A kind of method and application of improving cell phloroglucinol synthesis output

A phloroglucinol and cell technology, which is applied in the field of genetic engineering, can solve the problems that metabolism cannot be known, and the output and yield of phloroglucinol cannot be known, so as to achieve the effect of improving capacity and reducing production and emission.

Active Publication Date: 2017-05-24
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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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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  • A kind of method and application of improving cell phloroglucinol synthesis output
  • A kind of method and application of improving cell phloroglucinol synthesis output

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The multi-resistance factor gene MarA (GenBank accession number: 6060688), the polyketene anhydride synthetase gene PhlD (GenBank accession number: EU554263), the fructose 1,6-bisphosphatase gene fbp (GenBank: ACT45889.1), And phosphoketolase gene fxpk (GenBank: AY518212.1) were linked together by 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, the plasmid pA-accADBC was constructed, and the specific method was 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 coated with kanamycin and chlorin Positive clones were screened on the LB solid medium plate with two kinds of resistance, and recombinant E. coli LWNOGPG1 was obtained. The construction of the control bacteria was that the recombinant plasmids pET-phlD-marA and pA-accADBC were co-transformed into Escherichia coli BL21 (DE3) by the heat shock transformation method to obtain recombinant Escherichia coli LWNOGPG0 (all examples used this as the control bacteria).

Embodiment 3

[0034] Using the recombinant E. coli constructed in Example 2 to ferment to produce phloroglucinol, the steps are as follows:

[0035] Inoculate the recombinant cells at a volume ratio of 1% to add 50μg·mL -1 Kanamycin and 30μg·mL -1 Fermentation is carried out in the M9 fermentation medium of chloramphenicol, cultivated to OD under the conditions of culture temperature 30℃, stirring speed 400rpm, and pH 6.0 600 For 8, add the inducer IPTG to the final concentration of 0.1mmol·L -1 , Close the ventilation, continue to feed 40 wt% glucose fermentation for 12 hours;

[0036] The culture broth was centrifuged to separate the cells and the supernatant. The supernatant was extracted once with an equal volume of ethyl acetate; the extracted products were combined and concentrated by distillation under reduced pressure. The resulting solid powder was dissolved in deionized water and subjected to hydrogen spectrum carbon spectrum ( figure 1 with figure 2 ) Was identified as phloroglucinol,...

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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 improving the synthetic yield of cell phloroglucinol, and belongs to the technical field of genetic engineering. Background technique [0002] Phloroglucinol, also known as 1,3,5-trihydroxybenzene and pyrogallol, is an important fine chemical product that can be used as an intermediate for drug synthesis, fuel coupling agent, tire tackifier and azo composite ink Such raw materials are widely used in textile and leather dyeing processes, the production of plastic capsules, and the replacement of silver iodide for artificial rainfall. In addition, phloroglucinol itself is an excellent medicinal product, an anti-curing agent with superior performance, and has long been widely used in antibacterial and antiseptic applications. [0003] As early as the 1950s, the chemical synthesis process of phloroglucinol has been established and applied to industrial production, including 2,4,6-trinitrotoluene (TNT) pathway...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P7/22C12N1/21C12R1/19
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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