CN106755135B - Method for whole-cell transformation synthesis of caffeic acid by taking levodopa as substrate - Google Patents
Method for whole-cell transformation synthesis of caffeic acid by taking levodopa as substrate Download PDFInfo
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- CN106755135B CN106755135B CN201611158372.2A CN201611158372A CN106755135B CN 106755135 B CN106755135 B CN 106755135B CN 201611158372 A CN201611158372 A CN 201611158372A CN 106755135 B CN106755135 B CN 106755135B
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Abstract
The invention discloses a method for synthesizing caffeic acid by whole-cell transformation with levodopa as a substrate, belonging to the field of biochemical engineering. The invention takes levodopa as a substrate to biosynthesize caffeic acid. Compared with the prior conversion method using L-tyrosine as a substrate, the method has the advantages of high substrate solubility, high yield, high conversion rate and high production efficiency. Compared with the chemical synthesis method, the product of the method is single trans-caffeic acid, and further separation of isomers is not needed. After 6 hours of reaction, the yield of the caffeic acid can reach 910.90mg/L, and the conversion rate is 99.70%.
Description
Technical Field
The invention relates to a method for synthesizing caffeic acid by whole-cell transformation with levodopa as a substrate, belonging to the field of biochemical engineering.
Background
Caffeic acid is a high-value aromatic compound, which can be structurally classified as hydroxycinnamic acid, and has 2 functional groups of phenolic hydroxyl and acrylic acid. In vivo and in vitro studies indicate that caffeic acid has a series of physiological functions. For example, caffeic acid can inhibit cancer cell proliferation by an oxidative mechanism; caffeic acid has immunoregulatory and antiinflammatory activity; caffeic acid can also be used as antioxidant, and is superior to other natural compounds; in addition, caffeic acid also has antiviral, antidepressant, and diabetes treating effects.
Caffeic acid is present in almost all plants as a key intermediate metabolite of lignin synthesis. The pathway takes L-tyrosine or L-phenylalanine as a precursor, and relates to trans-cinnamic acid 4-monooxygenase (CYP73A), phenylalanine/tyrosine amino lyase, p-coumaric acid 3-hydroxylase and the like. However, caffeic acid is generally present in low levels in plants and is therefore difficult to extract. On the other hand, chemically synthesized caffeic acid is a mixture of homoeotic and trans caffeic acids; and because of the similarity of the structures, it is difficult to completely separate and purify any single compound.
The method is a main research idea at present by translating the synthetic pathway of caffeic acid in plants into a microbial chassis through a synthetic biological pathway strategy to construct engineering bacteria with caffeic acid synthesis capacity. However, because the solubility of substrates such as tyrosine is poor, the caffeic acid yield and the conversion rate of the substrates of the engineering bacteria are low. This problem is helped to be solved by using the L-tyrosine high-producing strain as a chassis of the caffeic acid heterologous synthesis pathway, however, the effect is not ideal, which is manifested by a longer production cycle and no significant increase in yield.
Disclosure of Invention
The problem to be solved by the invention is to provide a biosynthesis method of caffeic acid, and therefore, the invention firstly provides a recombinant Escherichia coli for expressing tyrosine amino lyase.
The tyrosine amino lyase RgTAL is derived from Rhodotorula glutinis (Rhodotorula glutinis), the amino acid sequence of the RgTAL is shown as SEQ ID NO.1, and the gene sequence of the coding tyrosine amino lyase is shown as SEQ ID NO. 2.
The Escherichia coli is Escherichia coli BL21(DE 3).
The expression vector used is pET-28a (PB), the DNA sequence of which is shown in SEQ ID NO.3, and the gene RgTAL is subcloned into the expression vector pET-28a (PB) through restriction enzyme cutting sites Bam HI/Hind III.
The invention also provides a method for producing caffeic acid by using the recombinant escherichia coli, which takes levodopa as a substrate and synthesizes the caffeic acid through whole cell transformation of the recombinant escherichia coli.
