Engineering bacterium for producing beta-caryophyllene, and construction method and application thereof
A construction method and caryophyllene technology, applied in the field of bioengineering, can solve problems such as low synthesis efficiency, affecting downstream applications of products, and complex conditions.
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Embodiment 1
[0061] Example 1. Cloning of tobacco β-caryophyllene synthase gene NtCAS.
[0062] Extract the RNA from the tobacco leaf tissue of the flue-cured tobacco variety Honghua Dajinyuan (purchased from the China Tobacco Germplasm Bank), and use reverse transcriptase to convert the RNA into a cDNA template. The cDNA obtained by the reverse transcription reaction is used as a template, and the forward primer is : 5'-ATGGATTTGAGCAAAGGCTTGCCGGT-3', the reverse primer is: 5'-TTATGGAACAGGATCAACCAATATTG-3', use TakaRa PrimeSTAR Max DNA polymerase for PCR amplification; PCR conditions: 95°C for 5min; 95°C for 30s, 55°C for 30s , 72°C for 30s, 35 cycles; 72°C for 5min. The PCR product was detected by 1% agarose gel electrophoresis, and the results were as follows: figure 1 As shown, lane M is the DNA marker DL10000, lanes 1 and 2 are the target gene NtCAS, and the fragment size of the gene NtCAS is about 1700bp, which is in line with the expected size.
[0063] Use the method of agarose ge...
Embodiment 2
[0064] Example 2. Biosynthesis of β-caryophyllene using β-caryophyllene synthase NtCAS.
[0065] By co-expressing the acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene (mvaE) from Enterococcus faecalis in Escherichia coli, 3-hydroxy-3-methylglutaryl-CoA synthesis Enzyme gene (mvaS); mevalonate kinase gene (ERG12), mevalonate-5-phosphate kinase gene (ERG8), mevalonate-5-diphosphate decarboxylase gene (ERG19) from Saccharomyces cerevisiae , isopentenyl pyrophosphate isomerase gene (IDI1); geranyl pyrophosphate synthase gene (GPPS2) from North American fir; farnesyl pyrophosphate synthase gene (ispA) from Escherichia coli and source Based on the β-caryophyllene synthase gene (NtCAS) of tobacco, caryophyllene is biosynthesized using glucose as a raw material. The specific method is as follows:
[0066] (1) Cloning the tobacco β-caryophyllene synthase gene NtCAS, the method refers to Example 1;
[0067] (2) Construction of expression vector:
[0068] Usin...
Embodiment 3
[0072] Example 3. Fermentative production of β-caryophyllene by engineering bacteria prepared in Example 2.
[0073] The white monoclonal engineered Escherichia coli strain obtained in Example 2 was picked and placed in 20 ml of LB liquid medium (containing Cm and Amp resistance), and cultured and activated on a shaker at 37° C. as seeds. When the concentration of the seed bacteria is about 1.0, take the expanded bacteria solution and insert it into 50ml of M9 medium according to the inoculum amount of 2% (wt), and cultivate it at 37°C. When the bacterial concentration grows to about 0.6, add IPTG with a final concentration of 0.2mM , continue culturing at 30°C.
[0074] (1) Product detection
[0075] After 24 hours of fermentation, the final fermentation liquid was taken to detect the yield, and the GC and GC-MS figures of the product were as follows: Figure 4 and 5 Shown, by GC and GC-MS figure with β-caryophyllene standard ( figure 2 and 3 ) for comparison, it was sh...
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