WO2020057308A1 - Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用 - Google Patents

Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用 Download PDF

Info

Publication number
WO2020057308A1
WO2020057308A1 PCT/CN2019/101521 CN2019101521W WO2020057308A1 WO 2020057308 A1 WO2020057308 A1 WO 2020057308A1 CN 2019101521 W CN2019101521 W CN 2019101521W WO 2020057308 A1 WO2020057308 A1 WO 2020057308A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
dehydrogenase
nadh
dependent
lysine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2019/101521
Other languages
English (en)
French (fr)
Inventor
陈振
刘德华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Original Assignee
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University filed Critical Tsinghua University
Priority to US17/277,661 priority Critical patent/US20220112471A1/en
Publication of WO2020057308A1 publication Critical patent/WO2020057308A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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)
    • C12N9/0016—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 NAD or NADP as acceptor (1.4.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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.)
    • C12N9/0008—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.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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)
    • 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.)
    • C12N9/0093—Oxidoreductases (1.) acting on CH or CH2 groups (1.17)
    • 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
    • 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
    • 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
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01011—Aspartate-semialdehyde dehydrogenase (1.2.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01026—Dihydrodipicolinate reductase (1.3.1.26)
    • 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)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01016—Diaminopimelate dehydrogenase (1.4.1.16)
    • 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)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01021—Aspartate dehydrogenase (1.4.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y117/00—Oxidoreductases acting on CH or CH2 groups (1.17)
    • C12Y117/01—Oxidoreductases acting on CH or CH2 groups (1.17) with NAD+ or NADP+ as acceptor (1.17.1)

