CN107460220A - A kind of preparation method of rhodioside and the like - Google Patents

A kind of preparation method of rhodioside and the like Download PDF

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CN107460220A
CN107460220A CN201610396521.2A CN201610396521A CN107460220A CN 107460220 A CN107460220 A CN 107460220A CN 201610396521 A CN201610396521 A CN 201610396521A CN 107460220 A CN107460220 A CN 107460220A
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sybe
glucose
rhodioside
gene
culture mediums
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赵广荣
李晓波
刘雪
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Tianjin University
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Tianjin University
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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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Abstract

The invention discloses a kind of preparation method of rhodioside and the like, step is：Recombinant bacterium SyBE 218014 and the bacterial strains of SyBE 218021 are seeded in LB culture mediums respectively, in OD600When reaching 1.0, it is transferred in the culture medium of the protease hydrolysate containing rhodiola plant or the M9 culture mediums containing glucose, is fermented jointly, when concentration of glucose is less than 0.5g/L, glucose is added to 2 10g/L, continuing fermentation, obtains rhodioside and the like.The method of the present invention uses engineering colon bacillus, and glucose is carbon source, and fermenting and producing rhodioside and the like is carried out using free induced expression and integrant expression Different Strategies.The present invention can solve the source problem that comes of rhodioside and the like, while reduce production cost to greatest extent, be advantageous to industrialized production.

Description

A kind of preparation method of rhodioside and the like
Technical field
Biomedicine technical field belonging to the present invention, it is related to a kind of preparation method of rhodioside and the like.
Background technology
Rhodioside (salidroside) is Crassulaceae (Crassulaceae) Rhodida plant rhodiola root The principle active component of (Rhodiola rosea), there is well anti-oxidant, radioresistance, antifatigue, immunological regulation, anti-plateau A variety of pharmacological activity such as hypoxemia.Its molecular formula is C14H20O7, molecular weight 300.30, off-white color or buff powder.In recent years, Research finds that rhodioside can be used for organ hypoxia and the ischemics such as cancer, the nervous system disease, cell ageing, brain and heart, recognize Many treatments such as obstacle, the skin wrinkle food in one's mouth, Metabolism regulation are known, hence in so that rhodioside is in medicine, health products, cosmetics etc. Widely used in industrial production.
So far, the main source of rhodioside is still wild rhodiola root, the amount of rhodioside in wild rhodiola root It is very low, and the complicated technique of extraction needs of natural salidroside, now the most frequently used sachalin rhodiola rhizome and rhodiola, The amount of rhodioside only has 0.5%-0.8% in its plant.And the rhodiola root cost of artificial cultivation is quite high and active ingredient amount is low. In recent years, in chemical synthesis, living things catalysis (enzymatic) synthesis rhodioside etc., correlation theory and practice all achieve weight Improve.Although chemical synthesis rhodioside and the like technology has reached its maturity, it is required for carrying out selective guarantor mostly Shield, activation use expensive metallic catalyst.Therefore above method is unfavorable for industrial production.
The biosynthesis of rhodioside is divided into 4 stages in plant：First stage is nascent metabolite phosphoenolpyruvate Pyruvic acid and E4P form shikimic acid through shikimic acid pathway；Second stage is anti-through a few step enzymatics again by shikimic acid Sieve's Ah acid should be formed；Phase III is by sour the synthesizing to tyrosol of sieve Ah；Fourth stage is combined to form by glucose and tyrosol Rhodioside.In this 4 stages, the l stages are the metabolic steps common to many higher plants, are the very clearly the 2nd The reaction mechanism in stage and the 4th stage has also been explored clear；On the 3rd stage, from sieve's Ah acid to the biosynthesis of tyrosol Approach, it has been proposed that 3 possible approach, i.e. phenylalanine metabolic pathways approach, tyrosine decarboxylation metabolic pathway and tyrosine turn Ammonia metabolism approach.
