CN106190881A - Bacterial strain and construction method, application - Google Patents
Bacterial strain and construction method, application Download PDFInfo
- Publication number
- CN106190881A CN106190881A CN201610594897.4A CN201610594897A CN106190881A CN 106190881 A CN106190881 A CN 106190881A CN 201610594897 A CN201610594897 A CN 201610594897A CN 106190881 A CN106190881 A CN 106190881A
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- dhcr7
- campesterol
- chassis
- gene
- 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.)
- Granted
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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
-
- 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
- C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
- C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
- C12Y103/01021—7-Dehydrocholesterol reductase (1.3.1.21)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to microorganism field, particularly to bacterial strain and construction method, application.The invention provides the bacterial strain of high yield campesterol, chassis bacterial strain imports external source functional gene DHCR7.The present invention utilizes the method for recombinant Saccharomyces cerevisiae and Ye Shi solution fat yeast two primary yeast chassis bacterial strain production campesterol relative to plant extract low cost, by-product is few and pollutes little, commercial production for campesterol provides a kind of practicable method, and the biosynthesis for steroid hormone class medicines such as downstream progesterone, 4AD lays the first stone, other kind of microbe high-yield campesterol had directive significance simultaneously.
Description
Technical field
The present invention relates to microorganism field, particularly to bacterial strain and construction method, application.
Background technology
At present, steroid hormone class medicine is the Equations of The Second Kind medicine being only second to antibiotic, is preparing health product, is treating and breathe system
The aspects such as system disease, endocrine disturbance, leukemic lymphoblastoid, rheumatism and dermatosis are widely used.Campesterol
(campesterol) as the important intermediate of steroid hormone class medicine, can be important as synthesis 4AD, ADD, 9 α-OH-AD etc.
Steroidal intermediate and the precursor of the important steroidal drug such as Progesterone, hydrocortisone, have major application in medicine synthesis field.
The preparation of campesterol uses plant extract mostly, but plant extract process steps is many, yield is low, by-product separates complexity, and
And easily to environment, significantly limit production and the application of campesterol.And Microbe synthesis is with low cost, high yield
Amount and Product Safety etc. are considered as promising production method.
Saccharomyces cerevisiae is as generally acknowledged safe mode microorganism, and genetic background understands, genetic manipulation is simple, can advise greatly
Mould fermenting and producing.And Ye Shi to solve fat yeast be a kind of of non-conventional yeasts it is considered to be safety, medicine can be used for and food is raw
On product.It is applicable to high density fermentation, and heterologous protein expression amount is high, and can utilize common carbohydrate, oils, alkane
Hydrocarbons is that substrate is allowed to more adapt to industrialization large-scale production.Campesterol synthesis in yeast cells, needs it
The path of endogenous production ergosterol is blocked, and is simultaneously introduced exogenous gene and then obtains campesterol.
But the content of currently acquired recombinant bacterial strain shake flask fermentation campesterol is relatively low, it is impossible to meet industrialized production need
Ask.
Summary of the invention
In view of this, the present invention provides bacterial strain and construction method, application.Recombinant Saccharomyces cerevisiae and Ye Shi solve fat yeast two
Primary yeast chassis bacterial strain produce the method for campesterol relative to plant extract low cost, by-product is few and pollutes little, for vegetable oil
The commercial production of sterol provides a kind of practicable method
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the bacterial strain of high yield campesterol, chassis bacterial strain imports external source functional gene DHCR7.
In some specific embodiments of the present invention, described external source functional gene includes Brachydanio rerio (Danio rerio)
The DHCR7 gene that the DHCR7 gene in source or chlamydia (Waddlia chondrophila WSU 86-1044) are originated.
In some specific embodiments of the present invention, the DHCR7 gene that described Brachydanio rerio (Danio rerio) is originated
Sequence is as shown in SEQ ID No.1.
In other specific embodiments of the present invention, described chlamydia (Waddlia chondrophila WSU
The sequence of DHCR7 gene 86-1044) originated is as shown in SEQ ID No.2.
In some specific embodiments of the present invention, described chassis bacterial strain is yeast.
In some specific embodiments of the present invention, described yeast includes that saccharomyces cerevisiae or Ye Shi solve fat yeast.
In some specific embodiments of the present invention, described saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1, described Ye Shi
Solving fat yeast is that Ye Shi solves fat yeast ATCC 201249.
In some specific embodiments of the present invention, the construction method of described bacterial strain is: with saccharomyces cerevisiae CEN.PK2-1
For chassis bacterial strain, utilize CRISPR technology to knock out endogenous ERG5 gene, described external source functional gene is integrated at the bottom of saccharomyces cerevisiae
Cup fungi genome.
