WO2021018170A1 - Préparation de micro-organisme transformé pour la production de pyridine carboxamide avec une faible teneur en sous-produits et son utilisation - Google Patents

Préparation de micro-organisme transformé pour la production de pyridine carboxamide avec une faible teneur en sous-produits et son utilisation Download PDF

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WO2021018170A1
WO2021018170A1 PCT/CN2020/105424 CN2020105424W WO2021018170A1 WO 2021018170 A1 WO2021018170 A1 WO 2021018170A1 CN 2020105424 W CN2020105424 W CN 2020105424W WO 2021018170 A1 WO2021018170 A1 WO 2021018170A1
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gene
seq
product
picolinamide
low
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董亢
袁晓路
方红新
居虎军
吴李瑞
夏斌
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安徽瑞邦生物科技有限公司
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01084—Nitrile hydratase (4.2.1.84)

Definitions

  • the invention relates to the technical field of microbial gene recombination, in particular to the preparation and application of a low by-product picolinamide transformed microorganism.
  • the nitrile hydratase-catalyzed microbial transformation method for the production of picolinamide from cyanopyridine has been widely used in industrial production.
  • it is usually due to the chassis cells or the microorganisms themselves.
  • the existence of genes that catalyze the conversion of picolinamide to carboxypyridine leads to the formation of by-product carboxypyridine in the biocatalysis process, and ultimately leads to excessively high carboxypyridine content in the finished product, which affects the use of picolinamide in food, medicine, and cosmetics.
  • the present invention mainly uses genetic knock-out or knock-in technology in molecular biology and microbiology to reduce or completely inactivate the key enzyme genes of microbial metabolism and reduce or eliminate microbial transformation through genetic modification at the genetic level.
  • the present invention provides the following technical solutions:
  • a preparation method of low by-product picolinamide transformed microorganisms is prepared by the following specific steps:
  • the knockout plasmid into the host microorganism, use homologous recombination or gene editing methods to mutate at least one enzyme activity-related gene in the target protein gene, and then knock out or inactivate the target gene or target protein, and finally make the target
  • the enzyme activity of the protein is reduced or completely lost;
  • the target gene or protein includes, but is not limited to, nicotinic acid phosphoribosyltransferase, pyridine amide deaminase, isochorisase, cysteine hydrolase, purine nucleic acid phosphatase, so
  • the main catalytic products of the target protein are 3-carboxypyridine and 4-carboxypyridine;
  • the nitrile hydratase gene is integrated into the genome as an episomal plasmid or gene knock-in through gene recombination, so that the microorganism can catalyze the conversion of cyanopyridine compounds such as 3-cyanopyridine and 4-cyanopyridine into 3-cyanopyridine.
  • cyanopyridine compounds such as 3-cyanopyridine and 4-cyanopyridine into 3-cyanopyridine.
  • carboxamide pyridine compounds such as picolinamide and 4-pyridine carboxamide can transform microorganisms into low by-product picolinamide.
  • the homologous recombination method is a single crossover or double crossover method
  • the gene editing method is Crispr/Cas9.
  • the host microorganisms include but are not limited to Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces marxianus, Rhodococcus, and Corynebacterium glutamicum.
  • the target gene or protein sequence includes but not limited to SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4, SEQ NO. 5, SEQ NO. 6, SEQ NO. 7, SEQ NO.8, SEQ NO.9, SEQ NO.10, SEQ NO.11, SEQ NO.12, SEQ NO.13, SEQ NO.14, SEQ NO.15, SEQ NO.16, SEQ NO.17, SEQ NO.18, SEQ NO.19, SEQ NO.20, SEQ NO.21, SEQ NO.22.
  • the target gene or protein further includes a gene or protein whose similarity to the target gene sequence or the primary sequence of the target protein is not less than 50%.
  • the gene mutations related to enzyme activity include, but are not limited to, gene point mutations, partial gene deletions, complete gene deletions, gene translocation from the original locus to a new locus, gene sequence inversion, and insertion at any position in the gene sequence Unrelated genes, inactivation or inactivation of upstream promoter sequences of genes, inactivation or inactivation of ribosome binding sites, etc.
  • the knocked-out or inactivated target gene sequence or target protein primary sequence can not be detected by methods such as Sanger sequencing, high-throughput genome sequencing, isopoint focusing-polyacrylamide gel, protein mass spectrometry, etc.