Also having studied in recent years confirms that tyrosine can pass through the steps such as decarboxylation, reduction and be directly translated into tyrosol.It is and red Red-spotted stonecrop glycosides biosynthesis final step reaction mechanism also clearly, the uridine diphosphoglucose based transferase in plant (UDP-glucosyltransferase, UDPGT, UGTs) is catalyzed using uridine diphosphoglucose (UDPG) and tyrosol as substrate Synthesize rhodioside.Therefore, by using synthetic biology technology, brand-new biosynthesis is built in engineered microbes and is led to Road, realize the de novo formation of rhodioside and the like.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided a kind of preparation side of rhodioside and the like Method.
Technical scheme is summarized as follows：
A kind of preparation method of rhodioside and the like, comprises the following steps：By recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strains are seeded in LB culture mediums respectively, in OD600When reaching 1.0, it is transferred to jointly containing rhodiola plant The culture medium of protease hydrolysate or the M9 culture mediums containing glucose in, fermented, when concentration of glucose is less than 0.5g/L, Glucose is added to 2-10g/L, continuing fermentation, obtains rhodioside and the like.
Recombinant bacterium SyBE-218014 is obtained with following methods：
(1) artificial fully synthetic ketone group decarboxylase gene synkdc, the nucleotides sequence of the ketone group decarboxylase gene synkdc Row are as shown in SEQ ID No.03；
(2) clone obtains t7 rna polymerase gene from e. coli bl21 (DE3) bacterium, passes through λ Red homologous recombination skills Art, which is incorporated on the strain chromosome of SyBE-002447 chassis, obtains SyBE-002447 (DE3) bacterial strain；
(3) by synkdc genes, it is incorporated into by λ Red homologous recombination techniques in Escherichia coli SyBE-002447 (DE3), Gene feaB is knocked out simultaneously, obtains SyBE-218014 bacterial strains.
Recombinant bacterium SyBE-218021 is obtained with following methods：
(1) artificial fully synthetic glycosyltransferase gene synyjic, the glycosyltransferase gene synyjic nucleotides sequences Row are as shown in SEQ ID No.04；
(2) clone to obtain pgm genes and galU genes from e. coli bl21 (DE3) strain, by Overlap extension PCR, Pgm-galU fragments are built, is incorporated on the strain chromosome of BL21 (DE3) chassis by λ Red homologous recombination techniques, knocked out simultaneously UhsA genes, obtain SyBE-218020 bacterial strains；
(3) by synyjic genes, it is incorporated into by λ Red homologous recombination techniques in Escherichia coli SyBE-218020 bacterial strains, Gene feaB is knocked out simultaneously, obtains SyBE-218021.
4. according to the method for claim 1, it is characterized in that the recombinant bacterium SyBE-218014 and SyBE-218021 Bacterial strain condition of culture in LB culture mediums is：30-37 DEG C, 200-250rpm, concussion and cultivate.
The culture medium of protease hydrolysate containing rhodiola plant, it is made of following methods：25g rhodiola root powder is weighed, is added Enter 100mg cellulases, 100mg pectases and 100mg hemicellulases, add distilled water and be settled to 500mL, 50 DEG C of enzymolysis 14h, filter, final concentration of 2g/L yeast extracts are added in obtained filtrate.
M9 culture mediums containing glucose, are made of following methods：Weigh 0.5g NaCl, 1.0g NH4Cl、3.0g KH2PO4、17.1g Na2HPO4·12H2O and 0.25g dusty yeasts, addition distilled water are settled to 1L；In 121 DEG C of 0.1Mpa pressure Lower sterilizing 20min；The final concentration of 5mM MgSO of film were added after having sterilized4、0.1mM CaCl2And sterilizing is final concentration of 5g/L glucose.
Advantages of the present invention：
Rhodioside can be used for many treatments such as cancer, the nervous system disease, cell ageing, hence in so that rhodiola root Glycosides is widely used in the industrial productions such as cosmetics, food, health products, medicine.The present invention uses engineering colon bacillus, grape Sugar is carbon source, and fermenting and producing rhodioside and the like is carried out using free induced expression and integrant expression Different Strategies.This Invention can solve the source problem that comes of rhodioside and the like, while reduce production cost to greatest extent, be advantageous to work Industry metaplasia is produced.