In other specific embodiments of the present invention, the construction method of described bacterial strain is: solve fat yeast with Ye Shi
ATCC 201249 is chassis bacterial strain, on the basis of not knocking out ERG5, utilizes integrative plasmid pYLEX1 at chassis bacterium genome
Upper integration described external source functional gene.
Present invention also offers the application in fermenting and producing campesterol of the described bacterial strain.
Present invention also offers the construction method of described bacterial strain, with saccharomyces cerevisiae CEN.PK2-1 for chassis bacterial strain, utilize
CRISPR technology knocks out endogenous ERG5 gene, and described external source functional gene is integrated into saccharomyces cerevisiae chassis bacterium genome.
Present invention also offers the construction method of described bacterial strain, solve fat yeast ATCC 201249 for chassis bacterium with Ye Shi
Strain, on the basis of not knocking out ERG5, utilizes integrative plasmid pYLEX1 to integrate described external source function on the bacterium genome of chassis
Gene.
The method that present invention also offers fermenting and producing campesterol, with described inoculation, fermentation, collects bacterium solution,
Extract and obtain campesterol.
Fermentation process:
The bacterial strain SyBE_Sc00080017-SyBE_Sc00080027 that the present invention provides is inoculated in the training of 2mL first order seed
Support in base, 30 DEG C, 250rpm cultivate 24h, with initial cell concentration OD600=0.2 is inoculated in secondary seed medium respectively
In, 30 DEG C, 250rpm cultivate 12h, then with initial cell concentration OD600=0.2 is inoculated in 50mL fermentation medium respectively,
In 30 DEG C, cultivate under the conditions of 250rpm, the cell density (OD in monitoring sweat600), end in 72 hours of fermenting, take all
Bacterium solution extracts campesterol.
Campesterol extracting method: take 42mL fermentation liquid, 6000rpm is centrifuged 5min and collects thalline, washes twice.Use liquid nitrogen
Frozen cell also grinds in mortar, until cell is ground into white superfine powder, is transferred in new 10mL centrifuge tube, adds
The 1.5MKOH of 2mL methanol preparation, 60 DEG C of water-bath saponifications are overnight.The concussion of 2mL analytical pure normal hexane vortex is added after saponification
Product is extracted by 10min, and 5000rpm is centrifuged 10min and collects organic facies vacuum lyophilization 2 hours, adds 400 μ L just own
Alkane dissolves lyophilization again 4 hours, adds 400 μ L chromatograph methanol and dissolves, uses efficient liquid phase after the 2 organic membrane filtrations of μm
Chromatograph (HPLC) detection campesterol content, flowing is acetonitrile mutually: isopropanol=3:1, and flow velocity is 1mL/min, detects wavelength
205nm, column temperature 35 DEG C, sample size 10 μ L.
Or
By the present invention provide bacterial strain SyBE_Yl02060002, SyBE_Yl02060004, SyBE_Yl02060005,
SyBE_Yl02060006, SyBE_Yl02060007, SyBE_Yl02060008 are inoculated in 5mL primary-seed medium, 30
DEG C, 250rpm cultivate 24h, with initial cell concentration OD600=0.2 is inoculated in secondary seed medium respectively, 30 DEG C,
250rpm cultivates 18h, then with initial cell concentration OD600=0.2 is inoculated in 50mL fermentation medium respectively, in 30 DEG C,
Cultivate under the conditions of 250rpm, the cell density (OD in monitoring sweat600), end in 144 hours of fermenting, take 10mL bacterium solution and carry
Take campesterol.
Campesterol extracting method: take 10mL fermentation liquid, 6000rpm is centrifuged 5min and collects thalline, washes twice.Use liquid nitrogen
Frozen cell also grinds in mortar, until cell is ground into white superfine powder, is transferred in new 10mL centrifuge tube, adds
The 1.5MKOH of 2mL methanol preparation, 60 DEG C of water-bath saponifications are overnight.The concussion of 2mL analytical pure normal hexane vortex is added after saponification
Product is extracted by 10min, and 5000rpm is centrifuged 10min and collects organic facies vacuum lyophilization 2 hours, adds 400 μ L just own
Alkane dissolves lyophilization again 4 hours, adds the 400 μ L derivatization reagent N-methyl silica-based trifluoroacetamides of-N-trimethyl
(MSTFA) 30 DEG C of water-baths 2 hours, detect campesterol content with utilizing GC-TOF/MS after the 2 organic membrane filtrations of μm.
