CN105886415B - A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid - Google Patents
A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid Download PDFInfo
- Publication number
- CN105886415B CN105886415B CN201610318425.6A CN201610318425A CN105886415B CN 105886415 B CN105886415 B CN 105886415B CN 201610318425 A CN201610318425 A CN 201610318425A CN 105886415 B CN105886415 B CN 105886415B
- Authority
- CN
- China
- Prior art keywords
- gene
- saccharomyces cerevisiae
- lup
- bplo
- engineered yeast
- 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.)
- Active
Links
Classifications
-
- 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
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- 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
- C12P33/00—Preparation of steroids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
- C12Y504/99041—Lupeol synthase (5.4.99.41)
-
- 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
- C12N2800/102—Plasmid DNA for yeast
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Botany (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of saccharomyces cerevisiae engineered yeasts, it is characterized in that, the saccharomyces cerevisiae engineered yeast is the expression cassette of LUP gene expression frame and BPLO gene to be incorporated in genome, the saccharomyces cerevisiae engineered yeast has preferably been knocked GAL80 gene based on saccharomyces cerevisiae strain WAT11.The invention further relates to the methods for constructing the Yeast engineering bacteria, and use the method for Yeast engineering bacteria production betulic acid.Through the invention, saccharomyces cerevisiae can be used, betulic acid is produced by way of microbial fermentation, easy to operate, yield is high, and it is with short production cycle, it takes up little area.
Description
Technical field
The present invention relates to the genetic modifications of microorganism, more specifically it relates to a kind of saccharomyces cerevisiae for producing betulic acid
(Saccharomyces cerevisiae) engineering bacteria and its construction method.
Background technique
Betulic acid (Betulinic acid, BA), also known as betulinic acid, are a kind of pentacyclic triterpenoids.Five rings three
Terpenoid is a kind of very important Secondary metabolites, and usually there is this kind of compound very extensive pharmacology to make
With and bioactivity.This kind of compound is widely present in plant kingdom, and the number of types for including is more, main five rings three
Terpene compound has oleanane type, Ursane, lupinane type and friedelane type several types, and betulic acid is lupin
The Typical Representative of alkane type five rings class triterpenoid.
In recent years a large number of studies show that, betulic acid has significant pharmacological action and bioactivity, is mainly reflected in
The effect of antitumor, AntiHIV1 RT activity, anti-inflammatory, antibacterial and anti-malarial etc..The above bioactivity that betulic acid is shown is (special
It is not the inhibiting effect of selective killing effect and anti-HIV-1 the type infection to human melanoma cells) plus its effect
The features such as mechanism is special and higher safety, becomes natural products very with researching value, has very wide
General application and development prospect.The main source of betulic acid is the direct extraction and purification from the bark of silver birch at present, is existed
Serious waste of resources the problem of.By the method for bioengineering, this kind of compound is synthesized as biopack using microorganism and is had
It holds out broad prospects.
Saccharomyces cerevisiae is the eucaryote that first genome completes sequencing, and by as eukaryon model organism, expression is adjusted
Control mechanism is relatively more clear and has the characteristics that genetic manipulation is simple, reproduction speed is fast, safe and reliable, is often used as extensive life
The eukaryotic expression bacterial strain of object fermentation.Wine brewing yeast strain WAT11 is the common Wine brewing yeast strain in laboratory, is in wild type strain
It is that arabidopsis reductase gene ATR1 (Urban P, Mignotte C, Kazmaier M, et are incorporated in W303
al.Cloning,yeast expression,and characterization of the coupling of two
distantly related Arabidopsis thaliana NADPH-cytochrome P450reductases with
P450CYP73A5[J].Journal of Biological Chemistry,1997,272(31):19176-19186)。
Betulic acid is by triterpenes common precursor substance 2,3- oxidosqualene by lupeol synthase and C-28
The effect of the P450 molecule of the single oxygenation in position generates.WAT11 itself can generate the precursor substance 2 of triterpene substance synthesis, 3- oxidation
Squalene, and with reductase needed for P450 molecular action.Therefore, inventor's expectation is raw by molecule based on WAT11
Object learns to do section and genetic engineering technology, constructs a kind of production betulic acid saccharomyces cerevisiae engineered yeast.