Brief description of the drawings
Fig. 1 is the synthetic route of rhodioside and the like (benzyl carbinol-β-D- glucopyranosides).
Fig. 2 is the HPLC collection of illustrative plates of rhodioside prepared by embodiment 6 and the like checking.
Fig. 3 is the HPLC collection of illustrative plates of rhodioside prepared by embodiment 7 and the like checking.
Embodiment
Utilize ketone group decarboxylase gene kdc and lichens gemma in Pichia yeast (Pichia pastoris GS115) Glycosyltransferase gene yjic in bacillus (Bacillus licheniformis), is realized with Portugal in engineering colon bacillus Grape sugar synthesizes rhodioside and the like for biomass carbon source, makes cost minimization.
Original chassis Escherichia coli are the bacterial strains of high yield tyrosine, entitled SyBE-002447, and Classification And Nomenclature large intestine angstrom is uncommon Salmonella (Escherichia coli) is now in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preservation Center numbering of registering on the books is CGMCC No.7962.The preservation time is on July 22nd, 2013, and address is Chaoyang District, Beijing City north No. 3 Institute of Microorganism, Academia Sinica of institute of occasion West Road 1, postcode 100101.
Coli strain E.coli BL21 (DE3) used in the present invention and E.coli DH5 α are bought from the full Shi Jinsheng in Beijing Thing Technology Co., Ltd..
PKD46 plasmids used in the present invention and pCP20 plasmids are bought from general such as spit of fland biotechnology (Beijing) Co., Ltd.
LB culture mediums form：10g/L NaCl, 10g/L peptones and 5g/L dusty yeasts, surplus are water, 0.1Mpa pressure Sterilize 20min at 121 DEG C.
The culture medium of protease hydrolysate containing rhodiola plant, it is made of following methods：25g rhodiola root powder is weighed, is added Enter 100mg cellulases, 100mg pectases and 100mg hemicellulases, add distilled water and be settled to 500mL, 50 DEG C of enzymolysis 14h, filter, final concentration of 2g/L yeast extracts are added in obtained filtrate.
M9 culture mediums containing glucose, are made of following methods：Weigh 0.5g NaCl, 1.0g NH4Cl、3.0g KH2PO4、17.1g Na2HPO4·12H2O and 0.25g dusty yeasts, addition distilled water are settled to 1L；In 121 DEG C of 0.1Mpa pressure Lower sterilizing 20min；The final concentration of 5mM MgSO of film were added after having sterilized4、0.1mM CaCl2And sterilizing is final concentration of 5g/L glucose.
With reference to specific embodiment, the present invention is further illustrated.
The biosynthesis pathway for the rhodioside that the present invention designs belongs to shikimic acid pathway, and detailed route of synthesis is as follows： Using glucose or other biological matter as carbon source, Single-chip microcomputer is synthesized by shikimic acid pathway.Single-chip microcomputer 4- phenylac epsilontaldshydes are synthesized under ketone group decarboxylase (synkdc) catalytic action, 4- phenylac epsilontaldshydes are in Escherichia coli Inner sources second The lower generation 4- hydroxylphenylethyl alcohols (tyrosol) of alcohol dehydrogenase (adh) catalysis, under glycosyl transferase (synyjic) catalysis, tyrosol Synthesis rhodioside and the like (benzyl carbinol-β-D- glucopyranosides), biosynthesis are combined with UDPG donor Approach schematic diagram is as shown in Figure 1.
Embodiment 1
Ketone group decarboxylase gene synkdc and glycosyltransferase gene synyjic designs
It is preferred that Pichia pastoris GS115 ketone group decarboxylase gene, bacillus licheniformis glycosyltransferase gene, its amino acid sequence Row are respectively as shown in SEQ ID No.01 in sequence table and SEQ ID No.02.Use the online codon optimization softwares of JCAT (http：//www.jcat.de) combine the online codon optimization instrument (http of OPTIMIZER://genomes.urv.es/ OPTIMIZER/), the Preference of codon is optimized with Escherichia coli, designs total length synkdc genes and synyjic bases Cause, respectively as shown in sequence table SEQ ID No.03 and SEQ ID No.04.