In the transformation system, the reaction medium for whole cell transformation is 10-200mM phosphate buffer solution, and the concentration of recombinant Escherichia coli in the whole cell transformation system is OD600The concentration of the substrate is 20 +/-1, the range of the concentration of the substrate is 0.01-10g/L, the pH range of the reaction system is 6.0-9.0, and the reaction temperature range is 25-42 ℃.
In one embodiment of the invention, the reaction medium is a phosphate buffer of 10 to 200 mM.
In one embodiment of the present invention, the reaction medium for whole-cell transformation is 50mM phosphate buffer, and the concentration of recombinant E.coli in the whole-cell transformation system is OD600The substrate concentration was 1g/L, the pH of the reaction system was 7.5, and the reaction temperature was 37 ℃.
In one embodiment of the present invention, the recombinant E.coli is cultured using TB medium.
The invention has the advantages that: the invention creates a biosynthesis method of caffeic acid with levodopa as a substrate. Compared with the prior conversion method using L-tyrosine as a substrate, the method has the advantages of high substrate solubility, high yield, high conversion rate and high production efficiency. Compared with the chemical synthesis method, the product of the method is single trans-caffeic acid, and further separation of isomers is not needed. Under the preferred reaction conditions, the yield of caffeic acid after 6 hours of reaction was 910.90mg/L, and the conversion was 99.70%. Is a biological method for synthesizing caffeic acid with the highest conversion rate.
Drawings
FIG. 1 chromatogram of caffeic acid. Wherein, 1 is a chromatographic peak corresponding to caffeic acid.
FIG. 2 is a mass spectrum of caffeic acid in the anionic mode. Wherein A is an extracted particle flow corresponding to 179.0350 m/z; b is a primary mass spectrogram of caffeic acid; c is a secondary mass spectrogram corresponding to caffeic acid.
Figure 3 caffeic acid production at various time points during the conversion process.
FIG. 4 shows the yield of caffeic acid under different temperature conditions.
FIG. 5 yield of caffeic acid at different pH conditions.
FIG. 6 yield and conversion of caffeic acid under different substrate concentrations.
Detailed Description
Materials and methods
Caffeic acid and levodopa (L-DOPA) standards were purchased from Sigma-Aldrich (st. louis, MO), Rhodotorula gluteninis tyrosine amino lyase gene RgTAL optimized and synthesized by tsingri biotechnology ltd, sequence SQ 2.
Preparation of TB culture Medium: 24g/L of yeast powder, 12g/L of tryptone, 4ml/L of glycerol, 17mM of monopotassium phosphate and 72mM of dipotassium phosphate. To prevent precipitation, potassium dihydrogen phosphate/dipotassium hydrogen phosphate is prepared into a mother liquor with a concentration 10 times that of the mother liquor, and the mother liquor is filtered to sterilize and added before use. Sterilizing the rest components with high pressure steam at 121 deg.C for 15 min.
Preparation of 50mM phosphate buffered saline PBS: 50mM NaH was prepared separately2PO4And 50mM Na2HPO4With NaH2PO4Titration of Na2HPO4To a different pH.
And (3) sample analysis: the sample was centrifuged at 12000rpm for 2min, and the supernatant was diluted 10-fold with methanol and filtered through a 0.22 μm filter. Sample analysis was performed using Shimadzu LC-MS/MS-IT-TOF with a sample volume of 10. mu.L using an autosampler. The samples were separated using a C18 reverse phase chromatography column (Thermo scientific, ODS-2HYPERSIL, Dim. (mm) 250X 4.6, particulate size5 μm). Mobile phase a was water and mobile phase B was methanol. Gradient elution was used, 0min 5% B, 8min 25% B, 9min 5% B, maintaining the concentration to 12 min. The flow rate was 1 mL/min. Column temperature: at 40 ℃. Caffeic acid and levodopa were measured using an ultraviolet detector at λ 323nm and 280 nm. Mass spectrometry uses a negative ion mode to detect caffeic acid with an extracted ion fluxes (EIC) m/z 179.0350 and levodopa with an extracted ion fluxes m/z 196.0615. The precursors for the secondary mass MS/MS analysis were: caffeic acid 179.0350m/z, levodopa 196.0615 m/z; the width is set to 1 Da. And determining the target substance by comparing the retention time with the retention time of the standard substance, the primary mass spectrum and the secondary mass spectrum. The peak areas of the liquid chromatogram were used for quantitative analysis of caffeic acid and levodopa.
