CN107805646A - A kind of method of E. coli whole cell catalytic production phloretin - Google Patents

A kind of method of E. coli whole cell catalytic production phloretin Download PDF

Info

Publication number
CN107805646A
CN107805646A CN201710938021.1A CN201710938021A CN107805646A CN 107805646 A CN107805646 A CN 107805646A CN 201710938021 A CN201710938021 A CN 201710938021A CN 107805646 A CN107805646 A CN 107805646A
Authority
CN
China
Prior art keywords
ala
leu
gly
pro
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710938021.1A
Other languages
Chinese (zh)
Inventor
陈贤情
王筱
王文
江会锋
刘晓楠
杨月
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiaxing Xin Baylet Biotechnology Co Ltd
Original Assignee
Jiaxing Xin Baylet Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiaxing Xin Baylet Biotechnology Co Ltd filed Critical Jiaxing Xin Baylet Biotechnology Co Ltd
Priority to CN201710938021.1A priority Critical patent/CN107805646A/en
Publication of CN107805646A publication Critical patent/CN107805646A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12N9/1037—Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13011—Trans-cinnamate 4-monooxygenase (1.14.13.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01074—Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01005—Phenylalanine ammonia-lyase (4.3.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • C12Y602/01012—4-Coumarate-CoA ligase (6.2.1.12)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a kind of method of E. coli whole cell catalytic production phloretin, for the synthetic method using phenylalanine as raw material, colibacillus engineering is Host Strains, is completed by the catalysis of some enzymes in host bacterial；The Host Strains can at least express phenylalanine lyase, the hydroxylase of cinnamic acid 4,4 coumarate CoA-ligases, double bond reductase and chalcone synthase.Advantages of the present invention：1) using phenylalanine as raw material, the technique cost of raw material is low.2) to transform microorganism as technical foundation, in microbial body carrying out biology enzyme catalyzes and synthesizes, and avoids large-scale extracting and developing, purge process, from production cost and it is environmentally friendly in terms of be easily controlled.3) this technique is fewer than other method in the scale and quantity of production equipment, is easy to industry conversion.4) this technique only isolates and purifies process in the final step of production, and purifying products technique is simple, and product quality is more preferable than conventional method purity, content is higher.

