CN109913508A - A method of phloretin is synthesized using cyanobacteria - Google Patents

A method of phloretin is synthesized using cyanobacteria Download PDF

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Publication number
CN109913508A
CN109913508A CN201810566561.6A CN201810566561A CN109913508A CN 109913508 A CN109913508 A CN 109913508A CN 201810566561 A CN201810566561 A CN 201810566561A CN 109913508 A CN109913508 A CN 109913508A
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gene
leu
phloretin
ala
chs
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Inventor
蒿飞
朱文博
陈贤情
夏文豪
杨月
王筱
王文
杨慧
江会峰
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Jiaxing Xin Baylet Biotechnology Co Ltd
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Jiaxing Xin Baylet Biotechnology Co Ltd
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Abstract

This present invention provides a kind of technical method that phloretin is synthesized using " photosynthetic bacteria "-cyanobacteria as chassis biology, have main steps that 4 coumaric acids of heterogenous expression-CoA ligase (4 coumarate Coenzyme A Ligase in Cells of Blue-green Algae, 4CL) and chalcone synthase (Chalcone synthase, CHS) the two phloretins synthesize key enzyme, generation phloretin is catalyzed so that para hydroxybenzene propionic acid (or phloretic acid) is substrate, 4CL is catalyzed a molecule para hydroxybenzene propionic acid and generates para hydroxybenzene propionyl coenzyme A, then the malonyl coenzyme A (malonyl coA) that CHS is catalyzed 3 molecules synthesizes a molecule phloretin with a molecule para hydroxybenzene propionyl coenzyme A.This method can carry out photosynthetic microorganism to produce phloretin by one kind, to create the phloretin biosynthesis pathway that a kind of raw material is cheap, equipment is simple, environmental pollution is low, yield is high, meet the direction of greenization production.

Description

A method of phloretin is synthesized using cyanobacteria
Technical field
The invention belongs to the synthesis technical field of phloretin more particularly to a kind of sides that phloretin is produced using cyanobacteria Method.
Background technique
Cyanobacteria (Cyanobacteria) is a kind of bacterium also known as blue-green alge, bluish-green bacterium that energy is obtained by photosynthesis Or cyanobacteria, morphosis is simple, and no fixed nucleus, the phycocyanin and allophycocyanin contained in vivo adds chloroplaset Albumen is allowed to show blue-green.Cyanobacteria is the algae occurred earliest on the earth, is most simple, most original unicellular organism, In very long evolutionary process, the earth is become the oxidized form earth by cyanobacteria, and by endocytosis at the chloroplaset of plant, it is believed that blue Algae is simplest Photosynthetic Unit.
It can be carried out photosynthetic physiological property since cyanobacteria is unique, many scientists regard cyanobacteria as a kind of " biology Photoreactor ", by it by air carbon dioxide and water become the chemical products of human needs and the day of some high values Right product.Genetic modification is carried out to cyanobacteria carry out these products of biosynthesis with many advantages: (1) many common cyanobacterias at present Genome, such as Synechococcus, cytoalgae, anabena, have all been sequenced and have finished, simultaneously because the genome of cyanobacteria is small, structure Simply, scientist has invented a set of mature, easy-operating gene editing system for Cells of Blue-green Algae；(2) relative to height Equal animals and plants, cyanobacteria have the characteristics that microorganism fast growing, are adapted for the large-scale production operation of product；(3) cyanobacteria Adaptability is very strong, can survive in many adverse circumstances, while the life condition that cyanobacteria needs is very simple, or even only needs sun Light and moisture can survive, therefore cost of material can not only be significantly saved in large-scale culture, and do not need to occupy and plough Ground resource avoids and produces the conflict of the competition land resource such as food grain.(4) cyanobacteria is absorbed in air by photosynthesis Carbon dioxide and generate oxygen and be discharged into the air, therefore large-scale culture cyanobacteria can effectively alleviate the mankind and continue on fossil " greenhouse effects " caused by raw material etc., play certain environmental-protection function.So using cyanobacteria as the chassis in synthetic biology Biology has vast prospect and huge value.