Summary of the invention
In order to solve the above technical problems, the present invention provides a kind of saccharomyces cerevisiae engineered yeast, the saccharomyces cerevisiae engineered yeasts
To incorporate LUP gene expression frame and BPLO gene expression frame in genome based on saccharomyces cerevisiae strain WAT11, wherein
The LUP gene expression frame is operably coupled to the first inducible promoters by LUP gene and is formed, the sequence of the LUP gene
As shown in SEQ ID NO:1, the BPLO gene expression frame is operably coupled to the second inducible promoters and shape by BPLO gene
At the sequence of the BPLO gene is as shown in SEQ ID NO:2.Here effective connection refers to that gene is located at the downstream of promoter
And it is controlled and is transcribed by promoter.
Preferably, first inducible promoters are GAL1 promoter, and sequence is as shown in SEQ ID NO:3.
Preferably, second inducible promoters are GAL10 promoter, and sequence is as shown in SEQ ID NO:4.
Preferably, the GAL80 gene of the engineering bacteria is partly or entirely knocked out, the sequence such as SEQ of the GAL80 gene
Shown in ID NO:16.
The present invention also provides a kind of construction methods for constructing saccharomyces cerevisiae engineered yeast, comprising the following steps:
1) LUP gene is inserted into the downstream of the GAL1 promoter of PESC-Trp carrier along the direction of GAL1 promoter,
It is in the LUP gene under the control of the GAL1 promoter, and BPLO gene is inserted along the direction of GAL10 promoter
The downstream for entering the GAL10 promoter to the PESC-Trp carrier makes the BPLO gene be in the control of the GAL10 promoter
Under system, thus obtain being integrated with the plasmid PESC-TRP-LUP-BPLO of LUP gene expression frame and BPLO gene expression frame, wherein
For the sequence of the LUP gene as shown in SEQ ID NO:1, the sequence of the GAL1 promoter is described as shown in SEQ ID NO:3
The sequence of BPLO gene is as shown in SEQ ID NO:2, and the sequence of the GAL10 promoter is as shown in SEQ ID NO:4；
2) by the method for rite-directed mutagenesis by GATATC in LUP gene described in the plasmid PESC-TRP-LUP-BPLO
In cytimidine sport thymidine；
3) the plasmid PESC-TRP-LUP-BPLO of the mutation obtained in EcoRV digestion step 2), makes its linearisation, so
The plasmid PESC-TRP-LUP-BPLO of the linearisation is transferred in saccharomyces cerevisiae WAT11 afterwards, by being sent out automatically in saccharomycete body
Raw homologous recombination has lacked the LUP base because expression cassette and the BPLO gene expression frame are integrated into the base of the saccharomycete
Because in group；
It 4) will be by the GAL80 gene 5 ' region sequence+loxP-KanMX-loxP+GAL80 gene 3 ' that is arranged with 5 ' to 3 ' directions
The GAL80 gene knockout frame of region sequence composition, which is converted, incorporates the LUP gene expression into genome obtained in step 3)
In the saccharomyces cerevisiae of frame and the BPLO gene expression frame, the saccharomyces cerevisiae is made by the homologous recombination occurred automatically into the cell
In the corresponding position of GAL80 gene replaced by the GAL80 gene knockout frame, the sequence of the GAL80 gene knockout frame is such as
Shown in SEQ ID NO:5；
5) in the saccharomyces cerevisiae for being knocked the GAL80 gene that Cre recombination enzymatic conversion to step 4) obtains, make the segment
It recombinates again, loses the loxP-KanMX-loxP segment, to obtain having the LUP gene expression frame and described
BPLO gene expression frame and the saccharomyces cerevisiae engineered yeast for having lacked the 80 gene encoder block of Gal.
The present invention also provides a kind of methods for producing betulic acid, comprising the following steps:
1) above-mentioned saccharomyces cerevisiae engineered yeast is cultivated to logarithmic phase；
2) induction step 1) saccharomyces cerevisiae engineered yeast cultivated；
3) the saccharomyces cerevisiae engineered yeast culture being induced from step 2) extracts betulic acid.
Preferably, saccharomyces cerevisiae engineering described in the SD-Trp- fluid nutrient medium culture added with glucose is used in step 1)
Bacterium.
