Recombinant escherichia coli and application thereof in synthesis of p-hydroxybenzaldehyde
A technology for recombining Escherichia coli and Escherichia coli, applied in the field of genetic engineering, can solve the problems of complex fermentation product composition, environmental pollution safety, high purification difficulty, etc., and achieve the effects of being beneficial to environmental protection, easy to popularize and apply, and easy to control.
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Embodiment 1
[0028] Example 1: Plasmid construction
[0029] Codon optimization is performed on the gene sequences encoding CYP199A4, HaPuR and HaPux, and after codon optimization, sequences such as cytochrome monooxygenase encoded by SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, The ferredoxin and ferredoxin reductase genes. The recombinant plasmids pRSFduet-1-CYP199A4-HaPux and pETduet-1-HaPuR-HaPux were obtained by one-step cloning. The CYP199A4 (primers P1, P2) and HaPux (primers P3, P4) genes were inserted into the restriction sites BamHI / SacI and NdeI / XhoI on the pRSFduet-1 plasmid, respectively, and the HaPuR (primers P5, P6) and HaPux (primers P7 , P8) genes were inserted into the restriction sites NdeI / XhoI and XbaI / EcoRI on the pETduet-1 plasmid, respectively. The PCR reaction conditions were: 95°C for 5min; 98°C for 10s, 55°C for 5s, and 72°C for 5s / kb (30 cycles); 72°C for 10min, and then verified and recovered the PCR amplification product by 1% agarose gel electrophoresis. Th...
Embodiment 2
[0038] Embodiment 2: Recombinant bacteria construction
[0039]Transform recombinant plasmids pRSFduet-1-CYP199A4-HaPux and pETduet-1-HaPuR-HaPux into Escherichia coli E.coli BL21 by heat shock method to obtain recombinant E.coli BL21 / pRSFduet-1-CYP199A4-HaPux / pETduet -1-HaPuR-HaPux.
[0040] Basic operation of heat shock method:
[0041] (1) Melt the Escherichia coli competent cells stored at -80°C in ice for 10 minutes;
[0042] (2) Add 0.1ng-10ng of the corresponding plasmid to E. coli competent cells, mix gently and place in ice for 30min;
[0043] (3) Heat shock in a water bath at 42°C for 45-90 seconds, and immediately place it in ice for 1-2 minutes;
[0044] (4) Add 890 μl of LB medium;
[0045] (5) Put it into a shaker at 37°C, 180rmp-300rmp, and incubate for 1h;
[0046] (6) Take an appropriate amount of bacterial liquid to coat a resistant plate containing 50ug / mL kanamycin and 100ug / mL ampicillin, and place the plate upside down in a 37°C incubator overnight; ...
Embodiment 3
[0051] Example 3: Recombinant Escherichia coli protein expression conditions
[0052] (1) Influence of the concentration of inducer IPTG on the conversion rate
[0053] The recombinant Escherichia coli E. coli BL21 / pRSFduet-1-CYP199A4-HaPux / pETduet-1-HaPuR-HaPux constructed in Example 2 was inoculated as a single colony in 30 mL of LB medium and cultured for 12 hours. Inoculate 10% of the inoculum into a 250mL container containing 30mL LB medium, cultivate at 37°C, 220r, and in the cell OD 600 When it reaches 0.6, add 0, 0.2, 0.4, 0.6, 0.8, 1.0mmol / L IPTG respectively, culture at 25°C for 20h, collect the bacteria by centrifugation, and resuspend the recombinant Escherichia coli with 15mL of LB medium. The transformation system was 15 mL, the temperature was 30° C., the pH was 7, and the substrate concentration was 1 g / L. After 3 hours of whole-cell transformation, HPLC detection of anisaldehyde and p-hydroxybenzaldehyde in the whole-cell transformation solution of recombina...
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