CN106755135A

CN106755135A - It is a kind of to synthesize caffeinic method by substrate resting cell of levodopa

Info

Publication number: CN106755135A
Application number: CN201611158372.2A
Authority: CN
Inventors: 周景文; 陈坚; 吕永坤; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2016-12-15
Filing date: 2016-12-15
Publication date: 2017-05-31
Anticipated expiration: 2036-12-15
Also published as: CN106755135B

Abstract

Synthesize caffeinic method by substrate resting cell of levodopa the invention discloses one kind, belong to biological chemical field.The present invention with levodopa be substrate biosynthesis caffeic acid.Compared with the conventional method for transformation with L tyrosine as substrate, the method substrate solubility is higher, and yield is high, conversion ratio, and production efficiency is high.Compared with chemical synthesis process, the product of the method is single anti-caffeic acid, it is not necessary to which isomer is further separated.Up to 910.90mg/L, conversion ratio is 99.70% to caffeinic yield after reacting 6 hours.

Description

It is a kind of to synthesize caffeinic method by substrate resting cell of levodopa

Technical field

Synthesize caffeinic method by substrate resting cell of levodopa the present invention relates to one kind, belong to biochemical industry Field.

Background technology

Caffeic acid is a kind of aromatic compounds of high value, and hydroxycinnamic acid can be divided into structure, and it has simultaneously 2 functional groups of phenolic hydroxyl group and acrylic acid.Research in vivo and in vitro shows that caffeic acid has a series of physiological functions.For example, passing through Oxidation mechanism, caffeic acid can suppress cancer cell multiplication；Caffeic acid has immunological regulation and anti-inflammatory activity；Caffeic acid can act also as Antioxidant, and better than other native compounds；In addition, caffeic acid also has antiviral, antidepression, treatment diabetes etc. living Property.

Used as the crucial mesostate of lignin synthesis, caffeic acid is present in nearly all plant.The approach with TYR or L-phenylalanine are precursor, are related to trans-Cinnamate 4-monooxygenase (CYP73A), phenylalanine/tyrosine Amino lyase, p-Coumaric Acid 3- hydroxylases etc..However, content of the caffeic acid in plant is universal 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 to be purified into single any compound all more difficult.

By synthetic biology approach strategy, the route of synthesis by caffeic acid in plant is moved in microorganism chassis, It is Research Thinking main at present to build the engineering bacteria with caffeic acid synthesis capability.However, molten due to substrates such as tyrosine Solution property is poor, the coffee acid yield of such engineering bacteria and relatively low to the conversion ratio of substrate.By with TYR superior strain As the chassis of the heterologous route of synthesis of caffeic acid, help to solve the problem, but effect is unsatisfactory, it shows as production week Phase is more long, and yield is not significantly improved.

The content of the invention

The problem to be solved in the present invention is to provide a kind of caffeinic biological synthesis method, therefore, present invention firstly provides A kind of recombination bacillus coli for expressing tyrosine amino lyase.

Described tyrosine amino lyase RgTAL derives from rhodotorula glutinis (Rhodotorula glutinis), its ammonia As shown in SEQ ID NO.1, the gene order of encoding tyrosine amino lyase is as shown in SEQ ID NO.2 for base acid sequence.

Described Escherichia coli are e. coli bl21 (DE3).

Expression vector used is pET-28a (PB), and as shown in SEQ ID NO.3, gene RgTAL passes through its DNA sequence dna Restriction enzyme site Bam HI/Hind III are subcloned to expression vector pET-28a (PB).

The present invention is also provided and produces caffeinic method with the recombination bacillus coli, is, with levodopa as substrate, to lead to Cross restructuring E. coli whole cell Synthesis caffeic acid.

In described transformation system, the reaction medium of resting cell is the phosphate buffer of 10-200mM, full cell The concentration of recombination bacillus coli is OD in transformation system₆₀₀=20 ± 1, substrate concentration range is 0.01-10g/L, described reaction The pH scopes of system are 6.0-9.0, and range of reaction temperature is 25-42 DEG C.

In one embodiment of the invention, described reaction medium is the phosphate buffer of 10-200mM.

