Recombinant bacterium for producing acetylacetone and construction method and application thereof
A technology of acetylacetone and a construction method, applied in the field of genetic engineering, can solve the problems of not conforming to low-carbon economy, high energy consumption, complex process and the like
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Embodiment 1
[0043] Embodiment 1 A kind of construction of the recombinant bacteria of synthetic production of acetylacetone by biological method
[0044] 1) Construction of the vector pACYCDuet-dke1
[0045] The acetylacetone lyase gene sequence derived from Acinetobacter johnsonii was codon-optimized, and the gene was synthesized by Suzhou Jinweizhi Company. Taking the synthetic gene as a template, it was obtained by PCR amplification (primers: 5'-CGGGATCCgATGGACTACTGCAACAAAAAACA-3' and 5'-CGGAATTCTTAAGCAGCTTCGTTTTTGGTAGC-3'), and then the target fragment was recovered by a recovery kit. The size of the target fragment was 462bp, and the obtained sequence was The dke1 gene fragment shown in SEQ ID NO.1.
[0046] The obtained dke1 gene fragment and plasmid pACYCDuet were digested with BamHI and EcoRI, and the digested product was recovered and then ligated. The vector and dke1 gene fragment were ligated at a molar ratio of 1:5 at 16 °C for more than 6 h, and the ligated product was trans...
Embodiment 2
[0056] Embodiment 2 Fermentation produces acetylacetone
[0057] The target recombinant bacteria 1 obtained in Example 1 was coated on an LB solid plate containing 100 μg·mL-1 of chloramphenicol; the coated plate was placed in a 37°C constant temperature incubator, and continued to grow until a single clone was grown.
[0058] The obtained engineering strain monoclone was activated in LB culture to obtain seed liquid, and the seed liquid was inoculated into a 500 mL baffle shake flask containing 100 mL of basic modified liquid medium at a ratio of 1:100 volume ratio of seed liquid to fermentation medium. medium (containing 100 μg·mL-1 chloramphenicol), shake cultured at 37 °C and 180 rpm. OD 600 When it reached about 0.6, the temperature was adjusted to 30°C, and IPTG with a final concentration of 0.05mM was added for induction. The pH was adjusted to about 7 with ammonia water every 12 h, and the fermentation was terminated 48 h after the initial induction.
Embodiment 3
[0059] Embodiment 3 Fermentation produces acetylacetone
[0060] The target recombinant bacteria 2 obtained in Example 1 was coated on an LB solid plate containing 100 μg·mL-1 of chloramphenicol; the coated plate was placed in a 37°C constant temperature incubator, and continued to grow until a single clone was grown.
[0061] The obtained single clone of engineered strain was activated in LB culture to obtain seed liquid, and the seed liquid was inoculated into a 500 mL baffle shake flask containing 100 mL of basic modified liquid medium at a ratio of 1:100 to the volume ratio of seed liquid to medium. (containing 100 μg·mL-1 chloramphenicol), shake culture at 37°C and 180rpm. OD 600 When it reached about 0.6, the temperature was adjusted to 30°C, and IPTG was added at a final concentration of 0.05mM for induction. The pH was adjusted to about 7 with ammonia water every 12 h, and the fermentation was terminated 48 h after the initial induction.
[0062] Detection of the pr...
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