CN106497990B - It is a kind of to convert caffeinic high-performance bio synthetic method for low value compound - Google Patents

It is a kind of to convert caffeinic high-performance bio synthetic method for low value compound Download PDF

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CN106497990B
CN106497990B CN201610928576.3A CN201610928576A CN106497990B CN 106497990 B CN106497990 B CN 106497990B CN 201610928576 A CN201610928576 A CN 201610928576A CN 106497990 B CN106497990 B CN 106497990B
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CN106497990A (en
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周景文
陈坚
吕永坤
堵国成
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Jiangnan University
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Abstract

Caffeinic high-performance bio synthetic method is converted by low value compound the invention discloses a kind of, belongs to biological chemical field.In the present invention, tyrosine benzene lyase synthesizes levodopa using catechol, pyruvic acid and ammonia as substrate, and levodopa is converted trans- caffeic acid and NH by tyrosine amino lyase3.Since catechol, Sodium Pyruvate and ammonium chloride are relatively inexpensive compound, caffeinic biological synthesis method is converted by low value compound invention creates a kind of, and be easy to be mass produced.Compared with chemical synthesis process, the product of this method is single trans- caffeic acid, does not need further to separate isomer.

Description

It is a kind of to convert caffeinic high-performance bio synthetic method for low value compound
Technical field
Caffeinic high-performance bio synthetic method is converted by low value compound the present invention relates to a kind of, belongs to biochemical industry Field.
Background technique
Caffeic acid is a kind of aromatic compounds of high value, can be divided into hydroxycinnamic acid in structure, have simultaneously 2 functional groups of phenolic hydroxyl group and acrylic acid.In vivo and in vitro studies have shown that caffeic acid has a series of physiological functions.For example, passing through Oxidation mechanism, caffeic acid can inhibit cancer cell multiplication；Caffeic acid has immunological regulation and anti-inflammatory activity；Caffeic acid can also be used as Antioxidant, and it is better than other native compounds；In addition, caffeic acid also has antiviral, antidepression, treatment diabetes etc. living Property.
As the crucial mesostate of lignin synthesis, caffeic acid is present in nearly all plant.The approach with L-tyrosine or L-phenylalanine are precursor, are related to trans-Cinnamate 4-monooxygenase (CYP73A), phenylalanine/tyrosine Amino lyase, p-Coumaric Acid 3- hydroxylase etc..However, content of the caffeic acid in plant is generally very low, therefore extract difficult. On the other hand, the caffeic acid being chemically synthesized is take advantage of a situation caffeic acid and trans- caffeinic mixture；And due to the phase of structure Like property, it is kept completely separate that be purified into single any compound all more difficult.By synthetic biology approach strategy, by caffeic acid Route of synthesis in plant is moved in microorganism chassis, and it is main at present that constructing, which has the engineering bacteria of caffeic acid synthesis capability, Research Thinking.However, since the dissolubility of the substrates such as tyrosine is poor, the coffee acid yield of such engineering bacteria and to substrate Conversion ratio is lower.By using l-tyrosine superior strain as the chassis of the heterologous route of synthesis of caffeic acid, facilitating to solve to be somebody's turn to do Problem, however the effect is unsatisfactory, it is longer to show as the production cycle, and yield does not significantly improve.
Summary of the invention
The problem to be solved in the present invention is to provide a kind of caffeinic biological synthesis methods, for this purpose, present invention firstly provides A kind of recombination bacillus coli co-expressing tyrosine benzene lyase EhTPL and tyrosine amino lyase TcXAL, wider Under substrate concentration range and reaction condition, it can be effectively synthesized caffeic acid.
The tyrosine benzene lyase EhTPL is from the raw Erwinia (Erwiniaherbicola) of grass, amino Acid sequence is as shown in SEQ ID NO.1, and the DNA sequence dna of encoding tyrosine benzene lyase is as shown in SEQ ID NO.3.