Phloretin English name: Phloretin；Molecular formula: C15H14O5；Chemical name: 3- (4- hydroxy phenyl) -1- (2, 4,6- trihydroxy phenyl) -1- acetone；CAS:60-82-2；Molecular weight: 274.28；Physical property: methanol, ethyl alcohol and third are soluble in Ketone is slightly soluble in chloroform, is insoluble in water, petroleum ether and benzene.
Phloretin is a kind of external new type natural skin-whitening agents for researching and developing out recently, be distributed mainly on apple, The pericarp and root skin of the rich fruits such as pears.Phloretin can inhibit the excessive secretion of sebaceous glands, secrete vigorous type acne for treating； It can inhibit melanocyte activity, have effect to various skin splash.Compared with similar natural component arbutin and kojic acid, on an equal basis The phloretin of concentration is better than them to the inhibiting effect of tyrosinase, and when it is compounded with arbutin and/or kojic acid When, product can be greatly improved to the inhibiting rate of tyrosinase, inhibiting rate is made to reach 100%.The latest researches have found that it is also anti- The effects of scorching, immunosupress, cardiovascular protection.The every annual consumption in the whole world is no less than 20 tons, and demand is every year with the increase of 20% rate, tool There is the larger market demand.
Currently, the production of phloretin mainly passes through chemical industry synthesis in the market, some is passed through using phloridzin crude product as raw material Simple purification phloridzin, then drag glycogen and crystallization to be recrystallized to give phloretin in strong lewis acid and metal catalytic.Also with Aurantiin is raw material, and Raney Ni is catalyst, and in sodium hydroxide solution, 10 hours synthesis aurantiin dihydros of catalytic hydrogenation look into ear Ketone, then in the hydrochloric acid solution at 85 DEG C -95 DEG C, aurantiin dihydrochalcone hydrolyzes 2 hours and obtains phloretin.These chemical industry Method synthesizes phloretin, and there are low efficiencys, the discharge of chemical waste fluid, serious the problems such as polluting environment.There are also develop some utilizations The method that the fermentations such as Escherichia coli or saccharomyces cerevisiae carry out biosynthesis phloretin, although these biological synthesis process solve low output The problems such as with component environment pollution, but since the costs such as raw material, equipment and water power in production process are too high, it cannot achieve big rule Mould production.
Summary of the invention
It is an object of the invention to solve the deficiencies in the prior art, a kind of biology conjunction using cyanobacteria synthesis phloretin is provided At method, have the advantages that yield is high, pollution-free, with short production cycle, at low cost and environmentally friendly.
A method of phloretin being synthesized using cyanobacteria, by 4 coumaric acids-CoA ligase gene (4CL) and chalcone Synthase gene (CHS) imports Cells of Blue-green Algae and obtains recombined blue algae cell, carries out photosynthesis using recombined blue algae cell and obtains Phloretin.
Preferably, the cyanobacteria is SynechococcusSynechococcus elongatus PCC7942。
The synthetic method specifically includes the following steps:
Construct integration vector NSI: described in selectionSynechococcus elongatus Gene order on PCC7942 genome Section, and the change of this section of gene order will not influenceSynechococcus elongatus The function of PCC7942 whole gene group Can, integration vector NSI is constructed and obtained with this;
Recombinant vector: according toSynechococcus elongatus PCC7942 cell codon Preference optimization gene 4CL and Then the two genes are inserted on carrier NSI by the codon of CHS, obtain the recombinant vector with chloramphenicol resistance gene NSI-CHS-4CL；
Homologous recombination: recombinant vector NSI-CHS-4CL is transformed intoSynechococcus elongatus PCC7942 cell It is interior, it is acted on by homologous recombination, two gene integrations of CHS and 4CL is arrivedSynechococcus elongatus PCC7942 base Because of the NSI gene loci in group, and control with inducible promoter Trc the expression of gene.
Screening confirmation: with chloramphenicol come screen conversion NSI-CHS-4CL carrier afterSynechococcus elongatus Then PCC7942 monoclonal cell extracts the cellular genome, determined by the test of PCR Molecular Identification and finally carry 4CL base Cause and CHS geneSynechococcus elongatus PCC7942。
Preferably, the 4CL is synthesized according to the amino acid sequence of gene in sunflower.
Preferably, the CHS is synthesized according to the amino acid sequence of gene in fleabane flower.