Preferably, saccharomyces cerevisiae engineering described in the SD-Trp- fluid nutrient medium culture added with galactolipin is used in step 2)
Bacterium is to induce the saccharomyces cerevisiae engineered yeast to generate betulic acid.
Preferably, it the described method comprises the following steps:
1) saccharomyces cerevisiae engineered yeast is inoculated in the SD-Trp- fluid nutrient medium added with 2% glucose, is placed in 30
DEG C, 250rpm/min is cultivated 16 hours；
2) thallus is collected, with sterile water washing, is resuspended in the SD-Trp- culture medium added with 2% galactolipin to starting
OD600Value is 0.8, is placed in 30 DEG C, Fiber differentiation 7 days under 250rpm/min；
3) yeast culture obtained in step 2) is adjusted to PH2.0 with 2M HCl, with isometric ethyl acetate extracting 3
It is secondary, merge this 3 ethyl acetate phases, dries up；
4) it will be redissolved after the middle drying of step 3) with a small amount of ethyl acetate, and be transferred to 2ml centrifuge tube, dry up to get white birch rouge is arrived
Acid.
Detailed description of the invention
Fig. 1 is the map of plasmid PESC-TRP-LUP-BPLO；
Fig. 2 is the map that yeast integrates YIPlac204-LUP-BPLO；
Fig. 3 is the electrophoresis photographs of PCR product, wherein swimming lane 1 and 3 be respectively with saccharomyces cerevisiae strain WAT11 (swimming lane 3) and
WAT11-LUP-BPLO (swimming lane 1) is template, expands the PCR product electrophoresis photographs of LUP gene, and swimming lane 2 and 4 is to make wine respectively
Yeast strainss WAT11 (swimming lane 4) and WAT11-LUP-BPLO (swimming lane 2) is template, and the PCR product electrophoresis of amplification BPLO gene shines
Piece；
Fig. 4 is the electrophoresis photographs of PCR product, and the PCR is with saccharomyces cerevisiae strain W80 (swimming lane 1) and WAT11-LUP-BPLO
The genome of (swimming lane 2) is template, is carried out using primer P7/P10；
Fig. 5 is the mass spectral analysis figure of saccharomyces cerevisiae strain WAT11-LUP-BPLO betulic acid produced, wherein arrow
The peak marked is betulic acid.
Fig. 6 is that display is produced using the betulic acid of saccharomyces cerevisiae strain W80 and saccharomyces cerevisiae strain WAT11-LUP-BPLO
The histogram of amount.
Specific embodiment
Principles and features of the present invention are described with reference to embodiments, the given examples are served only to explain the present invention,
It is not intended to limit the scope of the present invention.
Embodiment 1 produces the building of the saccharomyces cerevisiae strain WAT11-LUP-BPLO of betulic acid
1, the building of yeast integration plasmid YIPlac204-LUP-BPLO
By the LUP gene of arabidopsis (Arabidopsis thaliana) and come from white birch wood (Betula
Platyphylla BPLO gene) is connected under GAL1 the and GAL10 promoter of same PESC-Trp carrier, is obtained
Plasmid PESC-TRP-LUP-BPLO (Fig. 1).Because of the needs of restriction enzyme site, to one of the EcoRV restriction enzyme site in LUP gene
Base carries out point mutation (cytimidine in GATATC is sported thymidine).The specific method is as follows: with plasmid PESC-TRP-
LUP-BPLO is template, carries out first round PCR using two couples of primers P1/P4 and P2/P3 respectively.Then by two groups of the first round
The product mixed in equal amounts of PCR carries out the second wheel PCR as template, with primer pair P3/P4.Second wheel product is carried out agarose to coagulate
Gel electrophoresis recycles the segment of purpose band size, is connected to T and is loaded into capable sequencing.Sequencing is correctly cloned and uses PstI and XbaI
Segment comprising LUP expression casette and BPLO expression casette is connected to yeast integration plasmid YIplac204, obtained by digestion
To YIPlac204-LUP-BPLO (Fig. 2).