Description

A kind of method of E. coli whole cell catalytic production phloretin
Technical field
The present invention relates to a kind of biological synthesis method of phloretin, particularly a kind of E. coli whole cell catalysis The method for producing phloretin.
Background technology
Phloretin molecular formula：C15H14O5；English name：Phloretin；CAS：60-82-2；Molecular weight：274.28；Thing Rationality matter：Methanol, ethanol and acetone are soluble in, chloroform is slightly soluble in, is insoluble in water, petroleum ether and benzene, square prismatic or flaky crystal (ethanol or methanol).
Phloretin is a kind of external new type natural skin-whitening agents for researching and developing out recently, after the another day of ursin Right cosmetics, it is distributed mainly in the pericarp and root skin branches and leaves of the rich fruits such as apple, pears.It is natural that it is mainly used as skin Whitening agent, it can suppress tyrosinase activity, melanin activity, have desalination effect to various skin splash.Sent out according to current research Existing its also has the effect such as anti-inflammatory, immunosupress, cardiovascular protection.The whole world is no less than 20 tons per annual consumption, and demand is every year with 20% Speed increase, has the larger market demand.
At present, the technique of existing extraction phloretin, there are sour water solution, enzymolysis and directly extraction.It is more with shaddock ped in actual production Glycosides is raw material, and Raney Ni is catalyst, in sodium hydroxide solution, catalytic hydrogenation synthesis aurantiin dihydrochalcone, and Ran Hou In hydrochloric acid solution, aurantiin dihydrochalcone hydrolyzes to obtain phloretin.There is the discharge of chemical waste fluid in the method, chemical contamination is tight Weight.Also it is using apple skin as raw material, sour water solution extracts drying again after being extractant, concentrate by ethanol solution, obtains root Pi Su, the method uses sour water solution, seriously polluted.A kind of present invention seek to address that this technical problem, there is provided conditioned response temperature The high phloretin biological synthesis method with, combined coefficient.
The content of the invention
It is an object of the invention to solve the deficiencies in the prior art, there is provided a kind of E. coli whole cell catalytic production root Pi Su method.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of method of E. coli whole cell catalytic production phloretin, the synthetic method using phenylalanine as raw material, Colibacillus engineering is Host Strains, is completed by the catalysis of some enzymes in host bacterial；
The Host Strains can at least express phenylalanine lyase, cinnamic acid -4- hydroxylases, 4- coumaric acid coacetylases Ligase, double bond reductase and chalcone synthase.
The method of E. coli whole cell catalytic production phloretin, the synthetic route of the synthetic method are as follows：
Preferably, the phenylalanine lyase in the synthetic method, cinnamic acid -4- hydroxylases, 4- coumaric acid coenzyme The expressing gene of A ligases, double bond reductase and chalcone synthase be respectively derived from sunflower, thorn bud cynara scolymus, arabidopsis, Apple and fleabane flower.
Preferably, the expressing gene of the 4- coumarate CoA-ligases and chalcone synthase is implemented in carrier pET- On 28a, the expressing gene of phenylalanine lyase, cinnamic acid -4- hydroxylases and double bond reductase is implemented in carrier pET- On 22b, expressing gene in identical carrier shares T7 promoters, and the expressing gene of each enzyme has 6 before terminator codon × There are RBS train intervals, RBS sequences and the termination of the expressing gene of nearest enzyme between the expressing gene of his labels, enzyme and enzyme 3 and 7 bases are respectively separated between codon and initiation codon.
The method and step of E. coli whole cell catalytic production phloretin is as follows：
1) reduced according to phenylalanine lyase, cinnamic acid -4- hydroxylases, 4- coumarate CoA-ligases, double bond The amino acid sequence and e. coli codon Preference of enzyme and chalcone synthase, the nucleotide sequence of the corresponding enzyme of optimization, close Into correlated expression gene；