Definitions

  • the invention belongs to the technical fields of genetic engineering and biological fermentation, in particular to a NADH-dependent amino acid dehydrogenase and application thereof.
  • Lysine is an extremely important amino acid and is widely used in the fields of feed additives, health care and medicine. At present, the industrial production of lysine is mainly carried out by microbial fermentation.
  • the commonly used strains in the industry include Corynebacterium glutamicum and E. coli.
  • the traditional methods are to strengthen the supply of NADPH, such as increasing the metabolic flux of the pentose phosphate cycle pathway, or expressing NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase for balancing lysine synthesis.
  • Coenzyme demand which increases lysine production.
  • the object of the present invention is to provide a NADH-dependent amino acid dehydrogenase and its application.
  • coli detects its catalytic activity, and detects the specificity of the coenzymes NADH and NADPH and finds that it is derived from pseudomonas aeruginos' aspartate dehydrogenase (its amino acid sequence is shown in SEQ ID NO.1), aspartic acid semialdehyde dehydrogenase from Tistrella mobilis (its amino acid sequence is shown in SEQ ID NO.3) , A dihydropyridine dicarboxylic acid reductase derived from Mycobacterium tuberculosis (the amino acid sequence of which is shown in SEQ ID NO.
  • a diaminopimelate dehydrogenase derived from Tepidanaerobacter acetatoxydans (the amino acid sequence of which is shown in SEQ ID ID NO. (Shown in 7) NADH can be used to catalyze the above four-step reaction.
  • NADH-dependent amino acid dehydrogenase which is:
  • amino acid sequence is shown as SEQ ID No. 1, 3, 5, 7 or as shown in SEQ ID No. 1, 3, 5, 7 respectively, the amino acid sequence is substituted, deleted and / or added one or several amino acids without Amino acid sequence that affects its biological activity.
  • the present invention provides a gene encoding the above-mentioned NADH-dependent amino acid dehydrogenase, the nucleotide sequence of which contains the nucleotide sequence shown in SEQ ID No. 2, 4, 6, 8 or The nucleotide sequence shown in SEQ ID No. 2, 4, 6, 8 is substituted, deleted, and / or added with one or a few bases with a core encoding more than 90% homology and encoding the same functional amino acid dehydrogenase. Nucleotide sequence.
  • the present invention provides a biological material containing the above gene, and the biological material is a vector, a recombinant bacterium, a cell line, or an expression cassette.
  • the recombinant bacterium is a strain capable of fermenting and producing lysine or pentanediamine and contains or overexpresses the NADH-dependent amino acid dehydrogenase according to the present invention.
  • the strain capable of fermenting and producing lysine or pentanediamine is Corynebacterium glutamicum or E. coli.
  • the recombinant strain is a strain that can fermentively produce lysine or pentanediamine and contains or overexpresses aspartate dehydrogenase derived from Pseudomonas aeruginos and aspartate semialdehyde derived from Tistrella mobilis Dehydrogenase; or
  • the recombinant bacterium is a NADPH-dependent amino acid dehydrogenase in a strain that can fermentively produce lysine or pentanediamine by a NADH-dependent amino acid dehydrogenase according to the present invention.
  • the recombinant strain AM3 is a C. glutamicum ATCC21543 endogenous aspartate aminotransferase and is replaced with a NADH-dependent aspartate dehydrogenase (amino acid sequence SEQ ID NO. 1) Endogenous NADPH-dependent aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase (amino acid sequence SEQ ID NO. 3), and endogenous NADPH-dependent Type dihydropyridine dicarboxylic acid reductase was replaced with NADH-dependent dihydropyridine dicarboxylic acid reductase (amino acid sequence SEQ ID NO. 5), and the recombinant fermentation fermentation medium can significantly improve the lysine Yield reached 27.7g / L.
  • the recombinant bacteria AM2 and AM4 in the examples of the present invention also achieved excellent effects of increasing lysine production, reaching 25.8 g / L and 25.6 g / L, respectively.
  • the recombinant AM2 is a C. glutamicum ATCC21543 endogenous aspartate aminotransferase replaced with NADH-dependent aspartate dehydrogenase (amino acid sequence SEQ ID NO. 1), endogenous NADPH-dependent Type aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase (amino acid sequence SEQ ID NO. 3).
  • the recombinant AM4 is a C. glutamicum ATCC21543 endogenous aspartate aminotransferase replaced with a NADH-dependent aspartate dehydrogenase (amino acid sequence SEQ ID NO. 1), an endogenous NADPH-dependent Type aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase (amino acid sequence SEQ ID NO. 3), endogenous NADPH-dependent dihydropyridine dicarboxylic acid Reductase was replaced with NADH-dependent dihydropyridine dicarboxylic acid reductase (amino acid sequence SEQ ID NO. 5), and endogenous NADPH-dependent diaminopimelate dehydrogenase was replaced with NADH- Dependent diaminopimelate dehydrogenase.
  • the fermentation medium (g / L) is: glucose 80g, corn slurry 10g, urea 4.5g, ammonium sulfate 45g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, ferrous sulfate heptahydrate 10mg, tetrahydrate Manganese sulfate 10mg, ⁇ -alanine 5mg, niacin 5mg, thiamine-hydrochloric acid 5mg, biotin 0.3mg, calcium carbonate 30g, threonine 0.2g, leucine 0.2g, add water to 1L.
  • the present invention provides the use of the above NADH-dependent amino acid dehydrogenase or its encoding gene or the above-mentioned biological material containing the encoding gene in the preparation of lysine or pentanediamine.
  • the present invention provides an application of the above-mentioned NADH-dependent amino acid dehydrogenase or its encoding gene or the above-mentioned biological material containing the encoding gene in increasing lysine production or increasing pentanediamine production.
  • the invention provides the application of the NADH-dependent amino acid dehydrogenase or its encoding gene or the above-mentioned biological material containing the encoding gene in the preparation of feed additives.
  • the invention provides the application of the above NADH-dependent amino acid dehydrogenase or its encoding gene or the above-mentioned biological material containing the encoding gene in the preparation of a medicament.
  • the NADH-dependent amino acid dehydrogenase provided by the present invention can be used to replace the NADPH-dependent amino acid dehydrogenase of Corynebacterium glutamicum itself.
  • the special amino acid dehydrogenase screened by the present invention can be directly used NADH as a cofactor or using both NADH and NADPH to directly express such NADH-dependent amino acid dehydrogenases in lysine or pentanediamine producing strains or to replace the corresponding NADPH with NADH-dependent amino acid dehydrogenases -Dependent amino acid dehydrogenase can enable cells to use abundant intracellular NADH as a cofactor to synthesize lysine, thereby reducing the need for NADPH, and then significantly increasing the production of lysine or pentanediamine.
  • the present invention expresses genes of amino acid dehydrogenase from different sources in E. coli, detects its catalytic activity, and detects the specificity of the coenzymes NADH and NADPH and finds that it is derived from pseudomonas aeruginos' aspartate dehydrogenase (its amino acid sequence is shown in SEQ ID NO.1), aspartic acid semialdehyde dehydrogenase from Tistrella mobilis (its amino acid sequence is shown in SEQ ID ID NO.3) , A dihydropyridine dicarboxylic acid reductase derived from Mycobacterium tuberculosis (the amino acid sequence of which is shown in SEQ ID NO.
  • a diaminopimelate dehydrogenase derived from Tepidanaerobacter acetatoxydans (the amino acid sequence of which is shown in SEQ ID ID NO. (Shown in 7) can catalyze the above four-step reaction.
  • the present invention designs and gene synthesizes the corresponding codon-optimized gene sequence, as shown in the sequences SEQ ID No. 2, 4, 6, and 8.
  • the synthesized gene fragment was directly inserted into the EcoRI and SalI double-digestion sites of pET-28a, and the obtained plasmids were named pET-adh, pET-asd, pET-dapB, and pET-ddh, respectively.
  • the above plasmid was transferred into E.
  • coli BL21 (DE3) by chemical transformation method, and the recombinant bacteria were selected and screened on LB plate containing 50 mg / L kanamycin and named as BL21 / pET-adh, BL21 / pET-asd, respectively. , BL21 / pET-dapB, BL21 / pET-ddh.
  • the above BL21 strain was cultured in an LB liquid medium containing 50 mg / L kanamycin until the OD600 reached 0.6 (37 ° C, 150 rpm), 0.1 mM IPTG was added, and the culture was continued for 12 h to induce protein expression (20 ° C, 150 rpm) ).
  • the bacterial cells were centrifuged and washed twice with 100 ml of 100 mM PBS buffer (pH 7.0). Finally, the bacterial cells were suspended in 5 ml of 100 mM PBS buffer (pH 7.0). The resuspension was disrupted using ultrasound and centrifuged to obtain a supernatant (12000 rpm, 30 minutes).
  • the protein purification kit HisTrap (GE) was used to isolate and purify the enzyme for enzyme activity detection.
  • Aspartate dehydrogenase activity detection system includes: 100 mM Tris-HCl buffer (pH 8.2), 0.2 mM coenzyme NADH or NADPH, 4 mM oxaloacetate, 100 mM ammonium chloride, and an appropriate amount of enzyme .
  • the reaction was performed at 37 ° C, and the change in absorbance at 340 nm was measured.
  • Enzyme activity is defined as the amount of enzyme (U) required to consume 1 ⁇ M of NAD (P) H per minute. The experimental results are shown in Table 1.
  • Aspartate semialdehyde dehydrogenase activity detection system includes: 200 mM CHES buffer (pH 9.0), 50 mM KPi, 0.5 mM coenzyme NAD or NADP, 2 mM aspartate semialdehyde, and an appropriate amount of Enzyme. The reaction was carried out at 25 ° C, and the change in absorbance at 340 nm was measured. Enzyme activity is defined as the amount of enzyme (U) required to produce 1 ⁇ M of NAD (P) H per minute. The experimental results are shown in Table 1.
  • Dihydropyridine dicarboxylic acid reductase activity detection system includes: 100 mM HEPES buffer (pH 7.5), 0.2 mM coenzyme NADH or NADPH, 4 mM oxaloacetate, 1 mM pyruvate, 0.1 mM aspartic acid Acid semialdehyde, 25 ug / ml dihydropyridine dicarboxylic acid synthase, and appropriate amount of enzyme. The reaction was carried out at 25 ° C, and the change in absorbance at 340 nm was measured. Enzyme activity is defined as the amount of enzyme (U) required to consume 1 ⁇ M of NAD (P) H per minute. The experimental results are shown in Table 1.
  • the diaminopimelate dehydrogenase activity detection system includes: 100 mM glycine-KOH buffer (pH 10.0), 0.5 mM coenzyme NAD or NADP, 5 mM diaminopimelate, and an appropriate amount of enzyme. The reaction was performed at 30 ° C, and the change in absorbance at 340 nm was measured. Enzyme activity is defined as the amount of enzyme (U) required to produce 1 ⁇ M of NAD (P) H per minute. The experimental results are shown in Table 1.
  • This example expresses the NADH-dependent amino acid dehydrogenase selected in the present invention in a strain of Corynebacterium glutamicum LC298 (Applied and environmental microbiology, 2011, 02912-10), and also expresses Corynebacterium glutamicum The corresponding NADPH-dependent amino acid dehydrogenase was used as a control, and the effects of different coenzyme-specific enzymes on the promotion of lysine synthesis were compared.
  • pET-adh As a template to clone a NADH-dependent aspartate dehydrogenase gene fragment using acagctatgacatgattacgaaggagatatacatatgctgaacattgtgatgatcgg and tgcatgcctgcaggtcgactttagattgaatatcatcatgggca as primers, the fragment was purchased from Gibson Assembly Kit-EC2, and the fragment was added to the plasmid K2. Within the EcoRI and XbaI sites, the obtained plasmid was named pEC-adh_pa.
  • the pEC-adh_pa was electrotransformed into Corynebacterium glutamicum LC298 (the electroconversion conditions were a voltage of 2.5KV, 200 ⁇ , and a 2mm electrorotation cup), and the recombinant strains were obtained by screening on LB plates containing 50 mg / L kanamycin, which were named LC / pEC-adh_pa.
  • Corynebacterium glutamicum LC298 was used as a template, and acagctatgacatgattacgaaggagatatacatatgagttcagtttcgctgcagga and tgcatgcctgcaggtcgactttagttagcgtaatgctccgctgc were used as the primers to clone the aspartate aminotransferase gene fragment of Corynebacterium glutamicum, and this fragment was assembled with the Gibson plasmid kit 2 using the Gibson plasmid kit.
  • the resulting plasmid was named pEC-aspC_cg.
  • the pEC-aspC_cg was electrotransformed into Corynebacterium glutamicum LC298 (the electroporation conditions were a voltage of 2.5KV, 200 ⁇ , and a 2mm electrorotation cup), and the recombinant bacteria were screened on a LB plate containing 50 mg / L kanamycin, which were named LC / pEC-aspC_cg.
  • pEC-asd_tm Using pET-asd as a template and acagctatgacatgattacgaaggagatatacatatgcgtatcgggattgttgga and tgcatgcctgcaggtcgactttacaccagtaactctgcgatttgc as primers, buy NADH-dependent aspartate semialdehyde dehydrogenase gene fragment, and use the GibAdd assembly kit 2 from Gibson Plasmid Kit (GibAdd Add Kit 2) In EcoRI and XbaI sites, the plasmid obtained was named pEC-asd_tm.
  • the pEC-asd_tm was electrotransformed into Corynebacterium glutamicum LC298 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the kanamycin LB plates containing 50mg / L were screened to obtain recombinant bacteria, which were named LC. / pEC-asd_tm.