The invention provides the bacterial strain of high yield campesterol, chassis bacterial strain imports external source functional gene DHCR7.Implement
Example 1 test result indicate that, ferments 72 hours, and the yield of Sc_Dr_DHCR7 is the highest, reaches 24.66mg/L.It follows that screening
Optimum gene source produces campesterol for recombinant Saccharomyces cerevisiae positive effect.
Embodiment 2 test result indicate that, ferments 144 hours, also is able to synthesis not blocking bacterial strain on ERG5 gene basis
Campesterol, wherein the yield of Yl_Dr_DHCR7 is the highest, reaches 73.07mg/L, and the Ye Shi of report solves fat yeast vegetable oil steroid
It is 16.43mg/L that alcohol produces optimal DHCR7 gene source Yl_Xl_DHCR7 yield.It follows that the gene source that screening is optimum
Fat yeast production campesterol is solved for restructuring Ye Shi and has positive effect, and solve Brachydanio rerio in fat yeast at restructuring Ye Shi
(Danio rerio) source DHCR7 potency of gene is significantly better than the gene source reporting maximum output.
The present invention utilizes recombinant Saccharomyces cerevisiae and Ye Shi to solve fat yeast two primary yeast chassis bacterial strain and produces the side of campesterol
Method relative to plant extract low cost, by-product is few and pollutes little, provide one for the commercial production of campesterol and conscientiously may be used
The method of row, and the biosynthesis for the steroid hormone class medicine such as downstream progesterone, 4AD lays the first stone, simultaneously to other kind of micro-life
Thing high yield campesterol has directive significance.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 shows that recombinant Saccharomyces cerevisiae and Ye Shi solve the pathway figure of fat yeast synthesis campesterol；
Fig. 2 shows that saccharomyces cerevisiae ERG5 gene C RISPR knocks out plasmid construction procedure chart；
Fig. 3 shows that saccharomyces cerevisiae dissociates multicopy plasmid construction procedure chart；
Fig. 4 shows the campesterol shaking flask Yield compari@figure of recombinant Saccharomyces cerevisiae bacterial strain 11 kinds source DHCR7 gene；
Fig. 5 shows that Ye Shi solves fat yeast integrative plasmid building process figure；
Fig. 6 shows that restructuring Ye Shi solves the campesterol shaking flask Yield compari@figure of fat yeast strain 6 kinds source DHCR7 gene；
Fig. 7 shows embodiment 1 campesterol standard curve；
Fig. 8 shows embodiment 2 campesterol standard curve.
Detailed description of the invention
The invention discloses bacterial strain and construction method, application, those skilled in the art can use for reference present disclosure, suitably
Improvement technological parameter realizes.Special needs to be pointed out is, all similar replacements and change are for a person skilled in the art
It will be apparent that they are considered as being included in the present invention.Method and the application of the present invention are carried out by preferred embodiment
Describe, related personnel substantially can in without departing from present invention, spirit and scope to method described herein and apply into
Row is changed or suitably change and combination, realizes and applies the technology of the present invention.
In bacterial strain that the present invention provides and construction method, application, raw materials used and reagent all can be buied by market.
Below in conjunction with embodiment, the present invention it is expanded on further:
Embodiment 1
The source screening of recombinant Saccharomyces cerevisiae synthesis campesterol external source functional gene dhcr7
The construction method of the recombinant Saccharomyces cerevisiae bacterial strain producing campesterol of the present invention is as follows:
1, the acquisition of external source functional gene element
Exogenous gene is the key enzyme C7 position reductase DHCR7 gene for synthesizing campesterol: choose 11 kinds of differences
The gene source of the functional gene screening synthesis campesterol optimum in source, the gene source of DHCR7 includes Rattus norvegicus (Rattus
Norvegicus), mice (Mus musculus), people (Homo sapiens), common Adeps seu carnis Rhiopithecus roxellanae (Callithrix jacchus), cattle
(Bos taurus), jungle fowl (Gallus gallus), Africa xenopus (Xenopus laevis), flat algae (Tetraselmis
Sp.GSL018), chlamydia (Waddlia chondrophila WSU 86-1044), (Candidatus
Protochlamydia amoebophila UWE25), Brachydanio rerio (Danio rerio) etc., be abbreviated as Sc_Rn_ successively
DHCR7、Sc_Mm_DHCR7、Sc_Hs_DHCR7、Sc_Cj_DHCR7、Sc_Bt_DHCR7、Sc_Gg_DHCR7、Sc_Xl_
DHCR7, Sc_Ts_DHCR7, Sc_Wc_DHCR7, Sc_Cpa_DHCR7, Sc_Dr_DHCR7, concrete gene order is shown in Table 1.Above-mentioned
Gene is codon optimized through saccharomyces cerevisiae and suitably evades Bsa I restriction enzyme digestion sites, at gene two ends volume
Outer interpolation 5 ' end gcggccgcggtctcca；3 ' taaaggagaccgcggccgc are obtained by synthetic.