The present invention produce phloretin and the prior art the difference is that:
1) be transformed synthesis phloretin using cyanobacteria as chassis biology, raw materials for production only need sunlight and moisture etc., production equipment and Production environment building is simple, does not need a large amount of electricity consumptions, therefore compared to other production methods, production cost is greatly lowered；
2) microorganism is transformed as technical foundation, catalyzing and synthesizing for biological enzyme is carried out in microbial body, avoids mentioning on a large scale Take, separate, purification process, from production cost and it is environmentally friendly in terms of be easy to control；
3) this technique is lacked than other methods in the scale and quantity of production equipment, easy to operate, is easy to industry conversion；
4) this technique only isolates and purifies process in the final step of production, and purifying products simple process, product quality is than general Method purity is more preferable, content is higher；
5) synthesis process does not have discharging of waste liquid, environmental-friendly, pollution-free, sustainable production, present invention process from economy, environment and Occupational health angle is that excellent industrialized production shows the way line.
Detailed description of the invention
Fig. 1 is exogenous origin gene integrator schematic diagram in the present invention；
Fig. 2 is NSI plasmid map in the present invention；
Fig. 3 is recombinant vector NSI-CHS-4CL plasmid map in the present invention
Fig. 4 is phloretin biosynthesis route map in the present invention；
Fig. 5 is the liquid phase result figure of wild type Syn7942
Fig. 6 is the liquid phase result figure of embodiment in the present invention；
Fig. 7 is the mass spectrogram of embodiment in the present invention.
Specific embodiment
In order to enable the purposes, technical schemes and advantages of the embodiment of the present disclosure are clearer, below in conjunction with disclosure reality The technical solution of the embodiment of the present disclosure is clearly and completely described in the attached drawing for applying example.Obviously, described embodiment is A part of this disclosure embodiment, instead of all the embodiments.Based on described embodiment of the disclosure, this field is common Technical staff's every other embodiment obtained under the premise of being not necessarily to creative work, belongs to the model of disclosure protection It encloses.
The invention discloses a kind of methods using cyanobacteria synthesis phloretin, and it is fragrant to be included in heterogenous expression 4 in Cells of Blue-green Algae Beans acid-CoA ligase (4 coumarate Coenzyme A Ligase, 4CL) and chalcone synthase (Chalcone Synthase, CHS) the two phloretins synthesis key enzyme, life is catalyzed so that para hydroxybenzene propionic acid (also known as phloretic acid) is substrate At phloretin, 4CL is catalyzed a molecule para hydroxybenzene propionic acid and generates para hydroxybenzene propionyl coenzyme A, and then CHS is catalyzed 3 molecules Malonyl coenzyme A (malonyl coA) synthesizes a molecule phloretin with a molecule para hydroxybenzene propionyl coenzyme A, mainly includes structure It builds integration vector NSI, recombinant vector, homologous recombination, screening confirmation, catalyze and synthesize and detect six steps.
Using the synthesis mode, be transformed synthesis phloretin using cyanobacteria as chassis biology, raw materials for production only need sunlight and Moisture etc. meets the direction of current green production, and utilizes the biological means, compared with chemical purification mode, life of the invention It produces equipment and production environment building is simple, do not need a large amount of electricity consumptions, while production cost is greatly lowered, effective protection ring Border realizes greenization production.
Several embodiments of the present invention are described in detail below, but the invention is not limited to these to be embodied Example.
Embodiment one
The present embodiment provides vector plasmid building process: providing according to the present inventionSynechococcus elongatus PCC7942(abbreviation Syn7942) NSI gene order, construct integration vector NSI, then root according to the plasmid map in attached drawing 2 According to 4CL and CHS amino acid sequence provided in the present invention, according to Syn7942 cell codon Preference design corresponding gene Nucleic acid sequence send gene 4CL and CHS to company's synthesis with gene chemical synthesis ability on the market, design 20bp or so size Homology arm, it is using one-step cloning kit, the two are gene constructed in integration vector NSI according to the plasmid map in attached drawing 3 On.