  • Corynebacterium glutamicum LC298 was used as a template, acagctatgacatgattacgaaggagatatacatatgaccaccatcgcagttgtt and tgcatgcctgcaggtcgactttacttaaccagcagctcag were used as primers to clone the aspartate semialdehyde dehydrogenase gene fragment of Corynebacterium glutamicum, and the fragment was ligated to the plasmid with Gibson assembly kit pC Within the EcoRI and XbaI sites of K18mob2 (purchased from Addgene), the resulting plasmid was named pEC-asd_cg.
  • the pEC-asd_cg was electrotransformed into Corynebacterium glutamicum LC298 (the electroconversion conditions were a voltage of 2.5KV, 200 ⁇ , and a 2mm electrorotation cup), and a kanamycin LB plate containing 50mg / L was selected to obtain recombinant bacteria, which were named LC. / pEC-asd_cg.
  • pEC-dapB_mt Using pET-dapB as a template and acagctatgacatgattacgaaggagatatacat and atgcgggtaggcgtccttgg and tgcatgcctgcaggtcgactttacaaatttcagtgcagatcgagtagggg as primers, we cloned the NADH-dependent dihydropyridine dicarboxylic acid reductase gene fragment (using the EC-GibK18 reagent from the Add-GibK18 kit to assemble the fragment from Add-GibK18 In EcoRI and XbaI sites), the obtained plasmid was named pEC-dapB_mt.
  • the pEC-dapB_mt was electrotransformed into Corynebacterium glutamicum LC298 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the recombinant bacteria were selected by screening on LB plates containing 50mg / L kanamycin, which were named LC / pEC-dapB_mt.
  • Corynebacterium glutamicum LC298 was used as a template, and acagctatgacatgattacgaaggagatatacatatgggaatcaaggttggcgt and tgcatgcctgcaggtcgactttacaggcctaggtaatgctca were used as primers to clone the dihydropyridine dicarboxylic acid reductase gene fragment of Corynebacterium glutamicum, and this fragment was ligated to the plasmid Kb18p2b with Gibson assembly kit.
  • the obtained plasmid was named pEC-pEC-dapB_cg.
  • the pEC-dapB_cg was electrotransformed into Corynebacterium glutamicum LC298 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the recombinant bacteria were selected by screening on LB plates containing 50 mg / L kanamycin, which were named LC / pEC-dapB_cg.
  • NADH-dependent diaminopimelate dehydrogenase gene fragment was purchased, and the Gibson plasmid was assembled from the GibAdd 2 kit using Gibson 2 Within the EcoRI and XbaI sites, the obtained plasmid was named pEC-ddh_ta.
  • the pEC-ddh_ta was electrotransformed into Corynebacterium glutamicum LC298 (the electroconversion conditions were voltage 2.5KV, 200 ⁇ , 2mm electrorotation cup), and the recombinant bacteria were selected by screening on LB plates containing 50 mg / L kanamycin, which were named LC / pEC-ddh_ta.
  • Corynebacterium glutamicum LC298 was used as a template, and acagctatgacatgattacgaaggagatatacatatgaccaacatccgcgtagcta and tgcatgcctgcaggtcgactttagacgtcgcgtgcgatca were used as primers to clone the diaminopimelate dehydrogenase gene fragment of Corynebacterium glutamicum, and this fragment was ligated with the Gibson plasmid assembly kit 2 using the Gibsonb Kit EC2. Within the EcoRI and XbaI sites (purchased from Addgene), the obtained plasmid was named pEC-ddh_cg.
  • the pEC-ddh_cg was electrotransformed into Corynebacterium glutamicum LC298 (the electroconversion conditions were voltage 2.5KV, 200 ⁇ , 2mm electrorotation cup), and the recombinant bacteria were selected by screening on LB plates containing 50 mg / L kanamycin, which were named LC / pEC-ddh_cg.
  • the empty plasmid pEC-K18mob2 was electrotransformed into Corynebacterium glutamicum LC298 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm rotor), and the recombinant bacteria were screened on LB plates containing 50 mg / L kanamycin and named separately.
  • LC / pEC-K18 was used as a control strain.
  • the derived strains of Corynebacterium glutamicum LC298 obtained above were each cultured in a fermentation medium for 72 h (30 ° C, 200 rpm), and the yield of lysine was detected.
  • the components of the fermentation medium are (g / L): glucose 80g, corn slurry 10g, urea 4.5g, ammonium sulfate 45g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, ferrous sulfate heptahydrate 10mg, tetrahydrate Manganese sulfate 10 mg, ⁇ -alanine 5 mg, niacin 5 mg, thiamine-hydrochloric acid 5 mg, biotin 0.3 mg, calcium carbonate 30 g, and kanamycin 25 mg.
  • control strain LC / pEC-K18 lysine yield was 14.01 g / L, and the fermentation results of other strains are shown in Table 2, which indicates that overexpression of NADH-dependent amino acid dehydrogenase can significantly increase lysine yield , And the effect is better than the NADPH-dependent amino acid dehydrogenase derived from Corynebacterium glutamicum itself.
  • NADH-dependent amino acid dehydrogenase can also be used to directly replace the corresponding NADPH-dependent amino acid dehydrogenase in Corynebacterium glutamicum. Catalase to increase lysine production.
  • This example uses a method of homologous recombination in a Corynebacterium glutamicum ATCC21543 to replace the cell's own NADPH-dependent amino acid dehydrogenase one by one with the expression of the NADH-dependent amino acid dehydrogenase screened by the present invention. To investigate its effect on promoting lysine synthesis.
  • NADH-dependent aspartate dehydrogenase gene fragment adh was cloned using pET-adh as the template and gtacgcagttatgctgaacattgtgatgatcggatg and gctgtattcacttttagattgaaatggcatgggcatgattt as the primers, and the genome of ATCC 21543 as the template was acagctatgacatgtggggtcggtcggtcggtcggtcggtcggtcggtcggcggcggcggcggct Up, using the ATCC 21543 genome as a template to clone attcaatctaaaagtgaatacagcggagacagc and tgcatgcctgcaggtcgactctttcaacgattttcagcaaggc as primers, clone the gene fragment adh-down, and use the Gibson assembly kit to connect
  • PK18-adh was electrotransformed into Corynebacterium glutamicum ATCC 21543 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electrorotation cup), and the recombinant bacteria were obtained through two screenings.
  • Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin.
  • the recombinant bacteria was further cultured overnight in liquid LB medium, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strain was named AM1: aspC.
  • the main characteristic of this strain was endogenous Asparagus
  • the amino acid transaminase was replaced with a NADH-dependent aspartate dehydrogenase.
  • PK18-asd was electrotransformed into Corynebacterium glutamicum ATCC 21543 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the recombinant bacteria were obtained through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin. The recombinant bacteria was further cultured overnight in liquid LB medium, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strain was named AM1: asd. The main characteristic of this strain was endogenous NADPH- Dependent aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase.
  • PK18-dapB was electrotransformed into Corynebacterium glutamicum ATCC 21543 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the recombinant bacteria were obtained through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin. The recombinant bacteria was further cultured in liquid LB medium overnight, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strain was named AM1: dapB. The main characteristic of this strain was endogenous NADPH- The dependent dihydropyridine dicarboxylic acid reductase was replaced with a NADH-dependent dihydropyridine dicarboxylic acid reductase.
  • PK18-ddh was electrotransformed into Corynebacterium glutamicum ATCC 21543 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electrorotation cup), and the recombinant bacteria were obtained through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin. The recombinant strain was further cultured overnight in liquid LB medium, and then subjected to secondary screening on LB plates containing 100 g / L of sucrose, and the correct recombinant strain was named AM1: ddh. The main characteristic of this strain was endogenous NADPH- The diaminopimelate dehydrogenase dependent type was replaced with a NADH-dependent diaminopimelate dehydrogenase.
  • PK18-asd was electrotransformed into Corynebacterium glutamicum AM1: aspC (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electrorotation cup), and the recombinant bacteria were obtained through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin. The recombinant bacteria was further cultured in liquid LB medium overnight, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strain was named AM2.
  • the main characteristic of this strain was endogenous aspartic acid Transaminase was replaced with NADH-dependent aspartate dehydrogenase, and endogenous NADPH-dependent aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase .
  • PK18-dapB was electrotransformed into Corynebacterium glutamicum AM2 (the conditions of electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the recombinant bacteria were obtained through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin. The recombinant bacteria was further cultured in liquid LB medium overnight, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strain was named AM3.
  • the main characteristic of this strain was endogenous aspartic acid Transaminase was replaced with NADH-dependent aspartate dehydrogenase, and endogenous NADPH-dependent aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase , And the endogenous NADPH-dependent dihydropyridine dicarboxylic acid reductase was replaced with a NADH-dependent dihydropyridine dicarboxylic acid reductase.
  • PK18-ddh was electrotransformed into Corynebacterium glutamicum AM3 (the conditions for electroporation were 2.5KV, 200 ⁇ , 2mm electroporation cup), and the recombinant bacteria were obtained through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin. The recombinant bacteria was further cultured in liquid LB medium overnight, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strain was named AM4.
  • the main characteristic of this strain was endogenous aspartic acid Transaminase was replaced with NADH-dependent aspartate dehydrogenase, and endogenous NADPH-dependent aspartate semialdehyde transaminase was replaced with NADH-dependent aspartate semialdehyde dehydrogenase And the endogenous NADPH-dependent dihydropyridine dicarboxylic acid reductase was replaced with a NADH-dependent dihydropyridine dicarboxylic acid reductase, and the endogenous NADPH-dependent diaminopimelate was removed. Catalase was replaced with NADH-dependent diaminopimelate dehydrogenase.
  • the derived strains of Corynebacterium glutamicum ATCC 21543 obtained above were cultured in a fermentation medium for 72 h (30 ° C, 200 rpm), respectively, and the yield of lysine was detected.
  • the components of the fermentation medium are (g / L): glucose 80g, corn slurry 10g, urea 4.5g, ammonium sulfate 45g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, ferrous sulfate heptahydrate 10mg, tetrahydrate Manganese sulfate 10 mg, ⁇ -alanine 5 mg, niacin 5 mg, thiamine-hydrochloric acid 5 mg, biotin 0.3 mg, calcium carbonate 30 g, threonine 0.2 g, and leucine 0.2 g.
  • lysine decarboxylase in the above-mentioned NADH-dependent strain can achieve the direct biosynthesis of pentanediamine.
  • this example uses the method of homologous recombination to replace the lysE lysE efflux gene with E. coli-derived glutamic acid decarboxylase gene cadA to realize the conversion from glucose to pentyl.
  • E. coli-derived glutamic acid decarboxylase gene cadA to realize the conversion from glucose to pentyl.
  • the effects of NADH-dependent amino acid dehydrogenases on the promotion of pentanediamine synthesis were investigated.
  • E. coli MG1655 as a template, respectively TTCGTGGTGTTGCCCGTGGCCCGGTTGGTTGGGCAGGAGTATATTGGGATCCatgAACGTTATTGCAATATTGAATC and catcaacatcagttaTTTTTTGCTTTCTTCTTTCAATAC primers cloned lysine decarboxylase gene fragment cadA, genomic ATCC 21543 as a template, respectively acagctatgacatgattacgcgggcgaagaagtgaaaacc and GCCACGGGCAACACCACGAATGCGCTACCTTAACCGAAAAGTTACTTTcgtgacctatggaagtacttaa primers cloned gene fragments cadA-up, genome ATCC 21543 for The template used GCAAAAAAtaactgatgttgatgggttagttttttcgc and tgcatgcctgcaggtcgactttcaacg
  • PK18-cadA was electrotransformed into Corynebacterium glutamicum ATCC 21543, AM1: aspC, AM1: asd, AM1: dapB, AM1: ddh, AM2, AM3, AM4 (the conditions of electroporation are 2.5KV, 200 ⁇ , 2mm electroporation Cup) to obtain recombinant bacteria through two screenings. Primary recombinant bacteria were screened on LB plates containing 25 mg / L kanamycin.
  • the recombinant bacteria were further cultured overnight in liquid LB medium, and then subjected to secondary screening on LB plates containing 100g / L of sucrose, and the correct recombinant strains were obtained and named ATCC 21543-cadA, AM1: aspC-cadA, AM1: asd, respectively.
  • These strains are characterized by their endogenous lysE gene replaced by the lysine decarboxylase gene cadA of E. coli.
  • the obtained Corynebacterium glutamicum strains were respectively cultured in a fermentation medium for 72 h (30 ° C, 200 rpm), and the yield of lysine was detected.
  • the components of the fermentation medium are (g / L): glucose 80g, corn slurry 10g, urea 4.5g, ammonium sulfate 45g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.5g, ferrous sulfate heptahydrate 10mg, tetrahydrate Manganese sulfate 10 mg, ⁇ -alanine 5 mg, niacin 5 mg, thiamine-hydrochloric acid 5 mg, biotin 0.3 mg, calcium carbonate 30 g, threonine 0.2 g, and leucine 0.2 g.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Polymers & Plastics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Animal Husbandry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Physiology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