Table 1
2, the structure of the recombinant Saccharomyces cerevisiae of ERG5 gene is knocked out
Utilize CRSPR technology by existing for experiment plasmid pCRCT and the ERG5 gene knockout module Golden Gate of synthesis
Method builds, it is thus achieved that pCRCT-ERG5 plasmid, use Li-acetate method by Plastid transformation to CEN.PK2-1 chassis bacterial strain,
(synthetic yeast nitrogen source YNB6.7g/L, glucose 22g/L lack the kilnitamin powder of uracil to utilize Sc-URA solid plate
2g/L, the agar powder of 2%) to screen, the transformant obtained carries Yeast genome, to saltation zone after carrying out line point pure culture
Territory carries out PCR and sequence verification, and recombinant bacterial strain correct for checking accesses 30 DEG C, 250rpm cultivation in 5mLYPD fluid medium
12h, switching three times altogether in switching liquid YPD, utilize YPD and Sc-URA solid plate to screen, picking YPD flat board again
Upper growth not single bacterium colony of growth on Sc-URA flat board, preserves glycerol stock named SyBE_ to this recombinant bacterial strain
Sc00080007。
3, the recombinant Saccharomyces cerevisiae bacterial strain of production campesterol is built
First, by pRS425K-ENO2t-PDC1p-GPM1t plasmid in modular laboratory storehouse (can be at http: //
Synbioml.org/ Free Acquisition) and obtain external source DHCR7 gene Golden Gate method build, it is thus achieved that 11 kinds
The pRS425K-ENO2t-PDC1p-DHCR7-GPM1t saccharomyces cerevisiae restructuring sequestered multicopy plasmid of source dhcr7, uses vinegar
11 kinds of plasmids are transformed in the bacterial strain of SyBE_Sc00080007 chassis by acid lithium method respectively, use Sc-LEU solid plate (synthesis ferment
Female nitrogen source YNB6.7g/L, glucose 22g/L, lack leucic kilnitamin powder 2g/L, the agar powder of 2%) sieve
Choosing, the transformant obtained carries out bacterium colony PCR checking after carrying out line point pure culture, and the recombinant bacterial strain that checking is correct is preserved glycerol
Bacterium is also respectively designated as SyBE_Sc00080017, SyBE_Sc00080018, SyBE_Sc00080019, SyBE_
Sc00080020、SyBE_Sc00080021、SyBE_Sc00080022、SyBE_Sc00080023、SyBE_Sc00080024、
SyBE_Sc00080025, SyBE_Sc00080026 and SyBE_Sc00080027.Wherein, plasmid contained by each bacterial strain is as follows
SyBE_Sc00080017:pRS425K-ENO2t-PDC1p-Sc_Rn_DHCR7-GPM1t
SyBE_Sc00080018:pRS425K-ENO2t-PDC1p-Sc_Mm_DHCR7-GPM1t
SyBE_Sc00080019:pRS425K-ENO2t-PDC1p-Sc_Hs_DHCR7-GPM1t
SyBE_Sc00080020:pRS425K-ENO2t-PDC1p-Sc_Cj_DHCR7-GPM1t
SyBE_Sc00080021:pRS425K-ENO2t-PDC1p-Sc_Bt_DHCR7-GPM1t
SyBE_Sc00080022:pRS425K-ENO2t-PDC1p-Sc_Gg_DHCR7-GPM1t
SyBE_Sc00080023:pRS425K-ENO2t-PDC1p-Sc_Xl_DHCR7-GPM1t
SyBE_Sc00080024:pRS425K-ENO2t-PDC1p-Sc_Ts_DHCR7-GPM1t
SyBE_Sc00080025:pRS425K-ENO2t-PDC1p-Sc_Wc_DHCR7-GPM1t
SyBE_Sc00080026:pRS425K-ENO2t-PDC1p-Sc_Cpa_DHCR7-GPM1t
SyBE_Sc00080027:pRS425K-ENO2t-PDC1p-Sc_Dr_DHCR7-GPM1t
4, the campesterol shaking flask yield of bacterial strain SyBE_Sc00080017-SyBE_Sc00080027 is compared
Test material: bacterial strain SyBE_Sc00080017-SyBE_Sc00080027
Test method:
One-level, secondary seed medium: 20g/L glucose, 6.7g/L YNB (Yeast Nitrogen Base), 2g/L
Lack leucic kilnitamin powder, 100mg/L adenine；
Fermentation medium: 20g/L glucose, 20g/L peptone, 10g/L yeast leaching powder, 100mg/L adenine
Above-mentioned bacterial strains SyBE_Sc00080017-SyBE_Sc00080027 is inoculated in 2mL primary-seed medium,
30 DEG C, 250rpm cultivate 24h, with initial cell concentration OD600=0.2 is inoculated in secondary seed medium respectively, 30 DEG C,
250rpm cultivates 12h, then with initial cell concentration OD600=0.2 is inoculated in 50mL fermentation medium respectively, in 30 DEG C,
Cultivate under the conditions of 250rpm, the cell density (OD in monitoring sweat600), end in 72 hours of fermenting, take whole bacterium solution and extract
Campesterol.
