The Molecular Mechanism For Degradation Of Phenazine-1-carboxylic Acid In The Strain Sphingomonas Sp.dp58

Shenqinmycin, whose key component is pheazine-1-carboxylic acid (PCA), has a good effect on prevention pimiento epidemic and watermelon wilt diseases, which is broad-spectrum, high efficiency, low toxicity, good environmental compatibility and low cost. During the previous experimentation, it was found that PCA would be degraded easily in field and its protection efficiency was reduced. Therefore, it is important to investigate the biodegrading pathway to improve the prevention efficiency of Shenqinmycin. In our laboratory, a bacterial strain effectively capable of degrading PCA had been isolated and been named Sphingomonas sp. DP58. And the degradation characteristics of PCA by the strain Sphingomonas sp.DP58 were also studied.In this paper, the molecular mechanism for degradation of PCA were studied. Firstly, Sphingomonas sp.DP58 as the material, extract the entire genome, cut into 6-8k fragments by Gene Machines Shear. And construct 6-8Kb Shotgun gene library by the pUC118 vector. After gene library tested,we screened positive transformants using catechol as color indicator. Then the positive transformants were cultivated in PCA the mineral salt medium. We found the positive transformants can degrade PCA, so it was sent to Sangon Biological Engineering Technology & Services Company to test the sequence. According to sequencing results, using NCBI BLASTn tool and the network database (http://www.ncbi.Nlm.Nih.gov/BLAST/), a 6.4-kb fragment encompassing three entire open reading frames (ORFs; orf2 to orf4) and two truncated ones (orf1, orf5) was screened. The three entire open reading frames were 98% similar with three consecutive genes in EDO2 gene cluster of Sphingomonas sp.RW1, and the three genes were EDO2, ORFG3 and ORF G4.To verify the degradation function of each gene, we cloned the three or one or two genes into pEASY-E1 vector under the T7 promoter, and transformed into E.coli BL-21. After degradation test, it was found, the enzymes encoded by orf2 and orf4 genes work together for degradation of PCA and both of them used of the electron transport chain encoded by orf3 gene. Moreover,we isolated the unstable purple metabolites from PCA mineral salt medium of cultivating E.coli overexpression orf2-orf3-orf4 and detected the metabolites by liquid chromatography-mass spectrometry (LC-MS). The speculated oxidation product by orf2-orf3-orf4 was detected and the pathway was further completed.