CN108753852A - A kind of method that bioanalysis prepares raspberry ketone - Google Patents
A kind of method that bioanalysis prepares raspberry ketone Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
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- C12P7/26—Ketones
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Abstract
The invention discloses a kind of method that bioanalysis prepares raspberry ketone, it is included in Escherichia coli 4- coumaroyl A ligases, benzylacetone synthase and the benzylacetone reductase of induced expression plant origin simultaneously.The present invention is newly added to benzylacetone reductase (RiRZS1) in the metabolic pathway of biosynthesis raspberry ketone, increases transformation efficiency of the intermediate product to hydroxyl BENZYLIDENE ACETONE.In addition, the yield of shake flask fermentation raspberry ketone has reached 70.62mg/L, the yield of raspberry ketone is further significantly improved with biological synthesis process.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of method that bioanalysis prepares raspberry ketone.
Background technology
Raspberry ketone, also known as 4-(4-hydroxyphenyl)-2-butanone are the main aromatic components of Fructus Rubi, have characteristic hesperidium fragrance.
It is present in the fruit and vegetables such as raspberry, blackberry, blueberry, grape and rheum officinale.The purposes of raspberry ketone has diversity, as odor type
Essence, dressing agent or fixastive are largely used in fragrances, food flavor, daily chemical essence, flavouring essence for tobacco and cosmetics.Separately
Outer raspberry ketone can be used for the synthesis of drug, dyestuff and pesticide as a kind of intermediate of fine chemistry industry.Nowadays raspberry ketone
In perfume industry, it has also become be only second to a kind of fragrance with high economic value of vanillic aldehyde.
There are mainly three types of the preparation methods of raspberry ketone, plant extraction method, chemical synthesis and biological synthesis process, due to planting
Content is extremely low in object, only 0.11-0.12mg/kg, therefore the method cost extracted from plant is very high, so industry is raw at present
Production is mainly based on chemical synthesis.
Chemical synthesis is divided into the raspberry ketone synthesized with petrochemical materials from raw material sources and purposes and with natural
The raspberry ketone of equivalent anisic aldehyde synthesis.Mainly there are 4 kinds with petrochemical materials synthesis raspberry ketone：(1) with phenol and
Methyl vinyl ketone synthesizes raspberry ketone for raw material.(2) using phenol and 4- butanol -2- ketone as raw material, under acid catalysis into
Row alkylated reaction generates raspberry ketone.The method uses raw material butanol ketone, can be to avoid using the ethylene methacrylic being more toxic
Base ketone.(3) it by ethyl acetoacetate after the alpha-alkyl to methoxy base pitch chlorine replaces, carries out keto-acid and decomposes to obtain 4- to methoxyl group
Phenyl 2 butanone.Under concentrated hydrobromic acid effect, ether bond rupture ultimately produces 4-(4-hydroxyphenyl)-2-butanone (British patent
GB1094417).(4) hydroxy benzaldehyde carries out Claisen-Schmidt condensations with acetone, then hydrogenating reduction, then is steamed through decompression
The raspberry ketone (Chinese patent CN1097729) that post-processings obtain desired purity such as evaporate and recrystallize.Although chemical synthesis valence
Lattice are cheap, yield is very high, but obtained product its fragrance tends not to meet high-grade daily chemical essence and food flavor requirement,
It can only be used in the allotment of daily chemical essence and the raw material as synthesis insect attractant, meanwhile, there is also environment dirts for these methods
The problems such as dye, equipment burn into yield are low and post-processing is difficult is urgently to be resolved hurrily.
Chinese patent CN200910114390.4 is disclosed a kind of synthesizing raspberry ketone by natural equivalent anisic aldehyde
New method.Using natural equivalent anisic aldehyde as raw material, raspberry ketone, process conditions temperature can be prepared through three step process
With for reaction yield up to 67.5%, the more other synthesis type raspberry ketone odor types of product more faint scent, lasting is more lasting.By big
The raspberry ketone fragrance of anisaldehyde synthesis and the raspberry ketone naturally extracted are close, can be used for high-grade daily chemical essence and food is fragrant
In the formula of essence, but this semisynthesis depends on the supply of anisic aldehyde raw material.
Invention content
The purpose of this part is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferably to implement
Example.It may do a little simplified or be omitted to avoid our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, and this simplification or omit and cannot be used for limiting the scope of the invention.
In view of above-mentioned technological deficiency, it is proposed that the present invention.
Therefore, the present invention overcomes the deficiencies in the prior art, provides a kind of method that bioanalysis prepares raspberry ketone.