Campesterol extracting method: take 42mL fermentation liquid, 6000rpm is centrifuged 5min and collects thalline, washes twice.Use liquid nitrogen
Frozen cell also grinds in mortar, until cell is ground into white superfine powder, is transferred in new 10mL centrifuge tube, adds
The 1.5MKOH of 2mL methanol preparation, 60 DEG C of water-bath saponifications are overnight.The concussion of 2mL analytical pure normal hexane vortex is added after saponification
Product is extracted by 10min, and 5000rpm is centrifuged 10min and collects organic facies vacuum lyophilization 2 hours, adds 400 μ L just own
Alkane dissolves lyophilization again 4 hours, adds 400 μ L chromatograph methanol and dissolves, uses efficient liquid phase after the 2 organic membrane filtrations of μm
Chromatograph (HPLC) detection campesterol content, flowing is acetonitrile mutually: isopropanol=3:1, and flow velocity is 1mL/min, detects wavelength
205nm, column temperature 35 DEG C, sample size 10 μ L.
Result of the test:
Standard curve is shown in Fig. 7.
From the point of view of campesterol yield by Fig. 7 and Fig. 4 bacterial strain SyBE_Sc00080017-SyBE_Sc00080027, fermentation
72 hours, the yield of Sc_Dr_DHCR7 was the highest, reaches 24.66mg/L.It follows that the gene source of screening optimum is for weight
Group saccharomyces cerevisiae produces campesterol positive effect.
High performance liquid chromatography (HPLC) detection initial data is shown in Table 2.
Table 2
Embodiment 2
Restructuring Ye Shi solves the source screening of fat yeast synthesis campesterol external source functional gene dhcr7
The construction method that the restructuring Ye Shi producing campesterol of the present invention solves fat yeast strain is as follows:
1, the acquisition of external source functional gene element
Exogenous gene is the key enzyme C7 position reductase DHCR7 gene for synthesizing campesterol: choose 6 kinds of separate sources
Functional gene, the gene source that screening synthesis campesterol is optimum, wherein screen vegetable oil containing Yuan Yingjin seminar of University Of Tianjin
Africa xenopus (Xenopus laevis) the DHCR7 gene that sterol yield is the highest, the gene source of DHCR7 includes Africa xenopus
(Xenopus laevis), people (Homo sapiens), jungle fowl (Gallus gallus), Brachydanio rerio (Danio rerio), plan
South mustard (Arabidopsis thaliana), chlamydia (Waddlia chondrophila) etc., be abbreviated as Yl_Xl_ successively
DHCR7, Yl_Hs_DHCR7, Yl_Gg_DHCR7, Yl_Dr_DHCR7, Yl_At_DHCR7, Yl_Wc_DHCR7, concrete gene sequence
Row are shown in Table 3.Said gene is and is obtained by synthetic through Ye Shi solution fat yeast codons optimization.