NADH依赖性的氨基酸脱氢酶在提高赖氨酸产量中的应用，氨基酸脱氢酶分别为来源于Pseudomonas aeruginos的天冬氨酸脱氢酶，来源于Tistrella mobilis的天冬氨酸半醛脱氢酶，来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶，来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶，其氨基酸序列分别如SEQ ID NO.1、3、5、7所示。氨基酸脱氢酶能利用NADH或同时利用NADH和NADPH作为辅因子合成赖氨酸从而减少细胞对NADPH的需求，进而显著地提高赖氨酸或者戊二胺的产量。

Description

NADH依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用 技术领域
本发明属于基因工程和生物发酵技术领域，具体地说，涉及一种NADH依赖性的氨基酸脱氢酶及其应用。
背景技术
赖氨酸是一种极其重要的氨基酸，广泛应用于饲料添加剂、保健及医药领域。目前，赖氨酸的工业生产主要通过微生物发酵法进行，工业常用的菌种包括谷氨酸棒杆菌和大肠杆菌。在赖氨酸的生物合成过程中，有四步酶促反应过程需要消耗NADPH用于合成赖氨酸。因此，传统的方法都是通过强化NADPH的供应，例如提高戊糖磷酸循环途径的代谢通量，或者表达NADPH依赖型的甘油醛3-磷酸脱氢酶，用于平衡赖氨酸合成过程中的辅酶需求，从而提高赖氨酸的产量。在戊二胺的生产过程中同样存在高度依赖NADPH的问题，戊二胺是通过在赖氨酸生产菌株中表达赖氨酸脱羧酶脱而获得。在微生物细胞内，NADH的浓度一般远大于NADPH，且NADH的稳定性比NADPH更高，价格更加便宜，因此直接利用胞内的NADH合成赖氨酸或戊二胺具有极大的应用潜力。
发明内容
本发明的目的是提供一种NADH依赖性的氨基酸脱氢酶及其应用。
赖氨酸和戊二胺合成途径中有四步反应消耗NADPH：(1)草酰乙酸的氨基化生成天冬氨酸；(2)磷酸天冬氨酸还原生成天冬氨酸半醛；(3)二氢吡啶二羧酸还原生成哌啶二羧酸；(4)哌啶二羧酸还原生成二氨基庚二酸。目前大部分微生物体内发现的催化上述步骤的酶都是严格依赖于NADPH。本发明通过大量的生物信息学分析和反复对比实验，在大肠杆菌中表达不同来源的氨基酸脱氢酶的基因，检测其催化活性，及对辅酶NADH、NADPH的特异性进行检测发现，来源于Pseudomonas aeruginos的天冬氨酸脱氢酶(其氨基酸序列如SEQ ID NO.1所示)，来源于Tistrella mobilis的天冬氨酸半醛脱氢酶(其氨基酸序列如SEQ ID NO.3所示)，来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶(其氨基酸序列如SEQ ID NO.5所示)，来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶(其氨基酸序列如SEQ ID NO.7所示)可以利用NADH催化上述四步反应。
因此，本发明提供了NADH依赖性的氨基酸脱氢酶，所述氨基酸脱氢酶为：
来源于Pseudomonas aeruginos的天冬氨酸脱氢酶；
来源于Tistrella mobilis的天冬氨酸半醛脱氢酶；
来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶；和/或来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶；
其氨基酸序列分别如SEQ ID NO.1、3、5、7所示或如SEQ ID No.1、3、5、7所示氨基酸序列经取代、缺失和/或增加一个或几个氨基酸而不影响其生物活性的氨基酸序列。
经过密码子优化，本发明提供了编码上述述NADH依赖性的氨基酸脱氢酶的基因，其核苷酸序列分别含有如SEQ ID NO.2、4、6、8所示的核苷酸序列或SEQ ID No.2、4、6、8所示核苷酸序列经取代、缺失和/或增加一个或几个碱基的具有90％以上同源性的、编码相同功能氨基酸脱氢酶的核苷酸序列。
本发明提供了含有上述基因的生物材料，所述生物材料为载体、重组菌、细胞系或表达盒。
优选地，所述重组菌为可以发酵生产赖氨酸或戊二胺的菌株中含有或过表达本发明所述的NADH依赖性的氨基酸脱氢酶。
所述可以发酵生产赖氨酸或戊二胺的菌株为谷氨酸棒杆菌或大肠杆菌。
更优选地，所述重组菌为可以发酵生产赖氨酸或戊二胺的菌株中含有或过表达来源于Pseudomonas aeruginos的天冬氨酸脱氢酶和来源于Tistrella mobilis的天冬氨酸半醛脱氢酶；或
含有来源于Pseudomonas aeruginos的天冬氨酸脱氢酶、来源于Tistrella mobilis的天冬氨酸半醛脱氢酶和来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶；或
含有来源于Pseudomonas aeruginos的天冬氨酸脱氢酶、来源于Tistrella mobilis的天冬氨酸半醛脱氢酶、来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶和来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶。
最优选地，所述重组菌为可以发酵生产赖氨酸或戊二胺的菌株中NADPH-依赖性的氨基酸脱氢酶被本发明所述的NADH依赖性的氨基酸脱氢酶所替代。
在本发明的一个实施例中，重组菌AM3为谷氨酸棒杆菌ATCC21543内源的天冬氨酸转氨酶被替换成NADH依赖性的天冬氨酸脱氢酶(氨基酸序列SEQ ID NO.1)、内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶(氨基酸序列SEQ ID NO.3)，以及内源的NADPH-依赖型的二 氢吡啶二羧酸还原酶被替换成NADH-依赖型的二氢吡啶二羧酸还原酶(氨基酸序列SEQ ID NO.5)，该重组菌发酵发酵培养基能够显著提高赖氨酸的产量，达到27.7g/L。
另外，本发明实施例中的重组菌AM2和AM4也取得优异的提高赖氨酸产量的效果，分别达到25.8g/L和25.6g/L。所述重组菌AM2是谷氨酸棒杆菌ATCC21543内源的天冬氨酸转氨酶被替换成NADH依赖性的天冬氨酸脱氢酶(氨基酸序列SEQ ID NO.1)、内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶(氨基酸序列SEQ ID NO.3)。
所述重组菌AM4是谷氨酸棒杆菌ATCC21543内源的天冬氨酸转氨酶被替换成NADH依赖性的天冬氨酸脱氢酶(氨基酸序列SEQ ID NO.1)、内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶(氨基酸序列SEQ ID NO.3)，内源的NADPH-依赖型的二氢吡啶二羧酸还原酶被替换成NADH-依赖型的二氢吡啶二羧酸还原酶(氨基酸序列SEQ ID NO.5)，以及内源的NADPH-依赖型的二氨基庚二酸脱氢酶被替换成NADH-依赖型的二氨基庚二酸脱氢酶。
所述发酵培养基(g/L)为：葡萄糖80g，玉米浆10g，尿素4.5g，硫酸铵45g，磷酸二氢钾0.5g，七水硫酸镁0.5g，七水硫酸亚铁10mg，四水硫酸锰10mg，β-丙氨酸5mg，烟酸5mg，硫胺素-盐酸5mg，生物素0.3mg，碳酸钙30g，苏氨酸0.2g，亮氨酸0.2g，加水至1L。
进一步地，本发明提供了上述NADH依赖性的氨基酸脱氢酶或其编码基因或上述含有其编码基因的生物材料在制备赖氨酸或戊二胺中的应用。
本发明提供了上述NADH依赖性的氨基酸脱氢酶或其编码基因或上述含有其编码基因的生物材料在提高赖氨酸产量或提高戊二胺产量中的应用。
在发酵生产戊二胺时，发酵菌株中内源的lysE基因被大肠杆菌的赖氨酸脱羧酶基因cadA所替换，可以显著提高戊二胺的产量。
本发明提供了上述NADH依赖性的氨基酸脱氢酶或其编码基因或上述含有其编码基因的生物材料在制备饲料添加剂中的应用。
本发明提供了上述NADH依赖性的氨基酸脱氢酶或其编码基因或上述含有其编码基因的生物材料在制备药物中的应用。
本发明提供的NADH-依赖性的氨基酸脱氢酶可以用于取代谷氨酸棒杆菌自身的NADPH-依赖性的氨基酸脱氢酶，本发明所筛选的这类特殊的氨基酸脱氢酶能直 接利用NADH作为辅因子或者同时利用NADH和NADPH，在赖氨酸或者戊二胺生产菌株中直接表达这类NADH-依赖性的氨基酸脱氢酶或者用NADH-依赖性的氨基酸脱氢酶替代对应的NADPH-依赖性的氨基酸脱氢酶可以使细胞利用胞内丰富的NADH作为辅因子合成赖氨酸从而减少细胞对NADPH的需求，进而显著的提高赖氨酸或者戊二胺的产量。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 NADH-依赖型的氨基酸脱氢酶的表达及其催化性能验证
本发明通过大量的生物信息学分析和反复对比实验，在大肠杆菌中表达不同来源的氨基酸脱氢酶的基因，检测其催化活性，及对辅酶NADH、NADPH的特异性进行检测发现，来源于Pseudomonas aeruginos的天冬氨酸脱氢酶(其氨基酸序列如SEQ ID NO.1所示)，来源于Tistrella mobilis的天冬氨酸半醛脱氢酶(其氨基酸序列如SEQ ID NO.3所示)，来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶(其氨基酸序列如SEQ ID NO.5所示)，来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶(其氨基酸序列如SEQ ID NO.7所示)可以催化上述四步反应。
基于上述酶的氨基酸序列，本发明设计并基因合成了相应的密码子优化的基因序列，如序列SEQ ID NO.2、4、6、8所示。将合成后的基因片段直接插入到pET-28a的EcoRI和SalI双酶切位点，所获得的质粒分别命名为pET-adh，pET-asd，pET-dapB，pET-ddh。将上述质粒利用化学转化法转入到大肠杆菌BL21(DE3)中，在含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为BL21/pET-adh，BL21/pET-asd，BL21/pET-dapB，BL21/pET-ddh。
