DOI: 10.26083/tuprints-00005357
title: Characterization of flavin-dependent tryptophan halogenases and their application in plant metabolic engineering

description: 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 after translocation to the apoplast. Moreover, tandem halogenation of tryptophan, but not tryptamine, was observed when both enzymes were co-localized in the cytosol or chloroplasts. RebH wt alone also synthesized minor amounts of di-chlorotryptophan. Additionally, both enzymes were shown to efficiently catalyze bromide substitution of tryptophan, resulting in a variety of mono-brominated and di-brominated tryptophan molecules as well as chloro-bromotryptophan. The engineered 7-halogenase, RebH Y455W, reported to predominantly chlorinate tryptamine instead of tryptophan, showed only low catalytic activity in planta. This inefficiency could not be compensated by optimization of the metabolic flux through anchoring of the involved enzymes within a protein scaffold. On top of the aforementioned studies on MIA biosynthesis, the halogenases were also incorporated into a newly designed indoxyl biosynthetic pathway to synthesize chlorinated indican derivatives in planta. Introduction of tryptophanase TnaA from Escherichia coli, into a previously investigated artificial indican pathway resulted in high yields of 6- and 7-chloroindican. Moreover, subcellular localization of enzymes of this optimized metabolic route was shown to be very flexible and allowed both co-localization and separation of enzymes in the cytosol and chloroplasts. Remarkably, the human CYP450 2A6 mutant L240C/N297Q was also active in chloroplasts, which implies transport of electrons required for substrate oxidation, presumably from the photosystem I, to the cytochrome P450. Further optimization of this production system by introduction of additional enzymes or establishment of stable transgenic tobacco plants and cell cultures might enable efficient and ecological biosynthesis of a huge variety of highly valuable indigoids in planta. 
To reach the final goal of obtaining halogenated monoterpenoid indole alkaloids in a synthetic pathway, reconstitution and modification of the strictosidine biosynthetic pathway was analyzed in planta. In this regard, accumulation of 14 new metabolites was associated with transgene expression; biosynthesis of five of those was enhanced by co-infiltration of the initial precursor, geraniol, whereas four were synthesized exclusively upon geraniol supplementation. However, no actual pathway intermediates could be identified within this group. Even though biosynthesis of precursors was enhanced and constitutive gene expression was facilitated by establishing transgenic tobacco lines, no metabolites of interest were observed. Therefore, the biosynthetic track needs to be further optimized by elimination of potential bottlenecks and replacement of the inefficient RebH Y455W by wild type halogenases. Taken together, the analyzed flavin-dependent tryptophan halogenases represent promising tools for biosynthesis of valuable molecules. Their substantive efficiency enables economical production of halogenated metabolites in high yields in planta.