2) expressing gene is implemented in respective carrier, the wherein table of 4- coumarate CoA-ligases and chalcone synthase Up to gene constructed in carrier pET-28a, the expression base of phenylalanine lyase, cinnamic acid -4- hydroxylases and double bond reductase Because being implemented in carrier pET-22b；
3) carrier for building completion is transferred in Escherichia coli, Escherichia coli cultivated, induced expression；
4) phenylalanine substrate is added in the bacterium solution after expression, continues to react 20-26h.
5) the phloretin extracting and developing in reaction solution is purified.
Preferably, phloretin is extracted from zymotic fluid using methanol, the volume ratio 1: 1 of methanol and zymotic fluid.
The gene that this patent is related to is At4CL, EbCHS, MdDBR, HaPAL and CcC4H (being shown in Table 1).
The species used in the patent of table 1 and gene brief introduction
The beneficial effects of the invention are as follows：The present invention prepares phloretin difference from prior art and is：
1) using phenylalanine as raw material, the technique cost of raw material is low.
2) to transform microorganism as technical foundation, catalyzing and synthesizing for biology enzyme is carried out in microbial body, is avoided extensive Extracting and developing, purge process, from production cost and it is environmentally friendly in terms of be easily controlled.
3) this technique is fewer than other method in the scale and quantity of production equipment, simple to operate, is easy to industry and turns Change.
4) this technique only isolates and purifies process in the final step of production, and purifying products technique is simple, product quality ratio Conventional method purity is more preferable, content is higher.
5) building-up process does not have discharging of waste liquid, environment-friendly, pollution-free, sustainable production, present invention process from it is economical, Environment and occupational health angle are that excellent industrialized production shows the way line.
Brief description of the drawings
Plasmid pET-28a-EbCHS-Eb4CL collection of illustrative plates in Fig. 1 (a) present invention；
Plasmid pET-22b-Pal-C4H-DBR collection of illustrative plates in Fig. 1 (b) present invention；
6 × his label figures at expressing gene ending in Fig. 1 (c) present invention；
RBS joint figures in Fig. 1 (d) present invention；
The phloretin synthetic route chart of Fig. 2 present invention.
Embodiment
Below by specific embodiment, and with reference to accompanying drawing, technical scheme is described in further detail. The principles and features of the present invention are described below, and the given examples are served only to explain the present invention, is not intended to limit the present invention Scope.
The plasmid construction method of embodiment 1.
This patent is according to the amino acid sequence and e. coli codon Preference of corresponding expressing gene, optimization gene Nucleotide sequence, send business gene chemical synthesis company to synthesize expressing gene, by 2 or multiple gene integrations to expression vector On pET-28a or pET-22b, specifically, the expressing gene structure of 4- coumarate CoA-ligases and chalcone synthase It is built on carrier pET-28a, such as Fig. 1 (a), the table of phenylalanine lyase, cinnamic acid -4- hydroxylases and double bond reductase Up to gene constructed on carrier pET-22b, such as Fig. 1 (b), the expressing gene in identical carrier shares a T7 promoter, expression Modified at gene end, before terminator in N-terminal primer, 6 × his gene orders (CAT CAT are added on primer CAT CAT CAT CAT), see accompanying drawing 1 (c), connected between expressing gene with RBS sequences (AAGGAG), RBS sequences and upper one 3 bp (TAA), RBS and next expressing gene initiation codon are spaced between the terminator codon of individual expressing gene (MET) 7 bp (ATATACC) are spaced between, see accompanying drawing 1 (d).A commercial reagents box (step is used between genetic fragment and fragment Cloning Kit) connection, convert and enter Bacillus coli cells, correct plasmid is verified in the checking of picking monoclonal, and conversion enters In expression cell, induced expression is subsequently carried out.
The whole-cell catalytic of embodiment 2 synthesizes phloretin
A kind of method of E. coli whole cell catalytic production phloretin：
1) 37 DEG C, 220rpm, in it with the addition of the LB culture mediums of corresponding resistant, culture cell E. coli overnight；
2) transfer 20mL, 37 DEG C, 220rpm, in LB culture mediums, cultivates to OD600=1 or so；