In order to solve the above technical problems, the present invention provides following technical solutions：A kind of bioanalysis prepares raspberry ketone
Method comprising, the 4- coumaroyl A ligases of induced expression plant origin, benzylacetone close simultaneously in Escherichia coli
Enzyme and benzylacetone reductase.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention comprising,
The gene of 4- coumaroyl A ligases and the gene of benzylacetone synthase is enterprising in plasmid pACYC-Duet-1
Row coexpression, obtains expression vector pACYC-BAS-4CL；
The RiRZS1 genes of benzylacetone reductase are expressed on plasmid pET-Duet-1, obtain expression vector
pET-RZS1；
Expression vector pACYC-BAS-4CL, pET-RZS1 are imported into e. coli bl21 (DE3) simultaneously, it is big to obtain recombination
Enterobacteria Mu1.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：Encode the 4- tonka-beans
The nucleotide sequence of the gene 4CL of acyl coenzyme A ligases derives from plant parsley as shown in SEQ ID NO.1
(Petroselinum crispum) and codon optimize；Encode the nucleotide of the gene BAS of the benzylacetone synthase
Sequence is as shown in SEQ ID NO.2, from plant sorrel (Rheum palmatum) and codon optimization；Coding
The nucleotide sequence of the gene RiRZS1 of the benzylacetone reductase derives from plant raspberry as shown in SEQ ID NO.3
(Raspberry ketone), and codon optimizes.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：Further include, it will be described
Recombination bacillus coli Mu1 passes through IPTG Fiber differentiations, addition p-Coumaric Acid fermentation production raspberry ketone.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：It is described to obtain expression load
Body pACYC-BAS-4CL, including the 4- that is first cloned with EcoRI and HindIII double digestion plasmid pACYC-Duet-1 and PCR
Coumaroyl A connection enzyme genes obtain recombinant plasmid pACYC-4CL by one-step cloning, use KpnI and XhoI double digestions later
The benzylacetone synthase gene that plasmid pACYC-4CL and PCR is cloned obtains recombinant plasmid pACYC- by one-step cloning
BAS-4CL。
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：With BglII and KpnI
The benzylacetone reductase gene that double digestion plasmid pET-Duet-1 and PCR is cloned obtains recombinant plasmid by one-step cloning
pET-RZS1。
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：By plasmid pACYC-
BAS-4CL, pET-RZS1 thermal shock method are imported simultaneously in e. coli bl21 (DE3), obtain recombination bacillus coli Mu1.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：By recombination bacillus coli
It is transferred in seed LB culture mediums after Mu1 activation, and adds 10~30mg/mL chloramphenicol and 50~100mg/mL ammonia benzyl moulds
Element is incubated overnight with the rotating speed of 180~300rmp at 20~37 DEG C, and being inoculated into fresh fermentation later with 5% inoculum concentration trains
It supports in base, adds 10~30mg/mL chloramphenicol and 50~100mg/mL ampicillins, OD is arrived in 20~37 DEG C of cultures600For
When 1.0~1.2, the IPTG and substrate of final concentration of 0.1~1mM are added, continues culture in 20~37 DEG C to 72h.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：The p-Coumaric Acid adds
Final concentration of 100~the 150mg/L being added in fermentation medium.
A kind of preferred embodiment of the method for raspberry ketone is prepared as bioanalysis of the present invention：The fermented and cultured
Base, including TB culture mediums, component include 8~15g/L peptones, 15~30g/L yeast extracts, 1~5g/L glycerine, 1~5g/L
KH2PO4, 5~15g/L K2HPO4, 5~10g glucose.
Beneficial effects of the present invention：The present invention is newly added to benzylacetone in the metabolic pathway of biosynthesis raspberry ketone
Reductase (RiRZS1) increases transformation efficiency of the intermediate product to hydroxyl BENZYLIDENE ACETONE.In addition, shake flask fermentation raspberry ketone
Yield reached 70.62mg/L, the yield of raspberry ketone is further significantly improved with biological synthesis process.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment
Attached drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for this
For the those of ordinary skill of field, without having to pay creative labor, it can also be obtained according to these attached drawings other
Attached drawing.Wherein：
Fig. 1 is raspberry ketone biosynthesis pathway schematic diagram of the present invention.
Fig. 2 is plasmid pACYC-BAS-4CL of the present invention and pET-RZS1 schematic diagrames.
Fig. 3 product raspberry ketone GC-MS figures of the present invention.
