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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

Active Publication Date: 2020-02-14
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical synthesis methods generally have disadvantages such as high cost, complicated process, low yield, and high energy consumption, which do not meet the needs of today's low-carbon economy

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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PUM

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Abstract

The invention discloses a recombinant bacterium for producing acetylacetone and a construction method and application thereof, and belongs to the technical field of genetic engineering. The recombinant bacterium is obtained by taking Escherichia coli as a starting strain, introducing a gene encoding an acetylacetone lyase, a gene encoding a methylglyoxal synthase, and a gene encoding triose phosphate isomerase into the Escherichia coli. With this recombinant bacterium, biosynthesis of acetylacetone is achieved for the first time. The recombinant bacterium can synthesize acetylacetone by usingglucose as the sole carbon source, and can obtain 53 mg / L of acetylacetone by fermentation for 48 hours.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant bacterium for producing acetylacetone and a construction method and application thereof. Background technique [0002] Acetylacetone is an organic compound whose standard name is 2,4-pentanedione. Acetylacetone is widely used in the pharmaceutical industry, electronics, chemical industry, petroleum, materials, machinery and other industries, such as intermediates for the synthesis of diabetes drugs, synthesis of veterinary antibacterial drugs, and petroleum cracking catalysts. 90% of acetylacetone in China is used in the production of medicine and veterinary drugs, and 80% in Japan is used as catalyst and solvent for petrochemical reactions. Using acetylacetone as a raw material, tetrazine high nitrogen compounds can also be synthesized by reacting with guanidine and other substances. The latter is an energetic substance with excellent perfo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/26C12R1/19
CPCC12N9/0069C12N9/88C12N9/90C12N15/70C12P7/26C12Y113/1105C12Y402/03003C12Y503/01001
Inventor 赵广冯新军咸漠周怡斐高文杰
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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