Characterization of flavin-dependent tryptophan halogenases and their application in plant metabolic engineering

A huge variety of halogenated metabolites found in nature have a profound pharmacological effect or act as antimicrobials like the antibiotics, vancomycin and chloramphenicol, or the antitumor agent, rebeccamycin. Due to the high demand for halogenated compounds, which can be met only partially by chemical synthesis, intense research effort has been undertaken to characterize enzymes catalyzing halogenation reactions in nature and to uncover their reaction mechanisms with the aim to utilize biotechnological production strategies for the retrieval of these high-value compounds. Within the last 15 years, several bacterial flavin-dependent tryptophan halogenases have been characterized in terms of their regiospecific chlorine substitution of arenes. In this regard, halogenation of pharmacologically important secondary metabolites is of special interest, to introduce novel functions into given compounds or enable further modification of the skeleton by substitution. Also, capability of tryptophan-halogenases for application in plant biotechnology has been initially tested. Biosynthesis of chlorinated monoterpene indole alkaloids (MIAs) was previously demonstrated in Catharanthus roseus through halogenation of a precursor molecule by two tryptophan halogenases. Based on these findings, catalytic activity of three tryptophan halogenases, namely, RebH wt, RebH Y455W and Stth, was investigated in detail regarding subcellular localization, biosynthesis of valuable fine chemicals and modification of the precursor of all MIAs, strictosidine. In this regard, both the 7-halogenase, RebH wt as well as the 6-halogenase, Stth efficiently catalyzed chlorine substitution of tryptophan and tryptamine in the cytosol and chloroplasts of transiently transformed Nicotiana benthamiana. Halogenated products accumulated in high concentrations, up to 6.17 ± 2 ng/mg fresh weight (6-chlorotryptamine). Strikingly, both halogenases were active in chloroplasts without the partner reductase, RebF, whereas no enzymatic activity was observed ...