3) turn in the big bottle 2YT culture mediums of 800mL, 37 DEG C, 220rpm is cultivated to OD600=0.8 or so；
4) 16 DEG C, 220rpm, IPTG induction, 16h-18h, bacterium is received；
5) suspended with phosphate buffer, 20 times of concentrations；
6) substrate is added, 37 DEG C, 220rpm reacts 24h, phloretin caused by collection.
Note：LB (Luria-Bertani) culture medium：
Fluid nutrient medium：Tryptone：1%, yeast extract：0.5%, NaCl：1%, pH7.5
Solid medium：1.5% agar powder is added in LB fluid nutrient mediums
2YT culture mediums：Tryptone：1.6%, yeast extract：1%, NaCl：0.5%.
Described is only presently preferred embodiments of the present invention, is not intended to limit the invention, every in the spiritual and former of the present invention Within then, any modification, equivalent substitution and improvements done etc. all should be in the row of protection scope of the present invention.
Sequence table
<110>Jiaxing Xin Beilai bio tech ltd
<120>A kind of method of E. coli whole cell catalytic production phloretin
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 711
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 1
Met Gly Ala Gly His Ala Ala Gly Val Ala Ser Gly Leu Cys Ile Ala
1 5 10 15
Leu Ala Pro Leu Ala Thr Gly Val Ala Ala Gly Ala Leu Thr Gly Ser
20 25 30
His Leu Ala Gly Val Leu Ala Met Val Ala Gly Pro Ala Leu Pro Leu
35 40 45
Val Leu Leu Gly Gly Gly Thr Leu Thr Val Ser Gly Val Ala Gly Ile
50 55 60
Ala Ala Ala Gly Ala Ser Ser Val Val Leu Val Gly Leu Ser Gly Ser
65 70 75 80
Ala Ala Ala Gly Val Leu Ala Ser Ser Ala Thr Val Met Gly Ser Met
85 90 95
Ala Leu Gly Thr Ala Ser Thr Gly Val Thr Thr Gly Pro Gly Ala Thr
100 105 110
Ser His Ala Ala Thr Leu Ala Gly Gly Ala Leu Gly Leu Gly Leu Ile
115 120 125
Ala Pro Leu Ala Ala Gly Ile Pro Gly Ala Gly Thr Gly Ser Ser His
130 135 140
Thr Leu Pro His Ser Ala Thr Ala Ala Ala Met Leu Val Ala Ile Ala
145 150 155 160
Thr Leu Leu Gly Gly Thr Ser Gly Ile Ala Pro Gly Ile Leu Gly Ala
165 170 175
Ile Thr Leu Pro Leu Ala Thr Ala Ile Thr Pro Cys Leu Pro Leu Ala
180 185 190
Gly Thr Ile Thr Ala Ser Gly Ala Leu Val Pro Leu Ser Thr Ile Ala
195 200 205
Gly Leu Leu Thr Gly Ala Pro Ala Ser Leu Ala Val Gly Pro Ala Gly
210 215 220
Gly Val Leu Ala Ala Gly Leu Ala Pro Leu Leu Ala Gly Val Gly Gly
225 230 235 240
Gly Pro Pro Gly Leu Gly Pro Leu Gly Gly Leu Ala Leu Val Ala Gly
245 250 255
Thr Ala Val Gly Ser Gly Met Ala Ser Met Val Leu Pro Gly Ala Ala
260 265 270
Val Leu Ala Leu Leu Ser Gly Val Leu Ser Ala Ile Pro Ala Gly Val
275 280 285
Met Gly Gly Leu Pro Gly Pro Thr Ala His Leu Thr His Leu Leu Leu
290 295 300
His His Pro Gly Gly Ile Gly Ala Ala Ala Ile Met Gly Thr Ile Leu
305 310 315 320
Ala Gly Ser Ala Thr Val Leu Ala Ala Gly Leu Val His Gly Met Ala
325 330 335
Pro Leu Gly Leu Pro Leu Gly Ala Ala Thr Ala Leu Ala Thr Ser Pro
340 345 350
Gly Thr Leu Gly Pro Leu Ile Gly Val Ile Ala Ser Ser Thr Leu Met
355 360 365
Ile Gly Ala Gly Ile Ala Ser Val Ala Ala Ala Pro Leu Ile Ala Val
370 375 380
Ser Ala Ala Leu Ala Leu His Gly Gly Ala Pro Gly Gly Thr Pro Ile
385 390 395 400
Gly Val Ser Met Ala Ala Thr Ala Leu Ala Ile Ala Ala Ile Gly Leu
405 410 415
Leu Met Pro Ala Gly Pro Ser Gly Leu Val Ala Ala Pro Thr Ala Ala
420 425 430
Gly Leu Pro Ser Ala Leu Ser Gly Gly Ala Ala Pro Ser Leu Ala Thr
435 440 445
Gly Pro Leu Gly Gly Gly Ile Ala Met Ala Ser Thr Cys Ser Gly Leu
450 455 460
Gly Pro Leu Ala Ala Pro Val Thr Ala His Val Gly Ser Ala Gly Gly
465 470 475 480
His Ala Gly Ala Val Ala Ser Leu Gly Leu Ile Ser Ala Ala Leu Thr
485 490 495
Ala Gly Ala Val Ala Ile Leu Leu Leu Met Ser Ser Thr Thr Leu Val
500 505 510
Ala Leu Cys Gly Ser Val Ala Leu Ala His Leu Gly Gly Ala Met Leu
515 520 525
Ser Val Val Leu Ala Thr Val Ser Gly Val Ala Leu Leu Val Leu Thr
530 535 540
Met Gly Val Ala Gly Gly Leu His Pro Ser Ala Pro Cys Gly Leu Ala
545 550 555 560
Leu Leu Ala Val Val Ala Ala Gly Thr Val Pro Ala Thr Ile Ala Ala
565 570 575
Pro Cys Leu Ala Thr Thr Pro Leu Met Gly Leu Leu Ala Gly Val Leu
580 585 590
Val Ala His Ala Leu Ala Ala Gly Gly Thr Gly Leu Ala Thr Ala Thr
595 600 605
Ser Ile Pro Gly Leu Ile Ala Thr Pro Gly Gly Gly Leu Leu Ala Ile
610 615 620
Leu Pro Leu Gly Val Gly Ser Val Ala Val Ala Pro Gly Ala Ala Thr
625 630 635 640
Leu Ser Ile Pro Ala Ala Ile Leu Ala Cys Ala Ser Thr Pro Leu Thr
645 650 655
Ala Pro Val Ala Gly Gly Leu Gly Gly Ser Thr Leu Thr Gly Gly Leu
660 665 670
Val Thr Ser Pro Gly Gly Gly Pro Ala Ala Val Pro Thr Ala Met Cys
675 680 685
Leu Gly Gly Ile Ile Ala Pro Leu Leu Gly Cys Val Gly Gly Thr Ala
690 695 700
Gly Ala Pro Leu Pro Ile Cys
705 710
<210> 2
<211> 505