Fig. 4 is different culture media in the present invention to the yield effect of raspberry ketone
Specific implementation mode
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, with reference to specific embodiment pair
The specific implementation mode of the present invention is described in detail.
Many details are elaborated in the following description to facilitate a thorough understanding of the present invention, still the present invention can be with
Implemented different from other manner described here using other, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein refers to that may be included at least one realization side of the present invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiment.
Embodiment 1：
The nucleotide sequence of the gene 4CL of the 4- coumaroyls A ligases is encoded as shown in SEQ ID NO.1,
From plant parsley (Petroselinum crispum) and codon optimization；Encode the base of the benzylacetone synthase
Because the nucleotide sequence of BAS is as shown in SEQ ID NO.2, from plant sorrel (Rheum palmatum) and through overstocked
Numeral optimizes；The nucleotide sequence of the gene RiRZS1 of the benzylacetone reductase is encoded as shown in SEQ ID NO.3, source
In plant raspberry (Raspberry ketone), and codon optimizes.
The structure of plasmid pACYC-BAS-4CL：
Gene 4CL2 (SEQ ID are obtained by PCR primers A1 (SEQ ID No.4) and A2 (SEQ ID No.5) clones
NO.1 encodes 4- coumaroyl A ligases)：Pass through PCR primer B1 (SEQ ID No.6) and B2 (SEQ ID No.7) gram
It is grand to obtain gene BAS (SEQ ID NO.2 coding benzylacetones synthase).First use EcoRI and HindIII double digestion plasmids pACYC-
The 4CL genes (SEQ ID NO.1 coding 4- coumaroyl A ligases) that Duet-1 and PCR is cloned pass through one-step cloning
Recombinant plasmid pACYC-4CL is obtained, the BAS bases cloned later with KpnI and XhoI double digestion plasmid pACYC-4CL and PCR
Because (SEQ ID NO.2 coding benzylacetones synthase) obtains recombinant plasmid pACYC-4CL-BAS by one-step cloning.Wherein PCR
Reaction condition is：95℃5min；98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 5s/kb (30 cycles)；72 DEG C of 10min, later with 1% fine jade
Sepharose electrophoresis is verified and recycles pcr amplification product.One-step cloning specific method is by segment (0.04-0.08ng/bp) and carries
Body (0.005-0.01ng/bp) is mixed in same PCR pipe, and the recombinase of the recombination enzyme buffer liquid and 1-2 μ l of 2-4 μ l is added, adds
DdH2O polishings react 30min at 37 DEG C, obtain recombinant plasmid to 10-20 μ l.
A1：5'-CCACAGCCAGGATCCGAATTCATGGGTGATTGCGTTGCG-3’
(underscore is EcoRI restriction enzyme sites)
A2：5'-GCATTATGCGGCCGCAAGCTTTCATTTCGGCAGATCACCAGA-3’
(underscore is HindIII restriction enzyme sites)
B1：5'-GCGATCGCTGACGTCGGTACCAAGGAAACCGCCGCCGCA-3’
(underscore is KpnI restriction enzyme sites)
B2：5'-GGTTTCTTTACCAGACTCGAGTTAGCTAATAACCGGAACGC-3’
(underscore is XhoI restriction enzyme sites)
Embodiment 2：
The structure of plasmid pET-RZS1：
Select pET-Duet-1 as the expression vector of RiRZS1 genes.By PCR with primer C1 (SEQ ID NO.8) and
C2 (SEQ ID NO.9) clones obtain gene RiRZS1 (SEQ ID NO.3 coding benzylacetones reductase).By carrier pET-
RiRZS1 genes are inserted into matter by Duet-1 with after BglII and KpnI digestion with restriction enzyme with the method for one-step cloning
The corresponding restriction enzyme site of grain pET-Duet-1, obtains recombinant plasmid pET-RZS1.Wherein PCR reaction conditions are：95℃5min；
98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 5s/kb (30 cycles)；72 DEG C of 10min, are verified and are returned with 1% agarose gel electrophoresis later
Receive pcr amplification product.One-step cloning specific method is mixed by segment (0.04-0.08ng/bp) and carrier (0.005-0.01ng/bp)
In same PCR pipe, the recombinase of the recombination enzyme buffer liquid and 1-2 μ l of 2-4 μ l is added, adds ddH2O polishings to 10-20 μ l,
