Genetically engineered bacterium for improving yield of phloroglucinol as well as construction method and application of genetically engineered bacterium

A technology of genetic engineering bacteria and phloroglucinol, which is applied in the field of genetic engineering, can solve the problem of low phloroglucinol yield and achieve the effect of improving the synthesis ability

Active Publication Date: 2019-03-08
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the problem that the yield of phloroglucinol is too low in biological synthesis, the invention provides a genetically engineered bacterium with high phloroglucinol synthesis ability, the genetically engineered bacterium overexpresses triose phosphate isomerase gene tpiA, polysaccharide Keto synthase gene phlD, multiple resistance activator marA and acetyl CoA carboxylase gene ACCase, the starting strain is Escherichia coli

Method used

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  • Genetically engineered bacterium for improving yield of phloroglucinol as well as construction method and application of genetically engineered bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1. The construction method of the genetic engineering bacteria that improves phloroglucinol output.

[0040] The genetic engineering bacteria provided in this example overexpress triose phosphate isomerase gene tpiA, polyketide synthase gene phlD, multiple resistance activator marA and acetyl CoA carboxylase gene ACCase, and the starting strain is Escherichia coli.

[0041] The specific construction method is as follows:

[0042] 1). The specific process of preparing a recombinant plasmid containing the triose phosphate isomerase gene tpiA is as follows:

[0043] Using oligonucleotide 5'-ATCGCATATGCGACATCCTTTAGTGATG-3' as forward primer, denoted as tpiA-F, 5'-GAAGATCTTTAAGCCTGTTTAGCCGCTTC-3' as reverse primer, denoted as tpiA-R, Escherichia coli K-12[Escherichia coli K-12] genomic DNA is a template, and the gene tpiA is amplified by the polymerase chain reaction (PCR) method, and its nucleotide sequence is shown in SEQ ID NO.1, at the 5' end and 3' end of th...

Embodiment 2

[0047] Example 2. Utilize the genetically engineered bacteria obtained in the examples to ferment and produce phloroglucinol, and enhance the metabolic pathway of Escherichia coli to synthesize ATP and pyruvate by expressing the gene tpiA in the genetically engineered bacteria, thereby improving the synthesis of phloroglucinol by Escherichia coli Phenol production. The steps are as follows:

[0048] 1. Shake flask fermentation experiment

[0049] 1) Cultivation of primary seed liquid: Inoculate the recombinant Escherichia coli colony prepared in Example 1 on a solid LB plate in LB seed liquid medium, and add ampicillin and 34 μg / mL chloramphenicol at a final concentration of 100 μg / mL prime, and grow at 37°C for 10-12h to obtain the primary seed liquid of the genetically engineered bacteria of the present invention.

[0050] Control group: Inoculate Example 1 on a solid LB plate in LB seed liquid medium to prepare a single colony of the control strain, and add a final concen...

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Abstract

The invention provides a genetically engineered bacterium for improving the yield of phloroglucinol as well as a construction method and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The invention provides the genetically engineered bacterium for over-expression of a triosephosphate isomerase gene tpiA, a polyketide synthase gene phlD, a multiple resistance activation factor marA and an acetyl-CoA carboxylase gene ACCase, so that the yield of the phloroglucinol synthesized by phloroglucinol producing strains is obviously improved; through shake-flask fermentation, the yield of phloroglucinol synthesized by transformed strains is 1.85 times of the yield of phloroglucinol synthesized by contrast strains. Therefore, the genetically engineered bacterium can be used for industrial production of phloroglucinol.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium for increasing the yield of phloroglucinol, a construction method and application thereof. Background technique [0002] Phloroglucinol, also known as 1,3,5-trihydroxybenzene, is an important fine chemical product. Phloroglucinol itself is a drug that can be used as a smooth muscle relaxant with low toxicity; its derivative trimethoxybenzene is an effective sedative and has been used as a cosmetic additive. The nitro compound of phloroglucinol, trinitrophloroglucinol, is an important explosive and a synthetic intermediate for several other explosives. The chemical synthesis route of phloroglucinol was established at the beginning of the 20th century, and has been gradually improved by many researchers, but the chemical synthesis method still has many disadvantages, such as many by-products, difficult separation and purif...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/22C12R1/19
CPCC12N9/90C12N9/93C12N15/70C12P7/22C12Y503/01001C12Y604/01002
Inventor 张汝兵咸漠刘闻孙超
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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