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 (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.

Reconstitution of monoterpene indole alkaloid biosynthesis in genome engineered Nicotiana benthamiana

“…Wang et al 2016). Production of halogenated indican (Fräbel et al 2018) and vanillin (Gallage et al 2018) in N. benthmaiana also benefited from chloroplast localisation. In contrast, a recent report found that production of diterpenoids (typically synthesised in the chloroplast) was dramatically enhanced by co-opting the cytosolic mevalonate pathway to produce GGPP rather than the chloroplast MEP pathway (De La Peña and Sattely 2021).…”

Novel Arylindigoids by Late‐Stage Derivatization of Biocatalytically Synthesized Dibromoindigo

“…In more recent studies, Tischler and co‐authors reported on the conversion of haloindoles using a small array of styrene MOs [30] . One‐pot synthesis of indigoids either in bacteria or plant as the hosts was recently achieved: By introducing a tryptophan halogenase into the host strain along with a hydroxylase, production of indigoids from tryptophan was feasible, omitting the need for costly substituted indole substrates through exploiting the cellular metabolism [31, 32] . However, product titers remained low and the isolation of the pigment from the cultivation broth can become a tedious procedure.…”

Deploying Microbial Synthesis for Halogenating and Diversifying Medicinal Alkaloid Scaffolds

“…The recent development of novel, transcription factor-based biosensors returning a fluorescent output in response to 5-or 6-bromotryptophan (Ellefson et al, 2018) may, in combination with multiplex screening (e.g., fluorescence-activated cell sorting), underpin the screening of larger mutant libraries for optimization of the aforementioned parameters in the intracellular environment of yeast strains. In addition to the ever-expanding repertoire of halogenase substrate specificities, a recent study elegantly demonstrated that these halogenases can be used in combination with a tryptophanase for biosynthesis of a halogenated indole (Fräbel et al, 2018), which is a common biosynthetic precursor. While engineering Nicotiana benthamiana to produce chlorinated precursors of indican dyes, the authors noted that the three tryptophan halogenases (RebH, SttH, and PyrH) did not accept indole as a substrate.…”