Method for synthesizing phloroglucinol by microbial catalysis

A kind of phloroglucinol and microorganism technology, applied in the field of microbial catalytic synthesis of phloroglucinol

Active Publication Date: 2010-06-09
青岛生物能源与过程研究所
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AI Technical Summary

Problems solved by technology

Biosynthesis of phloroglucinol has not been reported in the past

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0024] (1) Screening of phloroglucinol-resistant mutants

[0025] Take 2ml bacterial solution and add 1ml 0.1mol / L NaNO 2 solution and 7ml pH4.4 HAC-NaAC buffer solution, cultured with shaking in a water bath at 37°C for 1-5min. Take 0.5ml and add to 9.5ml 0.07mol / L pH 8.6Na 2 HPO 4 Mix the buffer solution evenly, spread it on a plate containing 5g / L LiCl, and incubate at 37°C in the dark.

[0026] (2) PhlD1 gene cloning and acquisition of recombinant strains

[0027] Genomic DNA of Pseudomonas fluorescens Pf-5 was extracted, and using it as a template, primers were designed according to GenBank sequence, and polyketide synthase gene (phlD) was amplified by PCR.

[0028] (3) Site-directed mutation of the phlD gene

[0029] According to the preference of bacterial amino acid codons, the mutant gene phlD1 was obtained by using the TaKaRa MutanBEST kit and the site-directed mutagenesis method, and the phlD gene was mutated into phlD1. The mutation site rules are as follows: ...

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Abstract

A method for synthesizing phloroglucinol by microbial catalysis relates to a method for synthesizing phloroglucinol by catalysis of engineering escherichia coli cells. The method for synthesizing phloroglucinol by catalysis of engineering escherichia coli cells comprises the following steps of: compounding, inducing, selecting and optimizing anti-phloroglucinol mutant strains by employing HNO2 and LiCl; carrying out PCR amplification by taking the genome DNA of bacterium Pseudomonas fluorescens Pf-5 as a template to obtain a polyketide synthase gene (phlD); connecting with a pET carrier after cleavage, transferring to the anti-phloroglucinol mutant strains, and inudcing and expressing a target protein by using IPTG to obtain the engineering escherichia coli for catalyzing and synthesizing the phloroglucinol. The phloroglucinol can be obtained in the fermentation liquor after the engineering escherichia coli are fermented, and the phloroglucinol is extracted and concentrated to get the finished phloroglucinol. The invention first provides the method for synthesizing phloroglucinol by catalysis of the engineering escherichia coli, and lays a solid foundation for catalyzing and synthesizing phloroglucinol by an environment-friendly synthetic process in future.

Description

technical field [0001] The present invention constructs the phloroglucinol metabolic synthesis pathway in phloroglucinol-tolerant Escherichia coli, and uses microbial cells to catalyze the synthesis of phloroglucinol, which is a method for engineering Escherichia coli to biocatalyze the synthesis of phloroglucinol. Background technique [0002] Phloroglucinol is an important chemical intermediate, mainly used in drug synthesis, dye coupler, tire tackifier and azo composite ink and other raw materials; at the same time, it is used in textile and leather dyeing process, production of plastic capsules, and in place of silver iodide There are also important applications in artificial rainfall and preservatives for certain synthetic materials. [0003] The market price of phloroglucinol is about 500,000 yuan / ton, which is very expensive. The source of phloroglucinol is mainly through chemical synthesis. As early as the 1950s, M.L.Kasrens used 2,4,6-trinitrotoluene (TNT) as raw ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12N1/21C12N15/52C12N15/70C12R1/19
Inventor 咸漠杨建明李强徐鑫孟鑫张磊何玉财仝新利
Owner 青岛生物能源与过程研究所
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