The tyrosine amino lyase TcXAL derives from trichosporon cutaneum (Trichosporon cutaneum), Its amino acid sequence is as shown in SEQ ID NO.2, the gene order of encoding tyrosine amino lyase such as SEQ ID NO.4 institute Show.
The Escherichia coli are e. coli bl21 (DE3).
In one embodiment of the invention, expression vector used is pET28a (PB), which is used for independent table The coexpression structure of EhTPL and TcXAL is constructed up to EhTPL or by ePathBrick.Its DNA sequence dna is SEQ ID NO.5.
In one embodiment of the invention, gene EhTPL and TcXAL is different by ePathBrick construction of strategy Co-express structure.
In one embodiment of the invention, the recombination bacillus coli contains through pET28a (PB) with false maneuver Structural order expression EhTPL and TcXAL gene recombinant plasmid pTXe1.
In one embodiment of the invention, the recombination bacillus coli contains through pET28a (PB) with monocistron Structural order expression EhTPL and TcXAL gene recombinant plasmid pTXe3.
The present invention also provides the application recombination bacillus colis with the low values chemical combination such as catechol, Sodium Pyruvate and ammonium chloride Object synthesizes caffeinic method as substrate, by resting cell.
In one embodiment of the invention, the culture of recombination bacillus coli uses TB culture medium.
In one embodiment of the invention, the concentration of recombination bacillus coli is OD in resting cell system600=18 ±1；Substrate is the catechol of 10-100mM, NH4The ratio of the concentration of Cl, Sodium Pyruvate and catechol is 13:1:1；Instead Answering temperature is 25-42 DEG C.
In one embodiment of the invention, the concentration of recombination bacillus coli is OD in resting cell system600=18 ± 1,0.65M NH is also contained in transformation system4Cl, 50mM Sodium Pyruvate and 50mM catechol as substrate, 37 DEG C, Resting cell is carried out under the conditions of 220rpm.
In the present invention, tyrosine benzene lyase (tyrosine phenol-lyase, TPL) with catechol, pyruvic acid and Ammonia synthesizes 3,4- dihydroxy L-phenylalanine (L-DOPA, levodopa) as substrate.From trichosporon cutaneum Levodopa is converted trans- caffeic acid and NH by the tyrosine amino lyase TcXAL of (Trichosporon cutaneum)3, As shown in formula (1):
Since catechol, Sodium Pyruvate and ammonium chloride are relatively inexpensive compound, this method is a kind of tool There is the caffeic acid biological synthesis method of larger potentiality.Caffeinic biology is converted by low value compound invention creates a kind of Synthetic method.With it is previous using l-tyrosine compared with the method for transformation of substrate, substrate used in this method is more cheap and easily In large-scale production.Compared with chemical synthesis process, the product of this method is single trans- caffeic acid, is not needed to different with dividing Structure body is further separated.
Detailed description of the invention
Fig. 1, caffeinic technology path is converted by catechol, pyruvic acid and ammonia.
The structure of Fig. 2, pET28a (PB).
The different coexpression structures of Fig. 3, EhTPL and TcXAL.
The chromatogram and mass spectrogram of Fig. 4, levodopa.Wherein, A is chromatogram, and B is first mass spectrometric figure, and C is second order ms Figure.
Fig. 5, strCL-1 convert the production curve of levodopa synthesis.
Fig. 6, caffeinic chromatogram.Wherein, 1 is the corresponding chromatographic peak of caffeic acid.
Fig. 7, caffeinic mass spectrogram.Wherein, A is the corresponding extraction particle flux of m/z=179.0350 under negative ion mode, B is first mass spectrometric figure, and C is second order ms figure.
Fig. 8, the corresponding maximum coffee acid yield of the recombination bacillus coli containing different coexpression structures.