  • the sequence or protein sequence or the detected gene sequence or protein sequence does not have integrity.
  • a low-by-product picolinamide transformation microorganism is prepared by the above-mentioned preparation method of low-by-product picolinamide transformation microorganism.
  • the above-mentioned low by-product picolinamide conversion microorganisms are used to catalyze the reaction of cyanopyridine compounds to produce picolinamide.
  • the cyano group is converted into a formamide group after hydration, and it does not produce or only contains no more than 30ppm carboxyl-containing group A group of pyridine compounds; the parent structure of the cyanopyridine is a heterocyclic pyridine, and cyano addition occurs at the 3-position or the 4-position.
  • the method uses the low by-product picolinamide constructed in the present invention to transform microorganisms to catalyze the synthesis of picolinamide.
  • the synthesized products include 3-picolinamide and 4-
  • the deamination by-products of picolinamide, 3-carboxypyridine and 4-carboxypyridine have low content or no by-products.
  • the by-product 3-carboxypyridine, 3-pyridinecarboxamide and 4-pyridinecarboxamide with low content of 4-carboxypyridine were prepared in high yield.
  • the gene knockout plasmid obtained by recombinant construction was transferred into the host microorganism, and the nicotinic acid phosphoribosyltransferase, pyridine amide deaminase, and heterobranching of various microorganisms were mutated through double exchange, single exchange or Crispr/Cas9 method. Acidase, cysteine hydrolase, and purine nucleic acid phosphatase can effectively knock out or inhibit the expression and activity of the above-mentioned enzyme protein in the host microorganism, and reduce the production of carboxypyridine in the biocatalytic process.
  • a picolinamide-transformed microorganism in which the corresponding intracellular enzyme protein that catalyzes the conversion of picolinamide into carboxypyridine is missing or low, and the nitrile hydratase is highly expressed and has high activity.
  • FIG. 1 Schematic diagram of the principle of double crossover gene mutation.
  • FIG. 1 Schematic diagram of plasmid vector construction.
  • Figure 4 The target enzyme activity detection result of the ⁇ EKO-Bsi strain without transforming the nitrile hydratase gene plasmid;
  • Figure 8 The detection result of target enzyme activity of the ⁇ EKO-Epn strain without transforming the nitrile hydratase gene plasmid;
  • Figure 10 The target enzyme activity detection result of the ⁇ EKO-Epp strain without transforming the nitrile hydratase gene plasmid;
  • Figure 12 The target enzyme activity detection result of the ⁇ EKO-Scn strain without transforming the nitrile hydratase gene plasmid;
  • FIG. 17 to 26 agarose gel electrophoresis diagrams of PCR amplification products in Examples 1 to 10;
  • Figures 17 to 26 all use the same DNA marker, and the bands from top to bottom are 1500bp, 1000bp, 800bp, 600bp, 500bp, 400bp, 200bp:
  • Figure 27 The detection result of target enzyme activity of the ⁇ EKO-GBN strain without transforming the nitrile hydratase gene plasmid;
  • Figure 28 The detection result of the target enzyme activity of wild-type Corynebacterium glutamicum ATCC 13032.
  • the 3.5min peak in Figures 3-16 and Figures 27 and 28 is the by-product peak
  • the 5.5-6min peak is the main product peak.
  • Example 1 Preparation of low by-product picolinamide transformed microorganisms as follows:
  • Primer 1 TAAAAAGGATCATCGGATCCCAGCAACCGCATCAAGAGTAGT
  • Primer 2 CGCGCAGGAAATTCTTTTTTTCACCTCTTAAAATTTTTATACT
  • Primer 3 GGTGAAAAAAAGAATTTCCTGCGCGAACATAACG
  • Primer 4 GACCATGATTACGCCAAGCTTTTGGGTGCTTGAATACTAATCC
  • SEQ NO.11 is the protein primary sequence corresponding to SEQ NO.1.
  • the primer design must consider whether the change of amino acid residues in the protein primary sequence caused by gene mutation can lead to the loss or reduction of enzyme activity and avoid the generation of amino acid residues. Equivalent substitutions or changes in non-enzymatically related amino acid residues.
  • Primer 1 and Primer 2 respectively use Bacillus subtilis (subtilis subsp. subtilis 168) genome sequence as template to perform PCR amplification reaction.