37 DEG C of reaction 30min, obtain recombinant plasmid.
C1：5'-GCCGATATCCAATTGAGATCTATGGCGAGTGGTGGAGAAATG-3’
(underscore is BglII restriction enzyme sites)
C2：5'-CGCCTGCAGGTCGACAAGCTTTCACTCTCTGGAAACAACCACCA-3’
(underscore is KpnI restriction enzyme sites)
Embodiment 3：
Raspberry ketone produces the structure of bacterial strain Mu1：
By plasmid pACYC-BAS-4CL2, pET-RZS1 thermal shocks method is imported simultaneously in e. coli bl21 (DE3), gained
Positive strain is named as Mu1.Antibiotic is respectively chloramphenicol (25-50mg/mL), ampicillin (50-100mg/ in culture medium
mL).The wherein basic operation of thermal shock method：
1) the Escherichia coli bacillus competent cell that -80 DEG C preserve is placed in ice and is melted 10 minutes；
2) the corresponding plasmid of 0.1ng~10ng is added into competent escherichia coli cell, is gently put in ice after mixing
Set 30min；
3) 42 DEG C of water-bath 45~90s of thermal shock place 1~2min in ice immediately later；
4) the LB culture mediums of 890 μ l are added；
5) 37 DEG C are put into, cultivates 1h in 180rmp-300rmp shaking tables；
6) appropriate bacterium solution spread plate is taken, tablet is inverted in a night in 37 DEG C of incubators；
7) picking single bacterium colony carries out next step experiment.
Embodiment 4：
Colibacillus engineering Mu1 shake flask fermentations produce raspberry ketone：
The seed culture medium that engineered E. coli Mu1 shake flask fermentations produce raspberry ketone is LB culture mediums, nutrient media components
For：10g/LNaCL, 5g/L peptone, 10g/L yeast powders；The fermentation medium is TB culture mediums, and nutrient media components are：
8-15g/L peptones, 15-30g/L yeast extracts, 1-5g/L glycerine, 1-5g/L KH2PO4、5-15g/L K2HPO4, 5-10g grapes
Sugar.
Escherichia coli Mu1 is connected in seed culture medium and is activated, and adds corresponding antibiotic, with 180rmp-
The rotating speed of 300rmp is incubated overnight at 20-37 DEG C, is inoculated into later with 5% inoculum concentration in fresh fermentation medium, addition
Corresponding antibiotic (25mg/mL chloramphenicol, 100mg/mL ampicillins), when 20-37 DEG C of culture to OD600 is 1.0-1.2,
The IPTG and substrate for adding final concentration of 0.1-1mM continue culture in 20-37 DEG C to 72h.Thalline is centrifuged, with efficient liquid
Raspberry ketone (Waters, Agilent C18 columns, mobile phase 20 in phase chromatographic determination supernatant:80 acetonitrile：0.1%
Phosphate aqueous solution, flow velocity 1.0mL/min, Detection wavelength 260nm), the yield for measuring raspberry ketone is 70.62mg/L.It is empty
It carries plasmid control and does not produce raspberry ketone.
Embodiment 5：
Colibacillus engineering Mu1 uses M9 culture mediums fermentation production raspberry ketone：
To be transferred in seed LB culture mediums after recombination bacillus coli Mu1 activation, and add 10~30mg/mL chloramphenicol and
50~100mg/mL ampicillins are incubated overnight with the rotating speed of 180~300rmp at 20~37 DEG C, later by activated bacterium
Liquid is transferred to the inoculum concentration of 1-5% in fresh LB culture mediums, and adds corresponding antibiotic, with 180rmp-300rmp's
It is 0.6-0.8 that rotating speed, which is cultivated at 37 DEG C to OD600, then adds the IPTG of final concentration of 0.1-1mM, and 8- is cultivated in 20-37 DEG C
10h.Thalline is collected later, is resuspended in M9 culture mediums, and pair of IPTG, 100mg/L final concentration of final concentration of 0.1-1mM is added
Coumaric acid and corresponding antibiotic continue to cultivate 72h in 20-37 DEG C.The group of the wherein described M9 culture mediums becomes g/L：50ml
20x M9 buffer solutions, 1ml MgSO4 solution, 0.5g NaCL, 10g glucose；The 20x M9 buffer solution groups become g/L：20g
NH4CL,60g KH2PO4,125g Na2HPO4；MgSO4 solution concentrations are 1moL/L.
It is observed that generating raspberry ketone using M9 fermentations, ultimate output only has 12.47mg/L from Fig. 4；And make
Use TB as fermentation medium, the final concentration of raspberry ketone can reach 70.62mg/L, be 5.66 times using M9 fermentations.