Specific embodiment
Catechol, levodopa (L-DOPA) and caffeic acid standard items purchased from Sigma-Aldrich (St.Louis, MO), analyze pure catechol, levodopa (L-DOPA), ammonium chloride and Sodium Pyruvate has purchased from raw work bioengineering (Shanghai) Limit company.From the tyrosine benzene lyase genes EhTPL of the raw Erwinia Erwiniaherbicola of grass and from skin The tyrosine amino lyase genes TcXAL of shape trichosporon cutaneum Trichosporon cutaneum is had by Jin Sirui biotechnology Limit company optimizes and synthesizes.Its amino acid sequence is respectively SQ1 and SQ2, and DNA sequence dna is respectively SQ3 and SQ4.
TB culture medium: yeast powder 24g/L, tryptone 12g/L, glycerol 4ml/L, potassium dihydrogen phosphate 17mM, phosphoric acid hydrogen two Potassium 72mM.To prevent from precipitating, potassium dihydrogen phosphate/dipotassium hydrogen phosphate is configured to the mother liquor of 10 times of concentration, filtration sterilization, before use It is added.121 DEG C of high pressure steam sterilization 15min of other compositions.
50mM phosphate buffer PBS: the NaH of 50mM is respectively configured2PO4With the Na of 50mM2HPO4, with NaH2PO4Titration Na2HPO4To different pH.
The sample analysis of resting cell: being centrifuged 2min for sample 12000rpm, take supernatant, after 10 times of methanol dilution, Use 0.22 μm of membrane filtration.Sample analysis uses Shimadzu LC-MS/MS-IT-TOF, 10 μ L of sampling volume, using automatic Feeder.Using C18 reverse chromatograms column (Thermo scientific, ODS-2HYPERSIL, Dim. (mm) 250 × 4.6, particle 5 μm of size) sample is separated.Mobile phase A is water, and Mobile phase B is methanol.Using gradient elution, 0min 5%B, 8min 25%B, 9min 5%B, maintains the concentration to 12min.Flow velocity is 1mL/min.Column temperature: 40 DEG C.It uses UV detector, λ=323nm measure caffeic acid, and λ=280nm measures levodopa.Mass spectral analysis uses negative ion mode, with It extracts ion stream (extracted ion chromatograms, EIC) m/z=179.0350 and detects caffeic acid, to extract ion It flows m/z=196.0615 and detects levodopa.The precursor of second order ms MS/MS analysis is respectively as follows: caffeic acid 179.0350m/z, Levodopa 196.0615m/z；Width is set as 1Da.Pass through the retention time with standard items, first mass spectrometric, second order ms figure It is compared, determines target substance.Quantitative analysis is carried out to caffeic acid and levodopa using the peak area of liquid chromatogram.
The construction method of 1 recombination bacillus coli of embodiment
Gene EhTPL and TcXAL is cloned into pUC57- by Jin Sirui Biotechnology Co., Ltd optimum synthesis Simple, recombinant plasmid are respectively designated as pUC57-EhTPL and pUC57-TcXAL.PET28a (PB) is to be with pET-28a (+) The ePathBrick expression vector of fundamental construction, the carrier include isocaudarner Avr II, Xba I, the I, that is, downstream Spe I and Nhe Sal I, coexpression structure that can be different by ePathBrick construction of strategy.Structure such as Fig. 2, DNA of pET28a (PB) Sequence is SEQ ID NO.5.Respectively using restriction enzyme Bam HI/Hind III digestion recombinant vector pUC57-EhTPL, PUC57-TcXAL and expression vector pET28a (PB) does not cut product using agarose gel electrophoresis separation, and purpose is separately recovered Gene EhTPL (1371bp), TcXAL (2070bp) and expression vector pET28a (PB) (5371bp).It, will according to molar ratio 4:1 Two kinds of target gene are mixed with expression vector pET28a (PB) respectively, are connected under the conditions of 16 DEG C using T4 ligase overnight.It will Connection product converts escherichia coli jm109 competent cell, is coated with the LB plate containing 50 μ g/mL kanamycins.Pass through bacterium colony PCR verifies positive transformant, and