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 2
Met Ala Leu Leu Leu Leu Gly Leu Thr Leu Ile Ala Leu Pro Ala Thr
1 5 10 15
Ile Ile Ile Thr Ile Ile Ile Ser Leu Leu Ala Gly Leu Leu Pro Leu
20 25 30
Leu Pro Pro Gly Pro Thr Ala Val Pro Ile Pro Gly Ala Thr Leu Gly
35 40 45
Val Gly Ala Ala Leu Ala His Ala Ala Leu Thr Ala Leu Ala Leu Leu
50 55 60
Pro Gly Gly Ile Pro Leu Leu Ala Met Gly Gly Ala Ala Leu Val Val
65 70 75 80
Val Ser Ser Pro Ala Leu Ala Leu Gly Val Leu His Thr Gly Gly Val
85 90 95
Gly Pro Gly Ser Ala Thr Ala Ala Val Val Pro Ala Ile Pro Thr Gly
100 105 110
Leu Gly Gly Ala Met Val Pro Thr Val Thr Gly Gly His Thr Ala Leu
115 120 125
Met Ala Ala Ile Met Thr Val Pro Pro Pro Thr Ala Leu Val Val Gly
130 135 140
Gly Thr Ala Leu Gly Thr Gly Ala Gly Ala Ala Ala Val Val Gly Ala
145 150 155 160
Val Leu Leu Ala Pro Leu Ser Ala Thr Gly Gly Val Val Ile Ala Leu
165 170 175
Ala Leu Gly Leu Met Met Thr Ala Ala Met Pro Ala Ile Met Pro Ala
180 185 190
Ala Ala Pro Ala Ser Gly Ala Ala Pro Leu Pro Leu Leu Leu Leu Ala
195 200 205
Leu Ala Gly Gly Ala Ser Ala Leu Ala Gly Ser Pro Gly Thr Ala Thr
210 215 220
Gly Ala Pro Ile Pro Val Leu Ala Pro Pro Leu Ala Gly Thr Leu Leu
225 230 235 240
Leu Cys Leu Gly Val Leu Ala Leu Ala Leu Gly Leu Pro Leu Ala Thr
245 250 255
Pro Val Ala Gly Ala Leu Leu Ile Gly Ser Thr Leu Leu Leu Ala Ala
260 265 270
Ala Gly Leu Leu Cys Ala Ile Ala His Ile Leu Gly Ala Leu Ala Leu
275 280 285
Gly Gly Ile Ala Gly Ala Ala Val Leu Thr Ile Val Gly Ala Ile Ala
290 295 300
Val Ala Ala Ile Gly Thr Thr Leu Thr Ser Ile Gly Thr Gly Ile Ala
305 310 315 320
Gly Leu Val Ala His Pro Gly Ile Gly Ala Leu Leu Ala His Gly Leu
325 330 335
Ala Thr Leu Leu Gly Ser Gly Val Gly Ile Thr Gly Pro Ala Val Gly
340 345 350
Ala Leu Pro Thr Leu Gly Ala Val Val Leu Gly Thr Leu Ala Leu Ala
355 360 365
Met Ala Ile Pro Leu Leu Val Pro His Met Ala Leu His Ala Ala Leu
370 375 380
Leu Gly Gly Pro Ala Ile Pro Ala Gly Ser Leu Ile Leu Val Ala Ala
385 390 395 400
Thr Thr Leu Ala Ala Ala Pro Ala Gly Thr Leu Leu Pro Gly Gly Pro
405 410 415
Ala Pro Gly Ala Pro Leu Gly Gly Gly Ala Leu Val Gly Ala Ala Gly
420 425 430
Ala Ala Pro Ala Thr Leu Pro Pro Gly Val Gly Ala Ala Ser Cys Pro
435 440 445
Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Leu Gly Ala Leu
450 455 460
Val Gly Ala Pro Gly Leu Leu Pro Pro Pro Gly Gly Ser Leu Ile Ala
465 470 475 480
Thr Ser Gly Leu Gly Gly Gly Pro Ser Leu His Ile Leu Leu His Ser
485 490 495
Thr Ile Val Ala Leu Pro Ala Ser Pro
500 505
<210> 3
<211> 392
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 3
Met Ala Thr Thr Ala Ala Ser Ser Leu Gly Met Ala Thr Ala Ala Pro
1 5 10 15
Cys Ile Ser Ser Ser Ala Ala Ala Pro Gly Ser Ser Ser Leu Met Leu
20 25 30
Ala Ala Ala Pro Leu Val Ala Ser Thr Ser Leu Leu Ser Ser Thr Cys
35 40 45
His Thr Ser Ser Val Gly Ser Pro Gly Ala Ser Pro Thr Ser Ser Ser
50 55 60
Met Leu Met Ala Leu Pro Val Thr Ala Ala Met Ala Gly Ala Ser Gly
65 70 75 80
Ala Leu Pro Val Ser Gly Leu Pro Ile Ala Leu Leu Gly Leu Ala Ala
85 90 95
Pro Ile Ala Gly Val Ala Ala Ala Ala Gly Thr Gly Thr Ala Ile Ala
100 105 110
Leu Ser Leu Ala Ala Ala Gly Ala Gly Ile Leu Val Gly Thr Thr Val
115 120 125
Pro Ala Leu Ala Ile Pro Gly Ser Ser Leu Ala Ala Gly Leu Pro Ala
130 135 140
Gly Ser Ala Val Leu Pro Ala Gly Ser Leu Met Gly Ile Thr Leu Val
145 150 155 160
Thr Pro Leu Ala Ala Val Pro Ala Ala Pro Gly Ala Val Pro Gly Gly
165 170 175
Ile Leu Thr Ala Leu Ala Thr Ala Gly Ser Ser Ala Thr Thr Val Gly
180 185 190
Gly Ala Ala Gly Cys Val Leu Ala Ala Pro Gly Ser Ile Ala Ile Leu
195 200 205
Val His Ser Leu Ala Ala Gly Pro Gly Val Val Leu Pro Leu Leu Gly
210 215 220
Thr Ser Ala Leu Gly Thr Leu Ala Ala Ile Ser Ala Ser Ser Thr Ser
225 230 235 240
Thr Val Ser Leu Leu Leu His Pro Leu Pro Ile Ile Ala Pro Gly Gly
245 250 255
Ser Ser Ile Ser Leu Thr Thr Ile Ala Ser Gly Ala Ile Ile Pro Gly
260 265 270
Thr Gly Gly Gly Met Ser Ser Ala Leu Ala Ala Leu Gly Ser Ala Thr
275 280 285
Ala Val Leu Ala Pro Gly Ala Gly Ala Ala Leu Gly Ile Ala Val Ala
290 295 300
Thr Ile Ser Ala Gly Pro Leu Ala Ser Ala Ala Ala Leu Ala Ile Gly
305 310 315 320
Pro Ile Ala Met Met Ile Ala Thr Ser Ser Ala Ala Ala Pro Leu Gly
325 330 335
Leu Gly Leu Ser Ala Ala Gly Val Gly Ala Thr Ala Ala Pro Leu Ala
340 345 350
Ser Pro Leu Ala Ser Ala Ile Thr Gly Gly Val Ile Thr Val Ala Ala
355 360 365
Gly Leu Ala Ala Met Gly Val Gly Val Ala Ser Pro Ile Pro Gly Ala
370 375 380
Leu Ala Ile Pro Leu Ala Gly His
385 390
<210> 4
<211> 398
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 4
Met Ala Ser Ser Ile Ala Ile Ala Ala Ile Ala Gly Ala Gly Ala Ala
1 5 10 15