In one embodiment of the invention, the reaction medium of resting cell is the phosphate buffer of 50mM, entirely The concentration of recombination bacillus coli is OD in cell transformation system₆₀₀=20 ± 1, concentration of substrate is 1g/L, described reaction system PH scopes are 7.5, and range of reaction temperature is 37 DEG C.

In one embodiment of the invention, the culture of recombination bacillus coli uses TB culture mediums.

Usefulness of the present invention：Invention creates a kind of caffeinic biosynthesis side with levodopa as substrate Method.Compared with the conventional method for transformation with TYR as substrate, the method substrate solubility is higher, and yield is high, conversion ratio, And production efficiency is high.Compared with chemical synthesis process, the product of the method is single trans- caffeic acid, it is not necessary to different with dividing Structure body is further separated.Under this preferably reaction condition, caffeinic yield is 910.90mg/L after reacting 6 hours, Conversion ratio is 99.70%.Synthesize caffeinic method to be currently known a kind of bioanalysis of conversion ratio highest.

Brief description of the drawings

The caffeinic chromatograms of Fig. 1.Wherein, 1 is the corresponding chromatographic peak of caffeic acid.

Caffeinic mass spectrogram under Fig. 2 ion modes.Wherein, A is the corresponding extraction particle fluxes of 179.0350m/z；B is Caffeinic first mass spectrometric figure；C is the corresponding second order mses figure of caffeic acid.

The corresponding coffee acid yield of different time points in Fig. 3 conversion processes.

Caffeinic yield under Fig. 4 condition of different temperatures.

Caffeinic yield under Fig. 5 condition of different pH.

Caffeinic yield and conversion ratio under the conditions of Fig. 6 difference concentration of substrate.

Specific embodiment

Materials and methods

Caffeic acid and levodopa (L-DOPA) standard items are purchased from Sigma-Aldrich (St.Louis, MO), Rhodotorula glutinis tyrosine amino lyase genes RgTAL is optimized and is closed by Jin Sirui bio tech ltd Into sequence is SQ2.

The configuration of TB culture mediums：Dusty yeast 24g/L, tryptone 12g/L, glycerine 4ml/L, potassium dihydrogen phosphate 17mM, phosphorus Sour hydrogen dipotassium 72mM.To prevent precipitation, potassium dihydrogen phosphate/dipotassium hydrogen phosphate is configured to 10 times of mother liquors of concentration, filtration sterilization, Use preceding addition.121 DEG C of high pressure steam sterilization 15min of other compositions.

The configuration of 50mM phosphate buffers PBS：It is respectively configured the NaH of 50mM₂PO₄With the Na of 50mM₂HPO₄, with NaH₂PO₄Titration Na₂HPO₄To different pH.

Sample analysis：By sample 12000rpm be centrifuged 2min, take supernatant, with 10 times of methanol dilution after, use 0.22 μm filter Membrane filtration.Sample analysis uses Shimadzu LC-MS/MS-IT-TOF, the μ L of sampling volume 10, uses automatic sampler sample introduction. Use C18 reverse chromatograms post (Thermo scientific, ODS-2HYPERSIL, Dim. (mm) 250 × 4.6, particle Size5 μm) sample is separated.Mobile phase A is water, and Mobile phase B is methyl alcohol.Using gradient elution, 0min 5%B, 8min25%B, 9min 5%B, maintain the concentration to 12min.Flow velocity is 1mL/min.Column temperature：40℃.Using UV-detector, λ=323nm determines caffeic acid, and λ=280nm determines levodopa.Mass spectral analysis uses negative ion mode, to extract ion stream (extracted ion chromatograms, EIC) m/z=179.0350 detects caffeic acid, to extract ion stream m/z= 196.0615 detection levodopas.The precursor of second order mses MS/MS analyses is respectively：Caffeic acid 179.0350m/z, levodopa 196.0615m/z；Width is set to 1Da.Compared by the retention time with standard items, first mass spectrometric, second order mses figure, Determine target substance.Quantitative analysis is carried out to caffeic acid and levodopa using the peak area of liquid chromatogram.

