A kind of method of producing isoprene by biological enzymatic method
A technology of isoprene and purpose, applied in the field of biological enzymatic production of isoprene, can solve problems such as low yield, and achieve the effects of increased yield and high-efficiency expression
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Embodiment 1
[0030] Embodiment 1, the construction of the recombinant expression vector containing da-IspS gene
[0031] (1) PCR amplification of da-IspS gene
[0032] After removing the intron from the amino acid sequence of isoprene synthase from Dandelion, the amino acid sequence of isoprene synthase is SEQ ID NO: 2, and the specific sequence is as follows:
[0033]
[0034]
[0035] According to the codon preference of Pichia pastoris, the nucleotide sequence of isoprene synthase (Isps) was optimized, and the optimized nucleotide sequence was SEQ ID NO: 1, which was artificially synthesized. The specific sequence is as follows:
[0036]
[0037]
[0038] Using the DNA obtained in step (1) as a template, the specific sequences of upstream primers and downstream primers are added as follows:
[0039] Isps-F: 5'-CCG GAATTC atggctggtgacagattgtc-3'
[0040] Isps-R: 5'-ATTT GCGGCCG gtcccagtctggacaaccagt-3'
[0041] Primestar DNA polymerase (purchased from TaKaRa Company) and...
Embodiment 2
[0044] Embodiment 2 pPIC3.5-da-IspS transforms Pichia pastoris and fermentation culture
[0045] Digest pPIC3.5-Isps with restriction endonuclease SacI or SalI to linearize it, which is more conducive to the homologous recombination of the target gene into the chromosome of Pichia pastoris. After the yeast cells were treated with 1M sorbitol, they were mixed with the purified linear plasmid containing the da-IspS gene, and transformed with an electrotransformer. The revived yeast cells were spread on MD plate and cultured at 30°C for 3-4d. Single colonies grown on MD plates were picked and inoculated into 25mL BMGY, shaken at 30°C and 250-300rpm until OD600=3-5. At room temperature, centrifuge at 1500-3000 g for 5 min, collect the cells, remove the supernatant, resuspend the cells with BMMY to OD600=1.0, and add 0.5% methanol to induce expression. Add the above-mentioned culture into a 1L shaker flask, cover with two layers of sterilized gauze or cheese cloth, and put it in ...
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