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Recombined saccharomyces cerevisiae and construction method and application thereof

A technology for recombinant Saccharomyces cerevisiae and construction methods, applied in the direction of recombinant DNA technology, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of high price, long reaction time, low solubility, etc., and achieve mild transformation conditions and transformation The effect of high efficiency and simple operation

Inactive Publication Date: 2016-03-09
ZHEJIANG UNIV
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Problems solved by technology

At present, several fungi have been confirmed to complete the conversion of betulin to betulinic acid, namely Armillarialuteo-virens Sacc ZJUQH100-6, Cunninghamella blakesleeana, Aspergillus foetidus ) ZU-G1 and Aspergillus oryzae (Aspergillusoryzae) AS3.498, but the yield still cannot meet the commercial demand
[0004] The publication number is that the Chinese patent application of CN101457250A discloses that Armillaria luteo-virens Sacc (Armillarialuteo-virens Sacc) ZJUQH can catalyze betulin to produce betulinic acid in the aqueous phase system, but the substrate of the organic phase has low solubility in the aqueous phase system. High, not only affects the catalytic effect of the enzyme on the substrate, but also has certain toxicity to the microbial cells, resulting in low yield and long reaction time
Later, after improvement, in the Chinese patent with the publication number CN101709322A, ionic liquid or a two-phase system containing ionic liquid was used to catalyze the reaction, which improved the yield of the product and shortened the reaction time to 6-24h, but the ionic liquid itself was expensive. Although the yield of betulinic acid has been improved, it is still relatively low and cannot meet the needs of commercialization
[0005] The rapid development of metabolic engineering and synthetic biology has provided a good solution for obtaining high-yield natural products in microbial hosts, but there are few studies on the application of these technologies to the transformation of betulin

Method used

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  • Recombined saccharomyces cerevisiae and construction method and application thereof
  • Recombined saccharomyces cerevisiae and construction method and application thereof
  • Recombined saccharomyces cerevisiae and construction method and application thereof

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Effect test

Embodiment 1p

[0038] Embodiment 1 pESC-ura-CYP716A12-ATR1 plasmid construction

[0039] (1) Acquisition of CYP716A12 and ATR1 gene sequences

[0040] Using the total DNA from the four-week-old leaves of Medicago truncatula provided by Mr. Wang Yanzhang from the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, use primers C-F (see Table 1 for the primer sequence, the same below) and C-R to amplify CYP716A12 For the gene, the amplification conditions were: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 10 s, annealing at 56°C for 10 s, extension at 72°C for 40 s (35 cycles); complete extension at 72°C for 10 min.

[0041] Total RNA (OMEGA, E.Z.N.Z.PlantRNAKit) was extracted from the leaves of Arabidopsis thaliana seedlings, and ATR1 gene (TaKaRa, PrimeScriptOneStepRT-PCRKitVer.2) was amplified by reverse transcription with primers A-F and A-R. The amplification conditions were: reverse transcription at 50°C fo...

Embodiment 2

[0049] Example 2 Construction of Recombinant Saccharomyces cerevisiae W303-1b

[0050] The pESC-ura-CYP716A12-ATR1 plasmid was introduced into Saccharomyces cerevisiae by the lithium acetate transformation method, and the SC-ura solid medium plate was spread, and the single colony grown was streaked and purified, and a single colony was selected, and the preliminary identification of the colony was positive by PCR. Afterwards, the liquid medium was used to expand the culture, and the plasmid was extracted for sequencing to confirm that the plasmid was introduced successfully.

[0051] Add 25% sterilized glycerin to the successfully constructed recombinant Saccharomyces cerevisiae liquid, and store in a -80°C refrigerator. The recombinant Saccharomyces cerevisiae is named Saccharomyces cerevisiae W303-1b, and was deposited in the China Center for Type Culture Collection (CCTCC) located in Wuhan University, with storage time: November 5, 2015, and storage number: CCTCC NO: M2015...

Embodiment 3

[0052] Example 3 Microsomal protein preparation

[0053] 100 μL of recombinant Saccharomyces cerevisiae W303-1b bacteria liquid was inserted into 10mL LYPAD (GLu), and cultured at 30°C and 180rpm for 20h; 6 Inoculate 50mLYPAD (GLu) at a seeding density of one / mL, and culture at 200rpm at 30°C for 12h. Centrifuge at 3000g for 5min to precipitate the bacteria, and wash twice with normal saline. OD2.01:20 was resuspended in 50mLYPAD (GAL), cultured at 16°C and 180rpm for 24h.

[0054] Centrifuge at 2000g for 10min, add 50mLTEK buffer to the cell pellet, and centrifuge at 6100g for 3min. The bacterial pellet was resuspended in 10 mL of extraction buffer. Divide into 2mL centrifuge tubes with appropriate amount of glass beads, vibrate at a frequency of 65Hz, and vibrate for 49s, and vibrate continuously for 3 times. After the samples were placed on ice for 5 min, vibration grinding was repeated three times. Centrifuge at 6100g for 15min, filter, add MgCl with a final concentra...

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Abstract

The invention discloses recombined saccharomyces cerevisiae and a construction method and application thereof. The recombined saccharomyces cerevisiae is named (Saccharomyces cerevisiae) W303-1b, the preservation number is CCTCC NO:M 2015662, and the preservation data is November 5th, 2015. The invention further discloses a method for preparing microsome protein from the recombined saccharomyces cerevisiae. The method includes the following steps that firstly, inoculation is performed; secondly, thallus cells are cultured and collected; thirdly, the thallus cells are broken; fourthly, supernate is obtained through first-time centrifugation; fifthly, precipitant is added into the supernate, and a precipitate (namely the microsome protein) is obtained through second-time centrifugation. The invention discloses application of the microsome protein to the reaction generating betulinic acid by converting betulin. Betulinic acid can be generated by converting betulin through the microsome protein prepared by culturing recombined saccharomyces cerevisiae, the period of the method is short, post processing is convenient, and the maximum yield of betulinic acid can reach 27.5%.

Description

technical field [0001] The invention relates to the fields of bioengineering and microbial fermentation, in particular to a recombinant Saccharomyces cerevisiae capable of catalyzing the transformation of betulin into betulinic acid, its construction method and application. Background technique [0002] Betulinic acid (BA for short) is a class of pentacyclic lupane-type triterpenoids. Selective cytotoxicity against many specific tumor cells such as melanoma, prostate cancer, colon cancer, breast cancer, etc., different types of infectious agents such as human immunodeficiency virus, malaria and bacteria, and general inflammatory processes. Recently, a large number of in vitro and in vivo experiments have proved that betulinic acid also has other clinical functions. Betulinic acid can slow down ethanol-induced activation of hepatic stellate cells, alleviate hyperglycemia by inhibiting hepatic glucose production, enhance immune response, achieve weight loss by inhibiting panc...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/02C12P33/12C12P33/02C12R1/865
CPCC12N9/0004C12N9/0071C12N15/81C12P33/02C12P33/12
Inventor 陈启和李宏吉董亚晨牛永武
Owner ZHEJIANG UNIV
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