Engineering of new-to-nature halogenated indigo precursors in plants

Abstract

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 ( 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 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/262428 doi: bioRxiv preprint first posted online Feb. 8, 2018; nature offers an extended view on and, indeed, pushes forward the established frontiers of biosynthetic capacity of plants. KeywordsPlant specialized metabolism, indican, halogenase, Nicothiana benthamiana, modular cloning, pathway engineering Graphical abstract Highlights• Entirely novel combinatorial pathway yielded new-to-nature halogenated indigoids.• An array of specific chloro-and bromoindican regioisomers was generated.• Significant retrieval rates afforded unambiguous metabolite identification.• Engineered plants emerge as alternative manufacture chassis for fine chemicals. Abbreviations(i)DOSY, (integrated into) diffusion ordered spectroscopy; 2A6, human cytochrome P450 monooxygenase 2A6; BFP, blue fluorescent protein; Bx1, indole synthase from Zea mays;

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

“…This increase is possibly due to the isolation of metabolites from the cytosol where they may both impact viability and be exposed to unwanted derivatization by endogenous glycosyltransferases 11,12,56 . Production of halogenated indican 57 and vanillin 58 in N. benthamiana also benefited from chloroplast localization. In contrast, a recent report found that production of diterpenoids (typically synthesized in the chloroplast) was dramatically enhanced by co-opting the cytosolic mevalonate pathway to produce GGPP rather than the chloroplast MEP pathway 59 .…”

Strategies for the production of biochemicals in bioenergy crops

“…Alternatively, as shown in transient expression studies in tobacco, enhancement of indole synthesis can be achieved by expressing a tryptophanase (TnA) from E. coli, which also resulted in indican biosynthesis when co-expressed with CYP2A6 ( Fig. 2) [122]. Therefore, engineering strategies aiming at increasing tryptophan content could be leveraged for higher production of indican in bioenergy crops.…”

An overview of microbial indigo-forming enzymes

“…This would involve the breeding of plants with high indican content and other beneficial traits. Recently, obtained genetic insights in the natural indigo synthesis pathway in plants could be exploited for this (Jin et al 2016;Inoue et al 2017), and it has already been shown that through genetic engineering, plants can be bred that produce various indigo dyes (Fräbel et al 2018). However, production of indigo using plants would compete with food and feed production.…”