Fräbel Sabine, Wagner Bastian, Krischke Markus, Schmidts Volker, Thiele Christina M, Staniek Agata, Warzecha Heribert
Plant Biotechnology and Metabolic Engineering, Technische Universität Darmstadt, Schnittspahnstraße 4, 64287 Darmstadt, Germany.
Lehrstuhl für Pharmazeutische Biologie, Julius-von-Sachs-Institut der Universität Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany.
Metab Eng. 2018 Mar;46:20-27. doi: 10.1016/j.ymben.2018.02.003. Epub 2018 Feb 18.
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-β-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.
植物是多功能的化学家，能产生种类繁多的特殊化合物。在此，我们描述了在植物中构建全新代谢途径，从而能够生成卤代靛蓝前体作为非天然植物产物。吲哚苷（吲哚基-β-D-吡喃葡萄糖苷）是许多用于染色的植物所特有的次生代谢产物。其去糖基化及随后的氧化二聚作用会产生蓝色染料靛蓝。卤代吲哚苷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，它们的生产在很大程度上依赖于化学合成。为了在不含吲哚苷的植物系统中实现从头生物合成，我们采用了来自不同来源的一系列酶，包括三种微生物色氨酸卤化酶，它们可在吲哚部分的C5、C6或C7位取代氨基酸。随后，细菌色氨酸酶TnaA与人类细胞色素P450单加氧酶2A6的一个突变体协同作用对卤代色氨酸进行加工，并且通过内源性烟草糖基转移酶对所得吲哚酚衍生物进行糖基化，从而在瞬时转化的本氏烟草植株中产生了相应的卤代吲哚苷变体。当使用5-卤化酶PyrH时积累水平最高，5-氯吲哚苷达到0.93±0.089 mg/g干重。通过核磁共振分析明确证实了后者的身份。此外，我们的组合方法借助GoldenBraid克隆系统的模块化组装能力，并受植物细胞独特区室化的启发，为途径设计提供了多种亚细胞定位可供测试。结果，叶绿体被确认为卤代吲哚苷具有功能的生物合成场所，细胞器自身的催化系统满足了卤化酶以及细胞色素P450单加氧酶所需的还原增强。因此,我们的研究提出了一个可行的卤代精细化学品替代生产平台，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，在植物中生成自然界以前未知的卤代靛蓝类前体为植物生物合成能力提供了更广阔的视野，并且确实推进了已有的前沿界限。