Table 3
| The dhcr7 gene of separate sources | Sequence is numbered |
| Africa xenopus (Xenopus laevis) | SEQ ID No.12 |
| People (Homo sapiens) | SEQ ID No.13 |
| Jungle fowl (Gallus gallus) | SEQ ID No.14 |
| Brachydanio rerio (Danio rerio) | SEQ ID No.15 |
| Arabidopsis (Arabidopsis thaliana) | SEQ ID No.16 |
| Chlamydia (Waddlia chondrophila) | SEQ ID No.17 |
2, the restructuring Ye Shi building production campesterol solves fat yeast strain
Do not blocking introducing external source DHCR7 gene on ERG5 gene basis.First, the external source DHCR7 gene of acquisition is entered
Performing PCR amplification introduces I two restriction enzyme digestion sites of BglII, Kpn at gene two ends, respectively through BglII, Kpn I couple
Utilize In-Fusion cloning reaction to recombinate on BamH I, Kpn I linearizing pYLEX1 plasmid after enzyme action, obtain
The pYLEX1-DHCR7 Ye Shi obtaining 6 kinds of source dhcr7 solves fat yeast recombination and integration type list copy plasmid, uses Li-acetate method by 6
Plant plasmid to be transformed into respectively in the bacterial strain of ATCC 201249 chassis, be integrated in genome pBR322platform position, use Sc-
LEU solid plate (synthetic yeast nitrogen source YNB 6.7g/L, glucose 22g/L, lack leucic kilnitamin powder 2g/L,
The agar powder of 2%) to screen, the transformant obtained is extracted Yeast genome after carrying out line point pure culture and is carried out PCR checking,
The correct recombinant bacterial strain of checking is preserved glycerol stock and be respectively designated as SyBE_Yl02060002, SyBE_Yl02060004,
SyBE_Yl02060005、SyBE_Yl02060006、SyBE_Yl02060007、SyBE_Yl02060008.Wherein, each bacterial strain base
Because group genotype is as follows
SyBE_Yl02060002:pBR322::Yl_Xl_DHCR7
SyBE_Yl02060004:pBR322::Yl_Hs_DHCR7
SyBE_Yl02060005:pBR322::Yl_Gg_DHCR7
SyBE_Yl02060006:pBR322::Yl_Dr_DHCR7
SyBE_Yl02060007:pBR322::Yl_At_DHCR7
SyBE_Yl02060008:pBR322::Yl_Wc_DHCR7
3, bacterial strain SyBE_Yl02060002, SyBE_Yl02060004, SyBE_Yl02060005, SyBE_ are compared
The campesterol shaking flask yield of Yl02060006, SyBE_Yl02060007, SyBE_Yl02060008
Test material: bacterial strain SyBE_Yl02060002, SyBE_Yl02060004, SyBE_Yl02060005, SyBE_
Yl02060006、SyBE_Yl02060007、SyBE_Yl02060008
Test method:
One-level, secondary seed medium: 22g/L glucose, 20g/L peptone, 10g/L yeast leaching powder；
Fermentation medium: 50g/L glucose, 20g/L peptone, 10g/L yeast leaching powder
By above-mentioned bacterial strains SyBE_Yl02060002, SyBE_Yl02060004, SyBE_Yl02060005, SyBE_
Yl02060006, SyBE_Yl02060007, SyBE_Yl02060008 are inoculated in 5mL primary-seed medium, 30 DEG C,
250rpm cultivates 24h, with initial cell concentration OD600=0.2 is inoculated in secondary seed medium respectively, at 30 DEG C, 250rpm
Cultivate 18h, then with initial cell concentration OD600=0.2 is inoculated in 50mL fermentation medium respectively, in 30 DEG C, 250rpm condition
Lower cultivation, the cell density (OD in monitoring sweat600), end in 144 hours of fermenting, take 10mL bacterium solution and extract campesterol.
Campesterol extracting method: take 10mL fermentation liquid, 6000rpm is centrifuged 5min and collects thalline, washes twice.Use liquid nitrogen
Frozen cell also grinds in mortar, until cell is ground into white superfine powder, is transferred in new 10mL centrifuge tube, adds
The 1.5MKOH of 2mL methanol preparation, 60 DEG C of water-bath saponifications are overnight.The concussion of 2mL analytical pure normal hexane vortex is added after saponification
Product is extracted by 10min, and 5000rpm is centrifuged 10min and collects organic facies vacuum lyophilization 2 hours, adds 400 μ L just own
Alkane dissolves lyophilization again 4 hours, adds the 400 μ L derivatization reagent N-methyl silica-based trifluoroacetamides of-N-trimethyl
(MSTFA) 30 DEG C of water-baths 2 hours, detect campesterol content with utilizing GC-TOF/MS after the 2 organic membrane filtrations of μm.
Result of the test:
Campesterol standard curve is shown in Fig. 8.
By Fig. 8 and Fig. 6 bacterial strain SyBE_Yl02060002, SyBE_Yl02060004, SyBE_Yl02060005, SyBE_
From the point of view of the campesterol yield of Yl02060006, SyBE_Yl02060007, SyBE_Yl02060008, ferment 144 hours, not
Blocking bacterial strain on ERG5 gene basis also to be able to synthesize campesterol, wherein the yield of Yl_Dr_DHCR7 is the highest, reaches
73.07mg/L, the Ye Shi of report solve fat yeast campesterol and produce optimal DHCR7 gene source Yl_Xl_DHCR7 yield
For 16.43mg/L.Have actively it follows that the gene source of screening optimum solves fat yeast production campesterol for restructuring Ye Shi
Meaning, and in restructuring Ye Shi solution fat yeast, Brachydanio rerio (Danio rerio) originates the DHCR7 potency of gene significantly better than reporting
The gene source of road maximum output.
