Key enzyme for synthesizing butanediamine and application thereof

Abstract

The invention provides a key enzyme for synthesizing butanediamine and application of the key enzyme, and belongs to the technical field of bioengineering. The invention provides a gene for synthesizing ornithine decarboxylase. The nucleotide sequence of the ornithine decarboxylase is shown as SEQ ID NO. 7. By further optimizing the reaction system, the butanediamine synthesis capability is improved, and butanediamine can be synthesized more efficiently.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a key enzyme for synthesizing butanediamine and application thereof. Background Art

[0002] Butanediamine is the core monomer for producing nylon material PA46 and other high-temperature nylon materials. At present, the industrial production of butanediamine mainly relies on chemical production, which is synthesized by hydrogenation reaction of butanedinitrile and hydrogen cyanide, and the synthesis process is highly toxic and flammable. The biosynthesis of butanediamine mainly includes fermentation and whole-cell catalysis. The fermentation method synthesizes butanediamine by transforming the metabolic pathway of engineered strains, and obtains butanediamine by fermentation of strains. This method has the problems of complex metabolic pathways, low yield and low production efficiency. Compared with the fermentation method for synthesizing bio-based butanediamine, the whole-cell catalysis method uses arginine or ornithine as the reaction substrate, and uses arginine decarboxylase or ornithine decarboxylase to catalyze the substrate to produce butanediamine, which can shorten the reaction path and significantly improve the conversion efficiency. It is a relatively cheap and efficient method. However, due to the lack of efficient ornithine decarboxylase, the production of high-level putrescine based on whole-cell catalysis is still limited. Therefore, there is an urgent need to develop a key enzyme for efficient synthesis of butanediamine in this field to lay the foundation for large-scale biological production of butanediamine. Summary of the invention

[0003] The purpose of the present invention is to provide a key enzyme for synthesizing butanediamine and application thereof, and to improve the efficiency of synthesizing butanediamine by optimizing the reaction system.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] A gene sequence for synthesizing ornithine decarboxylase, wherein the nucleotide sequence of the ornithine decarboxylase is shown as SEQ ID NO.7.

[0006] The present invention also provides an ornithine decarboxylase synthesized by the gene sequence, and the amino acid sequence of the ornithine decarboxylase is shown as SEQ ID NO.3.

[0007] The present invention also provides an engineered strain for producing butanediamine, wherein the engineered strain is introduced with the gene sequence of the synthetic ornithine decarboxylase according to claim 1.

[0008] The present invention also provides an application of the gene sequence for synthesizing ornithine decarboxylase, the ornithine decarboxylase or the engineered strain in catalyzing the synthesis of butanediamine.

[0009] The present invention also provides a biosynthetic method of diaminobutane, which uses L-ornithine or L-ornithine hydrochloride as a substrate, PLP as a coenzyme, and utilizes the ornithine decarboxylase or the ornithine decarboxylase expressed by the engineered strain to catalyze the synthesis of diaminobutane.

[0010] Preferably, in the biosynthesis system, the concentration of L-ornithine or L-ornithine hydrochloride is 8-12 mM, the concentration of the coenzyme PLP is 0.05-0.15 mM, and the concentration of ornithine decarboxylase is 0.2-0.25 μg / mL.

[0011] Preferably, the reaction temperature during the catalytic synthesis is 35-40° C., and the reaction pH is 6-7.5.

[0012] The invention provides an ornithine decarboxylase mutant for synthesizing diaminobutane, comprising an amino acid sequence and a nucleotide sequence of the ornithine decarboxylase, a gene expression vector and a recombinant engineering strain. The diaminobutane synthesis ability is improved by optimizing the reaction system, and diaminobutane can be synthesized more efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The ornithine decarboxylase pETDuet-speC plasmid constructed in Example 1;

[0014] Figure 2 The catalytic results of the engineered strain mutant in Example 3 are shown. DETAILED DESCRIPTION

[0015] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0016] Example 1

[0017] Method for constructing engineered strains

[0018] The genes encoding ornithine decarboxylase speC1 (SEQ ID NO.6), speC2 (SEQ ID NO.7) and speC3 (SEQ ID NO.8) were inserted between the BamHI / SacI restriction enzyme sites of the pETDuet plasmid to construct plasmids pETDuet-speC1, pETDuet-speC2, and pETDuet-speC3, and were respectively transferred into E. coli BL21 (DE3) chassis cells to construct genetically engineered strains for the synthesis of diaminobutane, which were named SpeC1, SpeC2 and SpeC3, respectively. At the same time, a genetically engineered strain containing ornithine decarboxylase SpeC (SEQ ID NO.5) from Escherichia coli was constructed using the above method as a positive control group. The constructed plasmid pETDuet-speC Figure 1 As shown below. The nucleotide sequences of SpeC, SpeC1, SpeC2, and SpeC3 are as follows:

