Gene engineering bacterial strain used for producing phenazine-1-methanamide and its purpose

A genetically engineered strain and formamide technology, applied in the field of bioengineering, can solve the problems of low yield and efficiency of phenazine-1-carboxamide, achieve broad-spectrum antifungal activity, simple and easy production process and good stability Effect

Active Publication Date: 2014-03-19
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The object of the present invention is to overcome the defects of lower production and efficiency of phenazine-1-carboxamide produced by existing strains in the above-mentioned prior art, and provide a genetically engineered strain for producing phenazine-1-carboxamide and its application

Method used

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  • Gene engineering bacterial strain used for producing phenazine-1-methanamide and its purpose
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  • Gene engineering bacterial strain used for producing phenazine-1-methanamide and its purpose

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Embodiment 1

[0046] Preparation and identification of embodiment 1, phenazine-1-carboxamide

[0047] 1) Activation of Pseudomonas chloropinus strains: Prepare solid LB or KMB medium respectively; take out the frozen Pseudomonas chloropinus HT66, inoculate it on the solid medium and activate it at 26-32°C for 48 hours, then transfer to Plant on a new solid medium, and continue to pass for 2 to 3 times;

[0048] 2) Seed preparation of Pseudomonas chloropinus: prepare liquid LB or KMB medium; pick a ring of activated bacteria, inoculate into 50mL liquid medium (packed in a 250mL Erlenmeyer flask), and place at 26-32°C Shake culture on a constant temperature shaker for 10-20 hours, and the speed of the shaker is 150-5000 rpm. Then, the seed solution is further transferred to 1000-5000 mL of liquid medium, and cultivated under the same conditions to obtain a large number of vigorous and robust seeds;

[0049] Wherein, the composition of the LB medium is as follows: each liter medium contain...

Embodiment 2

[0056] Embodiment 2, the construction of rpeA gene in vitro mutant

[0057] Using the plasmid pUCGM as a template, the gentamicin resistance gene aacC1 was amplified. Design of primer 5'-TATTAA based on the sequence of rpeA in the genome of Pseudomonas chloropinus HT66 TCTAGAC CTGTTCAGCCGTTCCGAAT-3' (XbaI, such as SEQ ID NO.3) and 5'-AATTAT GAATTC - CCACGCCCAGTTGATCCT-3' (EcoRI, such as SEQ ID NO.4), using the genomic DNA as a template to amplify the corresponding fragment in the genome. The PCR product was connected to the pMD19T vector, transformed into Escherichia coli, and the plasmids were extracted for verification, and the plasmids pMD19TA and pMD19TG were obtained. These two plasmids were digested with ClaI respectively, purified through the column, and connected with T4 ligase to obtain the plasmid pMD19TAG. Using EcoRI The shuttle plasmids pEX18Tc and pMD19TAG were double digested with XbaI, and the digested products were purified through a column, and the obta...

Embodiment 3

[0059] Embodiment 3, the construction of psrA gene in vitro mutant

[0060] Using the pBS(kan) plasmid as a template, a 1.0KMB kan gene (Kana resistance gene) was amplified by PCR. Primers were designed according to the sequence of the psrA gene in the genome of Pseudomonas chloropinus HT66, 5'-TT AAGCTT ACGGTCGGGTCGTCGCTGCATA-3' (HindIII, such as SEQ ID NO.5) and 5'-AA GAGCTC CGCTGTTCATGCCACGGATAAAG-3' (SacI, such as SEQ ID NO. 6). Using HT66 genomic DNA as a template, a 1.5KMB psrA gene was amplified by PCR. After digestion with HindIII and SacI, it was cloned into the same site of the pEX18Tc plasmid to obtain the recombinant plasmid pEX18Tc-psrA. The competent cells of E.coli DH5α were transferred to make them resistant to tetracycline.

[0061] The kan gene and the recombinant plasmid pEX18Tc-psrA were digested with SphI respectively, and after recovery from rubber tapping, they were ligated overnight using T4 DNA ligase. The ligation product was directly heat-sho...

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Abstract

The invention discloses a gene engineering bacterial strain used for producing phenazine-1-methanamide and its purpose; which is characterized in that knockout of psrA or rpeA gene in Pseudomonas chlororaphis HT66(Pseudomonas chlororaphis HT66) CCTCC NO: M 2013467 genome is carried out. According to the invention, a strain of Pseudomonas chlororaphis is performed with seed culture, fermentation, extraction, concentration and crystallization to obtain phenazine-1-methanamide with purity of more than 96%. After the deletion of psrA and rpeA gene in the bacterium genome, the fermentation output of phenazine-1-methanamide can reach as high as 1800ppm. The agar plate bacteriostasis tests found that phenazine-1-methanamide has obvious inhibition effect on common plant pathogenic fungi such as Rhizoctonia solani, fusarium oxysporum, pythium wltmum and Stevia Septoria, has good stability and activity, and has good application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a genetically engineered bacterial strain for producing phenazine-1-carboxamide and its application. Background technique [0002] my country is a country with a large population, and the problem of grain production caused by pests and diseases is very serious. As a means of agricultural production, pesticides play an important and indispensable role in maintaining human life, the quality of life and the environment, and the sustainable use of land and biological resources. According to the data from the National Bureau of Statistics, the national pesticide technical production in November 2012 was 336,131 tons, an increase of 10.92% compared with the output in October (303,043 tons), and a year-on-year increase of 39.2% compared with the output in November last year (241,408 tons). However, due to the extensive use of chemical pesticides and fertilizers by humans, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/09C12P17/12C05F11/08A01N63/00A01N43/60A01P3/00C12R1/38
CPCY02E50/30Y02W30/40
Inventor 彭华松张雪洪张平原王威胡洪波
Owner SHANGHAI JIAO TONG UNIV
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