Embodiment 2
λ-red methods of homologous recombination is integrated on the t7 rna polymerase SyBE-002447 chromosomes of cup fungi strain on earth.
The chassis strain construction process integrated on the t7 rna polymerase SyBE-002447 chromosomes of cup fungi strain on earth walks in detail It is rapid as follows：
1st, primer T7-RNA F and T7-RNA R sequences are designed respectively such as sequence table SEQ ID NO.06, SEQ ID NO.07 Clone obtains t7 rna polymerase gene from e. coli bl21 (DE3) bacterium, and the t7 rna polymerase gene order is as such as sequence List SEQ ID NO.05.
2nd, primer T7F and T7R, T7-Chl F and T7-Chl R sequences are respectively such as sequence table SEQ ID NO.08, SEQ ID NO.09, SEQ ID NO.10, SEQ ID NO.11, Overlap extension PCR prepare the t7 rna polymerase piece with chlorampenicol resistant Section.
3rd, pKD46 plasmids are imported strain SyBE-002447, SyBE-002447/pKD46 is obtained, by the strain of activation SyBE-002447/pKD46, it is inoculated in 10ml LB fluid nutrient mediums, 30 DEG C, 200rpm, cultivates to OD600For 0.4-0.6. Final concentration of 10mM L-arabinoses are added, continue to cultivate 3h.4000rpm centrifuges 8min at 4 DEG C, collects cell.Abandon supernatant, 10% glycerine for adding ice precooling washs cell 2 times, is prepared into electricity and turns competent cell.The μ L of resistance fragments 2 are taken, are added to 100 μ L electricity turns in competent cell, and gently rotation mixes.Feed the mixture into 2mm ice precooling electric shock cup, 2.5KV electric shocks 4- 6ms.Then 1mL LB culture mediums are added, the flat board of corresponding resistance are coated with after 37 DEG C of recovery 3h, 42 DEG C are incubated overnight.
4th, picking PCR verifies correct single bacterium colony, and chlorampenicol resistant flat board passes on 3 times at 42 DEG C, amicillin resistance Flat board checking pKD46 missings, 37 DEG C of cultures preserve, obtain t7 rna polymerase fragment being incorporated into chassis bacterial strain SyBE-002447 Bacterial strain on chromosome.
5th, using the recombinant bacterium obtained in step 2, competent cell is prepared, same method electricity conversion plasmid pCP20,30 DEG C It is incubated overnight.Picking single bacterium colony, non-resistant plate streaking, 42 DEG C pass on 3 times, screen chloramphenicol and amicillin resistance missing Bacterial strain, 37 DEG C culture preserve, the optimization chassis bacterial strain SyBE-002447 (DE3) for seamless integration of succeeding.
Embodiment 3
λ-red methods of homologous recombination knocks out feaB genes and integrates synkdc genes cup fungi strain on earth SyBE-002447 simultaneously (DE3) on chromosome.
Knock out feaB genes while integrate the bottom on the cup fungi strain on earth of synkdc genes SyBE-002447 (DE3) chromosome Cup fungi strain building process detailed step is as follows：
1st, primer KdcF and Kdc R, Kan F and Kan R sequence are respectively such as sequence table SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, Overlap extension PCR prepare the Kdc-Kan fragments with kan resistances.
2nd, pKD46 plasmids are imported strain SyBE-002447 (DE3), SyBE-002447 (DE3)/pKD46 is obtained, by work Strain SyBE-002447 (DE3)/pKD46 of change, is inoculated in 10ml LB fluid nutrient mediums, 30 DEG C, 200rpm, and culture is extremely OD600For 0.4-0.6.Final concentration of 10mM L-arabinoses are added, continue to cultivate 3h, 4000rpm centrifuges 8min at 4 DEG C, receives Collect cell.Supernatant is abandoned, 10% glycerine for adding ice precooling washs cell 2 times, is prepared into electricity and turns competent cell.Take resistance fragments 7.5 μ L, it is added to 100 μ L electricity and turns in competent cell, gently rotation mixes.Feed the mixture into 2mm ice precooling electric shock cup In, 2.5KV electric shocks 4-6ms.Then 1mL LB culture mediums are added, the flat board of corresponding resistance, 42 DEG C of mistakes are coated with after 37 DEG C of recovery 2h Night cultivates.
