Volume 35 Number 11 November 2018 Pages 1113–1230 Natural Product Reports rsc.li/npr Themed issue: Understanding Biosynthetic Protein-Protein Interactions – Part 2 Guest Editors: David Ackerley, Gregory Challis and Max Cryle ISSN 0265-0568 REVIEW ARTICLE Tomas Laursen et al. Dynamic metabolic solutions to the sessile life style of plants Natural Product Reports REVIEW View Article Online View Journal | View Issue Dynamic metabolic solutions to the sessile life style of plants Cite this: Nat. Prod. Rep.,2018,35, 1140 Camilla Knudsen, abc Nethaji Janeshawari Gallage, abc Cecilie Cetti Hansen, abc Birger Lindberg Møller abcd and Tomas Laursen *abc Covering: up to 2018 Plants are sessile organisms. To compensate for not being able to escape when challenged by unfavorable growth conditions, pests or herbivores, plants have perfected their metabolic plasticity by having developed the capacity for on demand synthesis of a plethora of phytochemicals to specifically respond to the challenges arising during plant ontogeny. Key steps in the biosynthesis of phytochemicals are catalyzed by membrane-bound cytochrome P450 enzymes which in plants constitute a superfamily. In planta, the P450s may be organized in dynamic fi Creative Commons Attribution-NonCommercial 3.0 Unported Licence. enzyme clusters (metabolons) and the genes encoding the P450s and other enzymes in a speci cpathway may be clustered. Metabolon formation facilitates transfer of substrates between sequential enzymes and therefore enables the plant to channel the flux of general metabolites towards biosynthesis of specific phytochemicals. In the plant cell, compartmentalization of the operation of specific biosynthetic pathways in specialized plastids serves to avoid undesired metabolic cross-talk and offersdistinctstoragesitesformolar concentrations of specific phytochemicals. Liquid–liquid phase separation may lead to formation of dense biomolecular condensates within the cytoplasm or vacuole allowing swift activation of the stored phytochemicals as required upon pest or herbivore attack. The molecular grid behind plant plasticity offers an Received 19th April 2018 endless reservoir of functional modules, which may be utilized as a synthetic biology tool-box for engineering This article is licensed under a DOI: 10.1039/c8np00037a of novel biological systems based on rational design principles. In this review, we highlight some of the rsc.li/npr concepts used by plants to coordinate biosynthesis and storage of phytochemicals. Open Access Article. Published on 16 October 2018. Downloaded 10/2/2021 5:09:45 PM. 1 Introduction 5.1 The plant plasticity toolbox 2 Dynamic assembly of biosynthetic enzyme complexes 5.2 Engineering modules for production and storage of 2.1 Metabolons; the highways of plant metabolism phytochemicals 2.2 Organization and dynamics of metabolons 5.3 A bright green future of synthetic biology 2.3 Membrane dynamics shape phytochemical production 6 Conicts of interest 3 Classical compartments and phytochemical dynamics 7 Acknowledgements 3.1 Plastids for production and storage of phytochemicals 8 References 4 Formation of micro-compartments by liquid–liquid phase separation 4.1 Micro-compartments composed of natural deep eutectic 1 Introduction solvents (NADESs)? Phytochemicals play a key role in plant defense, communication 4.2 Vacuolar microcompartments and adaptation to abiotic and biotic stress. Derived from rela- 4.3 ER-derived microcompartments tively few general metabolites, phytochemicals diversify into an 5 Conned biosynthesis and storage as an eminent immense number of molecules through combinatorial enzyme synthetic biology toolbox cascade reactions.1 With enzymatic precision, plants produce complex phytochemicals with regio- and stereospecic functional groups matching or outperforming modern day synthetic chem- aPlant Biochemistry Laboratory, Department of Plant and Environmental Science, istry.2 Their biosynthesis oen involves assemblies of numerous University of Copenhagen, DK-1871 Frederiksberg C, Denmark. E-mail: tola@plen. ffi ku.dk enzymes, many of which appear to have low substrate a nity and bbioSYNergy, Center for Synthetic Biology, DK-1871 Frederiksberg C, Denmark speci city when studied in vitro.However,in planta the metabo- 3,4 cVILLUM Research Center for Plant Plasticity, DK-1871 Frederiksberg C, Denmark lism may be highly channeled and tends to work more efficient dCarlsberg Research Laboratory, DK-1799 Copenhagen V, Denmark than would be expected from the average cellular concentrations 1140 | Nat. Prod. Rep.,2018,35, 1140–1155 This journal is © The Royal Society of Chemistry 2018 View Article Online Review Natural Product Reports of the biosynthetic enzymes and their substrates.5,6 These serial ranges between 6 : 1 to 20 : 