[0019] SpeC SEQ ID NO.5

[0020] ATGAAGAGCATGAACATCGCAGCCAGCGGCGAACTGATTCCGCGT

[0021] CTTAGTACCCATCGCAATGTGGTGGCACTGGATAGTACCGACTTCACCG

[0022] ATGTGGCCGCAGTTGTTATTACCACCGCCGATAGTCGTAGCGGCATTCT

[0023] GGCACTGCTGAAACGTACCGGCTTCCATCTGCCGGTGTTCATGCTGGC

[0024] AGATGAACCGGTTAGTGCACCGGTGGGCGTTACCGCAGTTATTGTTGGT

[0025] AATGCCCAGGAATGGCTGGAACTGGAAAATGCCGCCTGCCGCTATGAA

[0026] GCAGAACTGCTGCCGCCGTTCTATGGCACCCTGACACAGTATGTTGATA

[0027] TGGGTAATAGTACCTTCGCCTGTCCGGGCCATCAGCATGGTGAGTTCTT

[0028] CCGCAAACATCCGGCAGATCGCCACTTCTATGACTTCTTCGGTGAAAAT

[0029] CTGTTCCGCGCAGATATGTGCAATGCCGATGTGAAACTGGGCGATCTGC

[0030] TGATTCATGAAGGTAGTGCAAAACATGCACAGAAATTCGCCGCAAAAG

[0031] TGTTCAATGCAGATAAAACCTACTTCGTTCTGAACGGTACCAGCGCAG

[0032] CAAATAAAGTGGTTACCAATGCCCTGCTGACCCGTGGTGATCTGGTTCT

[0033] GTTCGATCGTAATAATCATAAGAGCAACCACCACGGTGCACTGATTCAG

[0034] GCAGGTGCCACCCCTGTGTATCTGGAAGCCGCACGTAATCCGTTCGGC

[0035] TTCATTGGTGGTATTGATGCCCATTGCTTCGATGAAACCTATCTGCGCGA

[0036] TCAGATTCGCGATGTGATGCCGGAAAGTGCAGATGCAAGTCGTCCGTT

[0037] CCGTCTGGCAATTATTCAGCTGGGTACCTATGATGGCACCATCTATAATG

[0038] CCCGTCAGGTGGTTGATAAAATTGGTCATCTGTGCGATTACATCCTGTT

[0039] CGATAGCGCCTGGGTTGGTTATGAACAGTTCATTAATATGATGGCGGAC

[0040] ACCAGCCCGCTGCGTCTTGAATTAAATGAAAATGATCCGGGCATCTTCG

[0041] TTACCCAGAGTGTGCATAAACAGCAGGCAGGCTTCAGTCAGACCAGTC

[0042] AGATTCATAAAAAGGATAACCACATCCGCGGTCAGGCACGCTTCTGCC

[0043] CTCATAAACGTCTGAATAATGCCTTCATGCTGCATGCAAGTACCAGCCC

[0044] GTTCTATCCGCTGTTCGCCGCACTGGATGTGAATGCAAAAATTCATGAA

[0045] GGCGAAAGCGGTCGCCGTCTGTGGGCAGAGTGTGTTGCACTGGGTATT

[0046] GATGCACGCAAAGCCATTCTGGCACGCTGTAAACTGCTGCAACCGTTC

[0047] ATTCCGCTGGTGGTGGATGGTAAACCGTGGCAGGCCTATCCGACCGAA

[0048] ACCATTGCAAGCAATCGCCGCTTCTTCAGCTTCGAACCGGGTGCAAAA

[0049] TGGCATGGCTTCGAAGGTTATGCCGATGATCAGTACTTCGTGGACCCTT

[0050] GTAAACTGTTACTGACCACCCCGGGTATTGATGCTGATAGCGGTCGTTA

[0051] TACCGAGTTCGGCATTCCGGCCACCATTCTGGCCCATTATCTGCGTGAAAATGGTATTGTGCCGGAAAAATGCGATCTGAATAGCATTCTGTTCCTGCTGACCCCGGCCGAAAGTGAAGAAAAACTGGCACGTCTGGTGGCAATGCTGGCCCAATTCGAACGTCATATTGAAGATGATACCCCGCTGGCAGATGTTCTGCCGACCGTGTTCCAGAAATATCCGGTTCGTTATCGCGATTATACCCTGCGCGAACTGTGCCAGGAAATGCATAATCTGTATGTGAGCTTCGATGTGAAAGACTTACAGAAAGCAATGTTCCGCAAAGAAAGCCTGCCGCATGTGGCAATGAATCCGCAGGATGCAAATAGCGCATTCATTCGTGGCGATGTGGAACTGGTGCGCATTAGCGAAGCAGGTGGCCGTATTGCCGCCGAAGGTGCATTACCGTATCCGCCTGGTGTTCTGTGCGTTGTGCCGGGTGAAATCTGGGGTGGCGCAGCACAACGCTACTTCCTGGCACTGGAAGAAGGCATTAATCTGCTGCCGGGCTTCAGCCCGGAACTGCAAGGTGTGTATAGCGAAACCGATGCAGATGGCATTCAGCGTCTGTATGGTTATGTGCTGAAATAA

[0052] SpeC1 SEQ ID NO.6

[0053] ATGCCTAACCTGAAAATCGCATATAGCCCGAAAGTTGAACAGTACTTCAGTACCAATCGCGAACTGGTTGAAATTGCCAAAACCGACTTCACCGATGTTGCAGCAATCGTTCTGAGCAGTAGCGATGTGGGTGAATATCTGGATCGTATTAAAGCAACCAAGTTCGATGTGCCGGTGTTCATTGTTCAGACCGATGAACAGCAGGTGGACCCTAAATTCTATGATAGCATCTATCACATCCAGGATCTGAATGGCTATGATATTAAGCTGTACAGTCGCCAGATTGAAACCGCAGCCAAACTGTATGAAGAAAAAATGCTGCCGCCGTTCTTCAAAATGCTGAGCGAATATGTGGAAATGGGTAATATTGCATTCGACTGTCCGGGCCATCAGGGCGGTCAATATTATCGCAAACATCCGGCCGGTCGCTTCCTGTATGACTTCTATGGCGAAAATATCTTCCGTAGCGATATCTGTAACGCCGATGTGAAACTGGGTGATCTGCTGATTCATGAAGGCGCAGCCTGCGATGCACAGAAATATGCCGCCCAGGTGTTCAATGCAGATAAAACCTACTTCGTTCTGAACGGTACCAGTAGCAGCAATAAAGTGGCACTGAATGCAGTTCTGGCACCGGGCGATCTGGTGTTATTCGATCGTAATAATCACAAGAGCAACCA

