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

Abstract

Halogenation of natural compounds in planta is rare. Herein, a 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 is described. 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 FADH 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, the 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.

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

“…Recently, it has been shown that FDHs can be targeted to the chloroplasts in planta . Because chloroplasts contain naturally occurring reductases, there was no need for heterologous reductases (92). …”

Strategies to Produce Chlorinated Indole‐3‐Acetic Acid and Indole‐3‐Acetic Acid Intermediates

“…The tryptophan 5‐halogenase gene was chosen because PyrH showed the highest activity of the three halogenases with IAA (Figure S2). In previously published transformations of plants with bacterial halogenase genes, codon optimized halogenase genes had been used ,. In contrast to these experiments, we used native pyrH as well as pyrH with codons optimized for A. thaliana .…”

“…These experiments highlight the potential of combining the rich chemistry of plant metabolism with bacterial halogenases to generate unnatural products. Recently, the transient expression of another halogenase gene stth , coding for a tryptophan 6‐halogenase from Streptomyces toxytricini , in tobacco was reported and the authors showed that co‐expression of a tryptophan decarboxylase gene with this halogenase resulted in the production of chlorinated tryptamine …”

Halogenase engineering and its utility in medicinal chemistry

“…A similar study where RebH and Trp-6-halogenase SttH 44 were expressed in Nicotiana benthamiana revealed that the localization of the FDH/FR system to the chloroplasts could aid cofactor regeneration. 45 Localization of halogenases to the chloroplasts in the absence of exogenous FR led to comparable product formation indicating that the plastids provide a sufficient supply of reducing agents from native metabolic pathways. 46 The targeted coexpression of the Trp decarboxylase was required for production of halogenated tryptamine derivatives as the chloroplasts are devoid of this enzyme.…”