Gly Gly Pro Ala Thr Ile Leu Ala Ile Gly Thr Ala Thr Pro Ser Ala
20 25 30
Cys Val Thr Gly Ala Ala Thr Pro Ala Thr Thr Pro Ala Ile Thr Leu
35 40 45
Ser Gly His Met Val Ala Leu Leu Gly Leu Pro Leu Ala Met Cys Ala
50 55 60
Leu Ser Met Ile Ala Leu Ala Thr Met His Leu Thr Gly Gly Thr Leu
65 70 75 80
Leu Gly Ala Pro Ser Leu Cys Gly Thr Met Ala Pro Ser Leu Ala Ala
85 90 95
Ala Gly Ala Val Val Val Val Gly Val Pro Leu Leu Gly Leu Gly Ala
100 105 110
Ala Thr Leu Ala Ile Leu Gly Thr Gly Gly Pro Leu Ser Leu Ile Thr
115 120 125
His Leu Ile Pro Cys Thr Thr Ser Gly Val Ala Met Pro Gly Ala Ala
130 135 140
Thr Gly Leu Thr Leu Leu Leu Gly Leu Ala Pro Ser Val Leu Ala Pro
145 150 155 160
Met Met Thr Gly Gly Gly Cys Pro Ala Gly Gly Thr Val Leu Ala Leu
165 170 175
Ala Leu Ala Leu Ala Gly Ala Ala Leu Gly Ala Ala Val Leu Val Val
180 185 190
Cys Ser Gly Ile Thr Ala Val Thr Pro Ala Gly Pro Ala Ala Thr His
195 200 205
Leu Ala Ser Leu Val Gly Gly Ala Leu Pro Gly Ala Gly Ala Ala Ala
210 215 220
Val Ile Val Gly Ser Ala Pro Ala Leu Thr Thr Gly Ala Pro Leu Pro
225 230 235 240
Gly Met Ile Ser Ala Ala Gly Thr Ile Leu Pro Ala Ser Gly Gly Ala
245 250 255
Ile Ala Gly His Leu Ala Gly Val Gly Leu Thr Pro His Leu Leu Leu
260 265 270
Ala Val Pro Gly Leu Ile Ser Leu Ala Ile Gly Leu Ala Leu Thr Gly
275 280 285
Ala Pro Ser Pro Leu Gly Ile Ser Ala Thr Ala Ser Leu Pro Thr Ile
290 295 300
Ala His Pro Gly Gly Pro Ala Ile Leu Ala Gly Val Gly Leu Leu Leu
305 310 315 320
Gly Leu Leu Gly Gly Leu Met Ala Ala Thr Ala His Val Leu Ser Gly
325 330 335
Thr Gly Ala Met Ser Ser Ala Cys Val Leu Pro Ile Ile Ala Gly Met
340 345 350
Ala Leu Leu Ser Ala Gly Ala Gly Ala Ala Thr Thr Gly Gly Gly Leu
355 360 365
Ala Thr Gly Val Leu Pro Gly Pro Gly Pro Gly Leu Thr Val Gly Thr
370 375 380
Val Val Leu His Ser Leu Pro Thr Thr Thr Ala Ile Ala Thr
385 390 395
<210> 5
<211> 556
<212> PRT
<213>Escherichia coli (Escherichia coli)
<400> 5
Met Thr Thr Gly Ala Val Ile Val Ala Ala Gly Ala Ala Gly Leu Gly
1 5 10 15
Cys Ser Ala Ala Val Ile Pro Ala Ser Ala Leu Pro Ala Ile Thr Ile
20 25 30
Pro Ala His Leu Pro Leu His Ala Thr Ile Pro Gly Ala Ile Ser Gly
35 40 45
Pro Ala Ala Leu Pro Cys Leu Ile Ala Gly Pro Thr Gly Gly Val Thr
50 55 60
Thr Thr Ala Ala Val His Val Thr Ser Ala Leu Leu Ala Ala Gly Leu
65 70 75 80
His Ala Leu Gly Val Leu Gly His Ala Val Val Met Ile Leu Leu Pro
85 90 95
Ala Ser Pro Gly Val Val Leu Thr Pro Leu Ala Ala Ser Pro Ile Gly
100 105 110
Ala Ile Thr Thr Ser Ala Ala Pro Pro Pro Thr Pro Ala Gly Ile Ser
115 120 125
Leu Gly Ala Leu Ala Ser Ala Ala Leu Leu Ile Val Thr Gly Ser Ala
130 135 140
Thr Val Ala Leu Ile Leu Ala Leu Gly Ala Ala Gly Val Leu Ile Val
145 150 155 160
Thr Thr Ala Ser Ala Ala Ile Pro Gly Ala Cys Leu Ala Pro Ser Gly
165 170 175
Leu Thr Gly Ser Gly Gly Pro Ala Val Ala Ser Ile Pro Gly Leu Ile
180 185 190
Ser Pro Gly Ala Val Val Ala Leu Pro Pro Ser Ser Gly Thr Thr Gly
195 200 205
Leu Pro Leu Gly Val Met Leu Thr His Leu Gly Leu Val Thr Ser Val
210 215 220
Ala Gly Gly Val Ala Gly Gly Ala Pro Ala Leu Thr Pro Ala Ala Ala
225 230 235 240
Ala Val Ile Leu Cys Val Leu Pro Met Pro His Ile Thr Ala Leu Ala
245 250 255
Ser Ile Met Leu Cys Ser Leu Ala Val Gly Ala Thr Ile Leu Ile Met
260 265 270
Pro Leu Pro Gly Ile Thr Leu Leu Leu Gly Gly Ile Gly Ala Cys Leu
275 280 285
Val Thr Val Ala Met Val Val Pro Pro Ile Val Leu Ala Ile Ala Leu
290 295 300
Ser Pro Gly Thr Gly Leu Thr Ala Leu Ser Ser Val Ala Met Val Leu
305 310 315 320
Ser Gly Ala Ala Pro Leu Gly Leu Gly Leu Gly Ala Ala Ile Ser Ala
325 330 335
Leu Pro Pro Ala Ala Leu Leu Gly Gly Gly Thr Gly Met Thr Gly Ala
340 345 350
Gly Pro Val Leu Ala Met Ser Leu Gly Pro Ala Leu Gly Pro Pro Pro
355 360 365
Val Leu Ser Gly Ala Cys Gly Thr Val Val Ala Ala Ala Gly Met Leu
370 375 380
Ile Leu Ala Pro Ala Thr Gly Ala Ser Leu Pro Ala Ala Leu Pro Gly
385 390 395 400
Gly Ile Cys Ile Ala Gly Ala Gly Ile Met Leu Gly Thr Leu Ala Ala
405 410 415
Pro Leu Ala Thr Ala Ser Thr Ile Ala Leu Ala Gly Thr Leu His Thr
420 425 430
Gly Ala Val Gly Pro Ile Ala Ala Ala Ala Gly Leu Pro Ile Val Ala
435 440 445
Ala Leu Leu Gly Leu Ile Leu Thr Leu Gly Pro Gly Val Ala Pro Ala
450 455 460
Gly Leu Gly Ser Leu Leu Ile Gly His Pro Gly Ile Ala Ala Val Ala
465 470 475 480
Val Val Ala Met Leu Gly Gly Ala Ala Gly Gly Val Pro Val Ala Pro
485 490 495
Val Val Ala Ser Leu Ala Ser Ala Ile Ser Gly Ala Gly Ile Leu Gly
500 505 510
Pro Val Ser Leu Gly Val Val Pro Thr Leu Ala Ile Ala Leu Val Pro
515 520 525
Pro Thr Ala Ser Ile Pro Leu Ala Pro Ser Gly Leu Ile Leu Ala Leu
530 535 540
Ala Leu Ala Ala Ala Leu Ala Ala Gly Leu Met Ala
545 550 555