the primer sequence is SEQ ID NO.6/SEQ ID NO.7.Positive transformant is forwarded to and is contained Have a LB liquid medium of 50 μ g/mL kanamycins, 37 DEG C, 220rpm extract plasmid after being incubated overnight.Through Bam HI/Hind III double digestion verifies correct recombinant plasmid and is respectively labeled as pET-EhTPL and pET-TcXAL.According to 4 kinds of isocaudarner Avr The combination of II, Xba I, Spe I, Nhe I and Sal I pass through digestion, the coexpression vector of connection building different structure respectively. Double digestion is carried out to pET-CfTPL and pET-TcXAL by restriction enzyme site Spe I/Sal I and Xba I/Sal I respectively, respectively The segment that size is 6.7kb and 2.1kb is recycled, obtains recombinant plasmid pTXe1 after connecting by T4 nucleic acid ligase.Pass through respectively Restriction enzyme site Nhe I/Sal I and Avr II/Sal I carries out double digestion to pET-CfTPL and pET-TcXAL, is separately recovered big The small segment for 6.7kb and 2.1kb obtains recombinant plasmid pTXe3 after connecting by T4 nucleic acid ligase.Different expression structures As shown in Figure 3.Constructed coexpression vector converts e. coli bl21 (DE3) respectively after Sanger sequence verification is correct Competent cell, building obtain a series of engineered strains, as shown in table 1.
Recombinant escherichia coli strain of the table 1 containing different coexpression structures
The cultural method of recombination bacillus coli: the single colonie that plate streaking separates is forwarded to that is mould containing 50 μ g/mL cards The LB liquid medium of element, 37 DEG C, 220rpm is incubated overnight.It is forwarded to the inoculum concentration of 1% (v/v) and is cultivated equipped with 25mL TB In the 250mL triangular flask of base, while adding the kanamycins of final concentration of 50 μ g/mL.37 DEG C, 220rpm culture 4h after, addition The expression of final concentration of 500 μM of IPTG induction EhTPL and TcXAL, is transferred to 25 DEG C, 220rpm for triangular flask, continues to cultivate 10h.Bacterium solution is collected into centrifuge tube, 4000rpm, 4 DEG C of centrifugation 5min collect thallus.
Caffeinic resting cell method: the thallus collected with 25mL PBS washing is resuspended in isometric after centrifugation In PBS.0.65M NH is added simultaneously4Cl, 50mM Sodium Pyruvate and 50mM catechol are reacted as substrate, which exists It is carried out on constant-temperature table.Point sampling in different times is analyzed.
The analysis of 2 recombination bacillus coli strCL-1 catalyzing levorotatory DOPA synthesis capability of embodiment
In order to which recombination bacillus coli E.coli strCL-1 is catalyzed the energy of catechol synthesis levodopa in proof list 1 Power, with method for transformation described in embodiment 1, using the e. coli bl21 (DE3) containing empty plasmid pET28a (PB) as sky White control is used as reaction medium with PBS (50mM, pH 7.0), reacts under the conditions of 37 DEG C, 220rpm, particular point in time sampling, Measure the synthesis situation of levodopa.
The result shows that have the synthesis of levodopa in the reaction system being catalyzed with E.coli strCL-1, and blank control In levodopa is then not detected.The ability of result verification EhTPL catalysis catechol synthesis levodopa, and do not having The synthesis process spontaneous cannot carry out in the presence of tyrosine benzene lyase.The chromatogram and mass spectrogram of levodopa are as schemed 4, wherein A is the chromatogram of levodopa, and B is first mass spectrometric figure, and C is second order ms figure.Yield by levodopa is to taking The mapping of sample time point can determine and reach maximum production 1.64g/L in 0.5 hour levodopa of conversion, as shown in Figure 5.
