Screening, Cloning And Heterologous Expression Of FADH₂-Dependent Halogenases In Actinomycetes Islolated From The Arctic Ocean

Halogenated compounds are a important class of secondary metabolites, with wide applications in the field of pharmaceutical, agriculture and industry. Halogens introduced into molecular structure can significantly increase the biological activity of halogenased compounds. Traditional chemical synthesis can hardly obtain active halogenated compounds with substrate specificity and regioselectivity, while halogenases produced by many microorganisms are modifying enzymes. Among them FADH2-dependent halogenases are the most important class of halogenases. Study on halogenase genes of actinomycetes isolated from the Actic Ocean would be conducive to discover new halogenases and halometabolites, for better understanding of modification and catalytic mechanism of halogenase, as well as to lay the foundation for further combinational biosynthesis of novel types of halogenated compounds.Phylogenetic diversity analysis on31actinomycetes isolated from sediment samples of high latitude region in the Arctic Ocean showed that they belong to Streptomyces, Nocardiopsis, and Brevibacterium respectively. A total of22strains with bacterial and/or fungal inhibition activity were screened out by agar block method, among them72.7%of the positive strains showed inhibition activity against Candida albicans, and68.2%of them showed inhibition activity against Bacillus subtilis.Six halogenase gene-containing strains which all involves PKS (polyketide synthase) and NRPS (nonribosomal peptide synthetase) genes were obtained through PCR screening. Five of them showed strong inhibition activity against Candida albicans or against Bacillus subtilis. In the light of analysis of neighbor-joining tree, four of them belong to tryptophan halogenase, while the other two belong non-tryptophan halogenase. Based on the analysis of microbial inhibition activity assay and the presense of genes related to secondary metabolite production, Streptomyces sp.604F and Streptomyces sp.551F were selected as the representative strains for cloning and expression of non-tryptophan halogenase and tryptophan halogenase, respectively.The halogenase genes of604F (fdh604) and551F (fdh551) were cloned through high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). fdh604with1443bp and its flanking sequences were obtained. Sequence similarity and phylogenetic analysis indicated that FDH604was most closely related to halogenases involving in catalyzing the chlorination of glycopeptides, with the highest similarity of58%. The cloning and anslysis of fdh604provided guidance for searching target halometabolites, and laid the foundation for obtaining the biosynthetic gene cluster. fdh551with1605bp was cloned, showing79%similarity with tryptophan6-halogenase sequence from Streptomyces albogriseolus MJ286-76F7. FDH551was cloned into pET28a and heterologously expressed in E. coli. After optimizing expression conditions via orthogonal test and two-step purification, the pure FDH551(approximately65kDa) of electrophoresis purity was obtained, its recovery achieved26.35%.