[0054] TCATGGTGCCCTGATTCAGGCAGGCGCCACACCTATCTATCTGGAAACC

[0055] GCACGCAATCCGTTCGGCTTCATTGGCGGCATTGATAGCCATTGCTTCG

[0056] AAGAAGATTACCTGAAAAGTCTGATCAAGGAAGTGGCCCCGGAAAAA

[0057] CTGAATCAGAAACGTCCGTTCCGTCTGGCCGTTATTCAGCTGGGCACCT

[0058] ATGATGGCACCATCTATAATGCCCGTCAGGTTGTGGATAAAATTGGTCA

[0059] TCTGTGCGATTATATCCTGTTCGATAGTGCCTGGGTGGGCTATGAACAG

[0060] TTCATTCCGATGATGAAAGACTGTAGCCCGCTGCTGCTGGAACTGAAT

[0061] GAAAATGATCCGGGCATTCTGGTTACCCAGAGTGTGCATAAACAGCAG

[0062] GCAGGCTTCAGTCAGACCAGTCAGATTCATAAAAAGGATAAGCACATC

[0063] AAGGGCCAGGATCGTTATGTGAATCATAAACGCTTCAACAACGCATTCA

[0064] TGCTGCATGCAAGCACCAGTCCGTTCTATCCGCTGTTCGCCACCCTGGA

[0065] TGTTAATGCCAAAATTCAGGGCAGTGAAGCAGGTCGCCGTCTGTGGCA

[0066] TGAATGCGTGAAAGTGGGTATTGAAGCCCGTAAACTGGTGCTGAATCA

[0067] TTGCGAACTGATTCGTCCGTTCATTCCGACCACCATTAAAGGTAAAAAG

[0068] TGGCAGGATTACGACACCGAAGAAATTGCAACCAATCTGGAGTTCTTC

[0069] AAATTCCATCCGACCGATACCTGGCATAAATTCGAAGGCTATGCAGATG

[0070] AACAGTACTTCGTTGATCCGTGCAAATTCCTGCTGACCACCCCGGGTAT

[0071] TAGTCTGGAAACCGGTGAATATGAAGAGTTCGGCGTGCCGGCAACCAT

[0072] TCTGGCAAATTATCTGCGCGAAAATGGCATTATTCCGGAAAAATGCGAT

[0073] CTGAATAGCATTCTGTTCCTGCTGACACCGGCAGAAACCATTACCAAAA

[0074] TGCAGACCCTGGTTGCACAGATTGCACTGTTCGAAAAACATATTAAGC

[0075] AGGATAGCCTGCTGAAATATGTGCTGCCGACCGTGTATAAAAATAATGA

[0076] AGATCGCTACAAGGGTTACACCATTCGTCAGCTGTGCCAGGAAATGCA

[0077] TGATCTGTATGTTAGTCGCAATGTTAAGCAGCTGCAAAAAGACTTATTC

[0078] CGTAAAGCAACCCTGCCGGAATATGTTCTGAATCCGCATGATGCCAATA

[0079] TTGAACTGATTCGCAATAAGGTGGAGCTGGTTCCGCTGACCGATATTGT

[0080] TGGCCGCGTTGCAGCCGAAGGCGCTCTGCCTTATCCGCCTGGTGTTCT

[0081] GTGTGTGGTGCCGGGTGAAAAATGGAGCACCACCGCCCATCAGTACTT

[0082] CCTGGCACTGGAAGAAGGCATTAATACCCTGCCGGGCTTCGCCCCTGA

[0083] AATTCAGGGTGTGTATCTGCAAAAAGATCCGGATGGCCGTACCCGCGC

[0084] ATACGGTTATGTTCTGACCGATTATTAA

[0085] SpeC2 SEQ ID NO.7

[0086] ATGCTGCCTAGCCTGAATGGCGTGTTCGAACTGTGTGGTAATAATA

[0087] CCAACTTCTACGGCAAAACCTTAGAAACCGCCGCCAAAAAATATGAAA

[0088] GCGAACTGCTGCCGCCGTTCTTCAATACCCTGAAAAAATATGTGGAGAT

[0089] GGGCAATAGTACCTTCGCCTGTCCGGGCCATCAGGGTGGTCAATTCTTC

[0090] CGTAAACATCCGGCCGGTCGTCAGTTCTTCGACTTCTATGGCGAAGCAA

[0091] TCTTCCGTAGCGATATGTGTAATGCAGATGTGAAACTGGGTGATCTGCT

[0092] GATTCATGAAGGCGCCCCGTGTGATGCCCAGAAACATGCAGCCAAAGT

[0093] GTTCAATGCCGATAAAACCTACTTCGTTCTGAATGGCACCAGTGCCAGC

[0094] AATAAAGTGGCCACCAATGCACTGCTGGCACGCGGTGATCTGGTTCTG

[0095] TTCGATCGCAATAATCATAAAAGCAACCACCACGGTGCCCTGGTTCAG

[0096] GCAGGTGCAACACCTGTGTATCTGGAAACCGCACGTAATCCGTTCGGC

[0097] TTCATTGGTGGCATTGATGCACATTGCTTCGAAGAAAAATACCTGCGTG

[0098] AACAGATTCGCAGCGTTGCCCCGGAACGCGCAAATGAAGCCCGTCCGT

[0099] TCCGTCTGGCAATTATTCAGCTGGGTACCTATGATGGCACCATCTATAAT

[0100] GCACGTCAGGTGGTTGATAAAATCGGTCATCTGTGTGATTACATCCTGT

[0101] TCGATAGTGCATGGGTTGGCTATGAACAGTTCATTCCGATGATGAAAGA

[0102] CTGCTCACCGCTGCTGCTGGAACTGAATGAAAATGATCCGGGTATTATC

[0103] GTGACCCAGAGTGTTCATAAACAGCAGGCAGGCTTCAGCCAGACCAG

[0104] TCAGATTCATAAAAAAGATAAGCACATCAAGGGTCAGGATCGCTATTGC

[0105] AATCATAAACGCTTCAATAACGCCTTCATGATGCATGCCAGTACCAGTC

[0106] CGTTCTATCCGCTGTTCGCAGCACTGGATGTGAATGCCAAAATGCATGA

[0107] AGGTAAAAGTGGCCAGCGCATGTGGCTGGATTGCGTTAAAACCGGTAT

[0108] TGAAGCCCGTAAAATGATTCTGAGCACCTGTAATATGATCAAGCCGTTC