1,6,16–19 and the overall low abun- enzyme reactions require tight regulation to guide the metabo- dance suggest that these enzymes possess features guiding lism in a highly crowded environment.7,8 them towards specic preferential interactions thus circum- In 1978 Charles Tanford wrote: “Biological organization may venting bulk equilibrium.20–22 Metabolic channeling within be viewed as consisting of two stages: biosynthesis and biosynthetic pathways may be achieved through organization of assembly”.9 In plants, this is achieved through spatial organi- enzymes in complexes, termed metabolons.23 Efficient chan- zation of biosynthetic enzymes and metabolites in compart- neling requires close proximity (0.1–1 nm) between sequential ments dictating metabolic ux into streamlined coordinated enzymes24 as achieved by protein–protein interactions. Conse- highways.10,11 The classical view of compartments as clearly quently, metabolon assembly facilitates direct transfer of separated entities conned by membranes is being challenged substrates and products between sequential enzymes and with the advances of analytical tools with subcellular resolution. prevents leakage of potentially toxic and labile intermediates Instead, a dynamic intracellular web of the endoplasmic retic- and undesired metabolic cross talk. Dynamic assembly and ulum (ER) interacting with cellular compartments and forma- disassembly of metabolons offer an opportunity for swi tion of local microcompartments governs the plasticity of plant adaption to meet environmental challenges such as fungal or metabolism.12 Compartmentalization at the nanoscale level by insect attack (Fig. 1). The on-demand organization of POR with organization of enzymes in conned spaces at the membrane specic P450s would surpass the challenge of the stoichio- surfaces13 or in membrane-less assemblies obtained by liquid– metric imbalance between the enzymes. liquid phase separation (LLPS)14 provides dynamic assemblies necessary for metabolic adaptation. 2.1 Metabolons; the highways of plant metabolism In this review, we focus on new insights on plant compart- mentalization and the dynamic solutions plants use to cope Biosynthetic pathways for phytochemical production in the with their sessile life style. We also discuss how compartmen- plant cell can be highly branched sharing a few common steps. Assembly of sequential enzymes in metabolons provides a way Creative Commons Attribution-NonCommercial 3.0 Unported Licence. talization may be used as a synthetic biology tool to optimize the production of phytochemicals in heterologous hosts. of orchestrating the metabolic grid by guiding the metabolites towards a specic product. One key example is the phenyl- propanoid pathway, which directs the production towards 2 Dynamic assembly of biosynthetic monolignols used in lignin biosynthesis or towards bioactive enzyme complexes avonoids depending on the plant's need.6,25–27 The latter pathway is further differentially divided into sub-branches with Biosynthesis of phytochemicals typically involves multiple different end products such as vanilloids, isoavonoids, avo- enzymatic steps, and spatial connement of biosynthetic nols, avones, anthocyanins etc. This article is licensed under a enzymes governs the formation of metabolic highways. Most Assembly of metabolons facilitate channeling of phenylala- phytochemicals are products of biosynthetic pathways, in which nine through the core phenylpropanoid pathway28 and further enzymes of the cytochrome P450 (P450) superfamily catalyze downstream towards the monolignols,29 avonoids,25,26 sporo- one or more key steps.15 Microsomal P450 enzymes are tethered pollenin branches27,30 and vanilloids such as vanillin glucoside Open Access Article. Published on 16 October 2018. Downloaded 10/2/2021 5:09:45 PM. to the ER via a single transmembrane anchor and thereby (Fig. 2). Formation of enzymatic complexes in the phenyl- conned to the two-dimensional membrane lattice. Stoichio- propanoid pathway was rst proposed in 1974.31 Early research metric imbalances between P450s and their oxidoreductase, supported this hypothesis based on substrate channeling and NADPH-dependent cytochrome P450 oxidoreductase (POR), interactions between two enzymes in the core phenylpropanoid Fig. 1 Illustration of the dynamic assembly and disassembly of the dhurrin metabolon. Metabolon assembly involves recruitment of the soluble UGT to the ER-anchored P450s and POR and local accumulation of specific lipids resulting in biosynthesis of the insecticide dhurrin. Disassembly of the dhurrin metabolon results in release of the antifungal oxime intermediate. Thus, a single pathway confers resistance towards specific herbivores and pests governed by the dynamic assembly of enzymes in metabolons. This