3rd, picking PCR verifies correct single bacterium colony, and kalamycin resistance flat board passes on 3 times at 42 DEG C, ammonia benzyl kanamycins Resistant panel checking pKD46 missings, 37 DEG C of cultures preserve, obtain and knock out feaB genes simultaneously by synkdc gene integrations to chassis Bacterial strain on bacterial strain SyBE-002447 (DE3) chromosome.
4th, using the recombinant bacterium obtained in step 2, competent cell is prepared, same method electricity conversion plasmid pCP20,30 DEG C It is incubated overnight.Picking single bacterium colony, non-resistant plate streaking, 42 DEG C pass on 3 times, screen kanamycins and amicillin resistance lacks The bacterial strain of mistake, 37 DEG C of cultures preserve, the optimization chassis bacterial strain SyBE-218014 for seamless integration of succeeding.
Embodiment 4
Clone to obtain pgm genes and galU genes from e. coli bl21 (DE3) strain, pass through Overlap extension PCR, structure Pgm-galU, which is knocked out, integrates fragment, is incorporated into by λ Red homologous recombination techniques on the strain chromosome of BL21 (DE3) chassis, simultaneously UhsA genes are knocked out, obtain SyBE-218020 bacterial strains；
Knock out ushA genes while integrate the chassis bacterial strain on the cup fungi strain on earth of pgm-galU genes BL21 (DE3) chromosome Building process detailed step is as follows：
1st, primer Pgm F and PgmR, GalUF and GalUR, T7-Chl F and T7-Chl R sequences are respectively such as sequence table SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, weight Folded extension PCR obtains Pgm-GalU fragments, and Overlap extension PCR prepares the Pgm-GalU-Chl fragments with chlorampenicol resistant.
2nd, pKD46 plasmids are imported in strain Escherichia coli BL21 (DE3), BL21 (DE3)/pKD46 is obtained, by activation Strain BL21 (DE3)/pKD46, it is inoculated in 10ml LB fluid nutrient mediums, 30 DEG C, 200rpm, cultivates to OD600For 0.4- 0.6.Final concentration of 10mM L-arabinoses are added, continue to cultivate 3h.4000rpm centrifuges 8min at 4 DEG C, collects cell.Abandon Clearly, 10% glycerine for adding ice precooling washs cell 2 times, is prepared into electricity and turns competent cell.Take Pgm-GalU-Chl resistance pieces 2 μ L of section, are added to 100 μ L electricity and turn in competent cell, and gently rotation mixes.Feed the mixture into 2mm ice precooling electric shock cup In, 2.5KV electric shocks 4-6ms.Then 1mL LB culture mediums are added, the flat board of corresponding resistance, 42 DEG C of mistakes are coated with after 37 DEG C of recovery 3h Night cultivates.
3rd, picking PCR verifies correct single bacterium colony, and chlorampenicol resistant flat board passes on 3 times at 42 DEG C, ammonia benzyl chloride chloramphenicol resistance Flat board checking pKD46 missings, 37 DEG C of cultures preserve, and acquisition knocks out ushA genes and integrates the cup fungi strain on earth of pgm-galU genes simultaneously Bacterial strain on BL21 (DE3) chromosome.
4th, using the recombinant bacterium obtained in step 3, competent cell is prepared, same method electricity conversion plasmid pCP20,30 DEG C It is incubated overnight.Picking single bacterium colony, non-resistant plate streaking, 42 DEG C pass on 3 times, screen chloramphenicol and amicillin resistance missing Bacterial strain, 37 DEG C culture preserve, the optimization chassis bacterial strain SyBE-218020 for seamless integration of succeeding.
Embodiment 5
λ-red methods of homologous recombination knocks out feaB genes and integrates synyjic genes to Escherichia coli chassis bacterial strain simultaneously On SyBE-218020 chromosomes.
Knock out feaB genes and integrate synyjic genes to Escherichia coli chassis bacterial strain SyBE-218020 chromosomes simultaneously Strain construction process detailed step it is as follows：
1st, primer YjicF and YjicR, Yjic-Kan F and Yjic-Kan R sequences are respectively such as sequence table SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, Overlap extension PCR are prepared with kan resistances Yjic-Kan fragments.
2nd, pKD46 plasmids are imported in Escherichia coli SyBE-218020, SyBE-218020/pKD46 is obtained, by activation Strain SyBE-218020/pKD46, it is inoculated in 10ml LB fluid nutrient mediums, 30 DEG C, 200rpm, cultivates to OD600For 0.4- 0.6.Final concentration of 10mM L-arabinoses are added, continue to cultivate 3h, 4000rpm centrifuges 8min at 4 DEG C, collects cell.Abandon Clearly, 10% glycerine for adding ice precooling washs cell 2 times, is prepared into electricity and turns competent cell.The μ L of Yjic-Kan fragments 7.5 are taken, It is added to 100 μ L electricity to turn in competent cell, gently rotation mixes.Feed the mixture into 2mm ice precooling electric shock cup, 2.5KV electric shocks 4-6ms.Then 1mL LB culture mediums are added, the flat board of corresponding resistance, 42 DEG C of trainings overnight are coated with after 37 DEG C of recovery 2h Support.
3rd, picking PCR verifies correct single bacterium colony, and kalamycin resistance flat board passes on 3 times at 42 DEG C, ammonia benzyl kanamycins Resistant panel checking pKD46 missings, 37 DEG C of cultures preserve, obtain and integrate yjic genes cup fungi strain on earth SyBE-218020 dyeing Bacterial strain on body.
4th, using the recombinant bacterium obtained in step 3, competent cell is prepared, same method electricity conversion plasmid pCP20,30 DEG C It is incubated overnight.Picking single bacterium colony, non-resistant plate streaking, 42 DEG C pass on 3 times, screen kanamycins and amicillin resistance lacks The bacterial strain of mistake, 37 DEG C of cultures preserve, the chassis bacterial strain SyBE-218021 for seamless integration of succeeding.
Embodiment 6
Recombinant bacterium SyBE-218014 and SyBE-218021 ferment and detection
Recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strain are inoculated into 5ml LB culture mediums respectively and are incubated overnight. Then the bacterium solution being incubated overnight is transferred into the 250ml shaking flasks containing 50ml LB culture mediums respectively, initial OD600About 0.1,37 DEG C, 220rpm concussion and cultivates, in OD600When reaching 1.0, it is transferred to jointly in the M9 culture mediums containing glucose, 30 DEG C, 250rpm is fermented, and when concentration of glucose is less than 0.5g/L, adds glucose to 5g/L, continuing fermentation culture 72h.
Different time points during the fermentation, zymotic fluid 1ml is taken, then 12000r/min is centrifuged 10 minutes, takes supernatant, is used High performance liquid chromatography (HPLC) system detectio is carried out after 0.22 μm of filtering with microporous membrane.Chromatographic condition is as follows：C18(4.6× 250mm) chromatographic column；Mobile phase is the formic acid of -80% ultra-pure water solution of 20% methanol -0.1%；Flow velocity 1mL/min；The μ of sample size 20 L；Column temperature room temperature；UV-detector, Detection wavelength 280nm.
It is the HPLC figures that SyBE-218014 and SyBE-218021 train that product is verified in thing fermentation broth sample altogether as shown in Figure 2 Spectrum.
SyBE-218014 and SyBE-218021 trains thing and fermented 72 hours altogether, generation rhodioside and the like benzene second Alcohol-β-D- glucopyranosides.
Embodiment 7
Recombinant bacterium SyBE-218014 and SyBE-218021 ferment and detection
Recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strain are inoculated into 5ml LB culture mediums respectively and are incubated overnight. Then the bacterium solution being incubated overnight is transferred and entered in the 250ml shaking flasks containing 50ml LB culture mediums, initial OD600About 0.1,37 DEG C, 220rpm concussion and cultivates, in OD600When reaching 1.0, it is transferred to jointly in the culture medium containing rhodiola plant protease hydrolysate, 30 DEG C, 250rpm is fermented, and when concentration of glucose is less than 0.5g/L, adds glucose to 5g/L, continuing fermentation culture 72h。
It is the HPLC figures that SyBE-218014 and SyBE-218021 train that product is verified in thing fermentation broth sample altogether as shown in Figure 3 Spectrum.
SyBE-218014 and SyBE-218021 trains thing and fermented 72 hours altogether, generation rhodioside and the like benzene second Alcohol-β-D- glucopyranosides.
Embodiment 8
Recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strain are inoculated into 5ml LB culture mediums respectively and are incubated overnight. Then the bacterium solution being incubated overnight is transferred into the 250ml shaking flasks containing 50ml LB culture mediums respectively, initial OD600About 0.1,30 DEG C, 250rpm concussion and cultivates, in OD600When reaching 1.0, it is transferred to jointly in the M9 culture mediums containing glucose, 30 DEG C, 220rpm is fermented, and when concentration of glucose is less than 0.5g/L, adds glucose to 2g/L, continuing fermentation culture 72h.
Testing result is similar to the result of embodiment 6.
Embodiment 9
Recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strain are inoculated into 5ml LB culture mediums respectively and are incubated overnight. Then the bacterium solution being incubated overnight is transferred into the 250ml shaking flasks containing 50ml LB culture mediums respectively, initial OD600About 0.1,35 DEG C, 200rpm concussion and cultivates, in OD600When reaching 1.0, it is transferred to jointly in the M9 culture mediums containing glucose, 35 DEG C, 200rpm is fermented, and when concentration of glucose is less than 0.5g/L, adds glucose to 10g/L, continuing fermentation culture 72h.
Testing result is similar to the result of embodiment 6.
Embodiment 10
Recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strain are inoculated into 5ml LB culture mediums respectively and are incubated overnight. Then the bacterium solution being incubated overnight is transferred into the 250ml shaking flasks containing 50ml LB culture mediums respectively, initial OD600About 0.1,30 DEG C, 250rpm concussion and cultivates, in OD600When reaching 1.0, the protease hydrolysate containing rhodiola plant is transferred to jointly In culture medium, 30 DEG C, 220rpm is fermented, and when concentration of glucose is less than 0.5g/L, adds glucose to 2g/L, lasting hair Ferment culture 72h.
Testing result is similar to the result of embodiment 7.
Embodiment 11
Recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strain are inoculated into 5ml LB culture mediums respectively and are incubated overnight. Then the bacterium solution being incubated overnight is transferred into the 250ml shaking flasks containing 50ml LB culture mediums respectively, initial OD600About 0.1,35 DEG C, 200rpm concussion and cultivates, in OD600When reaching 1.0, the protease hydrolysate containing rhodiola plant is transferred to jointly In culture medium, 35 DEG C, 200rpm is fermented, and when concentration of glucose is less than 0.5g/L, is added glucose to 10g/L, is continued Fermented and cultured 72h.
Testing result is similar to the result of embodiment 7.
Exemplary description has been done to the present invention above, it should explanation, in the situation for the core for not departing from the present invention Under, any simple deformation, modification or other skilled in the art can not spend the equivalent substitution of creative work equal Fall into protection scope of the present invention.

Claims (6)

  1. A kind of 1. preparation method of rhodioside and the like, it is characterized in that comprising the following steps：By recombinant bacterium SyBE- 218014 and SyBE-218021 bacterial strains are seeded in LB culture mediums respectively, in OD600When reaching 1.0, it is transferred to jointly containing red In the culture medium of the protease hydrolysate of red-spotted stonecrop plant or M9 culture mediums containing glucose, fermented, when concentration of glucose is less than During 0.5g/L, glucose is added to 2-10g/L, continuing fermentation, obtains rhodioside and the like.