NSI is as Synechococcus provided by the inventionSynechococcus elongatus One on PCC7942 genome Section gene order, the effect of NSI is that the change of this section of gene order will not influence SynechococcusSynechococcus elongatus The function of PCC7942 whole gene group, so foreign gene (can not be Synechococcus by weSynechococcus elongatus PCC7942 intracellular gene) it is inserted into this gene order of this NSI, in this way Both exogenous origin gene integrator was arrivedSynechococcus elongatus On PCC7942 genome, while it will not influence original again There is the function of genome.
Exogenous origin gene integrator principle mainly passes through homologous recombination, as shown in Figure 1, the present invention is in NSI gene order On, respectively at 5 ' ends and 3 ' each segments for selecting 1000bp or so in end, the two segments are connected to the external source for needing to be inserted into (such as Fig. 1, top half is containing exogenous genetic fragment in figure, and top half is at gene both endsSynechococcus elongatus The NSI gene order of PCC7942), pass through the homologous sequence in this two segments and NSI gene order in this way Column just recombinate foreign gene onto NSI, and CHS and 4CL are the foreign genes for needing to express, wherein trc, lacl and Trrnb Be respectively promoter, operon and be transferred to terminate subcomponent be used to control gene expression jointly, CM is chloramphenicol resistance gene, use To screen the cyanobacteria containing foreign gene.
Embodiment two
The present embodiment provides recombinant conversion processes:
1) take the wild type Syn7942 that 2mL stand density OD730 is 0.8 ~ 1.2 in centrifuge tube, then 10000rpm, centrifugation 2min removes supernatant；
2) sediment in previous step is mixed with 1mL 10mM sodium chloride solution, then 10000rpm, is centrifuged 2min, removes supernatant Liquid；
3) sediment in previous step is mixed with the sterilized nonreactive BG11 fluid nutrient medium of 1mL, then 10000rpm, centrifugation 2min removes supernatant；
4) sediment in previous step is mixed with the sterilized nonreactive BG11 fluid nutrient medium of 100uL, be then added thereto The Plasmid DNA of 200ng.The centrifuge tube (being protected from light processing) is thoroughly sealed with masking foil, then in shaking table, 100rpm, 30 DEG C of trainings It supports 10 hours；
5) cyanobacteria in previous step centrifuge tube is all coated on the BG11 solid medium containing 25ug/mL chloramphenicol, will be put down Plate is put into 30 DEG C of illumination boxs, and subsequent authentication is carried out after growing cyanobacteria monoclonal 1 to 2 week.
It should be noted that remaining above operation must be completed in super-clean bench in addition to centrifugation, guarantee gnotobasis.
Embodiment three
The present embodiment provides the detection process of the screening confirmation and product of transgenic blue algae:
Picking 10 or so contain the monoclonal cyanobacteria bacterial plaque to grow on 25ug/mL chloramphenicol BG11 solid medium, access It is cultivated in 5mL 25ug/mL chloramphenicol BG11 liquid bulk culture medium, after cyanobacteria is cultivated, genome is extracted, with designed Primer PCR verifies two target gene of CHS and 4CL, and PCR send product to sequencing, determines whether gene order is correct；It will verifying Correctly turn illumination cultivation in the Syn7942 cyanobacteria access 100mL BG11 fluid nutrient medium of CHS gene and 4CL gene, to When OD730 is 1, substrate para hydroxybenzene propionic acid is added, while adding IPTG induction two gene expressions of CHS and 4CL, is then distinguished Transgenic blue algae when cultivating 1 day, 3 days, 5 days after inducing is taken, Cells of Blue-green Algae is crushed with high pressure cracker, to product separating-purifying Afterwards, it is detected with high performance liquid chromatograph.
Example IV
The present embodiment provides the synthesis of phloretin:
As shown in Fig. 4,4 coumaric acids of heterogenous expression-CoA ligase (4 coumarate in Syn7942 cell Coenzyme A Ligase, 4CL) and the synthesis of chalcone synthase (Chalcone synthase, CHS) the two phloretins Key enzyme is catalyzed generation phloretin so that para hydroxybenzene propionic acid (or phloretic acid) is substrate, and 4CL is catalyzed a molecule para hydroxybenzene Propionic acid generate para hydroxybenzene propionyl coenzyme A, then CHS be catalyzed 3 molecules malonyl coenzyme A (malonyl coA) with one point Sub- para hydroxybenzene propionyl coenzyme A synthesizes a molecule phloretin.
