Many halogenated compounds are used as intermediates in the synthesis of compounds of interest to the chemical, pharmaceutical industries; however, chemical halogenation involves the use of high-cost reagents that are often harmful to the environment. A large number of halogenated products produced by various organisms of all kingdoms have been found in nature, where these reactions are catalyzed by halogenases which can be classified according to their catalytic mechanism into: a) heme, vanadium and flavin-dependent halogenases, b) non-heme iron halogenases or c) S-adenosyl-L-methionine (SAM) halogenases that react through a nucleophilic pathway. This work focuses on flavin-dependent halogenases.
In this work we present the analysis by genomic mining in native strains for the search for new halogenases applicable to the functionalization of indole compounds of interest. From a collection of 235 strains of Streptomyces isolated from soil in Uruguay, 12 strains were selected for their biosynthetic potential of secondary metabolites from which their complete sequenced genomes were obtained. With data from the literature, a phylogenetic tree was created that allowed the classification of the different halogenases documented in groups of similar regioselectivity. Reference sequences were selected for each specific group, and homology searches were performed in the available genomes using pBLAST. Thus, candidate genes were identified that were analyzed by sequence alignment, detecting conserved motifs and essential amino acids for the mechanism of action of FADH-dependent halogenases. These genes were cloned into pET-28a(+) vectors for expression in E. coli BL21(DE3). The analysis of the reaction crudes by HPLC suggests that these biocatalysts are capable of halogenating the tryptophan indole core.