The primer sequence is respectively as follows:
P1:5 '-GCTTTCCATGATGTCAGCTGATATTGTCGGCCAGA-3 ' (SEQ ID NO:6)
P2:5 '-TCTGGCCGACAATATCAGCTGACATCATGGAAAGC-3 ' (SEQ ID NO:7)
P3:5 '-TCTCTGCAGGAGCGACCTCATGCTATACCTG-3 ' (SEQ ID NO:8) P4:5 '-TCTTCTAGA
CTTCGAGCGTCCCAAAACCT-3’(SEQ ID NO:9)
2, YIplac204-LUP-BPLO transformed saccharomyces cerevisiae WAT11
YIplac204-LUP-BPLO is linearized with EcoRV, after agarose gel electrophoresis recycles, uses lithium acetate transformation
Method converts WAT11 bacterial strain and plate is placed in 30 DEG C of growths, by well-grown by SD-Trp- deficiency plate screening transformant
Monoclonal access SD-Trp- deficiency fluid nutrient medium expand culture, mention genomic DNA and carry out that PCR identification is positive to be turned
Beggar.Resulting transformant is the saccharomyces cerevisiae strain WAT11-LUP-BPLO for being able to produce betulic acid.It is identified through PCR
(Fig. 3) does not have LUP gene and BPLO gene in saccharomyces cerevisiae strain WAT11, and the saccharomyces cerevisiae strain that the present invention constructs
There are LUP gene and BPLO gene in WAT11-LUP-BPLO, the two expression casettes are successfully integrated into genome, and
It is replicated with the duplication of genome.
The building of the saccharomyces cerevisiae strain W80 of 2 high yield betulic acid of embodiment
The saccharomyces cerevisiae strain WAT11-LUP-BPLO constructed in embodiment 1 has been able to production betulic acid, inventor
Continue to study on this basis, it has unexpectedly been found that after 80 gene of regulatory factor GAL mutates, product betulic acid contains
Amount is greatly enhanced.According to the discovery, inventor further change to saccharomyces cerevisiae strain WAT11-LUP-BPLO
Into having knocked out 80 gene of GAL in saccharomyces cerevisiae strain WAT11-LUP-BPLO genome by the method for homologous recombination.Tool
Body method is as follows:
1. the homologous building for knocking out segment
It is template by pUG6 plasmid (Euroscarf), expands loxP-KanMX-loxP selection markers using primer P5/P6
Segment (1724bp) uses 5th ' area of primer P7/P8 amplification GAL80 gene using saccharomyces cerevisiae WAT11 genomic DNA as template
Segment (393bp) uses 3 ' area's segments (393bp) of primer P9/P10 amplification GAL80 gene.
Expand the primer used:
P5:5'-cgaccagcgtatacaatctcgatagttggtttcccgttctttccactcccgtcAGGTCGACAA
CCCTTAATAT-3'(SEQ ID NO:10)
P6:5'-ttcgtttttataacgttcgctgcactgggggccaagcacagggcaagatgcttTATAGGGAG
ACCGGCAGATC-3'(SEQ ID NO:11)
P7:5'-GGATCCCCAATGCTAATCCGGTCACTG-3'(dashed part is BamHI restriction enzyme site) (SEQ ID
NO:12)
P8:5'-GACGGGAGTGGAAAGAACGGGAAACCAACTATCGAGATTGT AT-3'(SEQ ID NO:13)
P9:5'-GCCCTGTGCTTGGCCCCCAGTGCAGCGAACGTTATAAAAACG AA-3'(SEQ ID NO:14)
P10:5'-GAATTCATCAGTTTTTGAAGGCAGCCT-3'(dashed part is EcoRI restriction enzyme site) (SEQ ID
NO:15)
50 μ l PCR reaction systems are as follows: 10 × Buffer, 5 μ l, each 1 μ l (10pmol) of primer, 1 μ l of 10mM dNTP,
DdH is added in 1 μ l of cDNA template, 0.5 μ l of archaeal dna polymerase2O to 50 μ l of final volume；Reaction condition are as follows: 95 DEG C of initial denaturation 3min, 95
DEG C denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min, carry out 30 recycle, last 72 DEG C of extensions 10min.
