Bioengineering strain for production of novel microorganism-originated fungicides and uses thereof

Patent metadata

Field	Value
Publication number	US-9491943-B2
Application number	US-201114356636-A
Country	US
Kind code	B2
Filing date	Nov 21, 2011
Priority date	Nov 7, 2011
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

Abstract

The present invention relates to a bioengineering strain for production of novel microorganism-originated fungicides and uses thereof. The bioengineering strain for production of microorganism-originated fungicides of the present invention is obtained by transforming a phzH gene recombination expression plasmid into a strain producing phenazine-1-carboxylic acid, wherein the bioengineering strain produces phenazine-1-carboxamide. The present invention utilizes an existing strain producing phenazine-1-carboxylic acid to carry the phzH gene recombination expression plasmid, thereby achieving efficient expression of the phzH gene and transforming phenazine-1-carboxylic acid into phenazine-1-carboxamide. The present invention further discloses uses of the bioengineering strain, including a microorganism-originated fungicide produced by the bioengineering strain through fermentation, and preparation and application of the microorganism-originated fungicide. The antifungal activity of phenazine-1-carboxamide is not influenced by the acidity under which it is used, so that it has a stabilized antifungal activity and can be more effective in prophylaxis and treatment of crop diseases.

First claim

What is claimed is:

1. A bioengineering strain for production of microorganism-originated fungicides, wherein the bioengineering strain is obtained by transforming a phzH gene expression plasmid into a strain producing phenazine-1-carboxylic acid, wherein the bioengineering strain produces phenazine-1-carboxamide; the phzH gene expression plasmid is an expression plasmid cloned with a phzH gene fragment; the phzH gene expression plasmid expresses phzH gene in the strain producing phenazine-1-carboxylic acid where the phzH gene is encoded to produce PhzH (glutamine phenazine-1-carboxylic acid amidotransferase), the amino acid sequence of the PhzH is SEQ ID NO:1; the PhzH is a PhzH of Pseudomonas aeruginosa strains PAO1, and the phzH gene segment is a phzH gene segment of Pseudomonas aeruginosa strains PAO1; the nucleotide base sequence of the phzH gene fragment comprises a complete coding region of the phzH gene and a 5′-end noncoding region; the sequence of the 5′-end noncoding region comprises a polynucleotide segment starting from the first nucleotide base to the 683th nucleotide base of SEQ ID NO.2; and the strain producing phenazine-1-carboxylic acid is a growth-promoting antagonistic bacterium M18 or an engineering strain M18G.

2. The bioengineering strain of claim 1 , wherein the nucleotide base sequence of the phzH gene segment is SEQ ID NO: 2.

3. The bioengineering strain of claim 1 , wherein an expression vector for constructing the phzH gene expression plasmid is an Escherichia coli/Pseudomonas shuttle expression plasmid.

4. The bioengineering strain of claim 3 , wherein the Escherichia coli/Pseudomonas shuttle expression plasmid comprises a strong promoter, the phzH gene segment is cloned behind the strong promoter, and expression of the phzH gene segment is controlled by the strong promoter.

5. The bioengineering strain of claim 4 , wherein the Escherichia coli/Pseudomonas shuttle expression plasmid includes plasmids of pBBR1MCS series.

6. The bioengineering strain of claim 5 , wherein the Escherichia coli/Pseudomonas shuttle expression plasmid is pBBR1MCS-5 and the phzH gene is controlled by a phage promoter T3 of the pBBR1MCS-5 on the premise of ensuring a correct reading frame.

7. The bioengineering strain of claim 1 for use in the production of microorganism-originated fungicides through fermentation.

8. A microorganism-originated fungicide, which is a fermentation broth of the bioengineering strain of claim 1 .

9. The microorganism-originated fungicide of claim 8 , comprising the main fungicidal active component of the fermentation broth comprises phenazine-1-carboxamide.

10. The microorganism-originated fungicide of claim 9 , which is in the fermentation broth, and the content of phenazine-1-carboxamide is 2500-2800 mg/L.

11. The microorganism-originated fungicide of claim 8 , comprising the microorganism-originated fungicide which is obtained by fermenting and culturing the bioengineering strain of any one of claims 1 , 4 - 5 and 6 under conditions suitable for expression of PhzH and production of phenazine-1-carboxylic acid.

12. A pesticide composition for prophylaxis and treatment of plant diseases, which comprises a fungicidally effective amount of the microorganism-originated fungicide of claim 8 or a fungicidally active component from the microorganism-originated fungicide of claim 8 .

Assignees

• Xu Yuquan
• Shen Huifeng
• He Ya-Wen
• Zhou Wanping
• Univ Shanghai Jiaotong

Inventors

• Xu Yuquan
• Shen Huifeng
• He Ya-Wen
• Zhou Wanping

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• A01N43/60 Primary

  1,4-Diazines; Hydrogenated 1,4-diazines · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12Y603/05004

  Asparagine synthase (glutamine-hydrolyzing) (6.3.5.4) · CPC title

• C12P17/12

  containing a six-membered hetero ring · CPC title