Identification Of Aniline Dioxygenase Gene Cluster Ado From Sphingomonads

Aniline and its derivatives are very important chemical material in industrial production. Meanwhile, they are the intermediate of aniline-based herbicides such as phenylurea herbicides and their environmental detection rate are higher than phenylurea herbicides. Aniline compounds have toxicity that do harm to human health. In environment, degradation of aniline compounds mainly depends on microorganism. Currently, there are many studies on microbiological degradation, but most of them focus on isolation of aniline-degrading bacterium and metabolic pathway. However, no reported aniline-degrading bacteria have broad substrate spectrum toward aniline derivatives. The ideal strain for bioremediation of contaminated environments need to degrade more kinds of residual aniline derivatives. Therefore, to construct a broad-spectrum aniline-degrading strain, we need to isolate more strains with different substrate spectrum and reveal their molecular basis, which it is of great theoretical and practical significance to clean up the residual hytotoxicity and environmental pollution.Although, several different aniline-degrading gene clusters had been cloned, studies had little about degradation mechanism of aniline derivatives from degrading gene and enzyme. In particular, it is unclear that how does aniline dioxygenase gene cluster catalyze the substrate and what does influence the substrate spectrum. Sphingobium sp.YBL2, an excellent aniline-based herbicides degrading strain, belongs to sphingomonads, which contains Sphingomonas, Novosphingobium and Sphingopyxis. Sphingomonads is very similar to pseudomonad in classification and metabolism, but it has the conservative gene cluster which different from other aniline-degrading gene clusters. Now, we had got the no reported conservative aniline-degrading gene cluster.The ORF comparison show that structural genes of aniline degradation gene clusters from Sphingobium sp. YBL2 are similar to that of Pseudomonas reported. But the genetic composition and arrangement of aniline dioxygenase gene cluster are quite different. The oxidation of aniline by dioxygenase is the first step in aniline degradation process. So Far, most of genes’ functions in aniline dioxygenase gene cluster have not been determined. We think that the differences among aniline dioxygenase gene clusters affect their substrate spectrum. Therefore, we use the aniline dioxygenase gene cluster (ado) from Sphingobium sp. YBL2 as materials for further research on the transcriptional regulation mechanisms and functions of the genes comparing with typical aniline dioxygenase gene cluster reported.In this paper, we use the two ways that RT-PCR and promoter marker to preliminarily study the transcriptional regulation mechanisms of aniline dioxygenase gene cluster (ado). The results show that the gene cluster is constitutively expressed, where gene R plays a negative role. We use analytical methods such as UV and GC-MS to detect degradation of aniline derivatives. Compared with typical aniline dioxygenase gene clusters, we had identified the substrate spectrum of gene cluster and found the rules of substrate degradation. Refer to the substrate spectrum and literature reported, we researched on gene Q and found that gene Q is important to substrate spectrum. Finally, we builded a broad spectrum engineered bacteria for phenylurea herbicides degradation by connecting gene puhB and aniline-degrading gene cluster and verified its degradation.