Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Böttcher, Stephan; Thiem, Joachim

doi:10.3791/52442

“…Currently, indigoid precursors are synthesized chemically. The need for regiospecific halogenation of aromatic carbon molecules and the low reactivity of the hydroxyl function of indoxyl make the process rather difficult (Böttcher and Thiem, 2015). Therefore, a biosynthetic alternative would be beneficial, additionally limiting reliance on organic solvents and hazardous chemicals.…”

Section: Introductionmentioning

confidence: 99%

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

Fräbel

¹

,

Wagner

²

,

Krischke

³

et al. 2018

Metabolic Engineering

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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.

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“…We also considered the possible promiscuity of glucosidases as giving us a higher probability of identifying active GHs in our search. The synthetic strategy drew from reported methods for indolyl glycoside synthesis [ 18 , 23 , 24 , 25 ] and our previous experience at preparing septanose glycosides via nucleophilic displacement on anomeric bromides [ 26 ]. It leveraged a synthesis of the per- O -acetyl septanose precursors of the anomeric bromides that began from natural D-pyranosides such as D-glucose, suggesting that the route could be extended to other sugars.…”

Section: Resultsmentioning

confidence: 99%

Indolyl Septanoside Synthesis for In Vivo Screening of Bacterial Septanoside Hydrolases

Testolin

¹

,

Pascual

²

,

Planas

³

et al. 2021

IJMS

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Building-up and breaking-down of carbohydrates are processes common to all forms of life. Glycoside hydrolases are a broad class of enzymes that play a central role in the cleavage of glycosidic bonds, which is fundamental to carbohydrate degradation. The large majority of substrates are five- and six-membered ring glycosides. Our interest in seven-membered ring septanose sugars has inspired the development of a way to search for septanoside hydrolase activity. Described here is a strategy for the discovery of septanoside hydrolases that uses synthetic indolyl septanosides as chromogenic substrates. Access to these tool compounds was enabled by a route where septanosyl halides act as glycosyl donors for the synthesis of the indolyl septanosides. The screening strategy leverages the known dimerization of 3-hydroxy-indoles to make colored dyes, as occurs when the β-galactosidase substrate X-Gal is hydrolyzed. Because screens in bacterial cells would enable searches in organisms that utilize heptoses or from metagenomics libraries, we also demonstrate that septanosides are capable of entering E. coli cells through the use of a BODIPY-labeled septanoside. The modularity of the indolyl septanoside synthesis should allow the screening of a variety of substrates that mimic natural structures via this general approach.

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

Fräbel

¹

,

Wagner

²

,

Krischke³

et al. 2018

Preprint

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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;

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Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Cited by 3 publications

References 16 publications

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

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

Indolyl Septanoside Synthesis for In Vivo Screening of Bacterial Septanoside Hydrolases

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

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