将上述BL21菌株在含50mg/L卡那霉素的LB液体培养基中培养至OD600达到0.6(37℃，150rpm)，添加0.1mM的IPTG并继续培养12h以诱导蛋白的表达(20℃，150rpm)。将菌体离心分离，并用100ml 100mM的PBS缓冲液(pH7.0)洗涤两次，最后将菌体重悬于5ml的100mM的PBS缓冲液(pH7.0)中。将所述重悬液利用超声进行破碎，并离心获得上清液(12000rpm，30分钟)。利用蛋白纯 化试剂盒HisTrap(GE)分离纯化酶，用于酶活检测。
天冬氨酸脱氢酶的活性检测体系包括：100mM的Tris-HCl缓冲液(pH8.2)，0.2mM的辅酶NADH或者NADPH，4mM的草酰乙酸，100mM的氯化铵，和适量的酶。反应在37℃进行，测定其在340nm下的吸光值变化。酶活定义为每分钟消耗1μM的NAD(P)H所需要的酶量(U)。实验结果如表1所示。
天冬氨酸半醛脱氢酶的活性检测体系包括：200mM的CHES缓冲液(pH9.0)，50mM的KPi，0.5mM的辅酶NAD或者NADP，2mM的天冬氨酸半醛，和适量的酶。反应在25℃进行，测定其在340nm下的吸光值变化。酶活定义为每分钟产生1μM的NAD(P)H所需要的酶量(U)。实验结果如表1所示。
二氢吡啶二羧酸还原酶的活性检测体系包括：100mM的HEPES缓冲液(pH7.5)，0.2mM的辅酶NADH或者NADPH，4mM的草酰乙酸，1mM的丙酮酸，0.1mM的天冬氨酸半醛，25ug/ml的二氢吡啶二羧酸合成酶，和适量的酶。反应在25℃进行，测定其在340nm下的吸光值变化。酶活定义为每分钟消耗1μM的NAD(P)H所需要的酶量(U)。实验结果如表1所示。
二氨基庚二酸脱氢酶的活性检测体系包括：100mM的甘氨酸-KOH缓冲液(pH10.0)，0.5mM的辅酶NAD或者NADP，5mM的二氨基庚二酸，和适量的酶。反应在30℃进行，测定其在340nm下的吸光值变化。酶活定义为每分钟产生1μM的NAD(P)H所需要的酶量(U)。实验结果如表1所示。
表1.不同来源的氨基酸脱氢酶的酶活(单位U/mg)
Figure PCTCN2019101521-appb-000001
由表1可以看出，本发明所筛选获得的四种酶都可以高效的利用NADH作为辅因子来催化目标反应，而来自谷氨酸棒杆菌的酶基本不能利用NADH。
实施例2、过表达NADH-依赖型的氨基酸脱氢酶提高谷氨酸棒杆菌赖氨酸的产量
本实施例在一株谷氨酸棒杆菌LC298(Applied and environmental microbiology,2011，02912-10)中分别表达本发明所筛选的NADH-依赖性的氨基酸脱氢酶，同时也表达谷氨酸棒杆菌自身所对应的NADPH-依赖型的氨基酸脱氢酶作为对照，比较不同辅酶特异性的酶对于促进赖氨酸合成的影响。
以pET-adh为模板以分别以acagctatgacatgattacgaaggagatatacatatgctgaacattgtgatgatcgg和tgcatgcctgcaggtcgactttagattgaaatggcatgggca为引物克隆NADH-依赖型的天冬氨酸脱氢酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-adh_pa。将pEC-adh_pa电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-adh_pa。同时以谷氨酸棒杆菌LC298为模板，以acagctatgacatgattacgaaggagatatacatatgagttcagtttcgctgcagga和tgcatgcctgcaggtcgactttagttagcgtaatgctccgctgc为引物克隆谷氨酸棒杆菌自身的天冬氨酸氨基转移酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-aspC_cg。将pEC-aspC_cg电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-aspC_cg。
以pET-asd为模板以分别以acagctatgacatgattacgaaggagatatacatatgcgtatcgggattgttgga和tgcatgcctgcaggtcgactttacaccagtaactctgcgatttgc为引物克隆NADH-依赖型的天冬氨酸半醛脱氢酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-asd_tm。将pEC-asd_tm电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-asd_tm。同时以谷氨酸棒杆菌LC298为模板，以acagctatgacatgattacgaaggagatatacatatgaccaccatcgcagttgtt和tgcatgcctgcaggtcgactttacttaaccagcagctcag为引物克隆谷氨酸棒杆菌自身的天冬氨酸半醛脱氢酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2 (从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-asd_cg。将pEC-asd_cg电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-asd_cg。
以pET-dapB为模板以分别以acagctatgacatgattacgaaggagatatacat atgcgggtaggcgtccttgg和tgcatgcctgcaggtcgactttacaaatttcagtgcagatcgagtagggg为引物克隆NADH-依赖型的二氢吡啶二羧酸还原酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-dapB_mt。将pEC-dapB_mt电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-dapB_mt。同时以谷氨酸棒杆菌LC298为模板，以acagctatgacatgattacgaaggagatatacatatgggaatcaaggttggcgt和tgcatgcctgcaggtcgactttacaggcctaggtaatgctca为引物克隆谷氨酸棒杆菌自身的二氢吡啶二羧酸还原酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-pEC-dapB_cg。将pEC-dapB_cg电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-dapB_cg。
以pET-ddh为模板以分别以acagctatgacatgattacgaaggagatatacatatgccaaagaccaaagtgct和tgcatgcctgcaggtcgactttagaccagacgacaaattaattgttctaagtcg为引物克隆NADH-依赖型的二氨基庚二酸脱氢酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-ddh_ta。将pEC-ddh_ta电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-ddh_ta。同时以谷氨酸棒杆菌LC298为模板，以acagctatgacatgattacgaaggagatatacatatgaccaacatccgcgtagcta和tgcatgcctgcaggtcgactttagacgtcgcgtgcgatca为引物克隆谷氨酸棒杆菌自身的二氨基庚二酸脱氢酶基因片段，利用Gibson组装试剂盒将该片段连接到质粒pEC-K18mob2(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pEC-ddh_cg。将pEC-ddh_cg电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω， 2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-ddh_cg。
将空质粒pEC-K18mob2电转化到谷氨酸棒杆菌LC298中(电转条件为电压2.5KV，200Ω，2mm电转杯)，含50mg/L的卡那霉素LB平板上筛选获得重组菌，分别命名为LC/pEC-K18作为对照菌株。
将上述所获得的谷氨酸棒杆菌LC298的衍生菌株分别在发酵培养基中培养72h(30℃，200rpm)，检测赖氨酸的产量。发酵培养基的成分为(g/L)：葡萄糖80g，玉米浆10g，尿素4.5g，硫酸铵45g，磷酸二氢钾0.5g，七水硫酸镁0.5g，七水硫酸亚铁10mg，四水硫酸锰10mg，β-丙氨酸5mg，烟酸5mg，硫胺素-盐酸5mg，生物素0.3mg，碳酸钙30g，卡那霉素25mg。
其中对照菌株LC/pEC-K18赖氨酸的产量为14.01g/L,其他菌株的发酵结果如表2所示，说明过表达NADH-依赖型的氨基酸脱氢酶可以显著提高赖氨酸的产量，并且效果优于来源于谷氨酸棒杆菌自身的NADPH-依赖型氨基酸脱氢酶。
表2.表达不同辅酶特异性的氨基酸脱氢酶菌株的赖氨酸产量(单位g/L)
Figure PCTCN2019101521-appb-000002
实施例3利用NADH-依赖型的氨基酸脱氢酶替换NADPH-依赖型的氨基酸脱氢酶提高赖氨酸的产量
除了可以直接过表达NADH-依赖型的氨基酸脱氢酶提高赖氨酸产量以外，还可以直接用NADH-依赖型的氨基酸脱氢酶替换谷氨酸棒杆菌中对应的NADPH-依赖型的氨基酸脱氢酶以提高赖氨酸的产量。本实施例在一株谷氨酸棒杆菌ATCC21543中利用同源重组的方法分别将细胞自身NADPH-依赖型的氨基酸脱氢酶逐一替换为表达本发明所筛选的NADH-依赖性的氨基酸脱氢酶，考察其对于促进赖氨酸合成的影响。
以pET-adh为模板以分别以gtacgcagttatgctgaacattgtgatgatcggatg和gctgtattcacttttagattgaaatggcatgggcatgattt为引物克隆NADH-依赖型的天冬氨酸脱氢酶基因片段adh，以ATCC 21543的基因组为模板分别以acagctatgacatgattacggagttctttcttcagcgctgcg和tgttcagcataactgcgtacctccgcatgtg为引物克 隆基因片段adh-up，以ATCC 21543的基因组为模板分别以atttcaatctaaaagtgaatacagcggagacagc和tgcatgcctgcaggtcgactctcttcaacgattttcagcaaggc为引物克隆基因片段adh-down，利用Gibson组装试剂盒将这三个片段连接到质粒pK18-mobsacB(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pK18-adh。将pK18-adh电转化到谷氨酸棒杆菌ATCC 21543中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM1：aspC，该菌株的主要特征是内源的天冬氨酸转氨酶被替换成NADH-依赖型的天冬氨酸脱氢酶。
