Biological production method of betaxanthin
A technology for betaflavin and a production method, applied in the field of biomedicine, can solve problems such as large-scale innovative development and application of unfavorable betalain natural medicines and food additives, low purity, retention and the like
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Embodiment 1
[0031] Acquisition of 4,5-dopa-estradiol dioxygenase gene syndoda and endogenous tyrosine hydroxylase gene HpaBC:
[0032] The amino acid sequence of 4,5-dopaestradiol dioxygenase derived from Mirabilis jApa is shown in SEQ ID No. 1 in the sequence table, and E. coli BL2l(DE3) endogenous tyrosine hydroxylase The amino acid sequence of is shown in SEQ ID No. 2 in the sequence listing.
[0033] Use OPTIMIZER online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ) to optimize the codon preference of E. coli, design the full-length gene encoding MjDODA, named syndoda, its nucleotide The sequence is shown in SEQ ID No.4. The syndoda gene obtained by chemical total synthesis has NcoI and BamHI restriction sites on both sides. Using E. coli BL21 (DE3) bacterial solution as a template, primers F-HPNd and R-HPKp containing KpnI and HindIII restriction sites were PCR amplified to obtain the full-length HpaBC gene. Its nucleotide sequence is as SEQ ID No. 5 shown.
[0034] The pri...
Embodiment 2
[0038] Construction of recombinant expression vector pHTDH:
[0039] For syndoda gene fragments with restriction sites, use FastDigest endonucleases NcoI and BamHI for double restriction digestion, and cut the pHTrc expression vector with the same endonuclease (the nucleotide sequence of pHTrc is shown in SEQ ID No. 3. Shown). The reaction system is: 5μL 10×FastDigest Buffer, 2μL endonuclease 1, 2μL endonuclease 2, 41μL gene fragment. The reaction conditions are: 37°C, 1h. The plasmid vector and the target gene fragment obtained after the enzyme digestion reaction are purified and recovered by the operation method in the PCR product purification and recovery kit. The purified product is ligated into pHTD through a ligation reaction. The ligation system is: 6μL digested gene fragment, 2μL digested vector fragment, 1μL 10×T4 ligase buffer, 1μL T4 DNA ligase. The reaction conditions are: 22°C, 30 min. The ligated reaction system needs to be transformed into competent cells in t...
Embodiment 3
[0045] The pHTDH recombinant expression vector was electroporated into the SyBE-002444 strain with high L-tyrosine production to obtain the BTX1 recombinant strain.
[0046] The operation is as follows:
[0047] 1. The frozen E. coli SyBE-002444 was transferred to 5 mL liquid LB medium at a volume ratio of 1%, and activated and cultured overnight at 37°C and 220 rpm. The activated bacterial solution was continuously inserted into 10 mL LB liquid medium at a volume ratio of 1%, and cultured at 37°C and 220 rpm for about 4 hours.
[0048] 2. Pour all the bacterial liquid into a pre-cooled centrifuge tube, centrifuge at 4000 rpm, 4°C for 5 minutes, discard the supernatant medium and collect the bacterial cells. The cells were resuspended in pre-cooled 5mL 10% glycerol, mixed well and continued to 4500rpm, centrifuged at 4°C for 5min, discarded the supernatant, repeated this operation, and washed the cells with glycerol 3 times in total. Then add 200 μL of 10% glycerol to resuspend the...
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