Cloning Of Violacein Biosynthesis Gene Cluster And Crystal Structure Analysis Of Viod

Violacein, a blue-purple pigment, is produced by Gram-negative bacterium,such as Chromobacterium violaceum, Janthinobacterium lividu, Collomonas sp., Chromoba-cterium fluviatile, Pseudoalteromonas luteoviolacea, Duganella sp., and so on. Violacein consists of three structure units:5-hydroxyindole,2-pyrrolidone and oxindole. Violacein has received much attention in recent years due to its significant biological functions, such as broad spectrum antibacterial activity, anti-fungal, anti-viral, anti-tumoral, anti-protozoan, anti-oxidant activities, and so on. These characteristics make violacein have broad application in food, therapeutic and dying industry. Violacein is biosynthesized by five enzymes, VioA, VioB, VioC, VioD and VioE in the order of VioA→VioB→VioE→VioD→ VioC.In this study, the violacein producing bacterium ZLP-XI was isolated from the rhizosphere soil of Ferns. Based on its morphological; physiological, biochemical properties and combined with 16S rDNA homology analysis, the strain was characterized as Duganella and was given the name Duganella sp. ZLP-XI.In order to clone the violacein biosynthesis genes of the strain ZLP-XI, we used degeneration PCR and SEFA PCR method. We successfully cloned a 10431bp gene cluster for violacein biosynthesis. The gene cluster contains five orfs named as vioA, vioB, vioC, vioD and vioE respectively. The lengths of these orfs are 1305bp,3027bp,1290bp,1119bp and 582bp respectivley. Nucleotide BLAST result showed that either the gene cluster or the encoding genes VioABCDE has a closest relationship with those of Pseudoduganella violaceinigra DSM 15887.To elucidate the biosynthesis mechanism of violacein, we constructed the expression vector by inserting the target genes into pET28a vectors to overexpress the five enzymes resptectively. The five target proteins were all successfully expressed in E.Coli after induced with IPTG.Bying using vapor diffusion method, diamond-like crystals were obtained for VioD. A complete dataset was collected to 1.7A. Molecular replacement method was used to solve the crystal structure of VioD by using its homolog as the searching template. The result showed that the asymmetric unit contains two VioD molecules and the structure is under refinement.