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A kind of recombinant bacteria producing acetylacetone and its construction method and application

A technology of acetylacetone and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of high cost of chemical synthesis, incompatible with low-carbon economy, and high energy consumption

Active Publication Date: 2021-05-04
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 bacterium that produces acetylacetone synthetically by biological method

[0044] 1) Construction of vector pACYCDuet-dke1

[0045] Codon optimization was performed on the acetylacetone lyase gene sequence derived from Acinetobacter johnsonii, and the gene was synthesized by Suzhou Jinweizhi Company. Using the synthesized gene as a template, it was obtained by PCR amplification (primers: 5'-CGGGATCCgATGGACTACTGCAACAAAAAACA-3' and 5'-CGGAATTCTTAAGCAGCTTCGTTTTTGGTAGC-3'), and then recovered the target fragment with 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, the digested products were recovered, and then ligated. The vector and the dke1 gene fragment were ligated at a molar ratio of 1:5 at 16°C for more than 6 h, and the ligated pr...

Embodiment 2

[0056] Embodiment 2 fermentation produces acetylacetone

[0057] Spread the target recombinant bacteria 1 obtained in Example 1 on an LB solid plate containing 100 μg·mL-1 chloramphenicol; place the coated plate in a constant temperature incubator at 37°C, and continue culturing until a single colony grows.

[0058] The monoclonal engineering strain obtained is activated in LB culture to obtain seed liquid, and the seed liquid is inoculated into a 500 mL baffle shake flask containing 100 mL of basically improved liquid medium at a ratio of 1:100 by volume of the seed liquid to the fermentation medium medium (containing 100 μg·mL-1 chloramphenicol), shaking at 37°C and 180 rpm. OD 600 When it reaches about 0.6, adjust the temperature to 30°C, and add IPTG with a final concentration of 0.05mM for induction. The pH was adjusted to about 7 with ammonia water every 12 hours, and the fermentation was terminated 48 hours after the initial induction.

Embodiment 3

[0059] Embodiment 3 fermentation produces acetylacetone

[0060] Spread the target recombinant bacteria 2 obtained in Example 1 on an LB solid plate containing 100 μg·mL-1 chloramphenicol; place the coated plate in a constant temperature incubator at 37°C, and continue culturing until a single colony grows.

[0061] The single clone of the obtained engineering strain is activated in LB culture to obtain the seed solution, which is inoculated into a 500mL baffle shake flask containing 100mL of basic modified liquid medium at a ratio of 1:100 of the volume ratio of the seed solution to the medium (Containing 100 μg·mL-1 chloramphenicol), shaking culture at 37°C and 180rpm. OD 600 When it reaches about 0.6, adjust the temperature to 30°C, and add IPTG with a final concentration of 0.05mM for induction. The pH was adjusted to about 7 with ammonia water every 12 hours, and the fermentation was terminated 48 hours after the initial induction.

[0062] The detection of product (em...

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PUM

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Abstract

The invention discloses a recombinant bacterium for producing acetylacetone and its construction method and application, belonging to the technical field of genetic engineering. The recombinant bacterium uses E. The genes of aldehyde synthase and triose phosphate isomerase were introduced into Escherichia coli to obtain recombinant bacteria, and the biosynthesis of acetylacetone was realized for the first time. The recombinant bacteria constructed in the present invention can use glucose as the only carbon source to synthesize acetylacetone, and ferment for 48 hours to obtain 53 mg / L acetylacetone.

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, its construction method and application. Background technique [0002] Acetylacetone is an organic compound whose standard name is 2,4-pentanedione. Acetylacetone is widely used in many industries such as pharmaceutical industry, electronics, chemical industry, petroleum, materials, machinery, etc., for example, it can be used as an intermediate in the synthesis of drugs for treating diabetes, in the synthesis of veterinary antibacterial drugs, and as a catalyst for petroleum cracking. 90% of acetylacetone in China is used in the production of medicine and veterinary drugs, and 80% in Japan is used in catalysts and solvents 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 lat...

Claims

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Application Information

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Patent Type & Authority Patents(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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