The above experiment does negative control with wild type Syn7942, wherein phloretin liquid phase figure such as Fig. 5 institute of control group Show, the liquid phase result, mass spectral results in embodiment are as shown in attached drawing 6-7, and according to Fig. 5, it can be concluded that, wild type Syn7942 does root Skin element product detection occurs without peak figure, illustrates its not output phloretin, and according to Fig.6, the transgenosis in the present embodiment Syn7942 has the appearance of phloretin peak figure, illustrates to be possible to produce phloretin, it is also necessary to further confirm that product is exactly root with mass spectrum Pi Su, wherein phloretin molecular weight is 274.27, and can see by Fig. 7 has one 275 or so peak to occur in product, can To confirm that the product is exactly phloretin, thus prove that transgenosis Syn7942 disclosed in the present embodiment has production phloretin Effect, and the environmentally protective low cost of the process.
Related gene and amino acid sequence
The NSI gene order of Syn7942:
tatcaagattgctggggaagaaccgaccatccacaacgcgatcgagcggctgcttggcaaaaaccgtaagga aatcgagcaaattgccaaggagaccctcgaaggcaacttgcgtggtgttttagccagcctcacgccggagcagatc aacgaggacaaaattgcctttgccaaaagtctgctggaagaggcggaggatgaccttgagcagctgggtctagtcc tcgatacgctgcaagtccagaacatttccgatgaggtcggttatctctcggctagtggacgcaagcagcgggctga tctgcagcgagatgcccgaattgctgaagccgatgcccaggctgcctctgcgatccaaacggccgaaaatgacaag atcacggccctgcgtcggatcgatcgcgatgtagcgatcgcccaagccgaggccgagcgccggattcaggatgcgt tgacgcggcgcgaagcggtggtggccgaagctgaagcggacattgctaccgaagtcgctcgtagccaagcagaact ccctgtgcagcaggagcggatcaaacaggtgcagcagcaacttcaagccgatgtgatcgccccagctgaggcagct tgtaaacgggcgatcgcggaagcgcggggggccgccgcccgtatcgtcgaagatggaaaagctcaagcggaaggga cccaacggctggcggaggcttggcagaccgctggtgctaatgcccgcgacatcttcctgctccagaagctcgagtc cctgctcgtcacgctttcaggcaccgtgccagatatcgacgtggagtcgatcactgtgattggcgaaggggaaggc agcgctacccaaatcgctagcttgctggagaagctgaaacaaaccacgggcattgatctggcgaaatccctaccgg gtcaatccgactcgcccgctgcgaagtcctaagagatagcgatgtgaccgcgatcgcttgtcaagaatcccagtga tcccgaaccataggaaggcaagctcaatgcttgcctcgtcttgaggactatctagatgtctgtggaacgcacattt attgccatcaagcccgatggcgttcagcggggtttggtcggtacgatcatcggccgctttgagcaaaaaggcttca aactggtgggcctaaagcagctgaagcccagtcgcgagctggccgaacagcactatgctgtccaccgcgagcgccc cttcttcaatggcctcgtcgagttcatcacctctgggccgatcgtggcgatcgtcttggaaggcgaaggcgttgtg gcggctgctcgcaagttgatcggcgctaccaatccgctgacggcagaaccgggcaccatccgtggtgattttggtg tcaatattggccgcaacatcatccatggctcggatgcaatcgaaacagcacaacaggaaattgctctctggtttag cccagcagagctaagtgattggacccccacgattcaaccctggctgtacgaataaggtctgcattccttcagagag acattgccatgcc
Amino acid sequence
4CL:
<110>Jiaxing Xin Beilai Biotechnology Co., Ltd
<120>a kind of synthetic route of phloretin
<130> 456
<140> 999
<141> 2017-09-04
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<170> PatentIn version 3.3
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<213>sunflower (Helianthus annuus,Ha)
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Ser Lys Phe Leu Asp Arg Pro Cys Leu Ile Asn Gly Ala Thr Gly Glu
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Val His Thr Tyr Ala Asp Val Glu Leu Thr Ser Arg Lys Val Ala Ser
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Ala Leu His Gln Gln Gly Ile Ser Lys Gly Asp Val Ile Met Ile Leu
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Leu Pro Asn Ser Pro Glu Phe Val Tyr Ser Phe Ile Gly Ala Ser Tyr
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Leu Gly Ala Ile Ser Thr Met Ala Asn Pro Phe Phe Thr Ala Ala Glu
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Ile Ile Lys Gln Val Lys Ala Ser Asn Ser Lys Ile Ile Ile Thr Gln