Example 1
The construction method of the recombinant Escherichia coli comprises the following steps: RgTAL was optimally synthesized by Kinsley Biotechnology, Inc. and cloned into pUC57-Simple, and the recombinant plasmid was named pUC 57-TcXAL. The recombinant vector pUC57-TcXAL and the expression vector pET-28a (+) were digested with restriction endonuclease Bam HI/Hind III, the uncut product was separated by agarose gel electrophoresis, and the gene of interest RgTAL (2082bp) and the expression vector (5368bp) were recovered, respectively. The enzyme-cleaved target gene and the expression vector were mixed at a molar ratio of 4:1, and ligated with T4 ligase at 16 ℃ overnight. The ligation product was transformed into competent cells of Escherichia coli JM109 and plated on LB plates containing 50. mu.g/mL of kanamycin. Positive transformants were verified by colony PCR using the primer sequence SQ4/SQ 5. Transferring the positive transformant to a liquid LB culture medium containing 50 mu g/mL kanamycin, culturing overnight at 37 ℃ and 220rpm, extracting a plasmid, and converting the plasmid into escherichia coli BL21(DE3) competent cells to obtain the recombinant escherichia coli engineering strain expressing the RgTAL, which is named as E.
The culture method of the recombinant Escherichia coli comprises the following steps: the single colony streaked from the plate was transferred to liquid LB medium containing 50. mu.g/mL kanamycin and cultured overnight at 37 ℃ and 220 rpm. 1% (v/v) of the inoculum was transferred to a 250mL Erlenmeyer flask containing 25mL of TB medium, to which was added kanamycin to a final concentration of 50. mu.g/mL. After culturing at 37 ℃ and 220rpm for 4 hours, the expression of RgTAL was induced by adding IPTG at a final concentration of 500. mu.M, and the flask was transferred to 25 ℃ and 220rpm, and further cultured for 10 hours. Collecting the bacterial liquid into a centrifuge tube, centrifuging at 4000rpm and 4 ℃ for 5min, and collecting thalli.
The whole-cell transformation method of caffeic acid comprises the following steps: the collected cells were washed with 25mL PBS, centrifuged, and resuspended in an equal volume of PBS (50mM, pH 7.0) at OD60018 ± 1. While levodopa was added as a substrate at a final concentration of 1g/L to carry out a reaction at 37 ℃ on a 220rpm constant temperature shaker. To containColi BL21(DE3) having the empty plasmid pET-28a (PB) was used as a blank control. Samples were taken at specific time points to determine the synthesis of caffeic acid.
The results show that there was caffeic acid synthesis in the reaction system catalyzed by e. The results verify the ability of RgTAL to catalyze the synthesis of caffeic acid from levodopa, and the synthesis process cannot proceed spontaneously. The chromatogram of caffeic acid is shown in FIG. 1, wherein 1 is the peak corresponding to caffeic acid; the mass spectrum of caffeic acid is shown in FIG. 2, wherein A is the extracted particle stream, B is the primary mass spectrum, and C is the secondary mass spectrum. By plotting the caffeic acid production against the sampling time points, it was determined that the maximum production of 626.10mg/L was reached at 8 hours of conversion with a conversion of 68.53%, as shown in FIG. 3.
EXAMPLE 2 Effect of temperature on the conversion of synthetic caffeic acid
The temperature condition in the caffeic acid conversion process is optimized, recombinant escherichia coli E.coli DCA-2 is used as a catalyst, PBS (50mM, pH 7.0) is used as a reaction medium, and the addition concentration of levodopa is 1 g/L. The conversion was carried out at 20 ℃, 25 ℃, 30 ℃, 37 ℃ and 42 ℃ respectively, with the rotation speed set at 220 rpm. Since caffeic acid reached maximum yield at 6 hours, samples were taken at 6 hours and caffeic acid yield and conversion were determined.
As shown in fig. 4, the results showed that the yield of caffeic acid increased with increasing temperature, but did not increase but decreased after exceeding 37 ℃. This may be associated with the degradation of caffeic acid under higher temperature conditions. After 6 hours of conversion at 37℃, the maximum yield of caffeic acid was reached, 865.75mg/L, with a conversion of 94.76%.
Example 3 influence of pH on caffeic acid production results
The pH condition in the caffeic acid conversion process is optimized, recombinant Escherichia coli E.coli DCA-2 is used as a catalyst, and the addition concentration of levodopa is 1 g/L. The conversion conditions were set at 37 ℃ and 220 rpm. PBS (50mM) at different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5) was used as transformation medium. Samples were taken at 6 hours to determine caffeic acid production and conversion.
As shown in fig. 5, the results showed that the yield of caffeic acid increased with increasing pH, and that the yield of caffeic acid began to decrease when the pH exceeded 7.5. This may be associated with the ease with which caffeic acid is converted to benzoquinone like compounds under alkaline conditions. After 6 hours of conversion at 37 ℃, the caffeic acid achieves the maximum yield of 910.90mg/L, and the conversion rate is 99.70%, which is the highest conversion rate in the currently known biological method for synthesizing caffeic acid.
EXAMPLE 4 Effect of different substrate concentrations on caffeic acid production
The substrate concentration in the caffeic acid conversion process is optimized, recombinant escherichia coli E.coli DCA-2 is used as a catalyst, and the addition concentrations of levodopa are 2g/L, 5g/L and 10g/L respectively. The conversion conditions were at the optimum conditions described above, i.e., 37 deg.C, 220rpm, pH 7.5. Samples were taken at 6 hours and the caffeic acid production and conversion were determined.
As shown in FIG. 6, the results showed that the yield of caffeic acid was increased with increasing substrate concentration, with the highest caffeic acid yield at 10g/L, reaching 4.33 g/L. However, as the substrate concentration increases, the conversion of the substrate decreases. The reason may be that the substrate was not completely converted at 6 h. Therefore, it is possible to further increase the conversion rate of the substrate by prolonging the conversion time.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for whole-cell transformation and synthesis of caffeic acid by using levodopa as substrate
<160>5
<170>PatentIn version 3.3
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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>4
<211>20
<212>DNA
<213> Artificial sequence
<400>4
<210>5
<211>20
<212>DNA
<213> Artificial sequence
<400>5
Claims (3)
1. A high-efficiency whole-cell transformation method of caffeic acid is characterized in that the recombinant large intestine expressing tyrosine amino lyase RgTAL is usedBacillus, which catalyzes levodopa to convert into caffeic acid by whole cells; the amino acid sequence of the tyrosine amino lyase RgTAL is shown as SEQ ID NO.1, the recombinant Escherichia coli takes Escherichia coli BL21(DE3) as a host and pET-28a (PB) as an expression vector; the reaction medium of the whole cell transformation system is 10-200mM phosphate buffer solution, and the concentration of the recombinant Escherichia coli in the whole cell transformation system is OD600=20 + -1, substrate concentration range is 0.01-10g/L, pH range of the whole cell transformation system is 6.0-9.0, reaction temperature range is 25-42oC。
2. The method of claim 1, wherein the gene encoding tyrosine amino lyase is used as the gene encoding tyrosine amino lyaseRgTALBy restriction of the enzyme siteBamHI/HindIII subcloning into expression vector pET-28a (PB).
3. The method of claim 1, wherein the reaction medium for whole-cell transformation is 50mM phosphate buffer, and the concentration of recombinant E.coli in the whole-cell transformation system is OD600=20 ± 1, substrate concentration of 1g/L, pH range of the reaction system of 7.5, reaction temperature range of 37oC。
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| WO2008069958A2 (en) * | 2006-12-01 | 2008-06-12 | The Salk Institute For Biological Studies | Substrate switched ammonia lyases and mutases |
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