Claims (6)

  1. A kind of 1. method of E. coli whole cell catalytic production phloretin, it is characterised in that the synthetic method is with phenylpropyl alcohol ammonia Acid is raw material, and colibacillus engineering is Host Strains, is completed by the catalysis of some enzymes in host bacterial；
    The Host Strains can at least express phenylalanine lyase, cinnamic acid -4- hydroxylases, the connection of 4- coumaric acids coacetylase Enzyme, double bond reductase and chalcone synthase.
  2. 2. the method for E. coli whole cell catalytic production phloretin according to claim 1, it is characterised in that the conjunction Synthetic route into method is as follows：
  3. 3. the method for E. coli whole cell catalytic production phloretin according to claim 2, it is characterised in that the conjunction Into the phenylalanine lyase in method, cinnamic acid -4- hydroxylases, 4- coumarate CoA-ligases, double bond reductase and look into You are respectively derived from sunflower, thorn bud cynara scolymus, arabidopsis, apple and fleabane flower by the expressing gene of ketone synzyme.
  4. 4. the method for E. coli whole cell catalytic production phloretin according to claim 3, it is characterised in that the 4- The expressing gene of coumarate CoA-ligase and chalcone synthase is implemented on carrier pET-28a, phenylalanine lyase, The expressing gene of cinnamic acid -4- hydroxylases and double bond reductase is implemented on carrier pET-22b, the expression base in identical carrier Because sharing T7 promoters, the expressing gene of each enzyme has the expressing gene of 6 × his labels, enzyme and enzyme before terminator codon Between have a RBS train intervals, between the terminator codon and initiation codon of the expressing gene of RBS sequences and nearest enzyme respectively It is spaced 3 and 7 bases.
  5. 5. the method for E. coli whole cell catalytic production phloretin according to claim 1, it is characterised in that the conjunction It is as follows into method and step：
    1) according to phenylalanine lyase, cinnamic acid -4- hydroxylases, 4- coumarate CoA-ligases, double bond reductase and look into The amino acid sequence and e. coli codon Preference of your ketone synzyme, the nucleotide sequence of the corresponding enzyme of optimization, synthesis are related Expressing gene；
    2) expressing gene is implemented in the expression base of respective carrier, wherein 4- coumarate CoA-ligases and chalcone synthase Because being implemented in carrier pET-28a, the expressing gene structure of phenylalanine lyase, cinnamic acid -4- hydroxylases and double bond reductase It is built in carrier pET-22b；
    3) carrier for building completion is transferred in Escherichia coli, Escherichia coli cultivated, induced expression；
    4) phenylalanine substrate is added in the bacterium solution after expression, continues to react 20-26h.
    5) the phloretin extracting and developing in reaction solution is purified.
  6. 6. the method for E. coli whole cell catalytic production phloretin according to claim 5, it is characterised in that utilize first Alcohol extracts phloretin from zymotic fluid, the volume ratio 1: 1 of methanol and zymotic fluid.
CN201710938021.1A 2017-10-10 2017-10-10 A kind of method of E. coli whole cell catalytic production phloretin Pending CN107805646A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710938021.1A CN107805646A (en) 2017-10-10 2017-10-10 A kind of method of E. coli whole cell catalytic production phloretin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710938021.1A CN107805646A (en) 2017-10-10 2017-10-10 A kind of method of E. coli whole cell catalytic production phloretin

