Characterization of flavin-dependent tryptophan halogenases and their application in plant metabolic engineering by Sabine Fräbel.
A huge variety of halogenated metabolites found in nature have a profound pharmacological effect or act as antimicrobials like the antibiotics, vancomycin and chloramphenicol, antitumor agent, rebeccamycin. Due to high demand for compounds, which can be met only partially by chemical synthesis, intense research effort has been undertaken characterize enzymes catalyzing halogenation reactions uncover their reaction mechanisms with aim utilize biotechnological production strategies retrieval these high-value compounds. Within last 15 years, several bacterial flavin-dependent tryptophan halogenases characterized terms regiospecific chlorine substitution arenes. In this regard, pharmacologically important secondary is special interest, introduce novel functions into given compounds enable further modification skeleton substitution. Also, capability tryptophan-halogenases application plant biotechnology initially tested. Biosynthesis chlorinated monoterpene indole alkaloids (MIAs) was previously demonstrated Catharanthus roseus through precursor molecule two halogenases. Based on findings, catalytic activity three halogenases, namely, RebH wt, Y455W Stth, investigated detail regarding subcellular localization, biosynthesis valuable fine chemicals all MIAs, strictosidine. both 7-halogenase, wt well
6-halogenase, Stth efficiently catalyzed tryptamine cytosol chloroplasts transiently transformed Nicotiana benthamiana. Halogenated products accumulated concentrations, up 6.17 ± 2 ng/mg fresh weight (6-chlorotryptamine). Strikingly, were active without partner reductase, RebF, whereas no enzymatic observed after translocation apoplast. Moreover, tandem tryptophan, but not tryptamine, when co-localized chloroplasts. alone also synthesized minor amounts di-chlorotryptophan. Additionally, shown catalyze bromide resulting mono-brominated di-brominated molecules chloro-bromotryptophan. The engineered Y455W, reported predominantly chlorinate instead showed low planta. This inefficiency could compensated optimization metabolic flux anchoring involved within protein scaffold. On top aforementioned studies MIA biosynthesis, incorporated newly designed indoxyl biosynthetic pathway synthesize indican derivatives Introduction tryptophanase TnaA from Escherichia coli, artificial resulted yields 6- 7-chloroindican. localization optimized route very flexible allowed co-localization separation Remarkably, human CYP450 2A6 mutant L240C/N297Q chloroplasts, implies transport electrons required substrate oxidation, presumably photosystem I, cytochrome P450. Further system introduction additional establishment stable transgenic tobacco plants cell cultures might efficient ecological highly indigoids
To reach final goal obtaining monoterpenoid synthetic pathway, reconstitution strictosidine analyzed accumulation 14 new associated transgene expression; five those enhanced co-infiltration initial precursor, geraniol, four exclusively upon geraniol supplementation. However, actual intermediates identified group. Even though precursors constitutive gene expression facilitated establishing lines, interest observed. Therefore, track needs elimination potential bottlenecks replacement inefficient wild type Taken together, represent promising tools molecules. Their substantive efficiency enables economical