It follows that changing the fermentation medium of the present invention, the yield of raspberry ketone can be seriously affected.
Embodiment 6：
Express influence of the external source benzylacetone reductase gene to E. coli recombinant stain fermentation production raspberry ketone：
Plasmid pACYC-BAS-4CL is prepared as described in Example 1, and thermal shock imports in e. coli bl21 (DE3), obtains
The recombinant bacterial strain Mu0 containing pACYC-BAS-4CL plasmids, fermenting as described in Example 5, recombinant bacterial strain Mu0 with
Recombinant bacterial strain Mu1 carries out shake flask fermentation 72h respectively, and the 4.69mg/L of concentration containing raspberry ketone in recombinant bacterial strain Mu0 zymotic fluids is less than
Express the recombinant bacterial strain Mu1 fermented supernatant fluids 12.47mg/L of benzylacetone reductase (RiRZS1).Illustrate mistake in recombinant bacterial strain
The effect of amount expression benzylacetone reductase (RiRZS1), it makes 2.67 times of final product raspberry ketone output increased.
Embodiment 7：
4- coumaroyl A ligases (4CL) gene pairs E. coli recombinant stain fermentation production of different plant origins is covered
The influence of basin ketone：
PACYC-BAS-4CL plasmids are built as described in Example 1, wherein coding 4- coumaroyl A ligases (4CL)
Gene is selected from arabidopsis (Arabidopsis thaliana) 4CL1 genes and codon optimization, builds plasmid
PACYC-BAS-4CL1, the primer such as SEQ ID No.10, SEQ ID No.11, SEQ ID No.6 and SEQ ID No.7 institutes
Show.By plasmid pACYC-BAS-4CL1 and plasmid pET-RZS1 according to the method thermal shock in embodiment 3 into import Escherichia coli
In BL21 (DE3), recombinant bacterial strain Mu2 is obtained.Mu2 ferments by the method in embodiment 5, but ferments 72 hours, uses HPLC
Zymotic fluid is measured, does not detect raspberry ketone.Illustrate from object parsley coding 4- coumaroyls A connections enzyme gene more
Raspberry ketone is synthesized suitable for this exogenous route, plant origin is changed, the yield of raspberry ketone can be seriously affected.
D1：5'-CCACAGCCAGGATCCGAATTCATGGCACCGCAGGAACAG-3’
(underscore is EcoRI restriction enzyme sites)
D2：5'-AGCATTATGCGGCCGCAAGCTTTTACAGGCCATTTGCCAG-3’
(underscore is HindIII restriction enzyme sites)
B1：5'-GCGATCGCTGACGTCGGTACCAAGGAAACCGCCGCCGCA-3’
(underscore is KpnI restriction enzyme sites)
B2：5'-GGTTTCTTTACCAGACTCGAGTTAGCTAATAACCGGAACGC-3’
(underscore is XhoI restriction enzyme sites)
To sum up, the present invention is newly added to benzylacetone reductase in the metabolic pathway of biosynthesis raspberry ketone
(RiRZS1), transformation efficiency of the intermediate product to hydroxyl BENZYLIDENE ACETONE is increased.In addition, the yield of shake flask fermentation raspberry ketone
70.62mg/L is reached, the yield of raspberry ketone is further significantly improved with biological synthesis process.
The present invention by introducing 3 heterologous genes into Escherichia coli, 4CL genes that the present invention selects, BAS genes,
RiRZS1 genes are screened from specified plant and codon optimization, the plant of gene source of the present invention and codon optimization
For realize catalytic activity and generate high concentration raspberry ketone it is particularly significant, the present invention in encode the 4- coumaroyls A
The nucleotide sequence of the gene 4CL of ligase derives from plant parsley (Petroselinum as shown in SEQ ID NO.1
Crispum) and codon optimizes；Encode the nucleotide sequence such as SEQ ID of the gene BAS of the benzylacetone synthase
Shown in NO.2, from plant sorrel (Rheum palmatum) and codon optimization；Encode the benzylacetone
The nucleotide sequence of the gene RiRZS1 of reductase derives from plant raspberry (Raspberry as shown in SEQ ID NO.3
Ketone), and codon optimizes, and changes the plant origin of gene in the present invention, can cause reaction that can not carry out, meanwhile,
The solubility expression of each gene of the present invention and its it is active give full play to be specified plant source of the present invention gene with the present invention
The result that selected fermentation medium mutually acts synergistically.The present invention is built by the different of p-Coumaric Acid biosynthesis raspberry ketone
Source metabolic pathway, recombination bacillus coli concentration of shake flask fermentation production raspberry ketone in TB culture mediums reaches 70.62mg/L, than existing
There is technology to significantly improve, embodies the feasibility of Microbe synthesis basin ketone.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to preferable
Embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the technology of the present invention
Scheme is modified or replaced equivalently, and without departing from the spirit of the technical scheme of the invention and range, should all be covered in this hair
In bright right.
Sequence table
<110>Southern Yangtze University
<120>A kind of method that bioanalysis prepares raspberry ketone
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<170> SIPOSequenceListing 1.0
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<211> 1635
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gattacgcgg cggagaaaaa catccagatc atctgcattg acgacgcacc gcaagattgt 480
ctgcacttct ccaaactgat ggaagcggac gagtctgaaa tgccggaagt cgtcatcgat 540
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aacctgtaca tgcactccga ggacgttatg atctgcattc tgccgctgtt ccacatctac 720
agcctgaacg ctgttctgtg ttgcggttta cgcgctggcg ttaccattct gattatgcag 780
aaattcgaca tcgtcccgtt cctggaactg atccagaaat acaaagtcac catcggtccg 840
tttgttccgc cgattgttct ggctattgcg aaaagcccgg tcgttgacaa atacgacctg 900
agtagcgttc gtaccgttat gtccggcgca gcaccgttag gtaaagaact ggaagacgca 960
gttcgcgcaa aatttccgaa cgcgaaactg ggtcaaggtt acggtatgac cgaagcaggt 1020
ccggtcttag caatgtgttt agcgtttgcg aaagagccgt acgaaatcaa aagcggcgct 1080
tgcggcaccg tggttcgtaa cgcagaaatg aaaatcgtcg atccggaaac caacgcgagt 1140
ttaccgcgta atcaacgcgg cgaaatctgt attcgcggcg accagatcat gaaaggctac 1200
ttaaacgatc cggaaagtac ccgtaccacc attgacgaag aaggctggtt acataccggc 1260
gatatcggtt tcatcgacga cgacgacgaa ctgtttatcg tcgaccgcct gaaagagatc 1320
atcaaataca aaggcttcca ggtcgcaccg gcagaattag aagcactgct gctgacccat 1380
ccgaccattt ctgacgctgc tgttgttccg atgatcgacg aaaaagcagg cgaagttccg 1440
gttgcgtttg tcgttcgtac caacggcttt accaccaccg aagaagaaat taaacagttc 1500
gtcagcaaac aggtcgtctt ctacaaacgc atcttccgcg tcttcttcgt tgacgcgatt 1560
ccgaaaagtc cgagcggtaa aattctgcgt aaagatctgc gcgcgaaaat tgcgtctggt 1620
gatctgccga aatga 1635
<210> 2
<211> 1155
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atggccaccg aagaaatgaa aaaactggca accgttatgg ccattggtac agccaatccg 60
ccgaattgtt attatcaggc cgattttccg gatttttatt ttcgtgtgac caatagtgat 120
catctgatta atctgaaaca gaaattcaag cgcctgtgcg aaaatagccg tattgaaaaa 180
cgctatctgc atgttaccga agaaattctg aaagaaaatc cgaatatcgc cgcctatgaa 240
gcaaccagtc tgaatgtgcg ccataaaatg caggttaaag gtgtggccga actgggcaaa 300
gaagcagccc tgaaagcaat taaggaatgg ggccagccga aaagtaaaat tacccatctg 360
attgtgtgtt gcctggcagg cgttgatatg ccgggtgcag attatcagct gaccaaactg 420
ctggatctgg accctagcgt gaaacgtttt atgttttatc atctgggttg ctatgccggc 480
ggtacagttc tgcgtctggc caaagatatt gcagaaaata ataagggcgc ccgcgttctg 540
attgtttgca gtgaaatgac caccacctgc tttcgtggtc cgagcgaaac ccatctggat 600
agcatgattg gccaggccat tctgggcgat ggcgccgctg cagtgattgt tggtgccgat 660
ccggatctga ccgttgaacg tccgattttt gaactggtga gtaccgcaca gaccattgtg 720
ccggaaagcc atggcgccat tgaaggccat ctgctggaaa gcggtctgag ttttcatctg 780
tataaaaccg tgccgaccct gattagcaat aatattaaga cctgtctgag tgatgccttt 840
accccgctga atattagtga ttggaatagt ctgttttgga ttgcccatcc gggtggtccg 900
gcaattctgg atcaggttac cgccaaagtt ggtctggaaa aagaaaaact gaaagttacc 960
cgccaggtgc tgaaagatta tggcaatatg agtagtgcca ccgtgttttt cattatggat 1020
gaaatgcgta aaaagagcct ggaaaatggt caggccacca ccggtgaagg tctggaatgg 1080
ggtgtgctgt ttggttttgg tccgggtatt accgttgaaa ccgtggtgct gcgcagcgtt 1140
ccggttatta gctaa 1155
<210> 3
<211> 1047
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
atggccagcg gcggcgagat gcaggtgagc aacaagcagg tgatcttccg cgattacgtt 60
acaggcttcc cgaaagagag cgacatggaa ctgaccaccc gcagcatcac cctgaaactg 120
ccgcagggta gcaccggtct gctgctgaaa aacctgtacc tgagctgtga cccgtacatg 180
cgcgcccgta tgacaaacca ccatcgcctg agctacgtgg atagctttaa accgggcagc 240
ccgatcattg gctatggcgt ggcacgcgtg ttagaaagcg gcaacccgaa gttcaatccg 300
ggtgatctgg tttggggctt taccggttgg gaagagtata gcgtgatcac cgccaccgag 360
agcctgttca agattcacaa caccgatgtg ccgctgagct actacaccgg cctgctgggt 420
atgccgggta tgaccgccta tgccggcttt tacgaaattt gtagcccgaa aaagggcgag 480
acagtgtatg ttagcgcagc cagtggtgca gtgggtcaac tggttggcca gtttgccaag 540
ctgaccggct gctacgttgt gggcagcgcc ggtagcaaag agaaagtgga cctgctgaaa 600
aataaatttg gttttgatga agccttcaat tacaaagaag aagccgacct ggatgcagcc 660
ctgcgtcgct atttcccgga tggtattgat atttattttg aaaacgtggg cggcaagatg 720
ctggatgccg ttctgccgaa catgcgcccg aaaggccgca ttgcagtgtg cggcatgatc 780
agtcagtaca atctggagca gccggaaggt gtgcgcaatc tgatggccct gatcgtgaaa 840
caggtgcgca tggaaggctt catggtgttt agctattatc acctgtatgg caagttcctg 900
gaaacagtgc tgccgtacat caagcagggc aagatcacct acgtggaaga tgtggtggac 960
ggcctggata atgcaccggc cgccctgatt ggtctgtatt ctggtcgtaa tgtgggcaaa 1020
caagtggttg tggttagccg tgaataa 1047
<210> 4
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
ccacagccag gatccgaatt catgggtgat tgcgttgcg 39
<210> 5
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gcattatgcg gccgcaagct ttcatttcgg cagatcacca ga 42
<210> 6
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gcgatcgctg acgtcggtac caaggaaacc gccgccgca 39
<210> 7
<211> 41
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ggtttcttta ccagactcga gttagctaat aaccggaacg c 41
<210> 8
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gccgatatcc aattgagatc tatggcgagt ggtggagaaa tg 42
<210> 9
<211> 44
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
cgcctgcagg tcgacaagct ttcactctct ggaaacaacc acca 44
<210> 10
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
ccacagccag gatccgaatt catggcaccg caggaacag 39
<210> 11
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
agcattatgc ggccgcaagc ttttacaggc catttgccag 40
Claims (10)
1. a kind of method that bioanalysis prepares raspberry ketone, it is characterised in that：Including,
In Escherichia coli simultaneously induced expression plant origin 4- coumaroyl A ligases, benzylacetone synthase and benzyl
Acetone reductase.
2. the method that bioanalysis as described in claim 1 prepares raspberry ketone, it is characterised in that：Including,
The gene of the gene of 4- coumaroyl A ligases and benzylacetone synthase is total on plasmid pACYC-Duet-1
Expression, obtains expression vector pACYC-BAS-4CL；
The RiRZS1 genes of benzylacetone reductase are expressed on plasmid pET-Duet-1, obtain expression vector pET-
RZS1；
Expression vector pACYC-BAS-4CL, pET-RZS1 are imported into e. coli bl21 (DE3) simultaneously, obtain recombination large intestine bar
Bacterium Mu1.
3. the method that bioanalysis as claimed in claim 1 or 2 prepares raspberry ketone, it is characterised in that：Encode the 4- tonka-beans
The nucleotide sequence of the gene 4CL of acyl coenzyme A ligases derives from plant parsley as shown in SEQ ID NO.1
(Petroselinum crispum), and codon optimizes；Encode the nucleotide of the gene BAS of the benzylacetone synthase
Sequence derives from plant sorrel (Rheum palmatum), and codon optimizes as shown in SEQ ID NO.2；It compiles
The nucleotide sequence of the gene RiRZS1 of the code benzylacetone reductase covers basin as shown in SEQ ID NO.3 from plant
Sub (Raspberry ketone), and codon optimizes.
