Engineering of new-to-nature halogenated indigo precursors in plants
Engineering of new-to-nature halogenated indigo precursors in plants
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DOI：
10.1016/j.ymben.2018.02.003
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发表时间：
2018-03-01
影响因子：
8.4
通讯作者：
Warzecha, Heribert
中科院分区：
文献类型：
--
作者：
Fraebel, Sabine;Wagner, Bastian;Warzecha, Heribert
植物是多才多艺的化学家，可以产生各种各样的特殊化合物。在这里，我们描述了植物中全新代谢途径的工程设计，能够生成卤化靛蓝前体作为非天然植物产品。Indican（吲哚基-β-D-吡喃葡萄糖苷）是许多染色植物的次生代谢产物特征。它的脱糖基化和随后的氧化二聚化产生蓝色染料靛蓝。卤化二甲基衍生物通常用作组织化学和分子生物学应用中的检测试剂；然而，它们的生产很大程度上依赖于化学合成。为了在不含印度聚糖的植物系统中实现从头生物合成，我们使用了一系列来自不同来源的酶，包括三种微生物色氨酸卤化酶，取代了吲哚部分的 C5、C6 或 C7 处的氨基酸。随后，细菌色氨酸酶 TnaA 与人细胞色素 P450 单加氧酶 2A6 突变体一起加工卤代色氨酸，并通过内源烟草葡萄糖基转移酶对所得吲哚酚衍生物进行糖基化，在瞬时转化的本塞姆塞姆氏烟草植物中产生了相应的卤代吲哚变体。当使用 5-卤化酶 PyrH 时，积累水平最高，达到 0.93 +/- 0.089 mg/g 5-氯茚干重。核磁共振分析明确证实了后者的身份。此外，我们的组合方法在 GoldenBraid 克隆系统的模块化组装能力的推动下，并受到植物细胞独特分区的启发，为途径设计提供了多种替代亚细胞定位的测试。因此，叶绿体被验证为haloindican的功能性生物合成场所，并通过细胞器固有的催化系统实现卤化酶和细胞色素P450单加氧酶的必要减少增强。因此，我们的研究提出了卤化精细化学品的可行替代生产平台，避免了对化石燃料资源和有毒化学品的依赖。我们进一步认为，在植物中产生以前自然界未知的卤代靛蓝前体为植物生物合成能力的既定前沿提供了扩展的视野，并且实际上推动了植物生物合成能力的既定前沿。
Plants are versatile chemists producing a tremendous variety of specialized compounds. Here, we describe the engineering of entirely novel metabolic pathways in planta enabling generation of halogenated indigo precursors as non-natural plant products.Indican (indolyl-beta-D-glucopyranoside) is a secondary metabolite characteristic of a number of dyers plants. Its deglucosylation and subsequent oxidative dimerization leads to the blue dye, indigo. Halogenated indican derivatives are commonly used as detection reagents in histochemical and molecular biology applications; their production, however, relies largely on chemical synthesis. To attain the de novo biosynthesis in a plant-based system devoid of indican, we employed a sequence of enzymes from diverse sources, including three microbial tryptophan halogenases substituting the amino acid at either C5, C6, or C7 of the indole moiety. Subsequent processing of the halotryptophan by bacterial tryptophanase TnaA in concert with a mutant of the human cytochrome P450 monooxygenase 2A6 and glycosylation of the resulting indoxyl derivatives by an endogenous tobacco glucosyltransferase yielded corresponding haloindican variants in transiently transformed Nicotiana benthamiana plants. Accumulation levels were highest when the 5-halogenase PyrH was utilized, reaching 0.93 +/- 0.089 mg/g dry weight of 5-chloroindican. The identity of the latter was unambiguously confirmed by NMR analysis. Moreover, our combinatorial approach, facilitated by the modular assembly capabilities of the GoldenBraid cloning system and inspired by the unique compartmentation of plant cells, afforded testing a number of alternative subcellular localizations for pathway design. In consequence, chloroplasts were validated as functional biosynthetic venues for haloindican, with the requisite reducing augmentation of the halogenases as well as the cytochrome P450 monooxygenase fulfilled by catalytic systems native to the organelle.Thus, our study puts forward a viable alternative production platform for halogenated fine chemicals, eschewing reliance on fossil fuel resources and toxic chemicals. We further contend that in planta generation of halogenated indigoid precursors previously unknown to nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants.