CN117737102A - Lysine decarboxylase for synthesizing pentanediamine and application thereof - Google Patents
Lysine decarboxylase for synthesizing pentanediamine and application thereof Download PDFInfo
- Publication number
- CN117737102A CN117737102A CN202311789375.6A CN202311789375A CN117737102A CN 117737102 A CN117737102 A CN 117737102A CN 202311789375 A CN202311789375 A CN 202311789375A CN 117737102 A CN117737102 A CN 117737102A
- Authority
- CN
- China
- Prior art keywords
- lysine
- pentanediamine
- buffer solution
- lysine decarboxylase
- expression vector
- 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.)
- Pending
Links
- 108010048581 Lysine decarboxylase Proteins 0.000 title claims abstract description 68
- KJOMYNHMBRNCNY-UHFFFAOYSA-N pentane-1,1-diamine Chemical compound CCCCC(N)N KJOMYNHMBRNCNY-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- 241000588724 Escherichia coli Species 0.000 claims abstract description 33
- 239000013604 expression vector Substances 0.000 claims abstract description 33
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 claims abstract description 29
- 229960005337 lysine hydrochloride Drugs 0.000 claims abstract description 29
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- VHRGRCVQAFMJIZ-UHFFFAOYSA-N cadaverine Chemical compound NCCCCCN VHRGRCVQAFMJIZ-UHFFFAOYSA-N 0.000 claims description 36
- 239000002773 nucleotide Substances 0.000 claims description 35
- 125000003729 nucleotide group Chemical group 0.000 claims description 35
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 28
- 150000001413 amino acids Chemical group 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 21
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 18
- 230000001580 bacterial effect Effects 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 14
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 14
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 12
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 11
- -1 lysine pentylene diamine Chemical class 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 10
- 241000607626 Vibrio cholerae Species 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 8
- 229940118696 vibrio cholerae Drugs 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 241000607534 Aeromonas Species 0.000 claims description 6
- 101100136076 Aspergillus oryzae (strain ATCC 42149 / RIB 40) pel1 gene Proteins 0.000 claims description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 6
- 241000607471 Edwardsiella tarda Species 0.000 claims description 6
- 239000004472 Lysine Substances 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 229960003646 lysine Drugs 0.000 claims description 6
- 101150040383 pel2 gene Proteins 0.000 claims description 6
- 101150050446 pelB gene Proteins 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 241000193755 Bacillus cereus Species 0.000 claims description 5
- 241001453380 Burkholderia Species 0.000 claims description 5
- 241000588729 Hafnia alvei Species 0.000 claims description 5
- 241000588748 Klebsiella Species 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 5
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims description 5
- 238000007710 freezing Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 239000007974 sodium acetate buffer Substances 0.000 claims description 5
- 108090000084 Antiporters Proteins 0.000 claims description 4
- 241000607142 Salmonella Species 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 240000000233 Melia azedarach Species 0.000 claims description 3
- 241000282849 Ruminantia Species 0.000 claims description 3
- 241000607720 Serratia Species 0.000 claims description 3
- 230000002337 anti-port Effects 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 claims description 2
- 241000588807 Bordetella Species 0.000 claims description 2
- 101100378273 Brachyspira hyodysenteriae acpP gene Proteins 0.000 claims description 2
- 235000019687 Lamb Nutrition 0.000 claims description 2
- 101100278567 Lelliottia amnigena dsbL gene Proteins 0.000 claims description 2
- 101100098690 Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e) hly gene Proteins 0.000 claims description 2
- 101100084022 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) lapA gene Proteins 0.000 claims description 2
- 101100398653 Yersinia pestis lamB1 gene Proteins 0.000 claims description 2
- 101150042295 arfA gene Proteins 0.000 claims description 2
- 101150009558 dsbA gene Proteins 0.000 claims description 2
- 101150021605 hlyA gene Proteins 0.000 claims description 2
- 101150012518 lamB gene Proteins 0.000 claims description 2
- 101150087557 omcB gene Proteins 0.000 claims description 2
- 101150115693 ompA gene Proteins 0.000 claims description 2
- 101150073640 ompF gene Proteins 0.000 claims description 2
- 101150093139 ompT gene Proteins 0.000 claims description 2
- 210000001322 periplasm Anatomy 0.000 claims description 2
- 101150009573 phoA gene Proteins 0.000 claims description 2
- 230000028327 secretion Effects 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 241000588986 Alcaligenes Species 0.000 claims 1
- 241000589516 Pseudomonas Species 0.000 claims 1
- 230000010355 oscillation Effects 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 abstract description 15
- 238000010353 genetic engineering Methods 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 35
- 238000006555 catalytic reaction Methods 0.000 description 24
- 150000007523 nucleic acids Chemical group 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 101100276041 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) ctpD gene Proteins 0.000 description 10
- 229960000723 ampicillin Drugs 0.000 description 10
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 10
- 101150008667 cadA gene Proteins 0.000 description 10
- 108020004705 Codon Proteins 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000005457 optimization Methods 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000004677 Nylon Substances 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 6
- 229920001778 nylon Polymers 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 5
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 235000018977 lysine Nutrition 0.000 description 4
- 229920002302 Nylon 6,6 Polymers 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- LTMHNWPUDSTBKD-UHFFFAOYSA-N diethyl 2-(ethoxymethylidene)propanedioate Chemical compound CCOC=C(C(=O)OCC)C(=O)OCC LTMHNWPUDSTBKD-UHFFFAOYSA-N 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 241000006382 Bacillus halodurans Species 0.000 description 2
- 241000588879 Chromobacterium violaceum Species 0.000 description 2
- 241000533331 Salmonella bongori Species 0.000 description 2
- 241000605031 Selenomonas ruminantium Species 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000588881 Chromobacterium Species 0.000 description 1
- 241000193455 Clostridium cadaveris Species 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 101710172990 Inducible lysine decarboxylase Proteins 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- 241000982634 Tragelaphus eurycerus Species 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- BTGRAWJCKBQKAO-UHFFFAOYSA-N adiponitrile Chemical compound N#CCCCCC#N BTGRAWJCKBQKAO-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 101150063552 cadB gene Proteins 0.000 description 1
- 101150044474 calB gene Proteins 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 229920006351 engineering plastic Polymers 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 229920006118 nylon 56 Polymers 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000013585 weight reducing agent Substances 0.000 description 1
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
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
-
- 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
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01018—Lysine decarboxylase (4.1.1.18)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to lysine decarboxylase for synthesizing pentanediamine and application thereof, comprising a lysine decarboxylase gene and protein sequence, a constructed expression vector and a genetic engineering strain and application thereof in synthesizing bio-pentanediamine. Through constructing expression vector and genetic engineering bacteria, lysine decarboxylase is induced to be expressed, and the whole cell is catalyzed to synthesize the pentanediamine. The novel lysine decarboxylase developed by the invention can realize 100% conversion of high-concentration lysine hydrochloride, the production intensity of the pentanediamine can reach 204g/L/h, and the activity and the catalytic intensity of the novel lysine decarboxylase are obviously higher than those of the escherichia coli CadA and mutants thereof reported in the prior art, thereby being beneficial to efficiently synthesizing the high-concentration pentanediamine and having industrial application prospect.
Description
The present application is a divisional application of patent application No. 202010634482.1 (the application date of the original application is 2020, 07, 02, and the name of the present application is lysine decarboxylase for synthesizing pentanediamine and application thereof).
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to lysine decarboxylase for synthesizing pentanediamine and application thereof, in particular to a lysine decarboxylase gene sequence and protein sequence for stably and efficiently synthesizing pentanediamine, and an expression vector and recombinant engineering bacteria constructed by the same.
Background
Nylon has excellent mechanical properties, heat resistance, corrosion resistance and other properties, and is widely used in various fields such as fiber and engineering plastics, and in recent years, the yield, productivity and demand of chinese nylon are all growing. The nylon 66 has huge yield and consumption, but the synthesis technology of precursor adiponitrile of hexamethylenediamine, which is a core monomer of the nylon 66, is always monopolized by foreign enterprises, resources are tense, cost fluctuation is huge, and the nylon 66 is mainly derived from petroleum.
The pentylene diamine is a lysine decarboxylation product, and is a homolog with the hexylene diamine, and various nylon 5X products such as nylon 52, 5T, 54, 56, 510, 516, 518 and the like can be synthesized by using the pentylene diamine and the dibasic acid, so that the nylon has the characteristics of better light weight, weight reduction, moisture absorption, sweat release, temperature resistance, wear resistance, dyeing property, essential flame retardance and the like, and has wide development prospect. The synthesis of the bio-nylon 5X not only can reduce the dependence on petroleum resources, but also can break through monopoly of output and technology of hexamethylenediamine products by national enterprises, and has wide application prospect in the fields of aerospace and the like.
The key of the bio-based nylon 5X is the efficient synthesis of the core monomer of the pentanediamine. Efficient and stable lysine decarboxylase is the core of bio-based pentylene diamine synthesis. Lysine decarboxylase is widely available, and microorganisms reported to exist in lysine decarboxylase are mainly bacteria such as escherichia coli (E.coli), hafnia alvei (Hafnia alvei), alkaline Bacillus (Bacillus halodurans), bacillus cereus (Bacillus cereus), bacillus cadavermitilis (Bacterium cadaveris), burkholderia (Burkholderia vietnamensia), blue-violet Bacillus (Chromobacterium violaceum), vibrio cholerae (Vibrio cholerae), mao Lian mold (Streptomyces polosus), ruminant moon (Selenomonas ruminantium), salmonella typhimurium (Salmonella typhimurium) and the like, but only few sources of lysine decarboxylase such as lysine decarboxylase from escherichia coli and Hafnia alvei are studied intensively.
CN105316270B, CN105368766a and CN104498519a disclose the construction of different genetically engineered strains using the inducible lysine decarboxylase CadA of escherichia coli, whole cell catalytic synthesis of pentamethylene diamine. Meanwhile, the temperature regulation type promoter pR-pL and the signal peptide pelBs are used for modifying an over-expression vector, and T7CadB is integrated into chassis cell genome by the institute of microorganisms of China academy of sciences, so that the yield of pentanediamine is improved. CN106148373A, EP3118312B1 and US7189543 sequence-modified escherichia coli induced lysine decarboxylase CadA, and japanese monosodium glutamate company screened mutants with higher thermal stability by directed evolution of CadA, and triple well chemical company also disclosed mutants with 10-20% increased activity in its patents.
However, the mutant enzyme systems are only limited to transformation under the induction type lysine decarboxylase CadA of the escherichia coli, the source is single, the activity and the catalytic strength of the mutant strain in the high-concentration catalytic conversion process of the lysine are low, the cell cycle utilization rate is poor, the operation time and the production cost are increased, and the yield of the pentanediamine is reduced so as to restrict the industrialized development.
Disclosure of Invention
In view of the problems existing in the prior art, the invention aims to provide lysine decarboxylase for synthesizing pentanediamine and application thereof, wherein the lysine decarboxylase comprises an amino acid sequence of the lysine decarboxylase, a nucleotide sequence encoding the lysine decarboxylase, a gene expression vector and recombinant engineering bacteria.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a lysine decarboxylase for synthesizing pentamethylenediamine, which catalyzes the production of pentamethylenediamine from L-lysine, the lysine decarboxylase having any one of the amino acid sequences shown in (I), (II) or (III):
(I) An amino acid sequence as shown in any one of SEQ ID NO. 1-4;
(II) an amino acid sequence having a homology of not less than 75% with the amino acid sequence shown in any one of SEQ ID NO.1 to 4;
(II) an amino acid sequence obtained by modifying, substituting, deleting or adding at least one amino acid to the amino acid sequence shown in any one of SEQ ID NO. 1-4.
The invention provides a novel efficient lysine decarboxylase, which can catalyze L-lysine to generate pentanediamine, and the lysine decarboxylase can be obtained by constructing an expression vector and inducing expression after genetic engineering bacteria, and can catalyze lysine hydrochloride to synthesize the pentanediamine by whole cells. Meanwhile, the lysine decarboxylase provided by the invention is still stable at higher temperature and pH, and the yield is higher.
In the invention, the lysine decarboxylase represented by SEQ ID NO.1 is marked as LdcEdw, and the similarity of the amino acid sequence and the escherichia coli cadA is 86.7%; the lysine decarboxylase represented by SEQ ID No.2 was designated LdcAer, which has a similarity of 75.77% to E.coli cadA; the lysine decarboxylase represented by SEQ ID NO.3 was named LdcSal, and the similarity of the amino acid sequence to E.coli cadA was 92.3%; the lysine decarboxylase represented by SEQ ID No.4 was designated LdcKle, and the similarity of the amino acid sequence to E.coli cadA was 94.4%. While amino acid sequences have a large influence on the steric structure and enzymatic properties of enzymes, sometimes small amounts of amino acid differences may also lead to large differences in properties between the two enzymes, which effect is not to be expected. Thus, it is the differences that give LdcEdw different enzymatic properties than E.coli CadA, and that enable efficient conversion of lysine hydrochloride at high concentrations.
Meanwhile, in addition to the above four sequences, an amino acid sequence having homology of not less than 75% (for example, homology of more than 75%, 78%, 80%, 84%, 85%, 88%, 90%, 92%, 94%, 96%, 98% or 99% or the like) with the amino acid sequence shown in any one of SEQ ID NO.1 to 4 can also realize conversion of lysine hydrochloride.
Lysine decarboxylase genes derived from bacteria such as Hafnia alvei, alkali-resistant Bacillus (Bacillus halodurans), bacillus cereus (Bacillus cereus), cadaverium (Bacillus cadaveris), burkholderia (Burkholderia vietnamensia), chromobacterium cyanogen (Chromobacterium violaceum), edwardsiella tarda (Edwardsiella tarda), vibrio cholerae (Vibrio cholerae), mao Lian mold (Streptomyces polosus), zygomonas ruminant (Selenomonas ruminantium), salmonella typhimurium (Salmonella typhimurium), salmonella bongo (Salmonella bongori), serratia (Serratia), bordea (Bordetella), vibrio cholerae (Vibrio cholerae), aeromonas (Aeromonas), klebsiella (Klebsiella), and mutants thereof are disclosed.
Preferably, the lysine decarboxylase is derived from Edwardsiella tarda (Edwardsiella tarda), klebsiella, aeromonas (Aeromonas) or Salmonella bongolica (Salmonella bongori).
In a second aspect, a nucleotide encoding the lysine decarboxylase of the first aspect, the nucleotide having any one of the nucleotide sequences shown in (i), (ii) or (iii):
(i) A nucleotide sequence encoding the lysine decarboxylase of claim 1 or 2;
(ii) A nucleotide sequence encoding a lysine decarboxylase as set forth in any one of SEQ ID NO. 1-4;
(iii) Nucleotide sequence as shown in any one of SEQ ID NO. 5-8.
The nucleic acid sequence of LdcEdw is shown in SEQ ID NO. 5. The nucleic acid sequence is subjected to codon optimization, has a GC content of 43%, and has a similarity of 74.5% with the nucleic acid sequence of the escherichia coli CadA. The nucleic acid sequence of the LdcAer is shown in SEQ ID NO. 6. The nucleic acid sequence is subjected to codon optimization, the GC content is 45%, and the similarity with the nucleic acid sequence of the escherichia coli CadA is 69.2%. The LdcSal has a nucleic acid sequence shown in SEQ ID NO. 7. The nucleic acid sequence is subjected to codon optimization, has a GC content of 43%, and has a similarity of 78.1% with the nucleic acid sequence of the escherichia coli CadA. The nucleic acid sequence of LdcKle is shown in SEQ ID NO. 8. The nucleic acid sequence is subjected to codon optimization, has a GC content of 43%, and has a similarity of 78.6% with the nucleic acid sequence of the escherichia coli CadA.
In a third aspect, the invention also provides a gene expression vector. The gene expression vector includes: a nucleotide sequence encoding an amino acid sequence according to the first aspect or a nucleotide according to the second aspect.
Preferably, the gene expression vector is a pET plasmid, preferably a petdet plasmid. The expression vector can be pETDuet or various expression vectors commonly used in the field for expressing target genes in escherichia coli. Preferably, the gene expression vector further comprises a nucleotide sequence encoding a lysine pentylene diamine antiport protein.
In a fourth aspect, the present invention also provides a method for constructing a gene expression vector according to the third aspect, comprising the steps of:
inserting a nucleotide sequence encoding the amino acid sequence according to the first aspect or a nucleotide according to the second aspect between restriction sites of a plasmid to obtain the gene expression vector.
The construction method further comprises the operation of inserting a lysine-pentanediamine antiporter gene.
Preferably, the lysine-pentanediamine antiporter gene is preceded by a signal peptide.
Preferably, the signal peptide comprises a periplasmic space secretion signal peptide of E.coli.
The expression vector is exemplified by pelB signal peptide, but not limited to the signal peptide, and can be common to escherichia coli, such as dsbA, hlyA, lamB, malE, ompA, ompF, ompT, phoA and the like.
The construction method specifically comprises the following steps of:
the lysine decarboxylase gene ldc and the gene ca dB encoding the lysine-pentanediamine antiport protein are respectively inserted between NcoI/SacI and BglII/PacI restriction enzyme cutting sites of pETDuet plasmid to construct plasmid pETDuet-ldc-ca dB, pelB is introduced before the ca dB sequence, and the genes are connected through NdeI/BglII restriction enzyme cutting sites, and finally, the constructed expression vector is pETDuet-ldc-pelB-ca dB.
In a fifth aspect, a recombinant engineering bacterium for synthesizing pentanediamine, comprising the gene expression vector of the third aspect and/or a nucleotide encoding the lysine decarboxylase of the first aspect. The engineering bacteria can be E.coli BL21 (DE 3).
In a sixth aspect, the present invention also provides a method for producing pentamethylene diamine using the host cell as described in the fifth aspect, the method comprising the steps of:
and (3) culturing the recombinant engineering bacteria, centrifuging and re-suspending the bacterial liquid obtained after induction to obtain bacterial suspension, mixing the bacterial suspension with a buffer solution containing lysine hydrochloride and pyridoxal phosphate (PLP), reacting, and centrifuging to obtain the pentanediamine.
As a preferable embodiment of the present invention, the reaction temperature is 35 to 65 ℃, for example, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃, 60 ℃, 65 ℃ or the like, and the reaction time is 0.5 to 24 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 15 hours, 16 hours, 18 hours, 20 hours, 24 hours or the like, and preferably 1 to 4 hours.
Preferably, the shaking rate at the time of the reaction is 400 to 800rpm, and may be 400rpm, 500rpm, 550rpm, 600rpm, 650rpm, 700rpm, 800rpm, or the like, for example.
The rotational speed at the time of centrifugation is preferably 8000 to 12000rpm, and may be 8000rpm, 8500rpm, 9000rpm, 10000rpm, 10500rpm, 11000rpm, 12000rpm, or the like, and the time is preferably 1 to 3 minutes, and may be 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, or the like, for example.
Preferably, the molar concentration of lysine hydrochloride in the buffer is 0.1 to 3M, and may be, for example, 0.1M, 0.5M, 0.8M, 1M, 1.2M, 1.5M, 1.8M, 2M, 2.5M, or 3M, etc.
Preferably, the molar concentration of PLP in the buffer is 0.1 to 0.5mM, and may be, for example, 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, or the like.
Preferably, the buffer comprises any one of sodium acetate buffer, phosphate buffer, tris-HCl buffer or sodium carbonate buffer, preferably phosphate buffer.
Preferably, the pH of the buffer is 5 to 11, for example, 5, 6, 6.4, 7, 7.2, 7.5, 8, 9, 10 or 11, etc., preferably 6 to 10; the buffer is the same as the solution used in the resuspension.
Preferably, after the induced bacterial liquid is centrifuged, the method further comprises a freezing operation, wherein the freezing operation is as follows: freezing at-80deg.C for more than 1 hr.
Preferably, the method for preparing pentamethylene diamine comprises the following steps:
(1) Centrifuging the bacterial liquid obtained after the recombinant engineering bacteria are cultured and induced, and re-suspending the bacterial liquid after freezing to obtain bacterial suspension;
(2) Mixing the bacterial suspension with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, wherein the molar concentration of the lysine hydrochloride in the buffer solution is 0.1-3M, the molar concentration of the pyridoxal phosphate is 0.1-0.5 mM, and the buffer solution is phosphate buffer solution with the pH value of 6-10;
(3) Oscillating at 400-800 rpm at 35-65 deg.c for 1-4 hr, and centrifuging at 8000-12000 rpm for 1-3 min to obtain pentandiamine.
In the invention, after synthesizing the pentanediamine, the method for detecting lysine and the pentanediamine comprises the following steps:
(1) 600. Mu.L of 50mM boric acid buffer pH 9, 200. Mu.L of methanol, 60. Mu.L of diluted sample, 130. Mu.L of ddH were added to the reaction system 2 O and 10 mu L of 1M ethoxymethylene diethyl malonate (DEEMM) are placed at room temperature for reaction for 10min, and then the reaction is carried out by transferring to a water bath with the temperature of 60-80 ℃ for 1-2 h, and the reaction is terminated;
(2) Detecting by using a reversed-phase high performance liquid chromatography ultraviolet (nm) detector, wherein the mobile phase A is 100% acetonitrile; mobile phase B was 25mM sodium acetate buffer solution pH 4.8, flow rate 0.5mL/min; the detection column is C18; column detection temperature: 35 ℃; sample injection amount: 2-10 mu L; wavelength: 284nm.
Gradient elution is adopted: 0min A to B is 20:80;2min A: B is 25:75;27min A:B is 62.5:37.5;27.01min A:B is 20:80;37min A to B is 20 to 80; at the end of 37.01min, the gradient elution is not limited to the gradient elution ratio described above.
In a sixth aspect, the invention also provides the use of a lysine decarboxylase as described in the first aspect, a gene expression vector as described in the third aspect or a recombinant host cell as described in the fifth aspect in the synthesis of bio-based pentylene diamine.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The invention provides a novel lysine decarboxylase capable of efficiently synthesizing pentanediamine, which can be obtained by constructing an expression vector and inducing expression after genetic engineering bacteria, and can be kept stable at the pH value of 5-9 and the temperature of 40-60 ℃ when synthesizing the pentanediamine by catalyzing lysine hydrochloride through whole cells, and has higher catalytic efficiency and catalytic strength of 136-204 g/L/h;
(2) The novel lysine decarboxylase provided by the invention can realize near complete conversion of high-concentration lysine hydrochloride, the conversion rate of the pentanediamine is maximum and can reach 100% at the pH of 6.5 and the temperature of 50 ℃, the catalytic strength of the novel lysine decarboxylase can reach 204g/L/h, the activity and the catalytic strength of the novel lysine decarboxylase are obviously higher than those of the existing reported escherichia coli CadA and mutants thereof, the high-concentration pentanediamine can be efficiently synthesized, and the novel lysine decarboxylase has extremely high industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of the pETDuet-ldcEdw-pelB-calB expression vector constructed in example 1.
FIG. 2 is a graph showing the change in LdcEdw whole cell catalysis with reaction time in example 5.
FIG. 3 is a graph showing the change in LdcAer whole cell catalysis with reaction time in example 5.
Detailed Description
The following embodiments are further described with reference to the accompanying drawings, but the following examples are merely simple examples of the present invention and do not represent or limit the scope of the invention, which is defined by the claims.
In the present invention, there is no particular limitation on the type of expression vector, and various expression vectors commonly used in the art, such as plasmids, etc., capable of expressing a gene of interest in E.coli can be used. The construction method of the expression vector may employ various methods commonly used in the art, such as a method in which the desired gene is ligated into the vector after cleavage.
In the following examples, the HPLC detectors used were: an SPD-20A diode array detector; the detection column is as follows: c18 column (Shim-pack GIST-HP-C18 column, 2.1X100 mm,3 μm particleseize).
The detection method of lysine hydrochloride and pentanediamine used in the invention comprises the following specific steps:
600. Mu.L of 50mM boric acid buffer pH 9, 200. Mu.L of methanol, 60. Mu.L of the reaction solution and 130. Mu.L of ddH were added to the reaction system 2 O and 10. Mu.L of 1M diethyl ethoxymethylenemalonate (DEEMM) were reacted at room temperature for 10 minutes, then transferred to 70℃and allowed to stand for 2 hours to terminate the reaction, and detected by High Performance Liquid Chromatography (HPLC).
HPLC detector: an SPD-20A diode array detector; detection column: a C18 column; detecting the temperature: 35 ℃; sample injection amount: 5 μl, wavelength: 284nm.
Wherein mobile phase A is acetonitrile; mobile phase B was 25mm sodium acetate buffer at pH 4.8; the flow rate is 0.5mL/min; time program (mobile phase B ratio): 80% of 0 min; 2min 75%;22min51.7%;22.01min 80%;27min 80%.
Example 1
The present example provides a gene expression vector containing lysine decarboxylase and an engineering strain expressing the same.
The lysine decarboxylase is named as LdcEdw, the amino acid sequence of the lysine decarboxylase is SEQ ID NO.1, and the similarity with the escherichia coli cadA is 86.7%; after codon optimization, the nucleotide sequence of the synthesized LdcEdw is SEQ ID NO.5, and the GC content of the nucleotide sequence is 43 percent, and the nucleotide sequence has 74.5 percent of similarity with the nucleic acid sequence of the CadA of the escherichia coli.
The lysine decarboxylase is named as LdcAer, the amino acid sequence of the lysine decarboxylase is SEQ ID NO.2, and the similarity with the escherichia coli cadA is 75.77%; after codon optimization, the nucleotide sequence of the synthesized LdcAer is SEQ ID NO.6, the GC content of the nucleotide sequence is 45%, and the similarity with the nucleic acid sequence of the escherichia coli cadA is 69.2%.
The lysine decarboxylase is named as LdcSal, the amino acid sequence of the lysine decarboxylase is SEQ ID NO.3, and the similarity with the escherichia coli cadA is 92.3%; after codon optimization, the nucleotide sequence of LdcSal is synthesized as SEQ ID NO.7, and the GC content of the nucleotide sequence is 43 percent, and the similarity with the nucleic acid sequence of the escherichia coli cadA is 78.1 percent.
The lysine decarboxylase is named as LdcKle, the amino acid sequence of the lysine decarboxylase is SEQ ID NO.4, and the similarity with the escherichia coli cadA is 94.4%; after codon optimization, the nucleotide sequence of the synthesized LdcKle is SEQ ID NO.8, and the GC content of the nucleotide sequence is 43 percent, and the nucleotide sequence has 78.6 percent of similarity with the nucleic acid sequence of the CadA of the escherichia coli.
Meanwhile, in order to compare the functions of the lysine decarboxylase provided by the invention, the invention also uses EcCadA (GenBank: WP_ 001295383.1) from escherichia coli as comparison; and LdcEdw, ldcAer, ldcSal, ldcKle and CadA are respectively constructed between NcoI/SacI of the petdet plasmid; meanwhile, cadB (GenBank: WP_ 000092909.1) genes are constructed between BglII/PacI of the pETDuet plasmid; the His tag is inserted before the nucleotide sequence encoding the protein;
and introducing a signal peptide pelB before a calB sequence to construct pETDuet-ldcEdw-pelB-calB (shown in figure 1), pETDuet-ldcAer-pelB-calB, pETDuet-ldcSal-pelB-calB, pETDuet-ldcKle-pelB-calB and pETDuet-EccadA-pelB-calB plasmids, respectively transferring into E.coli BL21 (DE 3) chassis cells, constructing genetic engineering strains for synthesizing pentanediamine by whole cell catalysis, named EDW, AER, SAL, KLE and WT, and storing at-80 ℃.
Example 2
This example is a whole cell catalytic comparison of gene expression vectors containing lysine decarboxylase LdcEdw, ldcAer and ecada, respectively.
The engineering strains EDW, AER and WT obtained in example 1 were cultured overnight at 37℃in 5mL of LB medium to which 100mg/L of ampicillin was added to obtain a seed solution.
Then, the seed solution was transferred into 50mL of LB medium containing 100mg/L of ampicillin according to a transfer amount of 1% by volume, and cultured at 37℃until OD 600 IPTG (isopropyl-. Beta. -D-thiogalactopyranoside) was added at a final concentration of 0.1mM at 0.6 to induce and the culture was continued at 20℃for 20 hours. Centrifugation at 4000rpm, collection of the cells and storage at-80 ℃.
The cells were resuspended in 50mM sodium acetate buffer pH 6 at a pH of 0.1mM PLP, 1M lysine hydrochloride, OD 1.5 at 45℃and 500rpm for 1h, centrifuged at 12000rpm for 2min, and the supernatant was diluted and assayed for lysine hydrochloride and pentamethylenediamine content.
The final detection result can be seen as follows: the yield of EDW whole cell catalytic pentanediamine is 109g/L and is 1.6 times of the yield of WT; the yield of AER whole cell catalyzed pentamethylenediamine was 89.4 g/L1.3 times the yield of WT.
Example 3
Whole cell catalysis by lysine decarboxylases LdcEdw and LdcAer at different pH values was examined in this example
The engineering strain EDW obtained in example 1 was cultured overnight at 37℃in 5mL of LB medium containing 100mg/L of ampicillin to obtain a seed solution.
The seed solution is transferred into 50mL of LB culture medium added with 100mg/L of ampicillin antibiotics according to the transfer amount of 1% by volume, and is cultured at 37 ℃ until OD 600 At 0.6, IPTG was added at a final concentration of 0.1mM for induction, and after further culturing at 20℃for 20 hours, centrifugation was carried out at 4000rpm to collect the cells, which were stored at-80 ℃.
After the cells were resuspended in 50mM buffers of different pH, 500. Mu.L of whole cell catalysis was performed, wherein PLP was 0.1mM, lysine hydrochloride was 1M, and the OD of the bacterial suspension was 1.5.
The reaction was catalyzed at 45℃for 1 hour at 500rpm and centrifuged at 12000rpm for 2 minutes, and the supernatant was diluted and examined for the content of pentamethylene diamine. The whole cell catalysis results of EDW and AER at pH 4.5-7.5 are shown in the following table 1, wherein the EDW is highest when the pH of the reaction system reaches 6.5, the conversion rate can reach 99.47%, lysine hydrochloride is basically and completely converted, and then the yield of the pentamethylenediamine starts to decrease when the pH of the system is increased again; AER reached its maximum at pH 6 of the reaction system, at which point the conversion was 92.26%, and when pH increased to 7.5, the yield of pentamethylenediamine was reduced to 86.92%.
TABLE 1
Example 4
The whole cell catalytic effect of different temperatures on lysine decarboxylases ldcredw and LdcAer was examined in this example.
The engineering strain EDW obtained in example 1 was cultured overnight at 37℃in 5mL of LB medium containing 100mg/L of ampicillin to obtain a seed solution.
The seed solution is transferred into 50mL of LB medium added with 100mg/L of ampicillin antibiotics according to the transfer amount of 1% by volume, and is cultured at 37 ℃ until OD 600 At 0.6, IPTG was added at a final concentration of 0.1mM to induce, culturing was continued at 20℃for 20 hours, centrifugation was performed at 4000rpm, and the cells were collected and stored at-80 ℃.
The bacteria were resuspended in pH 8 buffer and subjected to 500. Mu.L whole cell catalysis, wherein PLP 0.1mM, lysine hydrochloride 1M, and the OD of the bacterial suspension was 1.5.
Catalyzing for 1h at 35-65 ℃ and 500rpm, centrifuging for 2min at 12000rpm, taking supernatant, diluting, and detecting the content of the pentanediamine. The whole cell catalysis results of EDW and AER at 35-65deg.C are shown in Table 2 below, and it is not difficult to find that the yield of pentamethylenediamine increases and decreases with increasing temperature, and the conversion of EDW pentamethylenediamine is maximum at 50deg.C and 95.95%.
TABLE 2
Example 5
Shake flask whole cell catalytic ability of lysine decarboxylases ldcredw and LdcAer was verified in this example.
The engineering strains EDW and AER obtained in example 1 were cultured overnight at 37℃in 5mL of LB medium containing 100mg/L of ampicillin to obtain a seed solution.
The seed solution is transferred into 50mL of LB medium added with 100mg/L of ampicillin antibiotics according to the transfer amount of 1% by volume, and is cultured at 37 ℃ until OD 600 At 0.6, IPTG was added at a final concentration of 0.1mM to induce, culturing was continued at 20℃for 20 hours, centrifugation was performed at 4000rpm, and the cells were collected and stored at-80 ℃.
The concentration of the bacterial cells in the system, OD of which is about 10 and the concentration of the substrate of which is 2M, is carried out by using a phosphate buffer solution with pH of 8 and a temperature of 50 ℃ to carry out 20mL shake flask whole cell catalysis, samples are taken at 0h, 1h and 2h respectively, and the content of the pentanediamine is detected after the supernatant is diluted.
The results of LdcEdw detection are shown in FIG. 2, in which the abscissa represents time (h) and the ordinate represents content (mM), and significant bubble generation was observed 30min before whole cell catalytic reaction. As can be seen from the figure, the amount of lysine hydrochloride gradually decreases with time, and the relative amount of pentamethylenediamine gradually increases, and after 2 hours of reaction, the lysine hydrochloride has been almost completely converted, and the pentamethylenediamine yield is 202.3g/L. The OD of the concentrated bacteria in the system is increased to 15, the substrate concentration is 2M, after 1h of reaction, the lysine hydrochloride is almost completely converted, the yield of the pentanediamine is 204g/L, and the production intensity of the pentanediamine is 204g/L/h.
The LdcAer detection result is shown in FIG. 3, the abscissa is time (h), the ordinate is content (unit mM), obvious bubbles are generated 30min before the whole cell catalytic reaction, the reaction lasts for 4 hours, the yield of the pentanediamine is 198.4g/L, and the catalytic rate can reach 136g/L/h.
Example 6
The in vitro catalytic capacity of lysine decarboxylases ldcredw and LdcAer at different concentrations was demonstrated in this example.
The engineering strains EDW and AER obtained in example 1 were cultured overnight at 37℃in 5mL of LB medium containing 100mg/L of ampicillin to obtain a seed solution.
The seed solution is transferred into 50mL of LB culture medium added with 100mg/L of ampicillin antibiotics according to the transfer amount of 1% by volume, and is cultured at 37 ℃ until OD 600 At 0.6, IPTG was added at a final concentration of 0.1mM for induction, and after further culturing at 20℃for 20 hours, centrifugation was carried out at 4000rpm to collect the cells, which were stored at-80 ℃.
The cells were disrupted by an ultrasonic disrupter at 40-60% of power, centrifuged at 8000rpm, the cell fragments were precipitated, filtered with a 0.22 μm filter, purified by a Hisnap purification column of 5ml on an AKTA protein purifier, the preservation solution was replaced with a 5mL HiTrap Desalting desalting column, and the concentrations of the purified lysine decarboxylases LdcEdw and LdcAer were measured by a BCA protein quantification method.
The in vitro catalytic reaction system of lysine decarboxylase LdcEdw was 500. Mu.L, lysine hydrochloride concentration was 1.5M, PLP concentration was 0.1mM, and after diluting LdcEdw pure enzyme 10-fold and 50-fold, 190. Mu.L of the diluted pure enzyme was added to the reaction system. Catalytic reaction was carried out at 50℃and pH 6.5 at 500rpm for 1 hour, centrifugation was carried out at 12000rpm for 2 minutes, and the content of pentamethylene diamine was measured after dilution of the supernatant. In the in-vitro enzyme catalysis of lysine decarboxylase LdcEdw, 60 mug of pure enzyme is added for catalytic reaction for 1h, and the conversion rate of the pentanediamine can reach 100%, namely, 60 mug of pure enzyme can catalyze 1.5M lysine hydrochloride, so that the lysine hydrochloride is completely converted into the pentanediamine.
In the in vitro enzyme catalysis of lysine decarboxylase LdcAer, the reaction system was 500. Mu.L, lysine hydrochloride at 1.5M and PLP at 0.1mM, and 190uL was added by 50-fold dilution of LdcAer pure enzyme. The reaction was catalyzed at 50℃and pH 6 at 500rpm for 1h, centrifuged at 12000rpm for 2min, the supernatant was taken and diluted, and the reaction was carried out as described in the embodiment, 60. Mu.g of pure enzyme was added for 1h, and the conversion of pentylene diamine could reach 94.43%.
In conclusion, the lysine decarboxylase LdcEdw provided by the invention can be used for efficiently catalyzing and synthesizing the pentanediamine, the optimal catalysis temperature of whole cell catalysis is 50 ℃, the optimal pH of a catalysis system is 6.5, the catalysis strength can reach 204g/L/h, and the complete conversion of high-concentration lysine hydrochloride can be realized; the optimal catalytic temperature of LdcAer whole-cell catalysis is 50 ℃, the optimal pH of a catalytic system is 6, and the conversion rate of the pentanediamine can reach 97.2%. In the invention, recombinant engineering bacteria are constructed by the amino acid sequences shown in SEQ ID NO.3 and SEQ ID NO.4, and can also express the pentanediamine with high efficiency, and only experimental results are written here for the sake of space and simplicity: the amino acid sequences shown in SEQ ID No.3 and SEQ ID No.4 are used for constructing strains by the methods of examples 1 and 2, in whole cell catalysis added with 1M lysine hydrochloride, the reaction is stable at the system pH of 5-10 and the temperature of 40-55 ℃, the optimal catalysis temperature is 50 ℃, the optimal pH of the catalysis system is 6 and 7.5 respectively, and the conversion rate of the pentanediamine can reach 72.5%.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (10)
1. A nucleotide encoding a lysine decarboxylase for synthesizing pentamethylenediamine, wherein the nucleotide has any one of the nucleotide sequences shown in (i) or (ii):
(i) A nucleotide sequence encoding a lysine decarboxylase as set forth in any one of SEQ ID NO. 1-4;
(ii) A nucleotide sequence encoding a lysine decarboxylase having at least 75% homology to the amino acid sequence shown in any one of SEQ ID No.1 to 4;
(iii) Nucleotide sequence as shown in any one of SEQ ID NO. 5-8.
2. The nucleotide according to claim 1, wherein the lysine decarboxylase is derived from any one of edwardsiella tarda, hafnia alvei, bacillus alcaligenes, bacillus cereus, bacillus cadavermitilis, burkholderia, pseudomonas, vibrio cholerae, mao Lian mold, thomonas ruminant, salmonella typhimurium, salmonella pangolicum, serratia, bordetella, vibrio cholerae, aeromonas or klebsiella.
3. The nucleotide according to claim 1, wherein the lysine decarboxylase is derived from edwardsiella tarda, klebsiella, aeromonas or salmonella pangolicum.
4. A gene expression vector for synthesizing pentamethylenediamine, the gene expression vector comprising: a nucleotide encoding any one of claims 1 to 3;
preferably, the gene expression vector is a pET plasmid, preferably a petdet plasmid;
preferably, the gene expression vector further comprises a nucleotide sequence encoding a lysine pentylene diamine antiport protein.
5. The method for constructing a gene expression vector according to claim 4, comprising the steps of:
inserting the nucleotide according to any one of claims 1 to 3 between restriction sites of a plasmid to obtain the gene expression vector.
6. The method according to claim 5, wherein the method further comprises inserting a lysine-pentanediamine antiporter gene;
preferably, the lysine-pentanediamine antiporter gene is preceded by a signal peptide;
preferably, the signal peptide comprises an escherichia coli periplasmic space secretion signal peptide;
preferably, the signal peptide comprises any one of dsbA, hlyA, lamB, malE, ompA, ompF, ompT, phoA or pelB, preferably pelB.
7. Recombinant engineering bacterium for synthesizing pentanediamine, characterized by comprising the gene expression vector of claim 4 and/or the nucleotide of any one of claims 1-3.
8. A method for preparing pentanediamine by using the recombinant engineering bacteria of claim 7, comprising the following steps:
and (3) culturing the recombinant engineering bacteria, centrifuging and re-suspending the bacterial liquid obtained after induction to obtain bacterial suspension, mixing the bacterial suspension with a buffer solution containing lysine hydrochloride and pyridoxal phosphate for reaction, and centrifuging to obtain the pentanediamine.
9. The method according to claim 8, wherein the reaction is carried out at a temperature of 35 to 65 ℃ for a time of 0.5 to 24 hours;
preferably, the mixing reaction time is 1-4 hours;
preferably, the oscillation rate at the time of the reaction is 400 to 800rpm;
preferably, the rotating speed during centrifugation is 8000-12000 rpm, and the time is 1-3 min;
preferably, the molar concentration of lysine hydrochloride in the buffer solution is 0.1-3M;
preferably, the molar concentration of pyridoxal phosphate in the buffer is 0.1 to 0.5mM;
preferably, the buffer solution comprises any one of sodium acetate buffer solution, phosphate buffer solution, tris-HCl buffer solution or sodium carbonate buffer solution, and is preferably phosphate buffer solution;
preferably, the pH of the buffer is 5 to 11, preferably 6 to 10;
preferably, the method comprises the steps of:
(1) Centrifuging the bacterial liquid obtained after the recombinant engineering bacteria are cultured and induced, and re-suspending the bacterial liquid after freezing to obtain bacterial suspension;
(2) Mixing the bacterial suspension with a buffer solution containing lysine hydrochloride and pyridoxal phosphate, wherein the molar concentration of the lysine hydrochloride in the buffer solution is 0.1-3M, the molar concentration of the pyridoxal phosphate is 0.1-0.5 mM, and the buffer solution is phosphate buffer solution with the pH value of 6-10;
(3) Oscillating at 400-800 rpm at 35-65 deg.c for 1-4 hr, and centrifuging at 8000-12000 rpm for 1-3 min to obtain pentandiamine.
10. Use of a nucleotide according to any one of claims 1 to 3, a gene expression vector according to claim 4 or a recombinant engineering bacterium according to claim 7 for bio-based synthesis of pentamethylenediamine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311789375.6A CN117737102A (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010634482.1A CN113881657B (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
| CN202311789375.6A CN117737102A (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010634482.1A Division CN113881657B (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117737102A true CN117737102A (en) | 2024-03-22 |
Family
ID=79012824
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010634482.1A Active CN113881657B (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
| CN202311789375.6A Pending CN117737102A (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010634482.1A Active CN113881657B (en) | 2020-07-02 | 2020-07-02 | Lysine decarboxylase for synthesizing pentanediamine and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN113881657B (en) |
| WO (1) | WO2022001121A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112746067B (en) * | 2021-01-26 | 2023-10-31 | 洛阳华荣生物技术有限公司 | Lysine decarboxylase mutants for preparing D-ornithine |
| WO2024000368A1 (en) * | 2022-06-30 | 2024-01-04 | 江南大学 | Recombinant escherichia coli and construction method therefor, and method for synthesizing 1,5-pentanediamine |
| CN114990045B (en) * | 2022-06-30 | 2023-08-22 | 江南大学 | Recombinant escherichia coli, construction method thereof and method for synthesizing 1, 5-pentanediamine |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK0796912T4 (en) * | 1994-12-09 | 2013-03-25 | Ajinomoto Kk | New lysine decarboxylase gene and method for preparing L-lysine |
| CN104498519A (en) * | 2014-12-19 | 2015-04-08 | 南京工业大学 | Expression recombinant vector and application thereof |
| CN107177641A (en) * | 2016-11-03 | 2017-09-19 | 中国科学院天津工业生物技术研究所 | New lysine decarboxylase and its application |
| WO2018120025A1 (en) * | 2016-12-30 | 2018-07-05 | Cathay R&D Center Co., Ltd. | Lysine decarboxylases having modifications at titratable amino acids |
| EP3562837A4 (en) * | 2016-12-30 | 2020-12-30 | Cathay Biotech Inc. | Modified lysine decarboxylase enzymes |
| EP3625339A4 (en) * | 2017-05-16 | 2021-01-20 | Cathay Biotech Inc. | Modifications to lysine decarboxylase enzymes |
| CN109402189B (en) * | 2018-12-06 | 2020-08-21 | 黑龙江伊品新材料有限公司 | Method for producing pentanediamine by fermentation and extraction method thereof |
| CN111117940B (en) * | 2019-12-04 | 2022-06-28 | 天津大学 | Escherichia coli engineering bacterium and method for high yield of pentamethylene diamine |
-
2020
- 2020-07-02 CN CN202010634482.1A patent/CN113881657B/en active Active
- 2020-07-02 CN CN202311789375.6A patent/CN117737102A/en active Pending
-
2021
- 2021-02-09 WO PCT/CN2021/076305 patent/WO2022001121A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| CN113881657A (en) | 2022-01-04 |
| WO2022001121A1 (en) | 2022-01-06 |
| CN113881657B (en) | 2024-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113881657B (en) | Lysine decarboxylase for synthesizing pentanediamine and application thereof | |
| US20200115695A1 (en) | Nitrilase mutants and application thereof | |
| CN107099516B (en) | 7 β -hydroxysteroid dehydrogenase mutant and application thereof in synthesis of ursodeoxycholic acid | |
| CN112662637B (en) | Formate dehydrogenase mutant and preparation method and application thereof | |
| CN112831488B (en) | Glutamic acid decarboxylase and gamma-aminobutyric acid high-yield strain | |
| CN109852644A (en) | A method of preparing Bu Waxitan intermediate | |
| CN104212850B (en) | The method that 1 cyanocyclohexanoic guanidine-acetic acid is prepared using nitrilase engineering bacteria | |
| CN116515802A (en) | Asparaase mutant and synthesis and application of engineering bacteria thereof | |
| CN113881719A (en) | Method for synthesizing 1, 5-pentanediamine through whole-cell catalysis | |
| CN115125229B (en) | Lysine decarboxylase mutant for synthesizing pentanediamine | |
| CN115725535B (en) | N-deoxyribotransferase and application thereof in preparation of deoxynucleosides | |
| CN111019915A (en) | Application of carbonyl reductase mutant in synthesis of chiral ortho-halogenated- α -phenethyl alcohol | |
| CN110452920A (en) | A kind of genetic engineering bacterium and with D, L- mandelic acid is the method that substrate prepares L- phenylglycine | |
| CN116004489B (en) | Recombinant escherichia coli for producing NMN and application thereof | |
| CN118222540B (en) | Heat-resistant plastic hydrolase AtPETase mutant and application thereof | |
| CN115247144B (en) | Genetically engineered bacterium for producing L-threo-3-hydroxy aspartic acid and application thereof | |
| CN112852912B (en) | Method for synthesizing 7-aminodesacetoxycephalosporanic acid | |
| CN114958894B (en) | Construction method and application of spermidine synthetic multienzyme complex based on CcmK2 fibrous protein | |
| CN115786286B (en) | Gamma-glutamine methylamine synthetase mutant, recombinant thereof and application thereof in continuous catalysis coupling ATP regeneration system | |
| CN116179499B (en) | Dipeptide synthetase mutant, recombinant thereof and application thereof in continuous circulation catalysis of high-concentration substrate | |
| CN115894639B (en) | Auxiliary invasin residue mutant protein, recombinant thereof and application thereof in exoenzyme immobilization process | |
| CN111575258B (en) | Carbonyl reductase EbSDR8 mutant and construction method and application thereof | |
| CN116949078A (en) | Construction method and application of engineering bacteria for high yield of pseudouridine | |
| CN118755652A (en) | Recombinant escherichia coli and method for synthesizing 1, 5-pentanediamine by using same | |
| CN116121310A (en) | Method for synthesizing dexketoprofen by amidase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |