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Method for increasing cell phloroglucinol synthesis yield and application

A phloroglucinol and cell technology, applied in the field of genetic engineering, can solve the problems of unknown metabolism, phloroglucinol production and yield, etc., and achieve the effect of improving capacity and reducing production and emission

Active Publication Date: 2015-02-18
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

Although this study demonstrated the feasibility of the NOG pathway, however, this study did not use glucose as a substrate to synthesize acetic acid in vivo using the NOG pathway.
The phosphoglucotransferase system (PTS) required for glucose degradation involves complex regulatory mechanisms, and whether the NOG pathway is suitable for in vivo metabolism using glucose as raw material is unknown
In addition, this study only reported the change of acetic acid. The synthesis of acetic acid from acetyl phosphate is only a one-step reaction, while the synthesis of acetyl-CoA from acetyl phosphate and the synthesis of phloroglucinol from acetyl-CoA as a substrate require a 7-step enzyme reaction. , whether overexpression of phosphoketolase gene (fxpk) and fructose 1,6 bisphosphatase gene (fbp) is suitable for such a complex metabolic pathway, whether it can improve the production and yield of this phloroglucinol is unknown

Method used

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  • Method for increasing cell phloroglucinol synthesis yield and application
  • Method for increasing cell phloroglucinol synthesis yield and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Gene multiple resistance factor gene MarA (GenBank accession number: 6060688), polyketene anhydride synthase gene PhlD (GenBank accession number: EU554263), fructose 1,6-bisphosphatase gene fbp (GenBank: ACT45889.1), And the phosphoketolase gene fxpk (GenBank: AY518212.1) was linked together by the method of overlap extension PCR, and then cloned on pET30a by homologous recombination to construct the recombinant plasmid pET-phlD-marA-fxpk-fbp. At the same time, construct the plasmid pA-accADBC, the specific method is the same as the patent CN 102787135B.

Embodiment 2

[0032] The recombinant plasmids pET-phlD-marA-fxpk-fbp and pA-accADBC constructed in Example 1 were co-transformed into Escherichia coli BL21 (DE3) by the heat shock transformation method, and then spread on the culture medium with kanamycin and chloramphenicol Positive clones were screened on LB solid medium plates resistant to two types of protein to obtain recombinant Escherichia coli LWNOGPG1. The control bacteria were constructed by co-transforming Escherichia coli BL21(DE3) with the recombinant plasmids pET-phlD-marA and pA-accADBC by the heat shock transformation method to obtain recombinant Escherichia coli LWNOGPGO (all examples use this as the control bacteria).

Embodiment 3

[0034] The recombinant Escherichia coli fermented and produced in Example 2 is used to produce phloroglucinol, and the steps are as follows:

[0035] Inoculate the recombinant cells at an inoculum volume of 1% by volume to add 50 μg·mL -1 Kanamycin and 30 μg·mL -1 Fermentation was carried out in the M9 fermentation medium of chloramphenicol, and cultured to OD under the conditions of culture temperature 30°C, stirring speed 400rpm, pH 6.0 600 8, add the inducer IPTG to a final concentration of 0.1mmol L -1 , close the ventilation, and continue feeding 40% by weight of glucose to ferment for 12 hours;

[0036] The culture medium was centrifuged to separate the cells and the supernatant, and the supernatant was extracted once with an equal volume of ethyl acetate; the extracted products were combined and concentrated by distillation under reduced pressure. figure 1 and figure 2 ) was identified as phloroglucinol, and the output per liter of culture medium reached 6.5 grams,...

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Abstract

The invention discloses a method for increasing the cell phloroglucinol synthesis yield and application, and belongs to the technical field of genetic engineering. According to the method provided by the invention, a recombined cell co-expresses a phosphoketolase fxpk gene, a fructose 1,6-biphosphatase fbp gene and a poly-ketene anhydride synthetase phld gene and also expresses any one or two of a multiple-resistance-factor MarA gene and an acetyl-coenzyme a carboxylase ACCase gene. According to the method provided by the invention, a new phloroglucinol synthesis way is built in the recombined cell, so that the yield of phloroglucinol is greatly increased; as for a contrast group, the phloroglucinol yield is increased by 68.9 percent; meanwhile, by the method, emission of carbon dioxide in a phloroglucinol synthesis process is also reduced; the method has high environmental benefits.

Description

technical field [0001] The invention relates to a method and application for increasing the synthesis yield of cell phloroglucinol, belonging to the technical field of genetic engineering. Background technique [0002] Phloroglucinol, also known as 1,3,5-trihydroxybenzene and pyroglucinol, is an important fine chemical product and can be used as an intermediate in drug synthesis, fuel coupling agent, tire tackifier and azo composite ink It is widely used in the dyeing process of textiles and leather, the production of plastic capsules, and the replacement of silver iodide for artificial rainfall. In addition, phloroglucinol itself is an excellent pharmaceutical product, an anti-curing agent with superior performance, and has been widely used in antibacterial and antiseptic. [0003] As early as the 1950s, the chemical synthesis process of phloroglucinol had been established and applied to industrial production, including 2,4,6-trinitrotoluene (TNT) pathway, 1,3,5-triiso Pr...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12N15/70C12N1/21C12R1/19
CPCC07K14/245C12N9/16C12N9/2402C12N9/88C12N9/93C12P7/22C12Y301/03011C12Y302/01134C12Y401/02009C12Y604/01002
Inventor 咸漠刘炜曹玉锦孙超
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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