Saccharomyces cerevisiae engineering bacteria with high betulinic acid production and its construction method and application
A technology of Saccharomyces cerevisiae and betulinic acid, which is applied in the fields of genetic engineering and metabolic engineering, can solve the problems of imperfect optimization, low betulinic acid production, and low oxidation efficiency, and achieve the effects of protecting the environment, solving the problem of drug sources, and saving plant resources
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Embodiment 1
[0111] Cloning of endogenous genes, expression elements and GDH genes in Bacillus subtilis in embodiment 1
[0112] 1. Extraction of yeast genome
[0113] (1) Take a single colony of Saccharomyces cerevisiae BY4741 (ATCC) and culture it in 5 mL of YPD medium for 16-24 hours, and centrifuge the bacterial liquid in a centrifuge at 4000 rpm for 5 minutes to collect the bacterial cells and put them in a mortar.
[0114] (2) Add liquid nitrogen and grind for 2 to 3 times. After the liquid nitrogen evaporates to dryness, add 1 mL of DNAiso Reagent (Baoriyi Biotechnology Co., Ltd.) to cover the bacteria.
[0115] (3) After standing for melting, continue to grind with a pestle until the lysate becomes transparent, and transfer the lysate to a centrifuge tube.
[0116] (4) Centrifuge the centrifuge tube containing the lysate at 4°C or room temperature for 10 minutes, transfer the supernatant to a new centrifuge tube, add 1 / 2 volume of absolute ethanol, invert it repeatedly for 2 minut...
Embodiment 2
[0140] The cloning of embodiment 2 plant source genes
[0141] 1. Extraction of RNA
[0142] (1) Take 100 mg sample with a medicine spoon (the samples are Arabidopsis thaliana, rosemary, and salvia miltiorrhiza, all of which can be purchased from the market) and add it to an EP tube containing 950 μL Trizol and 50 μL β-mercaptoethanol, vortex for 15 seconds, and Centrifuge at 12000 rpm for 10 min at 4°C.
[0143] (2) Pipette the supernatant into an EP tube containing 200 μL of chloroform, vortex for 15 s, and centrifuge at 12,000 rpm and 4° C. for 15 min.
[0144] (3) Use a pipette gun to absorb the supernatant, transfer it to an EP tube containing 500 μL of isopropanol, let it stand at room temperature for 10 minutes, and centrifuge at 12,000 rpm and 4° C. for 10 minutes.
[0145] (4) After centrifugation, remove the supernatant, add 1mL of 75% (v / v) ethanol solution to the EP tube, pipette with a pipette gun, centrifuge at 7500rpm, 4°C for 5min, and remove the supernatant....
Embodiment 3
[0156] Example 3, CRISPR-Cas9 system knockout of LPP1, DPP1, GDH1, PEP4 and PAH1 genes of Saccharomyces cerevisiae BY4741
[0157] 1. Design of crRNA spacer nucleic acid sequence and construction of vector
[0158] (1) By analyzing the gene sequence and according to the principle of CRISPR / Cas9 target design, design the target site sequence, take 50 bp base sequences at both ends of the PAM sequence as the homology arms, and the left and right homology arms are separated by 8 bp bases Base, and the 8bp base must include the PAM sequence. Homology arms are used to repair double-strand DNA breaks and cause frameshift mutations of target genes, specific crRNA spacer design references (Huimin Zhao et al, ACS Synth. Biol. 2015, 4, 585-594). in,
[0159] The crRNA spacer nucleic acid sequence of LPP1, DPP1, GDH1 gene is as described in SEQ ID NO.4;
[0160] The crRNA spacer nucleic acid sequence of PEP4 and PAH1 gene is as described in SEQ ID NO.5;
[0161] The crRNA spacer sequ...
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