Gas chromatography mass spectrometry (GC-TOF/MS) detection initial data is shown in Table 4.
Table 4
The present invention is disclosed in recombinant Saccharomyces cerevisiae and Ye Shi solves in fat yeast both of which bacterial strain, Brachydanio rerio (Danio
Rerio) C7 position reductase, source (DHCR7) is respectively provided with optimal effect, and this conclusion is also other kind of microbe high-yield dish simultaneously
Oil sterol provides directive significance.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (13)
1. the bacterial strain of high yield campesterol, it is characterised in that import external source functional gene DHCR7 in the bacterial strain of chassis.
Bacterial strain the most according to claim 1, it is characterised in that described external source functional gene includes Brachydanio rerio (Danio
Rerio) the DHCR7 base that the DHCR7 gene originated or chlamydia (Waddlia chondrophila WSU 86-1044) are originated
Cause.
Bacterial strain the most according to claim 2, it is characterised in that the DHCR7 base that described Brachydanio rerio (Danio rerio) is originated
The sequence of cause is as shown in SEQ ID No.11 or SEQ ID No.15.
Bacterial strain the most according to claim 2, it is characterised in that described chlamydia (Waddlia chondrophila WSU
The sequence of DHCR7 gene 86-1044) originated is as shown in SEQ ID No.9 or SEQ ID No.17.
5. according to the bacterial strain described in any one of Claims 1-4, it is characterised in that described chassis bacterial strain is yeast.
Bacterial strain the most according to claim 5, it is characterised in that described yeast includes that saccharomyces cerevisiae or Ye Shi solve fat yeast.
Bacterial strain the most according to claim 6, it is characterised in that described saccharomyces cerevisiae is saccharomyces cerevisiae CEN.PK2-1, described
It is that Ye Shi solves fat yeast ATCC 201249 that Ye Shi solves fat yeast.
8. according to the bacterial strain described in any one of Claims 1-4, it is characterised in that its construction method is: with saccharomyces cerevisiae
CEN.PK2-1 is chassis bacterial strain, knocks out endogenous ERG5 gene, and described external source functional gene is integrated into saccharomyces cerevisiae chassis bacterium base
Because of group.
9. according to the bacterial strain described in any one of Claims 1-4, it is characterised in that its construction method is: solve fat yeast with Ye Shi
ATCC 201249 is chassis bacterial strain, on the basis of not knocking out ERG5, utilizes integrative plasmid pYLEX1 at chassis bacterium genome
Upper integration described external source functional gene.
10. the bacterial strain as described in any one of claim 1 to 9 application in fermenting and producing campesterol.
11. according to the construction method of the bacterial strain described in any one of claim 1 to 9, it is characterised in that with saccharomyces cerevisiae
CEN.PK2-1 is chassis bacterial strain, knocks out endogenous ERG5 gene, and described external source functional gene is integrated into saccharomyces cerevisiae chassis bacterium base
Because of group.
12. according to the construction method of the bacterial strain described in any one of claim 1 to 9, it is characterised in that solve fat yeast with Ye Shi
ATCC 201249 is chassis bacterial strain, on the basis of not knocking out ERG5, utilizes integrative plasmid pYLEX1 at chassis bacterium genome
Upper integration described external source functional gene.