Embodiment 1

The construction method of recombination bacillus coli：RgTAL is cloned into by Jin Sirui bio tech ltd optimum synthesis PUC57-Simple, recombinant plasmid is named as pUC57-TcXAL.Weight is digested using restriction enzyme Bam HI/Hind III Group carrier pUC57-TcXAL and expression vector pET-28a (+), are separated using agarose gel electrophoresis and do not cut product, and returned respectively Receive genes of interest RgTAL (2082bp) and expression vector (5368bp).According to mol ratio 4:Genes of interest after 1 Double digestion and Expression vector, is connected overnight using T4 ligases under the conditions of 16 DEG C.Connection product conversion e. coli jm109 competence is thin Born of the same parents, are coated with the LB flat boards containing 50 μ g/mL kanamycins.Positive transformant is verified by bacterium colony PCR, the primer sequence is SQ4/SQ5.Positive transformant is forwarded to the LB liquid medium containing 50 μ g/mL kanamycins, 37 DEG C, 220rpm overnight trains Plasmid is extracted after supporting, plasmid is converted into e. coli bl21 (DE3) competent cell, that is, obtain expressing the restructuring large intestine of RgTAL Bacillus engineered strain, is named as E.coli DCA-2.

The cultural method of recombination bacillus coli：The single bacterium colony that plate streaking is separate is forwarded to containing 50 μ g/mL cards that is mould The LB liquid medium of element, 37 DEG C, 220rpm incubated overnights.It is forwarded to the inoculum concentration of 1% (v/v) and is cultivated equipped with 25mL TB In the 250mL triangular flasks of base, while the kanamycins of the final concentration of 50 μ g/mL of addition.37 DEG C, 220rpm culture 4h after, addition Final concentration of 500 μM of IPTG induces the expression of RgTAL, and triangular flask is transferred into 25 DEG C, 220rpm, continues to cultivate 10h.By bacterium Liquid is collected into centrifuge tube, 4000rpm, 4 DEG C of centrifugation 5min collects thallines.

Caffeinic resting cell method：With the thalline that 25mL PBS washings are collected, it is resuspended in after centrifugation isometric In PBS (50mM, pH 7.0), cell concentration is OD₆₀₀=18 ± 1.The levodopa of final concentration of 1g/L is added the bottom of as simultaneously Thing is reacted, and the reaction is carried out on 37 DEG C, 220rpm constant-temperature tables.With the large intestine bar containing empty plasmid pET-28a (PB) Bacterium BL21 (DE3) is used as blank.Particular point in time is sampled, and determines caffeinic synthesis situation.

Result shows, to have caffeinic synthesis in the reaction system that E.coli DCA-2 are catalyzed, and in blank then It is not detected by caffeic acid.Result verification RgTAL catalyzing levorotatories DOPA synthesizes caffeinic ability, and the building-up process can not It is spontaneous to carry out.Caffeinic chromatogram such as Fig. 1, wherein, 1 is the corresponding peak of caffeic acid；Caffeinic mass spectrogram as shown in Fig. 2 To extract particle flux, B is first mass spectrometric figure to wherein A, and C is second order mses figure.Sampling time point is made by caffeinic yield Figure, it may be determined that reach maximum production 626.10mg/L 8 hours caffeic acids are converted, conversion ratio is 68.53%, as shown in Figure 3.

The temperature of embodiment 2 is on the caffeinic influence of Synthesis

Temperature conditionss in caffeic acid conversion process are optimized, using recombination bacillus coli E.coli DCA-2 as catalysis Agent, with PBS (50mM, pH 7.0) as reaction medium, levodopa addition concentration is 1g/L.Respectively 20 DEG C, 25 DEG C, 30 DEG C, converted under the temperature conditionss of 37 DEG C and 42 DEG C, speed setting is 220rpm.Because caffeic acid reached maximum at 6 hours Yield, therefore sampled at the 6th hour, determine caffeinic yield and conversion ratio.

As shown in figure 4, as a result showing, caffeinic yield increases with the rising of temperature, after temperature is more than 37 DEG C, coffee The yield of coffee acid does not increase to be reduced on the contrary.This may be with caffeic acid under higher temperature conditions degraded it is relevant.Under the conditions of 37 DEG C After conversion 6 hours, caffeic acid reaches maximum production, i.e. 865.75mg/L, and conversion ratio is 94.76%.