3 recombination bacillus coli of embodiment is catalyzed catechol and synthesizes caffeic acid proficiency testing
In order to which the conversion of recombination bacillus coli listed by proof list 1 catechol, Sodium Pyruvate and ammonium chloride synthesis are caffeinic Ability is resuspended in isometric PBS (50mM, pH 7.0), cell concentration with the thallus that 25mL PBS washing is collected after centrifugation For OD600=18 ± 1.0.65M NH is added simultaneously4Cl, 50mM Sodium Pyruvate and 50mM catechol are reacted as substrate, The reaction carries out on 37 DEG C, 220rpm constant-temperature table.Made with the e. coli bl21 (DE3) containing empty plasmid pET28a (PB) For blank control.Particular point in time sampling, measures caffeinic synthesis situation.Made with maximum production caffeinic in 0-10 hours For the corresponding maximum production of the bacterial strain.
The result shows that recombination bacillus coli can be catalyzed catechol, Sodium Pyruvate and ammonium chloride synthesis caffeic acid；And Caffeinic synthesis cannot be detected in the transformation system of blank control.The chromatogram of sample analysis is as shown in fig. 6, wherein coffee The corresponding chromatographic peak of acid is (1)；The mass spectrogram of sample analysis is as shown in fig. 7, wherein A is m/z=under negative ion mode 179.0350 corresponding extraction particle fluxes, B are first mass spectrometric figure, and C is second order ms figure.The corresponding coffee acid yield of strTXe1 Highest reaches 214mg/L.Illustrate that the recombination bacillus coli corresponding EhTPL and TcXAL coexpression structure can balance substrate and arrive Caffeinic metabolic fluxes.
Foreign gene structure and coffee acid yield entrained by the different recombination bacillus colis of table 2
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
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gcaatggcaa ttggtctgcg cgaagcaatg cagtacgaat acatcgagca ccgcgtcaaa 960
caggttcgtt atctgggcga taaactgcgc gaagcaggcg ttccgattgt agaaccgacc 1020
ggcggtcacg cagtttttct ggacgcacgt cgtttttgtc cgcatctgac ccaggatcaa 1080
tttccggcac aaagcctggc agcatctatt tacatggaaa ccggcgtccg ttctatggaa 1140
cgtggtattg ttagcgcagg tcgtagcaaa gaaaccggcg aaaaccatag cccgaaactg 1200
gaaaccgttc gtctgaccat tccgcgtcgc gtttatacct acgcgcacat ggacgtcatc 1260
gcggacggta ttatcaaact gtaccagcac aaagaggaca ttcgcggcct gacctttgtt 1320
tacgaaccga aacagctgcg cttctttacc gcgcgtttcg acttcatcta a 1371
<210> 4
<211> 2070
<212> DNA
<213>trichosporon cutaneum
<400> 4
atgtttattg aaaccaacgt ggcaaaaccg gctagcacga aagcgatgaa tgccggctct 60
gcaaaagcgg ccccggtcga accgttcgct acctatgcgc atagtcaggc caccaaaacg 120
gtgtccatcg atggccacac gatgaaagtt ggtgacgtgg ttgctgttgc gcgtcatggc 180
gcgaaagttg aactggcagc tagtgttgct ggtccggtcc gtgcgtccgt ggattttaaa 240
gaaagcaaaa aacacacctc gatttatggc gtgaccacgg gtttcggcgg ttcagccgat 300
acccgtacgt cggacacgga agcactgcag atctctctgc tggaacatca actgtgcggc 360
tttctgccga ccgatgcgac gtacgagggt atgctgctgg cggccatgcc gattccgatc 420
gtgcgtggtg cgatggcggt ccgtgtgaac agctgtgttc gtggccactc tggtgttcgc 480
ctggaagtcc tgcagagctt tgccgatttc attaatcgtg gtctggttcc gtgcgtcccg 540
ctgcgtggta ccatcagtgc atccggtgac ctgtcaccgc tgtcgtatat tgctggcgcg 600
atctgtggtc atccggatgt taaagtcttc gacaccgcag cttcaccgcc gaccgttctg 660
acgtcgccgg aagcaattgc aaaatatggc ctgaaaaccg tcaaactggc gagcaaagaa 720
ggcctgggtc tggttaacgg tacggcagtc tctgcggcgg caggtgctct ggcactgtac 780
gatgccgaat gcctggcaat catgagtcag accaatacgg tgctgaccgt tgaagctctg 840
gacggccatg ttggttcctt tgcaccgttc attcaggaaa tccgtccgca cgcgggccaa 900
attgaagctg cgcgtaacat ccgccatatg ctgggcggtt caaaactggc cgtgcacgaa 960
gaatcggaac tgctggctga tcaggacgcg ggtattctgc gtcaagatcg ctacgccctg 1020
cgtaccagtg cacagtggat cggtccgcaa ctggaagccc tgggtctggc acgccagcaa 1080
attgaaacgg aactgaactc caccacggat aatccgctga tcgacgtgga aggcggtatg 1140
tttcatcacg gcggtaactt ccaggcgatg gcggtcacca gtgctatgga ttccgcgcgc 1200
attgtgctgc agaatctggg taaactgtca tttgcacaag tgaccgaact gatcaactgc 1260
gaaatgaatc atggcctgcc gtcgaacctg gcgggtagcg aaccgtctac caattatcat 1320
tgtaaaggcc tggatattca ctgcggtgcc tactgtgcag aactgggctt tctggcgaac 1380
ccgatgagca atcatgttca gtctaccgaa atgcacaacc agagcgtgaa cagcatggcg 1440
ttcgcaagcg cacgtcgcac gatggaagcg aacgaagttc tgagtctgct gctgggttcc 1500
cagatgtatt gtgctaccca agcgctggat ctgcgcgtca tggaagtgaa atttaaaatg 1560
gccattgtga aactgctgaa tgaaaccctg acgaaacatt ttgccgcatt cctgaccccg 1620