Three segments derived above are separately recovered purifying, and using the mixture of three segment as template, using drawing
Object P5/P10 is expanded, and 5 ' area segment+loxP-KanMX-loxP of the GAL80 gene by arranging with 5 ' to 3 ' directions are obtained
Selection markers segment+GAL80 gene 3 ' area's segments composition GAL80 gene knockout frame.GAL80 gene knockout frame is inserted into
To between BamHI the and EcoRI multiple cloning sites of carrier PMD-19T, E. coli DH5 is converted with obtained plasmid
α, monoclonal of the picking containing the plasmid, is sequenced.It extracts and correct plasmid is sequenced, with restriction endonuclease BamHI and the bis- enzymes of EcoRI
It cuts, recycles GAL80 gene knockout frame segment.
The knockout of 2.GAL80 gene
By lithium acetate transformation method, GAL80 gene knockout frame segment derived above is converted into the wine obtained to embodiment 1
In brewer yeast strain WAT11-LUP-BPLO.Make converted product in the SD-Trp- auxotrophy training comprising 200mg/L Geneticin
It supports and is grown at 30 DEG C on base plate to screen transformant, well-grown monoclonal is inoculated in the training of the liquid containing Geneticin
Feeding base expands culture, and extracts genomic DNA and carries out PCR identification positive transformant.Turned by lithium acetate transformation method heliotropism
Beggar is transferred to Cre recombinase, and second of homologous recombination occurs in transformant automatically, loses loxP-KanMX-loxP expression cassette.It will
The transformant that secondary homologous recombination occurs is inoculated in not antibiotic YPDA culture medium subculture and carries out losing Cre recombinase.It will
The transformant for losing loxP-KanMX-loxP expression cassette and Cre recombinase proposes genomic DNA progress PCR identification.The identification used
Primer is P7 and P10.
(Fig. 4) is identified through PCR, is successfully constructed the correct genetic engineering for having lacked 1308bp GAL80 gene encoder block and is changed
Bacterial strain is made, W80 is named as.
Embodiment 3 is using Wine brewing yeast strain production of the invention and extracts betulic acid
1. the production and extraction of betulic acid
Saccharomyces cerevisiae strain WAT11-LUP-BPLO or W80 obtained in embodiment 1 or 2 is inoculated in 2% glucose
In the SD-Trp- fluid nutrient medium of carbon source, to be placed in 30 DEG C, 250rpm/min is cultivated 16 hours, is then collected thallus and is used
It is resuspended in after sterile water washing using 2% galactolipin as extremely starting OD in the SD-Trp- culture medium of carbon source600Value is 0.8, is placed in 30
DEG C, 250rpm/min is carried out Fiber differentiation 7 days.Above-mentioned gained Yeast Cultivation liquid is acidified (2M HCl tune PH to 2.0), is used
Isometric ethyl acetate extracts 3 times.Merge 3 ethyl acetate phases, drying.It is redissolved, is turned with a small amount of ethyl acetate after drying
Enter 2ml centrifuge tube, dries up.Thus betulic acid is obtained
2. the identification and quantification of betulic acid
Betulic acid derived above is performed the derivatization, 80 DEG C with 50 μ l silylating reagent BSTFA, places 30min.Instead
It should be cooled to room temperature, be centrifuged to centrifuge tube later, suct clear layer and be transferred to clean anhydrous internal lining pipe, carry out GC-MS detection.GC-
MS detection uses Agilent 7890GC and 5975C mass detector and capillary chromatographic column HP-5MS.Carrier gas is helium, flow velocity
1.2ml/min, by not 1 μ l of shunt mode sample introduction, temperature is 250 DEG C, and temperature program is 80 DEG C of startings, is risen to 20 DEG C/min
310 DEG C, keep 15min.Mass Spectrometry Conditions: ionization source EI, electron energy 70eV, ion source temperature be 230 DEG C, full scan mode and
Choice ion pattern, full scan Mode scans range m/z are 50-600.GC-MS testing result confirms that the two bacterial strains can give birth to
It produces betulic acid (Fig. 5), yield is as shown in Figure 6.It can be seen that in conjunction with Fig. 5 and Fig. 6, saccharomyces cerevisiae strain WAT11-LUP-BPLO
Betulic acid can be generated with W80, and the betulic acid yield of W80 is the former 2-3 times.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of saccharomyces cerevisiae engineered yeast, which is characterized in that the saccharomyces cerevisiae engineered yeast is to be with saccharomyces cerevisiae strain WAT11
Basis incorporates LUP gene expression frame and BPLO gene expression frame in genome, wherein the LUP gene expression frame is by LUP base
It is formed because being operably coupled to GAL1 promoter, the sequence of the LUP gene is as shown in SEQ ID NO:1, the BPLO gene
Expression cassette is operably coupled to GAL10 promoter by BPLO gene and is formed, the sequence of the BPLO gene such as SEQ ID NO:2 institute
Show.