Publications (1)

Publication Number Publication Date
CN107805646A true CN107805646A (en) 2018-03-16

Family

ID=61592012

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710938021.1A Pending CN107805646A (en) 2017-10-10 2017-10-10 A kind of method of E. coli whole cell catalytic production phloretin

Country Status (1)

Country Link
CN (1) CN107805646A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913508A (en) * 2018-06-05 2019-06-21 嘉兴欣贝莱生物科技有限公司 A method of phloretin is synthesized using cyanobacteria
CN110117550A (en) * 2019-01-09 2019-08-13 嘉兴欣贝莱生物科技有限公司 Technique and saccharomyces cerevisiae based on fermentation by saccharomyces cerevisiae production phloretin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948794A (en) * 2010-09-06 2011-01-19 中国农业大学 Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof
WO2016193504A1 (en) * 2015-06-05 2016-12-08 Evolva Sa Biosynthesis of phenylpropanoid and dihydrophenylpropanoid derivatives

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101948794A (en) * 2010-09-06 2011-01-19 中国农业大学 Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof
WO2016193504A1 (en) * 2015-06-05 2016-12-08 Evolva Sa Biosynthesis of phenylpropanoid and dihydrophenylpropanoid derivatives

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
吴俊俊: "合成生物技术改造大肠杆菌生产黄酮骨架物质", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913508A (en) * 2018-06-05 2019-06-21 嘉兴欣贝莱生物科技有限公司 A method of phloretin is synthesized using cyanobacteria
CN110117550A (en) * 2019-01-09 2019-08-13 嘉兴欣贝莱生物科技有限公司 Technique and saccharomyces cerevisiae based on fermentation by saccharomyces cerevisiae production phloretin
CN110117550B (en) * 2019-01-09 2023-04-07 嘉兴欣贝莱生物科技有限公司 Process for producing phloretin based on saccharomyces cerevisiae fermentation and saccharomyces cerevisiae

Similar Documents

Publication Publication Date Title
CN104059872B (en) High-yield N-acetylglucosamine metabolic engineering bacterium, as well construction method and applications thereof
CN107586795A (en) A kind of method of fermentation by saccharomyces cerevisiae production phloretin
CN110066760B (en) Recombinant escherichia coli for expressing alpha-L-rhamnosidase and application thereof
CN106148265B (en) It is a kind of produce chondroitinase recombined bacillus subtilis and its application
CN104357418B (en) The application of a kind of glycosyl transferase and its mutant in ginseng saponin Rh 2 is synthesized
CN104651287B (en) A kind of engineering bacteria and application for synthetic glycerine glucoside
CN104531629B (en) A kind of yclodextrin glycosyltransferase mutant of raising AA 2G conversion ratios
US20240002892A1 (en) Compound enzyme and application thereof in preparation of l-ergothioneine
CN108795916A (en) A kind of lysine decarboxylase mutant, its encoding gene and its expression and application
CN106929462A (en) One kind accumulation N n acetylneuraminic acid ns recombined bacillus subtilis and its application
CN104789539B (en) A kind of mutant of trehalose synthase and its preparation method and application
CN107460138A (en) A kind of recombinant yeast pichia pastoris for the glucose dehydrogenase that production FAD is relied on and its construction method and application
CN103898177B (en) Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof
CN110229804A (en) A kind of limonene synzyme SynLS1 and its application
CN107805646A (en) A kind of method of E. coli whole cell catalytic production phloretin
CN110396508A (en) From the L- pantoic acid lactone dehydrogenase of Nocardia cyriacigeorgica and application
CN104480127B (en) Hyperthermophilic glucosides enzyme mutant and its application in prepared by Ginsenoside compound K
CN107460203A (en) A kind of recombinant bacterium and construction method and purposes for producing rhodioside and the like
CN113817782B (en) Full biosynthesis method of pimelic acid
CN107267478A (en) A kind of amylosucrase and its method for converting production α ursin
CN102690795B (en) Trehalose synthase of streptomyces griseochromogenes and coding gene and application of trehalose synthase
CN110396507A (en) L- pantoic acid lactone dehydrogenase from Cnuibacter physcomitrellae
CN104419688B (en) A kind of transfructosylase and its gene, its secreted expression carrier and application
CN104877983B (en) A kind of trehalose synthase mutant and its preparation and application
CN101250539B (en) Method for preparing recombinant heat-proof beta-glucuronic acid enzyme

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20180316

RJ01 Rejection of invention patent application after publication