4. the method that bioanalysis as claimed in claim 2 prepares raspberry ketone, it is characterised in that：Further include, by the recombination
Escherichia coli Mu1 passes through IPTG Fiber differentiations, addition p-Coumaric Acid fermentation production raspberry ketone.
5. the method that the bioanalysis as described in claim 1,2 or 4 are any prepares raspberry ketone, it is characterised in that：It is described to obtain
Expression vector pACYC-BAS-4CL, including first cloned with EcoRI and HindIII double digestion plasmid pACYC-Duet-1 and PCR
The 4- coumaroyls A connection enzyme genes arrived obtain recombinant plasmid pACYC-4CL by one-step cloning, use KpnI and XhoI later
The benzylacetone synthase gene that double digestion plasmid pACYC-4CL and PCR is cloned obtains recombinant plasmid by one-step cloning
pACYC-BAS-4CL。
6. the method that the bioanalysis as described in claim 1,2 or 4 are any prepares raspberry ketone, it is characterised in that：With BglII and
The benzylacetone reductase gene that KpnI double digestion plasmid pET-Duet-1 and PCR is cloned is recombinated by one-step cloning
Plasmid pET-RZS1.
7. the method that the bioanalysis as described in claim 1,2 or 4 are any prepares raspberry ketone, it is characterised in that：By plasmid
PACYC-BAS-4CL, pET-RZS1 thermal shock method are imported simultaneously in e. coli bl21 (DE3), obtain recombination bacillus coli
Mu1。
8. the method that the bioanalysis as described in claim 1,2 or 4 are any prepares raspberry ketone, it is characterised in that：It will recombinate big
It is transferred in seed LB culture mediums after enterobacteria Mu1 activation, and adds 10~30mg/mL chloramphenicol and 50~100mg/mL ammonia benzyls
Penicillin is incubated overnight with the rotating speed of 180~300rmp at 20~37 DEG C, is inoculated into fresh hair with 5% inoculum concentration later
In ferment culture medium, 10~30mg/mL chloramphenicol and 50~100mg/mL ampicillins are added, OD is arrived in 20~37 DEG C of cultures600
When being 1.0~1.2, the IPTG and substrate of final concentration of 0.1~1mM are added, continues culture in 20~37 DEG C to 72h.
9. the method that bioanalysis as claimed in claim 4 prepares raspberry ketone, it is characterised in that：The p-Coumaric Acid is added to
Final concentration of 100~150mg/L in fermentation medium.
10. the method that bioanalysis as claimed in claim 8 prepares raspberry ketone, it is characterised in that：The fermentation medium, packet
TB culture mediums are included, component includes 8~15g/L peptones, 15~30g/L yeast extracts, 1~5g/L glycerine, 1~5g/L
KH2PO4, 5~15g/L K2HPO4, 5~10g glucose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126614A (en) * | 2020-09-30 | 2020-12-25 | 江南大学 | Method for preparing raspberry ketone by whole cell transformation |
| CN112391418A (en) * | 2020-11-20 | 2021-02-23 | 厦门欧米克生物科技有限公司 | Industrialized fermentation production method of raspberry ketone |
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| GB2416769A (en) * | 2004-07-28 | 2006-02-08 | Danisco | Biosynthesis of raspberry ketone |
| WO2017207950A1 (en) * | 2016-06-03 | 2017-12-07 | Lesaffre Et Compagnie | Production of frambinone by a recombinant fungal microorganism |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126614A (en) * | 2020-09-30 | 2020-12-25 | 江南大学 | Method for preparing raspberry ketone by whole cell transformation |
| CN112391418A (en) * | 2020-11-20 | 2021-02-23 | 厦门欧米克生物科技有限公司 | Industrialized fermentation production method of raspberry ketone |
| CN112391418B (en) * | 2020-11-20 | 2022-02-18 | 厦门欧米克生物科技有限公司 | Industrialized fermentation production method of raspberry ketone |
| EP4001420A3 (en) * | 2020-11-20 | 2022-06-22 | Xiamen Oamic Biotech Co., Ltd. | Method for preparing raspberry ketone using industrial fermentation |
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| CN108753852B (en) | 2021-10-26 |
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