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Recombinant bacterium for synthesizing butantriol oleate and construction method and application thereof

A technology for synthesizing butanetriol oleate and recombinant bacteria, which is applied in the field of genetic engineering and can solve the problems of pollution, difficult separation of BT and harsh conditions.

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

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Problems solved by technology

However, the current methods of synthesizing BT are generally chemical methods, and there are problems such as harsh conditions and serious pollution in the synthesis of BT by chemical methods, so the development of new BT synthesis methods is a problem that needs to be solved at present.
At present, there have been studies using biological methods to synthesize BT, but there is still the problem that BT in the fermentation broth is difficult to separate

Method used

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  • Recombinant bacterium for synthesizing butantriol oleate and construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1 Construction of a recombinant bacterium for synthesizing butanetriol oleate

[0049] 1) Construction of vector pACYCDuet-atf

[0050] In this example, the acyltransferase gene atf (gene ID in NCBI is 947324) derived from E. coli was obtained, which was obtained by PCR amplification using the E. coli genome as a template (primer: 5'-GGAATTCAAGGAGATATACCATGAAAGACGTTGTGATTG-3 ' and 5'-GCGTCGACCTATTCGTCACGTTCAATG-3'), and then use the recovery kit to recover the target fragment, the target fragment size is 1182bp.

[0051] The obtained atf gene fragment and plasmid pACYCDuet-1 were digested with EcoRI and SalI, the digested product was recovered, and then ligated. The vector and the atf gene fragment were connected at a molar ratio of 1:5 at 16°C for more than 6 hours, and the ligated product was transformed E.coliDH5α, and then coated with 100μg·mL -1 On the LB solid plate of chloramphenicol, positive clones were screened by PCR. After the recombinant plasm...

Embodiment 2

[0068] Embodiment 2 fermentation produces butanetriol oleate

[0069] Apply the recombinant bacteria obtained in Example 1 to -1 Kanamycin and 100 μg·mL -1 Chloramphenicol LB solid plate; place the coated plate in a constant temperature incubator at 37°C, and continue to culture until a single colony grows.

[0070] Activate the single clone of the obtained engineering strain in LB culture, and cultivate it at 37°C and 180rpm for 12 hours to obtain a seed liquid, which is inoculated into LB liquid medium containing 100mL at a ratio of 1:100 to the volume of the medium In a 500mL shake flask (containing 100μg·mL -1 Kanamycin and 100 μg·mL -1 Chloramphenicol), cultured with shaking at 37°C and 180rpm. OD 600 When it reaches about 0.6, the temperature is adjusted to 30°C, and IPTG with a final concentration of 0.05mM is added for induction, and 1,2,4-butanetriol with a final concentration of 10g / L and sodium oleate with a final concentration of 10g / L are added at the same ti...

Embodiment 11

[0083] Example 1 1) Primer 1

[0084] 1

[0085] ggaattcaag gagatatacc atgaaagacg ttgtgattg 39

[0086] 2

[0087] 27

[0088] DNA

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Abstract

The invention discloses a recombinant bacterium for synthesizing butantriol oleate as well as a construction method and application of the recombinant bacterium, belonging to the technical field of gene engineering. For the recombinant bacterium, escherichia coli is used as an original strain, and a gene for encoding acyltransferase, a gene for encoding acyl CoA synthetase, a gene for encoding enoyl ACP reductase, a gene for encoding glycerol kinase, a gene for encoding 3-phosphoglycerol acyltransferase and a gene for encoding DAGP phosphatase are introduced into the escherichia coli to obtainrecombinant bacterium. Through fermentation, first synthesis of 1,2,4-butantriol oleate is realized. The recombinant bacterium constructed by the invention is fermented in an LB culture medium for 24hours, and the esterification of butantriol and sodium oleate can be realized to obtain the butantriol oleate, so that a new thought is provided for separation and purification of the butantriol.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant bacterium for synthesizing butanetriol oleate and its construction method and application. Background technique [0002] 1,2,4-Butanetriol (Butanetriol, BT) is a colorless and transparent substance similar to glycerin. Its main function is as a chemical intermediate in organic synthesis. It is widely used in JG, tobacco, cosmetics, Papermaking, medicine and polymer materials and other fields. Compared with nitroglycerin, its nitroglycerin has better thermal stability, less toxicity, less volatility, and less impact, and its demand is increasing year by year. At present, scholars from various countries have carried out a series of studies on the biological synthesis of BT, and have made great progress. Butanetriol is an alcohol substance, easily soluble in water, and difficult to separate from the fermentation broth. The Frost team once use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/64C12R1/19
CPCC12N9/1025C12N9/16C12N9/1029C12N9/1205C12N9/001C12P7/6445C12Y203/01086C12Y207/0103
Inventor 赵广冯新军咸漠李美洁徐超
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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