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Ser Ala His Ile Pro Lys Val Lys Asp Tyr Ala Ser Asp Asn Ser Ile
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Lys Leu Val Cys Ile Asp Ser Ala Pro Leu Gly Cys Leu His Phe Ser
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Glu Leu Thr Ser Ala Asp Glu Thr Lys Leu Pro Gln Ile Glu Val Ser
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Ser Asp Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr Thr Gly Leu
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Pro Lys Gly Val Met Leu Thr His Lys Gly Leu Val Thr Ser Val Ala
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Gln Gln Val Asp Gly Glu Asn Pro Asn Leu Trp Ile His Ser Glu Asp
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Val Leu Met Cys Ser Leu Pro Leu Phe His Ile Tyr Ser Leu Asn Ser
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Ile Leu Leu Cys Gly Leu Arg Ala Gly Ala Ala Ile Leu Leu Met Ser
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<213>fleabane flower (Erigeron breviscapus)
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Ala Thr Lys Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr
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Ile Asp Gly His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys
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Asp Val Pro Gly Leu Ile Ser Lys Asn Ile Glu Lys Ala Leu Thr Gln
275 280 285
Ala Phe Ser Pro Leu Gly Ile Ser Asp Trp Asn Ser Leu Phe Trp Ile
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Ala His Pro Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Leu Lys Leu
305 310 315 320
Gly Leu Lys Glu Glu Lys Met Arg Ala Thr Arg His Val Leu Ser Glu
325 330 335
Tyr Gly Asn Met Ser Ser Ala Cys Val Leu Phe Ile Ile Asp Glu Met
340 345 350
Arg Lys Lys Ser Ala Glu Asp Gly Ala Ala Thr Thr Gly Glu Gly Leu
355 360 365
Asp Trp Gly Val Leu Phe Gly Phe Gly Pro Gly Leu Thr Val Glu Thr
370 375 380
Val Val Leu His Ser Leu Pro Thr Thr Thr Ala Ile Ala Thr
385 390 395
Val Ile His Gly Ala Gln Ala Gly Gly Phe Ser Pro Ile Ser Ile Tyr
145 150 155
Gly Gly Ile Thr Asn Gln Ile Val Ala Lys Ala Gly Leu Pro Phe Ala
160 165 170 175
Pro Thr Ser Leu Phe Leu Ser Ser Phe Phe Phe Asn Leu Ala Ile Ala
180 185 190
Val Leu Val Phe Phe Val Phe Gly Gly Ala Arg Val Met Lys His Asp
195 200 205
Pro Ala Ser Leu Gly Pro Leu Pro Glu Leu His Pro Glu Gly Val Ser
210 215 220
Ala Ser Ile Arg Gly His Gly Gly Thr Pro Ala Lys Pro Ile Arg Glu
225 230 235
His Ala Tyr Gly Thr Ala Ala Asp Thr Ala Thr Thr Leu Arg Leu Asn
240 245 250 255
Asn Glu Arg Ile Thr Thr Leu Ile Gly Leu Thr Ala Leu Gly Ile Gly
260 265 270
Ala Leu Val Phe Lys Phe Asn Val Gly Leu Val Ala Met Thr Val Ala
275 280 285
Val Val Leu Ala Leu Leu Ser Pro Lys Thr Gln Lys Ala Ala Ile Asp
290 295 300
Lys Val Ser Trp Ser Thr Val Leu Leu Ile Ala Gly Ile Ile Thr Tyr
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Val Gly Val Met Glu Lys Ala Gly Thr Val Asp Tyr Val Ala Asn Gly
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340 345 350
Thr Gly Ala Ile Val Ser Ala Phe Ala Ser Ser Thr Ala Leu Leu Gly
355 360 365
Ala Ile Ile Pro Leu Ala Val Pro Phe Leu Leu Gln Gly His Ile Ser
370 375 380
Ala Ile Gly Val Val Ala Ala Ile Ala Ile Ser Thr Thr Ile Val Asp
385 390 395
Thr Ser Pro Phe Ser Thr Asn Gly Ala Leu Val Val Ala Asn Ala Pro
400 405 410 415
Asp Asp Ser Arg Glu Gln Val Leu Arg Gln Leu Leu Ile Tyr Ser Ala
420 425 430
Leu Ile Ala Ile Ile Gly Pro Ile Val Ala Trp Leu Val Phe Val Val
435 440 445
Pro Gly Leu Val
450
In conclusion above embodiment is not restricted embodiment of the invention, all those skilled in the art are at this The modification carried out on the basis of the substantive content of invention or equivalent deformation, in technology scope of the invention.

Claims (7)

1. a kind of method using cyanobacteria synthesis phloretin, which is characterized in that by 4 coumaric acids-CoA ligase gene (4CL) And chalcone synthase genes (CHS), it imports Cells of Blue-green Algae and obtains recombined blue algae cell, utilize recombined blue algae cell catalysis pair Hydroxy phenylpropionic acid obtains phloretin.
2. the method according to claim 1, wherein the cyanobacteria is Synechococcus Synechococcus elongatus PCC7942。
3. according to the method described in claim 2, characterized by comprising the following steps: building integration vector NSI: choosing institute The gene order section on Synechococcus elongatus PCC7942 genome is stated, and the change of this section of gene order is not It will affect the function of Synechococcus elongatus PCC7942 whole gene group, carried with this to construct and obtain integration Body NSI;Recombinant vector: according to Synechococcus elongatus PCC7942 cell codon Preference optimization gene Then the two genes are inserted on carrier NSI by the codon of 4CL and CHS, obtain the recombination with chloramphenicol resistance gene Carrier NSI-CHS-4CL；Homologous recombination: recombinant vector NSI-CHS-4CL is transformed into Synechococcus elongatus PCC7942 is intracellular, is acted on by homologous recombination, by two gene integrations of CHS and 4CL to Synechococcus NSI gene loci on elongatus PCC7942 genome, and control with inducible promoter Trc the expression of gene； Screening confirmation: with chloramphenicol come screen conversion NSI-CHS-4CL carrier after Synechococcus elongatus PCC7942 Then monoclonal cell extracts the cellular genome, determined by the test of PCR Molecular Identification and finally carry 4CL gene and CHS The Synechococcus elongatus PCC7942 of gene；It catalyzes and synthesizes: by the Synechococcus by screening Elongatus PCC7942 is cultivated, and substrate para hydroxybenzene propionic acid is added, and adds IPTG induction two genes of CHS and 4CL Expression, obtains phloretin.
4. method according to claim 1-3, which is characterized in that the 4CL is according to the ammonia of gene in sunflower Base acid sequence is synthesized.
5. method according to claim 1-3, which is characterized in that the CHS is according to the ammonia of gene in fleabane flower Base acid sequence is synthesized.
6. according to the method described in claim 3, it is characterized in that, the chloramphenicol screening process includes: S1: will be homologous heavy Cell after group is placed in chloramphenicol solid medium, the Synechococcus elongatus that preliminary screening is survived PCC7942；S2: Synechococcus elongatus PCC7942 switching is had to the Liquid Culture of chlorampenicol resistant Base extracts its genome after its growth, is determined by the test of PCR Molecular Identification and finally carries 4CL gene and CHS gene Synechococcus elongatus PCC7942.
7. according to the method described in claim 3, it is characterized by further comprising: after catalyzing and synthesizing phloretin, to having collected At phloretin carry out separating-purifying, and detected using high performance liquid chromatograph.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110960452A (en) * 2019-12-26 2020-04-07 嘉兴欣贝莱生物科技有限公司 Yeast extract containing phloretin and preparation process and application thereof
CN112899314A (en) * 2021-02-04 2021-06-04 陕西师范大学 Method for promoting recombinant yarrowia lipolytica to synthesize phloretin
CN114990086A (en) * 2022-06-16 2022-09-02 天津大学佐治亚理工深圳学院 Enzyme composition and application thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005084305A2 (en) * 2004-03-01 2005-09-15 Regents Of The University Of Minnesota Flavonoids
US20070232495A1 (en) * 2006-03-24 2007-10-04 Nappa Alvaro O Compositions and methods to add value to plant products, increasing the commercial quality, resistance to external factors and polyphenol content thereof
CN101748069A (en) * 2008-12-12 2010-06-23 中国科学院微生物研究所 recombinant blue-green algae
US20110093986A1 (en) * 2008-06-26 2011-04-21 Yeda Research And Development Co., Ltd At The Weizmann Institute Of Science Tomato simyb12 transcription factor and genetic selection thereof
CN102978229A (en) * 2012-11-23 2013-03-20 中国科学院微生物研究所 Blue-green algae integrant expression carrier and application thereof
CN104726505A (en) * 2015-03-31 2015-06-24 上海交通大学 Method for producing three-carbon compounds by using gene engineering cyanobacteria
WO2016193504A1 (en) * 2015-06-05 2016-12-08 Evolva Sa Biosynthesis of phenylpropanoid and dihydrophenylpropanoid derivatives
CN107805646A (en) * 2017-10-10 2018-03-16 嘉兴欣贝莱生物科技有限公司 A kind of method of E. coli whole cell catalytic production phloretin
CN107974484A (en) * 2017-11-10 2018-05-01 嘉兴欣贝莱生物科技有限公司 Chalcone synthase modeling method in phloretin biosynthetic process

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005084305A2 (en) * 2004-03-01 2005-09-15 Regents Of The University Of Minnesota Flavonoids
US20050208643A1 (en) * 2004-03-01 2005-09-22 Claudia Schmidt-Dannert Flavonoids
US20070232495A1 (en) * 2006-03-24 2007-10-04 Nappa Alvaro O Compositions and methods to add value to plant products, increasing the commercial quality, resistance to external factors and polyphenol content thereof
US20110093986A1 (en) * 2008-06-26 2011-04-21 Yeda Research And Development Co., Ltd At The Weizmann Institute Of Science Tomato simyb12 transcription factor and genetic selection thereof
CN101748069A (en) * 2008-12-12 2010-06-23 中国科学院微生物研究所 recombinant blue-green algae
CN102978229A (en) * 2012-11-23 2013-03-20 中国科学院微生物研究所 Blue-green algae integrant expression carrier and application thereof
CN104726505A (en) * 2015-03-31 2015-06-24 上海交通大学 Method for producing three-carbon compounds by using gene engineering cyanobacteria
WO2016193504A1 (en) * 2015-06-05 2016-12-08 Evolva Sa Biosynthesis of phenylpropanoid and dihydrophenylpropanoid derivatives
CN107805646A (en) * 2017-10-10 2018-03-16 嘉兴欣贝莱生物科技有限公司 A kind of method of E. coli whole cell catalytic production phloretin
CN107974484A (en) * 2017-11-10 2018-05-01 嘉兴欣贝莱生物科技有限公司 Chalcone synthase modeling method in phloretin biosynthetic process

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NCBI: "4-coumarate--CoA ligase 2-like [Helianthus annuus]", GENBANK, pages 021984256 - 3 *
楼士林 等: "蓝藻基因表达载体系统的构建和应用", 厦门大学学报, vol. 40, no. 2, pages 586 - 591 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110960452A (en) * 2019-12-26 2020-04-07 嘉兴欣贝莱生物科技有限公司 Yeast extract containing phloretin and preparation process and application thereof
CN112899314A (en) * 2021-02-04 2021-06-04 陕西师范大学 Method for promoting recombinant yarrowia lipolytica to synthesize phloretin
CN112899314B (en) * 2021-02-04 2022-06-17 陕西师范大学 Method for promoting recombinant yarrowia lipolytica to synthesize phloretin
CN114990086A (en) * 2022-06-16 2022-09-02 天津大学佐治亚理工深圳学院 Enzyme composition and application thereof
CN114990086B (en) * 2022-06-16 2024-05-07 天津大学佐治亚理工深圳学院 Enzyme composition and application thereof

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