以pET-asd为模板以分别以tagttttacaatgcgtatcgggattgttggag和atggcgggtttttacaccagtaactctgcgatttgcac为引物克隆NADH-依赖型的天冬氨酸半醛脱氢酶基因片段asd，以ATCC 21543的基因组为模板分别以acagctatgacatgattacgagcccaatctttcacgggc和cgatacgcattgtaaaactactcctttaaaactttagcgtccg为引物克隆基因片段asd，以ATCC 21543的基因组为模板分别以tactggtgtaaaaacccgccattaaaaactccg和tgcatgcctgcaggtcgactatttgtggtcattatctcggaaaaatgcg为引物克隆基因片段asd-down，利用Gibson组装试剂盒将这三个片段连接到质粒pK18-mobsacB(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pK18-asd。将pK18-asd电转化到谷氨酸棒杆菌ATCC 21543中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM1：asd，该菌株的主要特征是内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶。
以pET-dapB为模板以分别以aaggagcataatgcatgatgcaaacatccgc和tgaaatgagcctttacaaattattgagatcaagtacatctcgcatatcaaaaag为引物克隆NADH-依赖型的二氢吡啶二羧酸还原酶基因片段dapB，以ATCC 21543的基因组为模板分别以acagctatgacatgattacgctagatcgggctagatcgggctaa和catcatgcattatgctccttcattttcgtggggc为引物克隆基因片段dapB-up，以ATCC 21543的基因组为模板分别以aataatttgtaaaggctcatttcagcagcgg和tgcatgcctgcaggtcgactttaaaagtccatgacatacgggcttgt为引物克隆基因片段dapB-down，利用Gibson组装试剂盒将这三个片段连接到质粒 pK18-mobsacB(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pK18-dapB。将pK18-dapB电转化到谷氨酸棒杆菌ATCC 21543中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM1：dapB，该菌株的主要特征是内源的NADPH-依赖型的二氢吡啶二羧酸还原酶被替换成NADH-依赖型的二氢吡啶二羧酸还原酶。
以pET-ddh为模板以分别以ttacaagaacatgccaaagaccaaagtgctg和tcgagctaaattagaccagacgacaaattaattgttctaagtcg为引物克隆NADH-依赖型的二氨基庚二酸脱氢酶基因片段ddh，以ATCC 21543的基因组为模板分别以acagctatgacatgattacgatcgctcaaggctgctgctg和tctttggcatgttcttgtaatcctccaaaattgtggtgg为引物克隆基因片段ddh-up，以ATCC 21543的基因组为模板分别以tctggtctaatttagctcgaggggcaaggaa和tgcatgcctgcaggtcgactcttcccccgcaagacgatg为引物克隆基因片段ddh-down，利用Gibson组装试剂盒将这三个片段连接到质粒pK18-mobsacB(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pK18-ddh。将pK18-ddh电转化到谷氨酸棒杆菌ATCC 21543中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM1：ddh，该菌株的主要特征是内源的NADPH-依赖型的二氨基庚二酸脱氢酶被替换成NADH-依赖型的二氨基庚二酸脱氢酶。
将pK18-asd电转化到谷氨酸棒杆菌AM1：aspC中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM2，该菌株的主要特征是内源的天冬氨酸转氨酶被替换成NADH-依赖型的天冬氨酸脱氢酶，以及内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶。
将pK18-dapB电转化到谷氨酸棒杆菌AM2中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的 蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM3，该菌株的主要特征是内源的天冬氨酸转氨酶被替换成NADH-依赖型的天冬氨酸脱氢酶，以及内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶，以及内源的NADPH-依赖型的二氢吡啶二羧酸还原酶被替换成NADH-依赖型的二氢吡啶二羧酸还原酶。
将pK18-ddh电转化到谷氨酸棒杆菌AM3中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株命名为AM4，该菌株的主要特征是内源的天冬氨酸转氨酶被替换成NADH-依赖型的天冬氨酸脱氢酶，以及内源的NADPH-依赖型的天冬氨酸半醛转氨酶被替换成NADH-依赖型的天冬氨酸半醛脱氢酶，以及内源的NADPH-依赖型的二氢吡啶二羧酸还原酶被替换成NADH-依赖型的二氢吡啶二羧酸还原酶，以及内源的NADPH-依赖型的二氨基庚二酸脱氢酶被替换成NADH-依赖型的二氨基庚二酸脱氢酶。
将上述所获得的谷氨酸棒杆菌ATCC 21543的衍生菌株分别在发酵培养基中培养72h(30℃，200rpm)，检测赖氨酸的产量。发酵培养基的成分为(g/L)：葡萄糖80g，玉米浆10g，尿素4.5g，硫酸铵45g，磷酸二氢钾0.5g，七水硫酸镁0.5g，七水硫酸亚铁10mg，四水硫酸锰10mg，β-丙氨酸5mg，烟酸5mg，硫胺素-盐酸5mg，生物素0.3mg，碳酸钙30g，苏氨酸0.2g，亮氨酸0.2g。各菌株的发酵结果如表3所示，说明利用本发明所筛选的NADH-依赖型的氨基酸脱氢酶单独或者组合替换谷氨酸棒杆菌自身的NADPH-依赖型的氨基酸脱氢酶可以显著提高赖氨酸的产量。
表3.替换氨基酸脱氢酶重组菌株的赖氨酸产量(单位g/L)
ATCC 21543 AM1:aspC AM1:asd AM1:dapB AM1:ddh AM2 AM3 AM4
21.2 23.8 24.1 23.7 22.6 25.8 27.7 25.6
实施例4利用包含NADH-依赖型的氨基酸脱氢酶的谷氨酸棒杆菌提高戊二胺的产量
在上述表达NADH依赖型的菌株中表达赖氨酸脱羧酶可以实现戊二胺的直接生物合成。本实施例在实施例3所获得的菌株的基础上，利用同源重组的方法将赖氨酸的外排基因lysE替换为来源于大肠杆菌的谷氨酸脱羧酶基因cadA，实现从葡萄糖到戊二胺的直接生产，考察NADH依赖型的氨基酸脱氢酶对于促进戊二胺合 成的影响。
以大肠杆菌MG1655为模板分别以TTCGTGGTGTTGCCCGTGGCCCGGTTGGTTGGGCAGGAGTATATTGGGATCCatgAACGTTATTGCAATATTGAATC和catcaacatcagttaTTTTTTGCTTTCTTCTTTCAATAC为引物克隆赖氨酸脱羧酶基因片段cadA，以ATCC 21543的基因组为模板分别以acagctatgacatgattacgcgggcgaagaagtgaaaaacc和GCCACGGGCAACACCACGAATGCGCTACCTTAACCGAAAAGTTACTTTcgtgacctatggaagtacttaa为引物克隆基因片段cadA-up，以ATCC 21543的基因组为模板分别以GCAAAAAAtaactgatgttgatgggttagttttcgc和tgcatgcctgcaggtcgactttcaacgcagcgcagcatta为引物克隆基因片段cadA-down，利用Gibson组装试剂盒将这三个片段连接到质粒pK18-mobsacB(从Addgene购买)的EcoRI和XbaI位点内，所获得的质粒命名为pK18-cadA。将pK18-cadA分别电转化到谷氨酸棒杆菌ATCC 21543，AM1：aspC，AM1：asd，AM1：dapB，AM1：ddh，AM2，AM3，AM4中(电转条件为电压2.5KV，200Ω，2mm电转杯)，通过两次筛选获得重组菌。一次重组菌在含25mg/L的卡那霉素LB平板上筛选。该重组菌进一步在液体LB培养基里过夜培养，之后在含有100g/L的蔗糖LB平板上进行二次筛选，获得正确重组菌株分别命名为ATCC 21543-cadA，AM1：aspC-cadA，AM1：asd-cadA，AM1：dapB-cadA，AM1：ddh-cadA，AM2-cadA，AM3-cadA，AM4-cadA。这些菌株的特征是其内源的lysE基因被大肠杆菌的赖氨酸脱羧酶基因cadA所替换。
将上述所获得的谷氨酸棒杆菌菌株分别在发酵培养基中培养72h(30℃，200rpm)，检测赖氨酸的产量。发酵培养基的成分为(g/L)：葡萄糖80g，玉米浆10g，尿素4.5g，硫酸铵45g，磷酸二氢钾0.5g，七水硫酸镁0.5g，七水硫酸亚铁10mg，四水硫酸锰10mg，β-丙氨酸5mg，烟酸5mg，硫胺素-盐酸5mg，生物素0.3mg，碳酸钙30g，苏氨酸0.2g，亮氨酸0.2g。各菌株的发酵结果如表4所示，说明利用本发明所筛选的NADH-依赖型的氨基酸脱氢酶单独或者组合替换谷氨酸棒杆菌自身的NADPH-依赖型的氨基酸脱氢酶可以显著提高戊二胺的产量。
表4.替换氨基酸脱氢酶重组菌株的戊二胺产量(单位g/L)
Figure PCTCN2019101521-appb-000003
Figure PCTCN2019101521-appb-000004
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (10)

