Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes
Recombinant flavin-dependent halogenases are functional in tobacco chloroplasts without co-expression of flavin reductase genes
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DOI：
10.1002/biot.201600337
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发表时间：
2016-12-01
影响因子：
4.7
通讯作者：
Warzecha, Heribert
中科院分区：
文献类型：
--
作者：
Fraebel, Sabine;Krischke, Markus;Warzecha, Heribert
植物中天然化合物的卤化作用是罕见的。在此，一个成功的工程色氨酸6-卤化到植物环境中的异源表达的链霉菌toxytricini Stth基因和本地化的各种烟草细胞室中的酶的产品进行了描述。当与产气勒氏菌的黄素还原酶rebF共表达时，Stth在胞质溶胶中有效地产生氯化色氨酸。此外，补充KBr产生溴化代谢物。更引人注目的是，即使在没有伴侣还原酶的情况下，蛋白质靶向叶绿体也能有效地卤化色氨酸，为FADH 2辅因子的充分的细胞器特异性供应提供了关键证据，以驱动卤素整合。引入一种替代酶，来自L.气生菌进入代谢设置，导致形成6，7-二氯色氨酸。最后，色氨酸脱羧酶（tdc）的表达与stth一起导致6-氯色胺的产生，这是一种新的天然单萜吲哚生物碱的前体。总之，该报告强调了植物作为稀有和有价值的卤化天然产物工程的独特底盘的巨大应用潜力，叶绿体作为驱动代谢反应的还原当量的该高速缓存。
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 2 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.