  • PCR reaction conditions :
  • the target vector was cloned by Tiangen recombination kit to construct the final gene knockout vector pEKO-Bsi.
  • the amplified gene fragment is shown in Figure 1, and the final constructed vector is shown in Figure 2.
  • GM LB medium + 0.5M sorbitol
  • ETM 0.5M sorbitol, 0.5M mannitol, 10% glycerin, the balance is water;
  • RM LB medium + 0.5M sorbitol + 0.38M mannitol
  • Electrotransmitter gene-pulser produced by Bio-Rad
  • pEKO-Bsi After pEKO-Bsi is transformed into the bacteria, it recombines with the host target gene through the double exchange method. During the recombination process, point mutations, partial gene deletions, complete gene deletions, and gene transfer from the original locus to the new one are randomly introduced into the target gene. Locus, gene sequence inversion, insertion of unrelated genes at any position in the middle of the gene sequence, inactivation or inactivation of the upstream promoter sequence of the gene, inactivation or inactivation of the ribosome binding site, etc., which ultimately lead to the primary sequence of the target protein Change, which in turn causes the target protease activity to be lost or reduced.
  • the reaction system is 0.5mL, 50mM ammonium chloride-ammonia buffer (pH8.0), containing 100mM 3-cyanopyridine.
  • HPLC method adopts Agilent High Performance Liquid Chromatography (Agilent 1100, USA), the chromatographic column is Varian pursuit C18 reverse chromatographic column (4.6mm*250mm), the mobile phase is 15mM potassium phosphate buffer (pH2.8): acetonitrile 92 : 8(v/v), the flow rate is set to 0.5mL/min, the UV detector, the detection wavelength is 265mm.
  • the knockout strain ⁇ EKO-Bsi converts all 3-cyanopyridine to produce 3-pyridinecarboxamide.
  • the content of the by-product 3-carboxypyridine is less than 30ppm, while the control strain B.subtilis 168, the by-product 3-
  • the content of carboxypyridine exceeds 100 ppm.
  • the control strain B. subtilis 168 is only a positive clone for nitrile hydratase gene transformation.
  • Primer 1 CATGCCATATTCAAAACGATAAGATGG
  • Primer 2 TACATATTCACGAAAGCGTACGTCCACTCCTTAGA
  • Primer 4 ATAAGCACGCAAGAATTTTTAGCTCTCAAACAT
  • SEQ NO. 12 is the protein primary sequence corresponding to SEQ NO. 2.
  • Primer 1 and Primer 2 respectively use Bacillus cereus (ATCC 14579) genome sequence as a template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • GM MYP medium + 0.5M sorbitol
  • ETM 0.5M sorbitol, 0.5M mannitol, 10% glycerin, the balance is water;
  • Electrotransmitter gene-pulser produced by Bio-Rad
  • pEKO-Bci After pEKO-Bci is transformed into the bacteria, it recombines with the host target gene through a single exchange method. During the recombination process, point mutations, partial gene deletions, and complete gene deletions are randomly introduced into the target genes, and the genes are transferred from the original locus to the new one. Locus, gene sequence inversion, insertion of unrelated genes at any position in the middle of the gene sequence, inactivation or inactivation of the upstream promoter sequence of the gene, inactivation or inactivation of the ribosome binding site, etc., which ultimately lead to the primary sequence of the target protein Change, which in turn causes the target protease activity to be lost or reduced.
  • the knockout strain ⁇ EKO-Bci converts all 3-cyanopyridine to produce 3-pyridinecarboxamide.
  • the content of the by-product 3-carboxypyridine is less than 30 ppm, while the control strain Bacillus cereus ATCC 14579, the by-product 3- The content of carboxypyridine exceeds 100 ppm.
  • the control strain Bacillus cereus ATCC 14579 is only a positive clone for nitrile hydratase gene transformation.
  • Primer 1 AGAATTCCAGACTACACATTAATGCAGAAATGGGCGATTTCGCTG
  • Primer 2 GCTGACTTCGACATGGTGCGTATCGCAGATCGACGATACAATA
  • Primer 3 ATATCCGGCTACGTCGCTGCAGAATTTGGCGCGAA
  • SEQ NO. 13 is the protein primary sequence corresponding to SEQ NO. 3.
