA synthesis 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.
  • 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).
  • 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.
  • the production of GABA in C. glutamicum was made possible by heterologous overexpression of the genes patDA of the transaminase pathway from E.
  • FIG. 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.
  • GABA1 AargRF pVWExl-speC-5 '2i-argF pEKEx3patDA
  • 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.
  • 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.
  • patD gamma-aminobutyraldehyde dehydrogenase
  • PatA putrescine: 2-oxoglutarate aminotransferase
  • 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).
  • 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 ⁇ -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.
  • GABA5 FPGA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA1 + GABA5 + GABA5 + GABA5 + GABA5 + GABA6
  • GABA5 GABA5 + GABA5 + GABA6
  • PUT21 ⁇ cgl722AgabTDP pEKEx3-patDA
  • 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).
  • 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.
  • the yield could also be increased to a value of 0.20 ⁇ g "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.
  • 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%
  • the present invention relates to a process for the preparation of terminal amino aldehydes from diamines using recombinant microorganisms. Synthesis of ⁇ -aminobutyraldehyde (ABAL)
  • the patA gene from E. coli which codes for a putrescine: 2-oxoglutarate aminotransferase
  • 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.
  • GABA gamma-aminobutyric acid
  • PknA, PknB, PknG and PknL of Corynebacterium glutamicum evidence for non-essentiality and phosphorylation of Odhl and FtsZ by multiple kinases. Genetic and biochemical analysis of the serine / threonine protein kinases. Molecular Microbiology, 74 (3), 724-41. doi: 10.1111 / j.l365- 2958.2009.06897.x Shi, F., Jiang, J., Li, Y., Li, Y., & Xie, Y. (2013).

Abstract

L'invention concerne un procédé de préparation d'acides aminocarboxyliques et d'aldéhydes terminaux à partir de diamines en utilisant un micro-organisme recombinant. L'invention concerne en particulier un procédé de préparation d'acide gamma-aminobutyrique (GABA) ou de gamma-aminobutyraldéhyde (ABAL) en utilisant un micro-organisme recombinant. L'invention concerne également l'utilisation de micro-organismes recombinants qui comprennent des molécules d'ADN sous une forme dérégulée, qui améliorent la préparation des acides aminocarboxyliques et/ou aminoaldéhydes terminaux, par exemple l'acide gamma-aminobutyrique ou le gamma-aminobutyraldéhyde ainsi que des molécules d'ADN recombinantes et des polypeptides qui sont utilisés pour la préparation des micro-organismes.
PCT/EP2015/054330 2014-03-03 2015-03-02 Procédé de préparation d'acides aminocarboxyliques et d'aminoaldéhydes terminaux au moyen d'un micro-organisme recombinant WO2015132213A1 (fr)

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CN111635454B (zh) * 2020-06-04 2021-12-28 江南大学 利用生物传感器筛选精氨酸高产菌株的方法

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