The method of 13. fermenting and producing campesterols, it is characterised in that with the inoculation described in any one of claim 1 to 9,
Fermentation, collects bacterium solution, extracts and obtains campesterol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610594897.4A CN106190881B (en) | 2016-07-26 | 2016-07-26 | Bacterial strain and construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610594897.4A CN106190881B (en) | 2016-07-26 | 2016-07-26 | Bacterial strain and construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106190881A true CN106190881A (en) | 2016-12-07 |
| CN106190881B CN106190881B (en) | 2020-03-20 |
Family
ID=57496184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610594897.4A Active CN106190881B (en) | 2016-07-26 | 2016-07-26 | Bacterial strain and construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106190881B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083338A (en) * | 2017-05-15 | 2017-08-22 | 天津大学 | Recombinant bacterial strain and its construction method and the application in production campesterol |
| CN112852651A (en) * | 2020-11-25 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Method for increasing yield of hydrocortisone produced by saccharomyces cerevisiae biotransformation |
| CN113604470A (en) * | 2021-08-16 | 2021-11-05 | 西安海斯夫生物科技有限公司 | Recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805027A (en) * | 2015-05-27 | 2015-07-29 | 天津大学 | Recombinant yarrowia lipolytica bacterial strain as well as construction method and application thereof |
-
2016
- 2016-07-26 CN CN201610594897.4A patent/CN106190881B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805027A (en) * | 2015-05-27 | 2015-07-29 | 天津大学 | Recombinant yarrowia lipolytica bacterial strain as well as construction method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| HAO-XING DU等: "Engineering Yarrowia lipolytica for Campesterol Overproduction", 《PLOS ONE》 * |
| YU ZHANG等: "Improved campesterol production in engineered Yarrowia lipolytica strains", 《BIOTECHNOL LETT》 * |
| 谭思远等: "重组解脂亚罗酵母合成菜油甾醇", 《中国油脂》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107083338A (en) * | 2017-05-15 | 2017-08-22 | 天津大学 | Recombinant bacterial strain and its construction method and the application in production campesterol |
| CN112852651A (en) * | 2020-11-25 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Method for increasing yield of hydrocortisone produced by saccharomyces cerevisiae biotransformation |
| CN112852651B (en) * | 2020-11-25 | 2022-02-18 | 中国科学院天津工业生物技术研究所 | Method for increasing yield of hydrocortisone produced by saccharomyces cerevisiae biotransformation |
| CN113604470A (en) * | 2021-08-16 | 2021-11-05 | 西安海斯夫生物科技有限公司 | Recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof |
| CN113604470B (en) * | 2021-08-16 | 2022-04-12 | 西安海斯夫生物科技有限公司 | Recombinant yarrowia lipolytica T30pED for high yield of campesterol, construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106190881B (en) | 2020-03-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106635853B (en) | Recombinant saccharomyces cerevisiae for producing glycyrrhetinic acid, and construction method and application thereof | |
| CN106459985B (en) | A kind of Mycobacterium tuberculosis genes engineering bacteria is preparing the application in steroidal compounds | |
| Wang et al. | Systematic optimization of the yeast cell factory for sustainable and high efficiency production of bioactive ginsenoside compound K | |
| CN111690690B (en) | Saccharomyces cerevisiae for producing farnesene | |
| CN103468585B (en) | Monascus strain and application thereof in preparing functional monascus | |
| CN107841466B (en) | A method of by marine Penicillum category fungi large scale preparation penicillic acid | |
| CN106190881A (en) | Bacterial strain and construction method, application | |
| Wang et al. | Cloning and characterization of squalene synthase gene from Poria cocos and its up-regulation by methyl jasmonate | |
| CN112813013B (en) | Recombinant escherichia coli for producing hydroxytyrosol and application thereof | |
| CN108753627A (en) | A kind of marine aspergillus source oxa- anthraquinone analog compound and preparation method thereof and the application in preparing antitumor agent | |
| CN105969678A (en) | Low-yield high-grade alcohol high-yield ethyl lactate saccharomyces cerevisiae strain and construction method thereof | |
| CN105218447B (en) | Sclerotiorin derivatives and preparation method thereof and the application as anti-influenza A H 1 N 1 virus agent | |
| CN104805027B (en) | One kind restructuring Ye Shi solution fat yeast strains and its construction method and application | |
| CN112175848A (en) | Patchouli alcohol production yeast strain and construction method and application thereof | |
| CN103275997A (en) | Saccharomyces cerevisiae strain for producing 7-dehydrocholesterol and construction method | |
| CN102168099A (en) | 3-ketosteroid -delta 1-dehydrogenase, engineering bacterium and application thereof | |
| CN106318966B (en) | A method of 3-O- glucosyl group oleanolic acid and cellobiose oleanolic acid are synthesized using saccharomyces cerevisiae | |
| CN104388515A (en) | Method for producing 11alpha-OH-ADD from phytosterol by mixed fermentation | |
| CN106244615A (en) | A kind of engineering bacteria and construction method thereof and the application in preparing geraniol | |
| CN102168024B (en) | Meterhizium anisopliae and application thereof | |
| CN104278070B (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| WO2018233531A1 (en) | Microorganism and use thereof | |
| CN114774297B (en) | Recombinant saccharomyces cerevisiae for producing T-juniper alcohol and application thereof | |
| CN113956990B (en) | Recombinant saccharomyces cerevisiae for producing dihydronilotinib as well as preparation method and application thereof | |
| CN115786389A (en) | Saccharomyces cerevisiae engineering bacterium for high-yield santalol as well as construction method and application thereof |
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 |