  1. NADH依赖性的氨基酸脱氢酶，所述氨基酸脱氢酶为：
    来源于Pseudomonas aeruginos的天冬氨酸脱氢酶；
    来源于Tistrella mobilis的天冬氨酸半醛脱氢酶；
    来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶；和/或
    来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶；
    其氨基酸序列分别如SEQ ID NO.1、3、5、7所示或如SEQ ID No.1、3、5、7所示氨基酸序列经取代、缺失和/或增加一个或几个氨基酸而不影响其生物活性的氨基酸序列。
  2. 编码权利要求1所述NADH依赖性的氨基酸脱氢酶的基因，其特征在于，其核苷酸序列分别含有如SEQ ID NO.2、4、6、8所示的核苷酸序列或SEQ ID No.2、4、6、8所示核苷酸序列经取代、缺失和/或增加一个或几个碱基的具有90％以上同源性的、编码相同功能氨基酸脱氢酶的核苷酸序列。
  3. 含有权利要求2所述基因的生物材料，其特征在于，所述生物材料为载体、重组菌、细胞系或表达盒。
  4. 如权利要求3所述的生物材料，其特征在于，所述重组菌为可以发酵生产赖氨酸或戊二胺的菌株中含有或过表达权利要求1所述的NADH依赖性的氨基酸脱氢酶。
  5. 如权利要求4所述的生物材料，其特征在于，所述重组菌为可以发酵生产赖氨酸或戊二胺的菌株中含有或过表达来源于Pseudomonas aeruginos的天冬氨酸脱氢酶和来源于Tistrella mobilis的天冬氨酸半醛脱氢酶；或
    含有或过表达来源于Pseudomonas aeruginos的天冬氨酸脱氢酶、来源于Tistrella mobilis的天冬氨酸半醛脱氢酶和来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶；或
    含有或过表达来源于Pseudomonas aeruginos的天冬氨酸脱氢酶、来源于Tistrella mobilis的天冬氨酸半醛脱氢酶、来源于Mycobacterium tuberculosis的二氢吡啶二羧酸还原酶和来源于Tepidanaerobacter acetatoxydans的二氨基庚二酸脱氢酶。
  6. 如权利要求3-5任一所述的生物材料，其特征在于，所述重组菌为可以发酵生产赖氨酸或戊二胺的菌株中NADPH-依赖性的氨基酸脱氢酶被权利要求1所述的NADH依赖性的氨基酸脱氢酶所替代。
  7. 权利要求1所述的NADH依赖性的氨基酸脱氢酶或其编码基因或权利要求3-6任一所述的生物材料在制备赖氨酸或戊二胺中的应用。
  8. 权利要求1所述的NADH依赖性的氨基酸脱氢酶或其编码基因或权利要求3-6任一所述的生物材料在提高赖氨酸产量或提高戊二胺产量中的应用。
  9. 如权利要求7或8所述的应用，其特征在于，发酵生产戊二胺时，发酵菌株中内源的lysE基因被大肠杆菌的赖氨酸脱羧酶基因cadA所替换。
  10. 权利要求1所述的NADH依赖性的氨基酸脱氢酶或其编码基因或权利要求3-6任一所述的生物材料在制备药物或饲料添加剂中的应用。
PCT/CN2019/101521 2018-09-18 2019-08-20 Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用 Ceased WO2020057308A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/277,661 US20220112471A1 (en) 2018-09-18 2019-08-20 Nadh-dependent amino acid dehydrogenase and application thereof in increasing lysine yield