2. saccharomyces cerevisiae engineered yeast according to claim 1, which is characterized in that the GAL1 promoter sequence such as SEQ ID
Shown in NO:3.
3. saccharomyces cerevisiae engineered yeast according to claim 1, which is characterized in that the GAL10 promoter sequence such as SEQ
Shown in ID NO:4.
4. saccharomyces cerevisiae engineered yeast according to claim 1, which is characterized in that the GAL80 gene of the engineering bacteria is by portion
Divide or all knock out, the sequence of the GAL80 gene is as shown in SEQ ID NO:16.
5. a kind of method for constructing saccharomyces cerevisiae engineered yeast, which comprises the following steps:
1) LUP gene is inserted into the downstream of the GAL1 promoter of PESC-Trp carrier along the direction of GAL1 promoter, makes institute
It states LUP gene to be under the control of the GAL1 promoter, and BPLO gene is inserted into along the direction of GAL10 promoter
The downstream of the GAL10 promoter of the PESC-Trp carrier makes the BPLO gene be in the control of the GAL10 promoter
Under, to obtain being integrated with the plasmid PESC-TRP-LUP-BPLO of LUP gene expression frame and BPLO gene expression frame, wherein institute
The sequence of LUP gene is stated as shown in SEQ ID NO:1, the sequence of the GAL1 promoter is described as shown in SEQ ID NO:3
The sequence of BPLO gene is as shown in SEQ ID NO:2, and the sequence of the GAL10 promoter is as shown in SEQ ID NO:4；
It 2) will be in LUP gene described in the plasmid PESC-TRP-LUP-BPLO in GATATC by the method for rite-directed mutagenesis
Cytimidine sports thymidine；
3) the plasmid PESC-TRP-LUP-BPLO of the mutation obtained in EcoRV digestion step 2), makes its linearisation, then will
The plasmid PESC-TRP-LUP-BPLO of the linearisation is transferred in saccharomyces cerevisiae WAT11, passes through what is occurred automatically in saccharomycete body
Homologous recombination is integrated into the LUP gene expression frame and the BPLO gene expression frame in the genome of the saccharomycete.
6. according to the method described in claim 5, it is characterized in that, further comprising the steps of:
It 4) will be by the 3 ' Qu Xu of GAL80 gene 5 ' region sequence+loxP-KanMX-loxP+GAL80 gene that is arranged with 5 ' to 3 ' directions
Column composition GAL80 gene knockout frame convert incorporated into genome obtained in step 3) the LUP gene expression frame and
In the saccharomyces cerevisiae of the BPLO gene expression frame, made in the saccharomyces cerevisiae by the homologous recombination occurred automatically into the cell
The corresponding position of GAL80 gene is replaced by the GAL80 gene knockout frame, the sequence such as SEQ of the GAL80 gene knockout frame
Shown in ID NO:5；
5) in the saccharomyces cerevisiae for being knocked the GAL80 gene that Cre recombination enzymatic conversion to step 4) obtains, make the GAL80 base
It is recombinated again because knocking out frame, the loxP-KanMX-loxP segment is lost, to obtain that there is the LUP gene expression
Frame and the BPLO gene expression frame and the saccharomyces cerevisiae engineered yeast for having lacked the GAL80 gene encoder block.
7. a kind of method for producing betulic acid, which comprises the following steps:
1) saccharomyces cerevisiae engineered yeast of any of claims 1-4 is cultivated to logarithmic phase；
2) induction step 1) saccharomyces cerevisiae engineered yeast cultivated；
3) the saccharomyces cerevisiae engineered yeast culture being induced from step 2) extracts betulic acid.
8. the method according to the description of claim 7 is characterized in that using the SD-Trp- liquid added with glucose in step 1)
Saccharomyces cerevisiae engineered yeast described in culture medium culture.
9. the method according to the description of claim 7 is characterized in that using the SD-Trp- liquid added with galactolipin in step 2)
Saccharomyces cerevisiae engineered yeast described in culture medium culture is to induce the saccharomyces cerevisiae engineered yeast to generate betulic acid.
