Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes

Autor: Heribert Warzecha, Sabine Fräbel, Agata Staniek, Markus Krischke
Rok vydání: 2016
Předmět:
Zdroj: Biotechnology Journal. 11:1586-1594
ISSN: 1860-6768
DOI: 10.1002/biot.201600337
Popis: Halogenation of natural compounds in planta is rare. Herein, we describe successful engineering of tryptophan 6-halogenation into the plant context by heterologous expression of the Streptomyces toxytricini Stth gene and localization of its enzymatic product in various tobacco cell compartments. When co-expressed with the flavin reductase rebF from Lechevalieria aerocolonigenes, Stth efficiently produced chlorinated tryptophan in the cytosol. Further, supplementation of KBr yielded the brominated metabolite. More strikingly, targeting of the protein to the chloroplasts enabled effective halogenation of tryptophan even in absence of the partner reductase, providing crucial evidence for sufficient, organelle-specific supply of the FADH2 cofactor to drive halogen integration. Incorporation of an alternative enzyme, the 7-halogenase RebH from L. aerocolonigenes, into the metabolic set-up resulted in the formation of 6,7-dichlorotryptophan. Finally, expression of tryptophan decarboxylase (tdc) in concert with stth led to the generation of 6-chlorotryptamine, a new-to-nature precursor of monoterpenoid indole alkaloids. In sum, our report highlights the tremendous application potential of plants as a unique chassis for the engineering of rare and valuable halogenated natural products, with chloroplasts as the cache of reduction equivalents driving metabolic reactions.
Databáze: OpenAIRE