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811089319.0 2018-09-18
CN201811089319.0A CN109182283A (zh) 2018-09-18 2018-09-18 Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用

Publications (1)

Publication Number Publication Date
WO2020057308A1 true WO2020057308A1 (zh) 2020-03-26

Family

ID=64908249

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/101521 Ceased WO2020057308A1 (zh) 2018-09-18 2019-08-20 Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用

Country Status (3)

Country Link
US (1) US20220112471A1 (zh)
CN (1) CN109182283A (zh)
WO (1) WO2020057308A1 (zh)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109182283A (zh) * 2018-09-18 2019-01-11 清华大学 Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用
CN111518820B (zh) * 2019-02-01 2021-05-04 上海凯赛生物技术股份有限公司 用于发酵生产l-赖氨酸的重组dna、菌株及其应用
CN113564138B (zh) * 2021-09-03 2023-07-18 江南大学 一种二氨基庚二酸脱氢酶突变体及其应用
CN114752575B (zh) * 2022-04-07 2023-06-13 内蒙古工业大学 一种nad+依赖性脱氢酶基因及其在提高辅酶q10产量中的应用
CN116287046A (zh) * 2022-06-24 2023-06-23 吉林梅花氨基酸有限责任公司 一种氨基酸发酵培养基及半连续发酵生产赖氨酸的方法
CN115948313A (zh) * 2022-11-10 2023-04-11 清华大学 提高赖氨酸和戊二胺发酵产量的方法
CN116751700A (zh) * 2023-03-07 2023-09-15 中国石油大学(华东) 一株聚丙烯酰胺高效降解菌
CN118360265A (zh) * 2024-05-23 2024-07-19 华东理工大学 二氨基庚二酸脱氢酶突变体及在d-氨基酸合成中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109182283A (zh) * 2018-09-18 2019-01-11 清华大学 Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2010101135A (ru) * 2010-01-15 2011-07-20 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) Бактерия семейства enterobacteriaceae - продуцент l-аспартата или метаболитов, производных l-аспартата, и способ получения l-аспартата или метаболитов, производных l-аспартата
CN106190997B (zh) * 2016-07-20 2024-09-17 清华大学 一种nadh依赖性的二氨基庚二酸脱氢酶及其应用
CN107858318A (zh) * 2017-11-17 2018-03-30 江南大学 异源表达大肠杆菌中dhdpr提高赖氨酸产量的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109182283A (zh) * 2018-09-18 2019-01-11 清华大学 Nadh依赖性的氨基酸脱氢酶及其在提高赖氨酸产量中的应用

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 16 April 1997 (1997-04-16), PAVELKA: "Mycobacterium tuberculosis dihydrodipicolinate reductase (dapB) gene , complete cds", XP055696059, retrieved from NCBI Database accession no. U66101.1 *
DATABASE Nucleotide 16 April 1997 (1997-04-16), PAVELKA: "Mycobacterium tuberculosis dihydrodipicolinate reductase (dapB) gene", XP055696059, retrieved from NCBI Database accession no. U66101.1 *
DATABASE Protein 12 May 2013 (2013-05-12), "L-aspartate dehydrogenase [Pseudomonas aeruginosa", XP055696252, retrieved from NCBI Database accession no. WP_003453564.1 *
DATABASE Protein 13 July 2018 (2018-07-13), TULLY, B.J.: "aspartate-semialdehyde dehydrogenase [Tistrella sp.", XP055696254, retrieved from NCBI Database accession no. MAM72981.1 *
DATABASE Protein 19 May 2013 (2013-05-19), "aspartate-semialdehyde dehydrogenase [Tistrella mobilis", XP055696022, retrieved from NCBI Database accession no. WP_014746527.1 *
DATABASE Protein 21 July 2015 (2015-07-21), "MULTISPECIES: L-aspartate dehydrogenase [Pseudomonas]", XP055696019, retrieved from NCBI Database accession no. WP_003112905.1 *
DATABASE Protein 26 October 2017 (2017-10-26), CHRUSZCZ, M.: "Chain A, 4-hydroxy-tetrahydrodipicolinate Reductase", XP055696035, retrieved from NCBI Database accession no. 5TEK_A *
DATABASE Protein 30 January 2014 (2014-01-30), ZHANG, Y.: "dihydrodipicolinate reductase [Mycobacterium tuberculosis CCDC5079", XP055696258, retrieved from NCBI Database accession no. AEJ47734.1 *
DATABASE Protein 8 February 2016 (2016-02-08), "diaminopimelate dehydrogenase [Tepidanaerobacter acetatoxydans", XP055696040, retrieved from NCBI Database accession no. WP_013779049.1 *
LI, DONGXIA ET AL.: "Progress in Biosynthesis of Diaminopentane", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 30, no. 2, 25 February 2014 (2014-02-25), pages 161 - 174 *

Also Published As

Publication number Publication date
US20220112471A1 (en) 2022-04-14
CN109182283A (zh) 2019-01-11

Similar Documents

Publication Publication Date Title
US9121041B2 (en) Method for the preparation of diols
CN102165056B (zh) 生产l-氨基酸的微生物和使用其生产l-氨基酸的方法
US20220112471A1 (en) Nadh-dependent amino acid dehydrogenase and application thereof in increasing lysine yield
US10351839B2 (en) Lysine decarboxylase having improved stability with a pH change, microorganism comprising a polynucleotide encoding the same, and method for producing cadaverine using the same
KR101940647B1 (ko) 신규 라이신 디카르복실라제 및 이를 이용하여 카다베린을 생산하는 방법
KR20030078036A (ko) Ｌ-아미노산의 생산 방법
CN104894043B (zh) 一种产γ-氨基丁酸的工程菌及其构建和应用
WO2023169176A1 (zh) 表达天冬氨酸脱氢酶的工程菌及发酵生产维生素b5的方法
CN110272857B (zh) β-丙氨酸生产菌及其制备方法和用途
WO2022133917A1 (zh) 改造的磷酸烯醇丙酮酸羧化酶及其在提高谷氨酸棒杆菌氨基酸产量中的应用
CN104160015B (zh) 生产喹啉酸的重组微生物及利用该微生物的喹啉酸生产方法
Liu et al. Redesigning transamination and decarboxylation characteristics of L-aspartate aminotransferase by site directed mutation of non-active site
CN106978405B (zh) 天冬氨酸激酶/高丝氨酸脱氢酶突变体及其应用
JP7762226B2 (ja) 新規なプロモーター及びその使用
CN116286702A (zh) 一种天冬氨酸脱氢酶突变体及其应用
CN108546697B (zh) 酶法制备beta丙氨酸
CN106190997B (zh) 一种nadh依赖性的二氨基庚二酸脱氢酶及其应用
CN103667165B (zh) 高产l‑赖氨酸的生产菌株及其应用
CN116333953A (zh) 一种高产胞嘧啶核苷的基因工程菌及其应用
CN112921012A (zh) 谷氨酸棒杆菌meso-2,6-二氨基庚二酸脱氢酶突变体及其应用
Wang et al. Highly efficient spermidine production system in Escherichia coli BL21 (DE3) based on precursor metabolic modules optimization and carboxyaminopropylagmatine pathway construction
CN118931813A (zh) 表达活性d-脯氨酸还原酶的微生物以及生产活性d-脯氨酸还原酶的方法
CN121518416A (zh) 一种琥珀酸脱氢酶突变体及其应用
CN117431283A (zh) 一种烟酰胺腺嘌呤二核苷酸类化合物的生物合成方法
JP2026501793A (ja) Ｌ－セリンヒドロキシメチルトランスフェラーゼ活性が増強された微生物を使用したセリンからの発酵生産グアニジノ酢酸（ｇａａ）

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19861939

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19861939

Country of ref document: EP

Kind code of ref document: A1