  • Primer 1 and Primer 2 respectively use Escherichia coli K-12 MG1655 genome sequence as template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • GM LB medium + 0.5M sorbitol
  • ETM 0.5M sorbitol, 0.5M mannitol, 10% glycerol; the balance is water.
  • RM LB medium + 0.5M sorbitol + 0.38M mannitol
  • Electrotransmitter gene-pulser produced by Bio-Rad
  • step 2 Transform the nitrile hydratase gene plasmid pET28-NHase into ⁇ EKO-Epn according to the method of step 2 (pET28-NHas e, kanamycin resistance, plasmid source reference, Efficient cloning and expression of a thermostable nitrile hydratase inEscherichia coli using an auto-induction fed-batch strategy);
  • the knockout strain ⁇ EKO-Epn transforms all 3-cyanopyridine to produce 3-pyridinecarboxamide.
  • the content of the by-product 3-carboxypyridine is less than 30ppm, while the control strain Escherichia coli K-12 MG1655 is a by-product
  • the content of 3-carboxypyridine exceeds 100 ppm.
  • the control strain Escherichia coli K-12 MG1655 is only a positive clone for nitrile hydratase gene transformation.
  • Primer 1 ATTTCGACACATTACTGTGCTTTTGCCGGGAGA
  • Primer 2 TAAAGATGTCAAAGTGCGAGACGTCATAGATTAACATA
  • Primer 4 AAACGACAGCGGAAGAGCGTCAAACGATCAGACCATTTTA
  • SEQ NO.14 is the protein primary sequence corresponding to SEQ NO.4.
  • Primer 1 and Primer 2 respectively use Bacillus subtilis (subtilis subsp. subtilis 168) genome sequence as a template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • GM LB medium + 0.5M sorbitol
  • ETM 0.5M sorbitol, 0.5M mannitol, 10% glycerol; the balance is water.
  • RM LB medium + 0.5M sorbitol + 0.38M mannitol
  • Electrotransmitter gene-pulser produced by Bio-Rad
  • the knockout strain ⁇ EKO-Epp converts all 4-cyanopyridine to produce 4-pyridinecarboxamide, the by-product 4-carboxypyridine content is less than 30ppm, while the control strain B.subtilis 168, the by-product 4- The content of carboxypyridine exceeds 100 ppm.
  • the control strain B. subtilis 168 is only a positive clone for nitrile hydratase gene transformation.
  • Embodiment 5 The rest are the same as Embodiment 3, the difference lies in:
  • Design mutation primers with the nicotinamidase/pyrazinamidase (E C:3.5.1.19) sequence SEQ NO.5 of Escherichia coli as the target gene:
  • Primer 1 ACATAAATTAACTCGCGCCCTGTTACTGGTCGATTTACAAAA
  • Primer 3 ATCATCCTATTTTGAAGTTTACCGTGCTGGACGCGTTACA
  • SEQ NO. 15 is the protein primary sequence corresponding to SEQ NO. 5.
  • Primer 1 and Primer 2 respectively use Escherichia coli genome sequence as template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • 4-cyanopyridine is used as a raw material to catalyze the reaction to generate 4-pyridinecarboxamide.
  • the knockout strain ⁇ EKO converted all 4-cyanopyridine to produce 4-pyridinecarboxamide, the by-product 4-carboxypyridine content was less than 30ppm, while the control strain Escherichia coli, the by-product 4-carboxypyridine content exceeded 100ppm.
  • the control strain Escherichia coli is only a positive clone for nitrile hydratase gene transformation.
  • Example 6 Preparation of low by-product picolinamide transformed microorganisms according to the following method:
  • Design mutation primers with the nicotinamidase/pyrazin amidase (EC:3.5.1.19) sequence SEQ NO.6 of Saccharomyces cerevisiae as the target gene:
  • Primer 1 ATTGTTGTTGATATGCAAAATGATTTTATTTCA
  • Primer 2 AAGCCACCCGACATATCCGCATTAGAA
  • SEQ NO.16 is the protein primary sequence corresponding to SEQ NO.6.
  • Primer 1 and Primer 2 respectively use Saccharomyces cerevisiae genome sequence as a template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • YEPD 1% yeast extract, 2% bacteriological peptone, 2% glucose
  • Electrotransmitter gene-pulser produced by Bio-Rad
  • step 1 Construct the pYES2-NHase expression vector (amplify the nitrile hydratase gene from pET28-NHase and clone it into the pYES2 vector) according to the method of step 2 to transform the nitrile hydratase gene plasmid into ⁇ EKO-Scn;
  • the knockout strain ⁇ EKO-Scn converts all 3-cyanopyridine to produce 3-pyridinecarboxamide, the by-product 3-carboxypyridine content is less than 30ppm, while the control strain Saccharomyces cerevisiae, the by-product 3-carboxypyridine The content exceeds 100ppm.
  • the control strain Saccharomyces cerevisiae is only a positive clone for nitrile hydratase gene transformation.
  • Example 7 Preparation of low by-product picolinamide transformed microorganisms according to the following method:
  • Primer 1 AAAATTCATTAGCCGTGCAAGATGGGGAT
  • Primer 3 ACCTTGTGTTATGGACACTGCTATAA
  • SEQ NO.17 is the protein primary sequence corresponding to SEQ NO.7.
  • Primer 1 and Primer 2 use Kluyveromyces marxianus genomic sequence as a template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • YEPD 1% yeast extract, 2% bacteriological peptone, 2% glucose
  • Electrotransmitter gene-pulser produced by Bio-Rad
  • the knockout strain ⁇ EKO-Kmm converts all 4-cyanopyridine to produce 4-pyridinecarboxamide, the by-product 4-carboxypyridine content is less than 30ppm, while the control strain Kluyveromyces marxianus, the by-product 4-carboxypyridine The content exceeds 100ppm.
  • the control strain Kluyveromyces marxianus is only a positive clone for nitrile hydratase gene transformation.
  • Embodiment 8 the rest are the same as embodiment 3, the difference lies in:
  • Primer 1 AGTCGCTAACCGCCTTATTGACTGGTACCAGTC
  • Primer 3 AATTCAGGCGAGCATCGCAGTAGTT
  • SEQ NO.18 is the protein primary sequence corresponding to SEQ NO.8.
  • Primer 1 and Primer 2 use the EHEC genome sequence as a template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • 4-cyanopyridine is used as a raw material to catalyze the reaction to generate 4-pyridinecarboxamide.
  • the knockout strain ⁇ EKO-Enp converts all 4-cyanopyridine to produce 4-pyridinecarboxamide, the by-product 4-carboxypyridine content is less than 30ppm, while the control strain EHEC, the by-product 4-carboxypyridine content exceeds 100ppm .
  • the control strain EHEC is only a positive clone for nitrile hydratase gene transformation.
  • Embodiment 9 The rest are the same as Embodiment 3, the difference is:
  • Primer 2 CTGATGGATTTTGGCACCCGTCGCCGCGATCAA
  • Primer 3 AATTTCAGCATTGTCGAGTTCGCTAGTCGGTA
  • SEQ NO.19 is the protein primary sequence corresponding to SEQ NO.9.
  • Primer 1 and Primer 2 respectively use Escherichia coli K-12 MG1655 genome sequence as template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • 3-cyanopyridine is used as a raw material to catalyze the reaction to generate 3-pyridinecarboxamide.
  • the knockout strain ⁇ EKO-Eno converts all 3-cyanopyridine to produce 3-pyridinecarboxamide.
  • the content of the by-product 3-carboxypyridine is less than 30ppm, while the control strain Escherichia coli K-12 MG1655, the by-product 3- The content of carboxypyridine exceeds 100 ppm.
  • the control strain Escherichia coli K-12 MG1655 is only a positive clone for nitrile hydratase gene transformation.
  • Embodiment 10 The rest are the same as Embodiment 1, except that:
  • Primer 1 ATCAGCAAGAATATTTGCGCGGCTTATCTTTT
  • SEQ NO.20 is the protein primary sequence corresponding to SEQ NO.10.
  • Primer 1 and Primer 2 respectively use the Bacillus subtilis subsp.subtilis 168 genome sequence as a template to perform PCR amplification reaction.
  • the PCR reaction conditions are as follows:
  • 4-cyanopyridine is used as a raw material to catalyze the reaction to generate 4-pyridinecarboxamide.
  • the knockout strain ⁇ EKO-Bpp transforms all 4-cyanopyridine into 4-pyridinecarboxamide.
  • the content of 4-carboxypyridine as a by-product is less than 30ppm, while the control strain Bacillus subtilis subsp.subtilis 168, vice The content of the product 4-carboxypyridine exceeds 100 ppm.