Embodiment 3pH produces the influence of result to caffeic acid

PH conditions in caffeic acid conversion process are optimized, using recombination bacillus coli E.coli DCA-2 as catalysis Agent, levodopa addition concentration is 1g/L.Conversion condition is set as 37 DEG C, 220rpm.Respectively with different pH (6.0,6.5,7.0, 7.5th, 8.0, PBS (50mM) 8.5) is used as conversion medium.Sampled at the 6th hour respectively, determine caffeinic yield and conversion Rate.

As shown in figure 5, as a result showing, caffeinic yield increases with the rising of pH, after pH is more than 7.5, coffee The yield of acid starts to reduce.It is relevant that this may in the basic conditions be easily converted to benzoquinone compound with caffeic acid.At 37 DEG C Under the conditions of conversion 6 hours after, caffeic acid reaches maximum production, i.e. 910.90mg/L, and conversion ratio is 99.70%, to be currently known A kind of method of conversion ratio highest in bioanalysis synthesis caffeic acid.

The influence that the different concentration of substrate of embodiment 4 are produced to caffeic acid

Concentration of substrate in caffeic acid conversion process is optimized, using recombination bacillus coli E.coli DCA-2 as urging Agent, levodopa addition concentration is respectively 2g/L, 5g/L and 10g/L.Conversion condition be above-mentioned optimal conditions, i.e., 37 DEG C, 220rpm、pH 7.5.Sampled at the 6th hour, determine caffeinic yield and conversion ratio.

As shown in fig. 6, as a result showing, with the increase of concentration of substrate, caffeinic yield is improved constantly, during 10g/L Coffee acid yield highest, reaches 4.33g/L.But, with the raising of concentration of substrate, the conversion ratio of substrate is constantly reduced.Reason May is that, substrate is not yet converted completely in 6h.Therefore, extension transformation time is advantageously possible for further improving turning for substrate Rate.

Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes with modification, therefore protection model of the invention Enclose being defined of being defined by claims.

SEQUENCE LISTING

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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

aagaaagcga aaggagcggg 20

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence

<400> 5

ccatacccac gccgaaacaa 20

Claims

1. a kind of caffeinic efficient resting cell method, it is characterised in that to express tyrosine amino lyase RgTAL's Recombination bacillus coli, whole-cell catalytic levodopa Synthesis caffeic acid.

2. a kind of caffeinic bioconversion method according to claim 1, it is characterised in that described tyrosine amino The amino acid sequence of lyase RgTAL is as shown in SEQ ID NO.1.

3. a kind of caffeinic biological synthesis method according to claim 1 and 2, it is characterised in that described large intestine bar Bacterium is e. coli bl21 (DE3).

4. according to any described a kind of caffeinic biological synthesis method of claims 1 to 3, it is characterised in that table used It is pET-28a (PB) up to carrier.

5. a kind of caffeinic biological synthesis method according to claim 4, it is characterised in that encoding tyrosine amino splits The gene RgTAL of synthase is subcloned to expression vector pET-28a (PB) by restriction enzyme site Bam HI/Hind III.

6. according to any described a kind of caffeinic biological synthesis method of Claims 1 to 5, it is characterised in that full cell turns The reaction medium of change system is the phosphate buffer of 10-200mM, and the concentration of recombination bacillus coli is in resting cell system OD₆₀₀=20 ± 1, substrate concentration range is 0.01-10g/L, and the pH scopes of described resting cell system are 6.0-9.0, instead It is 25-42 DEG C to answer temperature range.

7. a kind of caffeinic biological synthesis method according to claim 6, it is characterised in that described reaction medium is The phosphate buffer of 10-200mM.

8. a kind of caffeinic biological synthesis method according to claim 6, it is characterised in that the reaction of resting cell Medium is the phosphate buffer of 50mM, and the concentration of recombination bacillus coli is OD in resting cell system₆₀₀=20 ± 1, substrate Concentration is 1g/L, and the pH scopes of described reaction system are 7.5, and range of reaction temperature is 37 DEG C.

9. a kind of recombination bacillus coli, it is characterised in that expression tyrosine amino lyase RgTAL.

10. a kind of recombination bacillus coli according to claim 9, it is characterised in that described tyrosine amino lyase As shown in SEQ ID NO.1, e. coli bl21 (DE3) is host to the amino acid sequence of RgTAL, and expression vector is pET-28a (PB)。