  2. 2. according to the method for claim 1, it is characterized in that the recombinant bacterium SyBE-218014 is obtained with following methods：
    (1) artificial fully synthetic ketone group decarboxylase gene synkdc, the nucleotide sequence of the ketone group decarboxylase gene synkdc is such as Shown in SEQ ID No.03；
    (2) clone obtains t7 rna polymerase gene from e. coli bl21 (DE3) bacterium, whole by λ Red homologous recombination techniques Close and SyBE-002447 (DE3) bacterial strain is obtained on the strain chromosome of SyBE-002447 chassis；
    (3) by synkdc genes, it is incorporated into by λ Red homologous recombination techniques in Escherichia coli SyBE-002447 (DE3), simultaneously Gene feaB is knocked out, obtains SyBE-218014 bacterial strains.
  3. 3. according to the method for claim 1, it is characterized in that the recombinant bacterium SyBE-218021 is obtained with following methods：
    (1) artificial fully synthetic glycosyltransferase gene synyjic, the glycosyltransferase gene synyjic nucleotide sequences are such as Shown in SEQ ID No.04；
    (2) clone to obtain pgm genes and galU genes from e. coli bl21 (DE3) strain, pass through Overlap extension PCR, structure Pgm-galU fragments, it is incorporated into by λ Red homologous recombination techniques on the strain chromosome of BL21 (DE3) chassis, while knocks out uhsA Gene, obtain SyBE-218020 bacterial strains；
    (3) by synyjic genes, it is incorporated into by λ Red homologous recombination techniques in Escherichia coli SyBE-218020 bacterial strains, simultaneously Gene feaB is knocked out, obtains SyBE-218021.
  4. 4. according to the method for claim 1, it is characterized in that the recombinant bacterium SyBE-218014 and SyBE-218021 bacterial strains Condition of culture is in LB culture mediums：30-37 DEG C, 200-250rpm, concussion and cultivate.
  5. 5. according to the method for claim 1, it is characterized in that the culture medium of the protease hydrolysate containing rhodiola plant, is used Following methods are made：25g rhodiola root powder is weighed, adds 100mg cellulases, 100mg pectases and 100mg hemicelluloses Enzyme, add distilled water and be settled to 500mL, 50 DEG C of enzymolysis 14h, filter, final concentration of 2g/L yeast extracts are added in obtained filtrate.
  6. 6. the method according to claim 11, it is characterized in that the M9 culture mediums containing glucose, with following method systems Into：Weigh 0.5g NaCl, 1.0g NH4Cl、3.0g KH2PO4、17.1g Na2HPO4·12H2O and 0.25g dusty yeasts, addition Distilled water is settled to 1L；Sterilize 20min at 121 DEG C of 0.1Mpa pressure；The final concentration of 5mM of film was added after having sterilized MgSO4、0.1mM CaCl2And the final concentration of 5g/L glucose of sterilizing.
CN201610396521.2A 2016-06-03 2016-06-03 A kind of preparation method of rhodioside and the like Pending CN107460220A (en)

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CN108998460A (en) * 2018-07-10 2018-12-14 天津大学 A kind of biological production of rhodioside
CN109303786A (en) * 2018-11-27 2019-02-05 山东省药学科学院 A kind of composition of resist oxygen lack and its application
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WO2020001166A1 (en) * 2018-06-27 2020-01-02 上海和黄药业有限公司 Glycoside compound and preparation method therefor, composition, application, and intermediate
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CN108998460A (en) * 2018-07-10 2018-12-14 天津大学 A kind of biological production of rhodioside
CN108998460B (en) * 2018-07-10 2021-10-15 天津大学 Biological production method of salidroside
CN109303786A (en) * 2018-11-27 2019-02-05 山东省药学科学院 A kind of composition of resist oxygen lack and its application

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Application publication date: 20171212