[0109] GTTCCGGTTGAAGTGGATGGTAAACCGTGGCAGGAATATGATACCGAA

[0110] GCAATGGCACAGGATCTGCGCTTCTTCAACTTCATTCCGGGCGAAAAA

[0111] TGGCATGCATTCGAAGGCTATGAAGAAAGCCAGTACTTCGTGGACCCT

[0112] TGTAAACTGCTGCTGACCACCCCGGGCATTGATACCAATACCGGCGAAT

[0113] ATACCGAAAGTGGTATTCCGGCAACCATTCTGGCAAACTTCCTGCGCG

[0114] AAAATGGCATTGTTCCGGAAAAATGCGATCTGAATAGTATTCTGTTCCT

[0115] GCTGACCCCGGCAGAAGACTTAGCCAAAATGCAGCATCTGGTTGCCCA

[0116] GATTGCACGCTTCGAACGCTTCATTGAAGAAGATGAACTGCTGAGTGT

[0117] TGTGCTGCCGACCGTGTATAGCAATAATGAAACCAGATACAAGGACTA

[0118] CACCATTGGTAAACTGTGCCAGGAAATGCATGATCTGTATGTTAGCTAT

[0119] GACGTTAAGCAGCTGCAAAAAGAAATGTTCCGTAAACAGCACTTCCCG

[0120] AAAGTTATGATGAATCCGCAGGATGCAAATATTGAGTTCGTGCGTGGTC

[0121] ATGCCGAACTGGTTCCGCTGTGTAAAGCAGAAGGTCGTATTGCCGCAG

[0122] AAGGTGCCCTGCCTTATCCGCCTGGTGTTCTGTGTGTTGTTCCGGGTGA

[0123] AGTGTGGGGCGGTGCTGCACAGAGATACTTCCTGGCACTGGAAGAAA

[0124] GTATTAATCTGCTGCCGGGCTTCGCCCCGGAATTACAAGGTGTGTATCT

[0125] GCAAGTGGATGAAGATGGCTGGAATCGCGCATACGGTTATATGATGAA

[0126] ACAGTAA

[0127] SpeC3 SEQ ID NO.8

[0128] ATGAAGAGCATGAACATCGCCGCAAGTAGCGAACTGGTGAGCCGT

[0129] CTGAGTAGCCATCGCCGTGTTGTGGCCCTGGGTGACACAGATTTTACC

[0130] GATGTGGCAGCCGTTGTGATTACCGCCGCAGATAGTCGTAGTGGTATTC

[0131] TGGCCCTGCTGAAACGCACCGGTTTTCATCTGCCGGTGTTTCTGTATAG

[0132] TGAACATGCAGTTGAACTGCCGGCAGGTGTGACCGCAGTGATTAATGG

[0133] CAATGAACAGCAGTGGCTGGAACTGGAAAGCGCCGCATGTCAGTATG

[0134] AAGAAAATCTGCTGCCGCCGTTTTATGATACCCTGACCCAGTATGTGGA

[0135] AATGGGCAATAGCACCTTTGCCTGCCCGGGCCATCAGCATGGTGCATTT

[0136] TTCAAAAAACACCCGGCAGGTCGTCATTTTTATGATTTCTTTGGCGAAA

[0137] ACGTGTTCCGCGCAGATATGTGCAATGCAGATGTTAAACTGGGTGACCT

[0138] GCTGATTCATGAAGGTAGTGCCAAAGATGCCCAGAAATTTGCAGCAAA

[0139] AGTTTTTCATGCCGACAAAACCTATTTCGTTCTGAATGGTACCAGCGCA

[0140] GCAAATAAGGTGGTTACCAATGCACTGCTGACCCGCGGCGATCTGGTT

[0141] TTATTTGATCGCAATAATCACAAGAGCAACCATCATGGTGCCCTGATTC

[0142] AGGCAGGTGCCACCCCTGTTTATCTGGAAGCAAGCCGCAATCCGTTTG

[0143] GTTTTATTGGCGGTATTGATGCACATTGTTTCAATGAAGAGTACCTGCG

[0144] TCAGCAGATTCGCGATGTGGCACCGGAAAAAGCAGATTTGCCGCGTCC

[0145] GTATCGTCTGGCAATTATTCAGCTGGGTACCTATGATGGTACCGTGTATA

[0146] ATGCCCGTCAGGTTATTGATACCGTGGGTCATCTGTGCGATTATATTCTG

[0147] TTTGATAGCGCCTGGGTGGGCTATGAACAGTTTATTCCGATGATGGCCG

[0148] ATAGCAGCCCGCTGTTACTGGAACTGAATGAAAATGATCCGGGTATTTT

[0149] CGTTACCCAGAGTGTGCATAAACAGCAGGCAGGTTTTAGTCAGACCAG

[0150] CCAGATTCATAAAAAGGATAATCACATCCGCGGTCAGGCCCGCTTTTGC

[0151] CCTCATAAACGTCTGAATAATGCCTTTATGCTGCATGCAAGTACCAGCC

[0152] CGTTTTATCCGCTGTTTGCCGCACTGGATGTTAATGCCAAAATTCATGA

[0153] AGGCGAAAGCGGTCGTCGCCTGTGGGCAGAGTGTGTTGAAATTGGCAT

[0154] TGAAGCCCGTAAAGCCATTCTGGCACGCTGTAAACTGTTTCGCCCGTTT

[0155] ATTCCGCCGGTGGTTGATGGCAAACTGTGGCAGGATTATCCGACCAGC

[0156] GTGCTGGCCAGCGATCGTCGTTTCTTTAGTTTTGAACCGGGTGCAAAAT

[0157] GGCATGGTTTTGAAGGTTATGCAGCCGATCAGTATTTTGTTGATCCGTG

[0158] CAAACTGCTGCTGACCACCCCGGGTATTGATGCCGAAACCGGCGAATA

[0159] TAGTGATTTTGGCGTTCCGGCCACCATTCTGGCACATTATCTGCGCGAA

[0160] AATGGTATTGTGCCGGAAAAATGCGATCTGAATAGTATTCTGTTCCTGC

[0161] TGACCCCGGCCGAAAGCCATGAAAAACTGGCACAGCTGGTTGCAATG

[0162] CTGGCACAGTTTGAACAGCATATTGAAGATGATAGCCCGCTGGTTGAA

[0163] GTGCTGCCGAGTGTTTATAATAAGTATCCGGTTCGCTACCGTGATTATAC

[0164] CCTGCGCCAGCTGTGTCAGGAAATGCATGATCTGTATGTTAGCTTTGAC

[0165] GTGAAAGATTTGCAGAAAGCCATGTTTCGTCAGCAGAGCTTTCCGAGT

[0166] GTGGTTATGAATCCGCAGGATGCACATAGCGCCTATATTCGCGGCGATG

[0167] TTGAACTGGTTCGTATTCGCGATGCCGAAGGCCGCATTGCCGCTGAGGGTGCATTACCTTATCCGCCGGGTGTGCTGTGCGTTGTTCCGGGTGAAGTGTGGGGCGGTGCAGTTCAACGCTA TTTTCTGGCCCTGGAAGAAGGTGTGAATCTGCTGCCTGGTTTTAGCCCGGAACTGCAAGGGCTGTATAGCGAAACCGATGCAGATGGTGTTAAACGTCTGTATGGCTATGTGCTGAAATAA

[0168] Example 2

[0169] Liquid Phase Quantitative Analysis Method for L-Ornithine (Hydrochloride) and Diaminodiphenylamine

[0170] (1) Add 600 μL of boric acid buffer (50 mM pH = 9), 200 μL of methanol, 60 μL of diluted sample (L-ornithine (hydrochloride) and diaminobenzene standard sample, diluted to different concentrations: 20 mM, 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM, 0.15625 mM, 0.078125 mM), 130 μL of ddH2O and 10 μL of diethyl ethoxymethylenemalonate (Aladdin, prepared as a 1 M methanol solution) to the reaction system, place the reaction at room temperature for 10 min, transfer to a 70°C metal bath for 2 h, and terminate the reaction;

[0171] (2) Reverse phase high performance liquid chromatography UV (284 nm) detector was used for detection, mobile phase A was 100% acetonitrile; mobile phase B was sodium acetate buffer solution (25 mM pH 4.8), flow rate was 0.4 mL / min; detection column was C18; column detection temperature was 35°C; injection volume was 10 μL; wavelength was 284 nm. Gradient elution was used, and the gradient elution program was as follows: 0-5 min, 87% B-80% B; 5-22 min, 80% B-51.7% B; 22-27 min, 51.7%-87% B.

[0172] L-ornithine or L-ornithine hydrochloride peaks at 10.8 min, while diaminobutane peaks at 19.8 min.

[0173] Example 3

[0174] Comparison of activity of engineered strain mutants

[0175] The engineered strains SpeC, SpeC1, SpeC2 and SpeC3 obtained in Example 1 were cultured in 5 mL of LB medium containing 100 mg / L ampicillin at 37°C overnight to obtain seed solution. The seed solution was transferred into 50 mL of LB medium containing 100 mg / L ampicillin at a transfer volume of 1% by volume and cultured at 37°C. When the OD 600 When the pH value was 0.6, IPTG with a final concentration of 0.1 mM was added for induction. After further cultivation at 20°C for 20 h, the bacteria were collected by centrifugation at 4000 rpm and stored in a freezer at -80°C for no less than 24 h.

[0176] The frozen cells were resuspended in 50 mM phosphate buffer (pH = 7.5) for whole-cell catalysis experiments. In the 500 μL whole-cell catalysis system, the concentration of substrate L-ornithine hydrochloride was 250 mM, the concentration of coenzyme PLP was 0.1 mM, and the OD of the mutant genetic engineering strain was 600 The reaction was carried out at 37°C and 500 rpm for 1 hour, and then centrifuged at 12000 rpm for 2 minutes. The supernatant was taken to detect the content of diaminobutane. The catalytic results of the engineered strain mutants are shown in Figure 2 As shown, the whole-cell catalytic results of the mutant SpeC2 were better than those of the positive control, while SpeC1 and SpeC3 did not produce diaminobutane.

[0177] Example 4

[0178] The engineered strains SpeC and SpeC2 obtained in Example 1 were cultured and protein induced for expression according to the method shown in Example 3, and stored at -80°C. The protein was purified in an AKTA protein purifier by using 4°C pre-cooled buffer A (20mM potassium phosphate buffer, 0.1mM PLP, 500mM NaCl, 20mM imidazole, pH=7.4), buffer B (20mM potassium phosphate buffer, 0.1mM PLP, 500mM NaCl, 500mM imidazole, pH=7.4), and preservation solution (20mM potassium phosphate buffer, 0.1mM PLP, 150mM NaCl, 10% glycerol, pH=7.4). The -80°C frozen bacteria were resuspended in precooled Binding buffer (buffer A), and ultrasonically disrupted at a power of 55%, ultrasonic for 2s, and paused for 3s. The disrupted liquid was centrifuged at 4°C and 12000rpm for 20min to obtain the supernatant, and the supernatant was filtered through a 0.22μm filter membrane, and then protein was purified on an AKTA protein purifier using a 5mL Histrap purification column, and then the preservation solution was replaced with a 5mL HiTrap Desalting column to finally obtain pure enzymes dissolved in the preservation solution. The obtained enzymes were the isolated and purified ornithine decarboxylases SpeC, SpeC1, SpeC2 and SpeC3, and the concentration of the purified ornithine decarboxylase was determined by the BCA protein quantification method and stored at -80°C.

[0179] Example 5

[0180] Determination of the catalytic ability of ornithine decarboxylase SpeC2 in buffers with different pH values

[0181] The different pH buffers are: sodium acetate buffer (50 mM, pH=5-6.5), phosphate buffer (50 mM, pH=7-8), and sodium carbonate buffer (50 mM, pH=9-11).

[0182] In 500 μL sodium acetate buffer reaction system, phosphate buffer reaction system, and sodium carbonate buffer reaction system, the substrate L-ornithine hydrochloride was added at a concentration of 10 mM and the coenzyme PLP concentration was 0.1 mM. The ornithine decarboxylase SpeC2 and SpeC obtained in Example 4 were diluted to 35 μg / mL, respectively, and added to the above three reaction systems so that the concentrations of ornithine decarboxylase SpeC2 and SpeC in the system were both 0.236 μg / mL. The reaction was catalyzed at 37°C and 500 rpm for 1 h, centrifuged at 12000 rpm for 2 min, and the supernatant solution was taken to detect the content of diaminobutane.

[0183] The reaction results of ornithine decarboxylase SpeC2 and positive control SpeC in a buffer reaction system of pH 5 to 11 are shown in Table 1, wherein the catalytic ability of ornithine decarboxylase SpeC2 is the highest when the pH of the reaction system reaches 7, and then the catalytic ability decreases with the increase of pH, while the catalytic ability of positive control SpeC is the highest when the pH of the reaction system reaches 9. Therefore, the pH characteristics of ornithine decarboxylase SpeC2 are significantly different from those of the existing ornithine decarboxylase SpeC.

[0184] Table 1 Catalytic ability of SpeC2 in different pH buffers

[0185]

[0186] Example 6

[0187] Determination of the catalytic ability of ornithine decarboxylase SpeC2 at different temperatures

[0188] The buffers of the reaction system are phosphate buffer (50mM, pH=7) and sodium carbonate buffer (50mM, pH=9). In a 500μL buffer reaction system, the concentration of the substrate L-ornithine hydrochloride is 10mM, the concentration of the coenzyme PLP is 0.1mM, then the ornithine decarboxylase SpeC2 and SpeC obtained in Example 4 are diluted to 35μg / mL and added to the reaction system, the concentration of lysine decarboxylase SpeC2 and SpeC in the system is 0.236μg / mL, at different temperatures and optimal pH (SpeC2: pH=7; SpeC: pH=9), after catalytic reaction at 500rpm for 1h, centrifugation at 12000rpm for 2min, and the supernatant solution is taken to detect the content of diaminobutane.

[0189] The results of free enzyme catalysis of ornithine decarboxylase SpeC2 and SpeC at 30-65°C are shown in Table 2. It is not difficult to find that the production of diaminobutane increases first and then decreases with the increase of temperature. Ornithine decarboxylase SpeC2 has the highest catalytic activity at 37°C and the strongest diaminobutane synthesis ability, showing similar temperature characteristics to the positive control SpeC.

[0190] Table 2 Catalytic ability of SpeC2 at different temperatures

[0191]

[0192] Example 7

[0193] Determination of the kinetic properties of ornithine decarboxylase SpeC2

[0194] The reaction system buffers were phosphate buffer (50 mM, pH=7) and sodium carbonate buffer (50 mM, pH=9). In a 500 μL buffer reaction system, the concentration of substrate L-lysine hydrochloride was 1-30 mM, and the concentration of coenzyme PLP was 0.1 mM. The ornithine decarboxylase SpeC2 and SpeC obtained in Example 4 were diluted to 35 μg / mL, respectively, and added to the reaction system. The concentrations of lysine decarboxylase SpeC2 and SpeC in the system were both 0.936 μg / mL. The reaction was catalyzed at 500 rpm for 1 hour under the optimal conditions (SpeC2: pH = 7, 37°C; SpeC: pH = 9, 37°C), and then centrifuged at 12000 rpm for 2 minutes. The supernatant solution was taken to detect the content of diaminobutane. The enzyme kinetics calculation module "Enzyme kinetics-Michaelis-Menten" in the nonlinear regression program GraphPad Prism 8.0 (https: / / www.graphpad.com / ) was used to fit and calculate Vmax, kcat, and Km. The kinetic parameter results of ornithine decarboxylase SpeC2 and the positive control group SpeC are shown in Table 3. Compared with the positive control SpeC, ornithine decarboxylase SpeC2 has a lower Km and a higher kcat, that is, it has a stronger substrate affinity and product synthesis rate, and can synthesize diaminobutane more efficiently.

[0195] Table 3 Kinetic properties of SpeC2

[0196]

[0197]

[0198] Example 8

[0199] Detection of the thermal stability of ornithine decarboxylase SpeC2 by differential scanning fluorimetry

[0200] The reaction system buffers were phosphate buffer (50 mM, pH = 7) and sodium carbonate buffer (50 mM, pH = 9). The total volume of the thermal stability detection system was 20 μL, in which the fluorescent dye Protein ThermalShift was diluted 125 times. TM The volume of Dye (ThermoFisher, 4461146) was 2.5 μL, and the thermal denaturation buffer was Protein Thermal Shift TMThe volume of the buffer was 5 μL. Then the ornithine decarboxylase SpeC2 and SpeC obtained in Example 4 were diluted to 433 μg / mL. 2.7 μL of the diluted enzyme solution was added to the detection system. Finally, 9.8 μL of the corresponding buffer (SpeC2: pH = 7; SpeC: pH = 9) was added. TM 1Plus Real-time Fluorescence Quantitative PCR System (Applied Biosystems TM , USA) was used to test the thermal stability of ornithine decarboxylase SpeC2 and positive control SpeC, and the heating program was as follows: 25°C (retained for 2 min), then heated to 90°C at 0.05°C / s (retained for 2 min). The thermodynamic stabilities of ornithine decarboxylase SpeC2 and positive control SpeC are shown in Table 4, where the Tm value of ornithine decarboxylase SpeC2 is 57.2°C, and the Tm value of positive control ornithine decarboxylase SpeC2 is 69.4°C.

[0201] Table 4 Thermal stability results of SpeC2

[0202]

[0203] The amino acid sequences of SpeC, SpeC1, SpeC2, and SpeC3 genes are as follows:

[0204] SpeC SEQ ID NO.1

[0205] MKSMNIAASGELIPRLSTHRNVVALDSTDFTDVAAVVITTADSRSGIL

[0206] ALLKRTGFHLPVFMLADEPVSAPVGVTAVIVGNAQEWLELENAACRYEA

[0207] ELLPPFYGTLTQYVDMGNSTTFACPGHQHGEFFRKHPADRHFYDFFGENLF

[0208] RADMCNADVKLGDLLIHEGSAKHAQKFAAKVFNADKTYFVLNGTSAAN

[0209] KVVTNALLTRGDLVLFDRNNHKSNHHGALIQAGATPVYLEAARNPFGFIG

[0210] GIDAHCFDETYLRDQIRDVMPESADASRPFRLAIIQLGTYDGTIYNARQV

[0211] VDKIGHLCDYILFDSAWVGYEQFINMMADTSPLRLELNENDPGIFVTQSV

[0212] HKQQAGFSQTSQIHKKDNHIRGQARFCPHKRLNNAFMLHASTSPPYPLFA

[0213] ALDVNAKIHEGESGRRLWAECVALGIDARKAILARCKLLQPFIPLVVDGK

[0214] PWQAYPTETIASNRRFFSFEPGAKWHGFEGYADDQYFVDPCKLLLTTPGI

[0215] DESCRYTEFGIPATILAHYLRENGIVPEKCDLNSILFLLTPAESEEKLARLV

[0216] AMLAQFERHIEDDTPLADVLPTVFQKYPVRYRDYTLRELCQEMHNLYVS

[0217] FDVKDLQKAMFRKESLPHVAMNPQDANSAFIRGDVELVRISEAGGRIAAE

[0218] GALPYPPGVLCVVPGEIWGGAAQRYFLALEEGINLLPGFSPELQGVYSET

[0219] DADGIQRLYGYVLK

[0220] SpeC1 SEQ ID NO.2

[0221] MPNLKIAYSPKVEQYFSTNRELVEIAKTDFTDVAAIVLSSSDVGEYLD

[0222] RIKATKFDVPVFIVQTDEQQVDPKFYDSIYHIQDLNGYDIKLYSRQIETAA

[0223] KLYEEKMLPPFFKMLSEYVEMGNIAFDCPGHQGGQYYRKHPAGRFLYDF

[0224] YGENIFRSDICNADVKLGDLLIHEGAACDAQKYAAQVFNADKTYFVLNG

[0225] TSSSNKVALNAVLAPGDLVLFDRNNHKSNHHGALIQAGATPIYLETARNPF

[0226] GFIGGIDSHCFEEDYLKSLIKEVAPEKLNQKRPFRLAVIQLGTYDGTIYNAR

[0227] QVVDKIGHLCDYILFDSAWVGYEQFIPMMKDCSPLLLELNENDPGILVTQ

[0228] SVHKQQAGFSQTSQIHKKDKHIKGQDRYVNHKRFNNAFMLHASTSPFYP

[0229] LFATLDVNAKIQGSEAGRRLWHECVKVGIEARKLVLNHCELIRPFIPTTIK

[0230] GKKWQDYDTEEIATNLEFFKFHPTDTWHKFEGYADEQYFVDPCKFLLTTP

[0231] GISLETGEYEEFGVPATILANYLRENGIIPEKCDLNSILFLLTPAETITKMQTL

[0232] VAQIALFEKHIKQDSLLKYVLPTVYKNNEDRYKGYTIRQLCQEMHDLYVS

[0233] RNVKQLQKDLFRKATLPEYVLNPHDANIELIRNKVELVPLTDIVGRVAAE

[0234] GALPYPPGVLCVVPGEKWSTTAHQYFLALEEGINTLPGFAPEIQGVYLQK

[0235] DPDGRTRAYGYVLTDY

[0236] SpeC2 SEQ ID NO.3

[0237] MLPSLNGVFELCGNNTNFYGKTLETAAKKYESELLPPFFNTLKKYVE

[0238] MGNSTFACPGHQGGQFFRKHPAGRQFFDFYGEAIFRSDMCNADVKLGDL

[0239] LIHEGAPCDAQKHAAKVFNADKTYFVLNGTSASNKVATNALLARGDLVL

[0240] FDRNNHKSNHHGALVQAGATPVYLETARNPFGFIGGIDAHCFEEKYLREQ

[0241] IRSVAPERANEARPFRLAIIQLGTYDGTIYNARQVVDKIGHLCDYILFDSA

[0242] WVGYEQFIPMMKDCSPLLLELNENDPGIIVTQSVHKQQAGFSQTSQIHKK

[0243] DKHIKGQDRYCNHKRFNNAFMMHASTSPFYPLFAALDVNAKMHEGKSG

[0244] QRMWLDCVKTGIEARKMILSTCNMIKPFVPVEVDGKPWQEYDTEAMAQ

[0245] DLRFFNFIPGEKWHAFEGYEESQYFVDPCKLLLTTPGIDTNTGEYTESGIPA

[0246] TILANFLRENGIVPEKCDLNSILFLLTPAEDLAKMQHLVAQIARFERFIEED

[0247] ELLSVVLPTVYSNNETRYKDYTIGKLCQEMHDLYVSYDVKQLQKEMFRK

[0248] QHFPKVMMNPQDANIEFVRGHAELVPLCKAEGRIAAEGALPYPPGVLCV

[0249] VPGEVWGGAAQRYFLALEESINLLPGFAPELQGVYLQVDEDGWNRAYG

[0250] YMMKQ

[0251] SpeC3 SEQ ID NO.4

[0252] MKSMNIAASSELVSRLSSHRRVVALGDTDFTDVAAVVITAADSRSGIL

[0253] ALLKRTGFHLPVFLYSEHAVELPAGVTAVINGNEQQWLELESAACQYEEN

[0254] LLPPFYDTLTQYVEMGNSTFACPGHQHGAFFKKHPAGRHFYDFFGENVF

[0255] RADMCNADVKLGDLLIHEGSAKDAQKFAAKVFHADKTYFVLNGTSAAN

[0256] KVVTNALLTRGDLVLFDRNNHKSNHHGALIQAGATPVYLEASRNPFGFIG

[0257] GIDAHCFNEEYLRQQIRDVAPEKADLPRPYRLAIIQLGTYDGTVYNARQVI

[0258] DTVGHLCDYILFDSAWVGYEQFIPMMADSSPLLLELNENDPGIFVTQSVH

[0259] KQQAGFSQTSQIHKKDNHIRGQARFCPHKRLNNAFMLHASTSPFYPLFAA

[0260] LDVNAKIHEGESGRRLWAECVEIGIEARKAILARCKLFRPFIPPVVDGKLW

[0261] QDYPTSVLASDRRFFSFEPGAKWHGFEGYAADQYFVDPCKLLLTTPGIDA

[0262] ETGEYSDFGVPATILAHYLRENGIVPEKCDLNSILFLLTPAESHEKLAQLVA

[0263] MLAQFEQHIEDDSPLVEVLPSVYNKYPVRYRDYTLRQLCQEMHDLYVSF

[0264] DVKDLQKAMFRQQSFPSVVMNPQDAHSAYIRGDVELVRIRDAEGRIAAE

[0265] GALPYPPGVLCVVPGEVWGGAVQRYFLALEEGVNLLPGFSPELQGVYSE

[0266] TDADGVKRLYGYVLK

[0267] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gene sequence for synthesizing ornithine decarboxylase, characterized in that: The nucleotide sequence of the ornithine decarboxylase is shown in SEQ ID NO.

7.

2. An ornithine decarboxylase synthesized from the gene sequence of claim 1, characterized in that: The amino acid sequence of the ornithine decarboxylase is shown in SEQ ID NO.

3.

3. An engineered strain for producing diaminobutane, characterized in that: The engineered strain is introduced with the gene sequence of the synthetic ornithine decarboxylase according to claim 1.

4. Use of the gene sequence for synthesizing ornithine decarboxylase according to claim 1, or the ornithine decarboxylase according to claim 2, or the engineered strain according to claim 3 in catalytic synthesis of butanediamine.

5. A biosynthetic method of butanediamine, characterized in that: The method uses L-ornithine or L-ornithine hydrochloride as a substrate, PLP as a coenzyme, and utilizes the ornithine decarboxylase described in claim 2 or the ornithine decarboxylase expressed by the engineered strain described in claim 3 to catalyze the synthesis of diaminobutane.

6. The biosynthesis method according to claim 5, characterized in that In the biosynthesis system, the concentration of L-ornithine or L-ornithine hydrochloride is 8-12 mM, the concentration of the coenzyme PLP is 0.05-0.15 mM, and the concentration of the ornithine decarboxylase is 0.2-0.25 μg / mL.

7. The biosynthesis method according to claim 6, characterized in that The reaction temperature during the catalytic synthesis is 35-40° C., and the reaction pH is 6-7.5.

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