10. the method according to the description of claim 7 is characterized in that the following steps are included:
1) saccharomyces cerevisiae engineered yeast is inoculated in the SD-Trp- fluid nutrient medium added with 2% glucose, is placed in 30 DEG C,
250rpm is cultivated to logarithmic phase；
2) thallus is collected, with sterile water washing, is resuspended in the SD-Trp- culture medium added with 2% galactolipin to starting OD600Value
It is 0.8, is placed in 30 DEG C, Fiber differentiation under 250rpm；
3) yeast culture obtained in step 2) is adjusted to PH2.0 with 2M HCl, is extracted 3 times with isometric ethyl acetate,
Merge this 3 ethyl acetate phases, dries up.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610318425.6A CN105886415B (en) | 2016-05-13 | 2016-05-13 | A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610318425.6A CN105886415B (en) | 2016-05-13 | 2016-05-13 | A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105886415A CN105886415A (en) | 2016-08-24 |
| CN105886415B true CN105886415B (en) | 2019-07-19 |
Family
ID=56716369
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610318425.6A Active CN105886415B (en) | 2016-05-13 | 2016-05-13 | A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105886415B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110511879A (en) * | 2018-05-22 | 2019-11-29 | 暨南大学 | The building of saccharomyces cerevisiae engineered yeast and the preparation method of betulinic acid |
| CN109628543B (en) * | 2019-01-18 | 2020-12-25 | 浙江大学 | Method for preparing betulinic acid by using recombinant saccharomyces cerevisiae and inonotus obliquus mixed bacteria system |
| CN111304104A (en) * | 2020-02-10 | 2020-06-19 | 天津大学 | Recombinant yarrowia lipolytica for heterologous synthesis of betulinic acid and construction method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110067297A (en) * | 2009-12-14 | 2011-06-22 | 한국에너지기술연구원 | Extraction, crystallization method as well as 4-beds eluent swing adsorptive continuous unit and their operation methods for the simultaneous production of ultra pure betulin and high pure betulinic acid from birch barks |
| CN102433347A (en) * | 2011-11-03 | 2012-05-02 | 中国科学院武汉植物园 | Lupeol C28 oxidase gene and preparation method and application thereof |
| CN105255924A (en) * | 2015-09-14 | 2016-01-20 | 东北林业大学 | Betula platyphylla cycloartenol synthase gene BPX3 and application for regulation and control of betula platyphylla triterpene content |
| CN105296372A (en) * | 2015-12-10 | 2016-02-03 | 江南大学 | Engineered strain of saccharomyces cerevisiae capable of efficiently expressing macrobrachium rosenbergii lectin (Mr Lectin) |
| CN105385614A (en) * | 2015-12-17 | 2016-03-09 | 浙江大学 | Recombined saccharomyces cerevisiae and construction method and application thereof |
-
2016
- 2016-05-13 CN CN201610318425.6A patent/CN105886415B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110067297A (en) * | 2009-12-14 | 2011-06-22 | 한국에너지기술연구원 | Extraction, crystallization method as well as 4-beds eluent swing adsorptive continuous unit and their operation methods for the simultaneous production of ultra pure betulin and high pure betulinic acid from birch barks |
| CN102433347A (en) * | 2011-11-03 | 2012-05-02 | 中国科学院武汉植物园 | Lupeol C28 oxidase gene and preparation method and application thereof |
| CN105255924A (en) * | 2015-09-14 | 2016-01-20 | 东北林业大学 | Betula platyphylla cycloartenol synthase gene BPX3 and application for regulation and control of betula platyphylla triterpene content |
| CN105296372A (en) * | 2015-12-10 | 2016-02-03 | 江南大学 | Engineered strain of saccharomyces cerevisiae capable of efficiently expressing macrobrachium rosenbergii lectin (Mr Lectin) |
| CN105385614A (en) * | 2015-12-17 | 2016-03-09 | 浙江大学 | Recombined saccharomyces cerevisiae and construction method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| "Cloning and characterization of the Arabidopsis Thaliana lupeol synthase gene";Jennifer B.R.等;《Phytochemistry》;19981205;第49卷(第7期);第1905-1911页 * |
| "Increase of betulinic acid production in Saccharomyces cerevisiae by balancing fatty acids and betulinic acid forming pathways";Jing Li等;《Applied Gentics and Molecular Biotechnology》;20140105;第98卷(第7期);第3081-3089页 * |