gaacagctgg cgaaactgaa cacccacgct gcgatcacgc tgtacaaacg tctgaatcag 1680
accccgtcat gggattcggc accgcgcttt gaagacgccg caaaacatct ggtgggcgtt 1740
attatggatg cgctgatggt taacgatgac atcaccgacc tgacgaatct gccgaaatgg 1800
aagaaagaat ttgccaaaga agcaggtaac ctgtatcgta gcattctggt ggctaccacg 1860
gcggatggcc gcaatgacct ggaaccggcc gaatatctgg gtcagacccg tgccgtgtac 1920
gaagcagttc gcagcgaact gggcgtcaaa gtgcgtcgcg gtgatgttgc ggaaggcaaa 1980
agcggtaaat ctattggcag ctctgtcgct aaaatcgtgg aagcaatgcg tgacggtcgc 2040
ctgatgggtg cagtgggtaa aatgttttaa 2070
<210> 5
<211> 5371
<212> DNA
<213>artificial sequence
<400> 5
gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg 60
atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc 120
gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg 180
ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc 240
cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc 300
ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg 360
atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta 420
acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg 480
atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa 540
tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg 600
ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa 660
tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt 720
cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc 780
agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt 840
gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 900
cctgaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc 960
tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 1020
gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 1080
tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 1140
ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 1200
agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 1260
aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 1320
accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggagt 1380
cgactcctcc tttcgctagc aaaaaacccc tcaagacccg tttagaggcc ccaaggggtt 1440
atgctagtta ttgctcagcg gtggcagcag ccaactcagc ttcctttact agtttgttag 1500
cagccggatc tcagtggtgg tggtggtggt gctcgagtgc ggccgcaagc ttgtagacgg 1560
agctcgaatt cggatccgcg acccatttgc tgtccaccag tcatgcttgc catatggctg 1620
ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc catggtatat 1680
ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc 1740
ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc 1800
atcgtggccg gcatcaccgg cgcctaggtg cggttgctgg cgcctatatc gccgacatca 1860
ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta 1920
tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc 1980
ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt 2040
cgcataaggg agagcgtcga gatcccggac accatcgaat ggcgcaaaac ctttcgcggt 2100
atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg 2160
ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac 2220
caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg 2280
aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc 2340
gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct 2400
cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa 2460
gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac 2520
tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg 2580
ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac 2640
ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta 2700
gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc 2760
actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt 2820
tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc 2880
aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt 2940
gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg 3000
ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg 3060
caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa 3120
agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 3180
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 3240
taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt 3300
caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac 3360
tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg 3420
cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat 3480
cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa 3540
gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt 3600
gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg 3660
cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat 3720
ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat 3780
tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt 3840
attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac 3900
cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg 3960
tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc 4020
ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat 4080
cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc 4140
agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg 4200
tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 4260
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 4320
gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 4380
tatgcggcat cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc 4440
acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact 4500
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4560
ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4620
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4680
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4740
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4800
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 4860
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4920
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4980
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5040
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 5100
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5160
cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 5220
ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 5280
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct 5340
tacataaaca gtaatacaag gggtgttatg a 5371
<210> 6
<211> 20
<212> DNA
<213>artificial sequence
<400> 6
aagaaagcga aaggagcggg 20
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<400> 7
ccatacccac gccgaaacaa 20

Claims (6)

1. a kind of caffeinic biological synthesis method, which is characterized in that with a kind of coexpression tyrosine benzene lyase EhTPL and junket The recombination bacillus coli of histidine amino group lyase TcXAL, catalysis substrate catechol, Sodium Pyruvate and ammonium chloride synthesize coffee Acid；The tyrosine benzene lyase EhTPL is from the raw Erwinia (Erwinia herbicola) of grass, amino acid sequence Column are as shown in SEQ ID NO.1；The tyrosine amino lyase TcXAL derives from trichosporon cutaneum (Trichosporon cutaneum), amino acid sequence is as shown in SEQ ID NO.2；
The concentration of recombination bacillus coli is OD in resting cell system600=18 ± 1；Substrate is the catechol of 10-100mM, NH4The ratio of the concentration of Cl, Sodium Pyruvate and catechol is 13:1:1；Reaction temperature is 25-42 DEG C.
2. a kind of caffeinic biological synthesis method according to claim 1, which is characterized in that the Escherichia coli are E. coli bl21 (DE3).
3. a kind of caffeinic biological synthesis method according to claim 1, which is characterized in that expression vector used is pET28a(PB)。
4. any a kind of caffeinic biological synthesis method according to claim 1~3, which is characterized in that the recombination Escherichia coli contain the recombinant plasmid for expressing EhTPL and TcXAL gene with the structural order of false maneuver by pET28a (PB) pTXe1。
5. any a kind of caffeinic biological synthesis method according to claim 1~3, which is characterized in that the recombination Escherichia coli contain the recombinant plasmid for expressing EhTPL and TcXAL gene with the structural order of monocistron by pET28a (PB) pTXe3。
6. a kind of caffeinic biological synthesis method according to claim 1, which is characterized in that in resting cell system The concentration of recombination bacillus coli is OD600=18 ± 1,0.65 M NH is also contained in transformation system4Cl, 50mM Sodium Pyruvate and 50mM catechol carries out resting cell under the conditions of 37 DEG C, 220rpm as substrate.
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex;Yuheng Lin等;《Microbial Cell Factories》;20120404;第42卷(第11期);第1-9页
ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli;Peng Xu等;《ACS Synth Biol》;20120504;第1卷(第7期);第256-266页
Gordon V. Louie等.Structural Determinants and Modulation of Substrate Specificity in Phenylalanine-Tyrosine Ammonia-Lyases.《Chemistry & Biology》.2006,第13卷第1327-1338.
微生物酪氨酸解氨酶的研究进展;张峰等;《食品与生物技术学报》;20100731;第29卷(第4期);第496-499
重组大肠杆菌合成左旋多巴条件的优化;李华钟等;《工业微生物》;20021231;第32卷(第2期);第5-9页

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