Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes.
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 FADH2cofactor 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.
Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Darmstadt, Germany.
Sabine
S
Fräbel
Lehrstuhl für Pharmazeutische Biologie, Julius-von-Sachs-Institut der Universität Würzburg, Würzburg, Germany.
Markus
M
Krischke
Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Darmstadt, Germany.
Agata
A
Staniek
Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Darmstadt, Germany.
Heribert
H
Warzecha
D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D038181:FMN Reductase / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering / Q000379:methods*; D054879:Halogenation; D010088:Oxidoreductases / Q000235:genetics; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014363:Tryptamines / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism
C533763:6,7-dichlorotryptophan; D011994:Recombinant Proteins; D014363:Tryptamines; C026380:6-chlorotryptophan; C030820:tryptamine; D014364:Tryptophan; D010088:Oxidoreductases; D038181:FMN Reductase; D001142:Aromatic-L-Amino-Acid Decarboxylases
Combinatorial biosynthesis; Flavin-dependent tryptophan halogenases; Plant metabolic engineering; Plant natural products; Synthetic biology