| "Modulating betulinic acid in Saccharomyces cerevisiae by managing the intracellular of the co-factor NADPH and oxygen";Jing Li等;《Journal of Bioscience and Bioengineering》;20140717;第119卷(第1期);第77-81页 * |
| "Molecular characterization of the pentacyclic triterpenoid biosythetic pathway in Catharanthus roseus";Lili Huang等;《Planta》;20121130;第236卷(第5期);第1571-1581页 * |
| "白桦脂酸的研究进展";徐军等;《生命科学》;20110531;第23卷(第5期);第503-510页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105886415A (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105886415B (en) | A kind of saccharomyces cerevisiae engineered yeast and its construction method producing betulic acid | |
| CN112280698B (en) | Saccharomyces cerevisiae engineering bacteria for high-yield yacholanol type sesquiterpene and construction method and application thereof | |
| CN109988722A (en) | A kind of method of recombinant Saccharomyces cerevisiae bacterial strain and its application and production tyrosol and/or rhodioside | |
| CN106459985A (en) | Hydroxyacyl-coenzyme a dehydrogenase gene and acyl-coenzyme a thiolase gene, and genetically-engineered bacteria and use thereof | |
| WO2023173565A1 (en) | Method for simultaneously enhancing and inhibiting multiple key genes during synthesis of 7-dehydrocholesterol in saccharomyces cerevisiae | |
| CN101565710B (en) | 3-sterone-Delta[1]-dehydrogenase gene, relevant carriers, engineering strains and applications thereof | |
| CN104017738A (en) | High-yield engineering strain kmust-SQS for ganoderic acid | |
| CN111484961B (en) | Genetically engineered bacterium for producing 5 alpha-androstanedione and application thereof | |
| CN106119180B (en) | Mycobacterium recombinant genetic engineering bacterium and application thereof | |
| CN108138126B (en) | Mycobacterium genetic engineering bacteria and application thereof in preparation of steroid compound | |
| CN108929884A (en) | The method for synthesizing ganoderic acid by synthetic biology means heterologous organisms | |
| CN107488638B (en) | 15 α -hydroxylase and preparation method and application thereof | |
| CN104611303B (en) | A kind of fused protein that can improve dammarendiol transformation efficiency and construction method and application | |
| CN111454871B (en) | Recombinant mycobacterium with high androstenedione yield, construction method and application | |
| CN108588108B (en) | Preparation method and application of bacillus for efficiently metabolizing glycerol | |
| CN115948265A (en) | Kluyveromyces marxianus haploid yeast and construction method and application thereof | |
| CN114736918A (en) | Recombinant escherichia coli for producing salidroside through integrated expression and application thereof | |
| CN107903227B (en) | Succinic anhydride compound, gene and protein related to succinic anhydride compound and preparation method of succinic anhydride compound | |
| CN113684191A (en) | Pear head mould steroid 11 beta-hydroxylase CYP5311B2 mutant construction and application thereof | |
| CN105886413A (en) | High-yield engineering strain kmust-Lae of ganoderic acid | |
| CN113913448B (en) | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application | |
| CN110540943A (en) | Gene engineering strain for producing emodin and construction method and application thereof | |
| CN110331101A (en) | The construction method of purple Monascus mokH gene deletion strains | |
| CN106010989A (en) | High-ganodenic-acid-yield engineering strain kmust-Crz | |
| CN114507684B (en) | Method for inhibiting target gene expression in Mediterranean salt-rich bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220701 Address after: 433200 No. 1, Baili Changqu Road, Honghu Economic Development Zone, Hubei Province Patentee after: Hubei ji'antang Pharmaceutical Co.,Ltd. Patentee after: Wuhan Botanical Garden, Chinese Academy of Sciences Address before: 433300 East Industrial Park, Yusha Avenue, Rongcheng Town, Jianli County, Jingzhou City, Hubei Province Patentee before: HUBEI RENYUE PHARMACEUTICAL CO.,LTD. Patentee before: Wuhan Botanical Garden, Chinese Academy of Sciences |