  • the control strain Bacillus subtilis subsp.subtilis 168 is only a positive clone for nitrile hydratase gene transformation.
  • Example 11 Preparation of low by-product picolinamide to transform microorganisms as follows:
  • SEQ NO. 22 is the protein primary sequence corresponding to SEQ NO. 21.
  • step two Transform the nitrile hydratase gene plasmid pET28-NHase into ⁇ EKO-GBN according to the method of step two (pET28-NHase, kanamycin resistance, plasmid source reference, Efficient cloning and expression of a thermos table nitrile hydratase inEscherichia coli using an auto-induction fed-batch strategy y);
  • the knockout strain ⁇ EKO-GBN converts all 3-cyanopyridine to produce 3-pyridinecarboxamide, the content of the by-product 3-carboxypyridine is less than 30 ppm, while the control strain Corynebacterium glutamicum, by-product 3 -The content of carboxypyridine exceeds 100 ppm.
  • the control strain Corynebacterium glutamicum is only a positive clone for nitrile hydratase gene transformation.
  • the above-mentioned microorganisms have reduced or inactivated enzyme catalytic activity that catalyzes the deamination of 3-pyridinecarboxamide and 4-pyridinecarboxamide to produce 3-carboxypyridine and 4-carboxypyridine-related genes.
  • 3-cyanopyridine and 4-cyanopyridine After adding 3-cyanopyridine and 4-cyanopyridine in batches or continuous streams, it is converted into the final product 3-pyridinecarboxamide with a content of not less than 20%, and 4-pyridinecarboxamide with a content of not less than 10%.
  • the method uses the low by-product picolinamide constructed in the present invention to transform microorganisms to catalyze the synthesis of picolinamide, and the synthesized product contains 3-picolinamide,
  • the deamination by-products of 4-pyridinecarboxamide, 3-carboxypyridine and 4-carboxypyridine have low content or no by-products.
  • the by-product 3-carboxypyridine, 3-pyridinecarboxamide and 4-pyridinecarboxamide with low content of 4-carboxypyridine were prepared in high yield.
  • the gene knockout plasmid obtained by recombination was transferred into the host microorganism, and the nicotinic acid phosphoribosyl transferase and pyridine amide depletion were mutated through homologous recombination or gene editing (one of double exchange, single exchange, Crispr/Cas9).
  • the enzyme activity-related genes of ammoniaase, isochorisase, cysteine hydrolase, and purine nucleic acid phosphatase effectively knock out or inhibit the expression and activity of the above-mentioned enzyme proteins in the host microorganism, and reduce the by-products generated in the biocatalytic process The formation of carboxypyridine.
  • a picolinamide-transformed microorganism in which the corresponding intracellular enzyme protein that catalyzes the conversion of picolinamide to carboxypyridine is missing or low, and the nitrile hydratase is highly expressed and has high activity.

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Abstract

L'invention concerne la préparation d'un micro-organisme transformé pour la production de pyridine carboxamide avec un faible sous-produit et son utilisation, la préparation comprenant les étapes suivantes: construction d'un plasmide avec inactivation génique ; transformation d'un micro-organisme hôte avec le plasmide avec inactivation génique ; transformation d'un gène de nitrile hydratase, etc. La préparation peut efficacement inactiver ou inhiber la nicotinate phosphoribosyltransférase, picolinamide désaminase, isochorismatase, la cystéine hydrolase et la phosphatase d'acide nucléique de purine dans divers hôtes microbiens, réduire l'activité de la zymoprotéine mentionnée ci-dessus ou l'inactiver directement, et réduire la génération d'un sous-produit, la carboxyle pyridine, lors de la biocatalyse, de sorte que les produits de synthèse, 3-pyridine carboxamide et 4-pyridine carboxamide, ont une faible teneur en sous-produits de désamination, la 3-carboxyle pyridine et la 4-carboxyle pyridine, ou ne contiennent pas les sous-produits. Les micro-organismes recombinés peuvent être utilisés pour préparer catalytiquement des produits de 3-pyridine carboxamide et de 4-pyridine carboxamide avec une faible teneur en sous-produits et un rendement élevé.
PCT/CN2020/105424 2019-08-01 2020-07-29 Préparation de micro-organisme transformé pour la production de pyridine carboxamide avec une faible teneur en sous-produits et son utilisation WO2021018170A1 (fr)

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