Stabilization of Dhurrin Biosynthetic Enzymes from Sorghum Bicolor Using a Natural Deep Eutectic Solvent

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Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a natural deep eutectic solvent

Knudsen, Camilla; Bavishi, Krutika; Viborg, Ketil Mathiasen; Drew, Damian Paul; Simonsen, Henrik Toft; Motawia, Mohammed Saddik; Møller, Birger Lindberg; Laursen, Tomas

Published in: Phytochemistry

Link to article, DOI: 10.1016/j.phytochem.2019.112214

Publication date: 2020

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Citation (APA): Knudsen, C., Bavishi, K., Viborg, K. M., Drew, D. P., Simonsen, H. T., Motawia, M. S., Møller, B. L., & Laursen, T. (2020). Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a natural deep eutectic solvent. Phytochemistry, 170, [112214]. https://doi.org/10.1016/j.phytochem.2019.112214

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Phytochemistry 170 (2020) 112214

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Phytochemistry

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Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a T natural deep eutectic solvent Camilla Knudsena,b,c, Krutika Bavishia,b,c,d, Ketil Mathiasen Viborga,b,c, Damian Paul Drewa,e, ∗∗ Henrik Toft Simonsena,f, Mohammed Saddik Motawiaa,b,c, Birger Lindberg Møllera,b,c,g, , ∗ Tomas Laursena,b,c, a Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark b Center for Synthetic Biology “bioSYNergy”, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark c VILLUM Research Center “Plant Plasticity”, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark d Department of Molecular Biology and Genetics, Structural Biology, Gustav Wieds Vej 10, 8000, Aarhus C, Denmark e Lyell McEwin Hospital, Elizabeth Vale, SA 5112, Australia f Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, DK-2800, Kgs. Lyngby, Denmark g Carlsberg Research Laboratory, J. C. Jacobsen Gade, DK-1799, Copenhagen V, Denmark

ARTICLE INFO ABSTRACT

Keywords: In recent years, ionic liquids and deep eutectic solvents (DESs) have gained increasing attention due to their Sorghum bicolor (L.) Moench ability to extract and solubilize metabolites and biopolymers in quantities far beyond their solubility in oil and Poaceae water. The hypothesis that naturally occurring metabolites are able to form a natural deep eutectic solvent Natural deep eutectic solvents (NADES), thereby constituting a third intracellular phase in addition to the aqueous and lipid phases, has Metabolons prompted researchers to study the role of NADES in living systems. As an excellent solvent for specialized Cytochromes P450 metabolites, formation of NADES in response to dehydration of plant cells could provide an appropriate en- Specialized metabolites Liquid-liquid phase separation vironment for the functional storage of enzymes during drought. Using the enzymes catalyzing the biosynthesis of the defense compound dhurrin as an experimental model system, we demonstrate that enzymes involved in this pathway exhibit increased stability in NADES compared with aqueous buffer solutions, and that enzyme activity is restored upon rehydration. Inspired by nature, application of NADES provides a biotechnological approach for long-term storage of entire biosynthetic pathways including membrane-anchored enzymes.

1. Introduction renders harvest and extraction unsustainable. Some unique plant species have the ability to accumulate Today, we rapidly need to move into The Planthropocene Era where extraordinarily high levels of natural products stored in soluble form in petrochemicals are replaced by sustainable green bio-production their tissues. In the black flower petals of the gentiana “flower of death” (Møller, 2014, 2017). Green photosynthetic organisms are instrumental (Lisianthius nigrescens Schltdl. & Cham.), the anthocyanin content con- in this transition. Using solar energy as the energy source and carbon stitutes 24% of the dry mass (Markham et al., 2004). In the buds and dioxide from the atmosphere as the sole carbon source, photosynthetic flowers of sophora (Sophora japonica L, Pagoda tree), the content of the organisms provide food, biomaterials and bioenergy. At a minor scale, anthocyanin rutin constitutes up to 30% of the dry mass (Chen et al., they also serve as a rich source of bio-active natural products used by 2018; Koja et al., 2018). This is equivalent to a 12,000 fold higher humanity as nutraceuticals, condiments, flavors, colorants as well as solubility in the plant tissue in comparison to aqueous media (Dai et al., medicinal compounds (Jensen and Scharff, 2019). These precious 2016; Horosanskaia et al., 2017). Anthocyanins may self-organize and natural products are typically found in small amounts and are present accumulate in anthocyanoplasts or in anthocyanic vacuolar inclusions, in native plants that are not easily grown at a commercial scale. This which may or may not be membrane encapsulated (Chanoca et al.,

∗ Corresponding author. Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Copenhagen, Denmark. ∗∗ Corresponding author. Plant Biochemistry Laboratory, Department of Plant and Environmental Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark. E-mail addresses: [email protected] (B.L. Møller), [email protected] (T. Laursen). https://doi.org/10.1016/j.phytochem.2019.112214 Received 20 August 2019; Received in revised form 14 November 2019; Accepted 16 November 2019 0031-9422/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). C. Knudsen, et al. Phytochemistry 170 (2020) 112214

2015; Conn et al., 2010; Fernandes et al., 2015; Grotewold and Davies, water. NADESs composed of specific subsets of these crystalline 2008; Irani and Grotewold, 2005; Kallam et al., 2017; Markham et al., constituents have been demonstrated to be excellent solvents for 2000; Nielsen et al., 2004; Pecket and Small, 1980; Pourcel et al., 2010; sparsely soluble natural products such as vanillin glucoside and rutin Snyder and Nicholson, 1990). In mature vanilla (Vanilla planifolia Jacks. and related anthocyanins (Dai et al., 2013a, 2013b, 2015, 2016; ex Andrews) pods, vanillin glucoside is present in 4 M concentrations González et al., 2018; Peng et al., 2018; Vetrova et al., 2017; Zhao stored in senescent chloroplasts termed phenyloplasts (Brillouet et al., et al., 2015). Accordingly, NADESs are being used as green solvents 2014; Gallage et al., 2014, 2018; Gallage and Møller, 2015). In Indian for the industrial-scale extraction of bio-active natural products from coleus (Coleus forskohlii (Willd.) Briq.), forskolin accumulates in dense plants. However, no in planta functions of NADESs have been single droplets in the root cortex cells (Pateraki et al., 2014, 2017). In demonstrated, nor is the mechanism of their possible formation the upper part of sorghum seedlings (Sorghum bicolor (L.) Moench), the understood. cyanogenic glucoside dhurrin constitutes 30% of the dry mass (Halkier Sorghum (Sorghum bicolor (L.) Moench) (Poaceae) is a drought-tol- and Møller, 1989; Kojima et al., 1979). Dhurrin had previously been erant cereal that maintains growth even under severe drought stress reported to be stored in the main vacuole (Saunders and Conn, 1978), (Pavli et al., 2013). Metabolite profiling of sorghum plants exposed to but this could not be verified by Raman spectrometry, which pointed to different levels of drought revealed a significant accumulation of storage in the cytosol or apoplast (Heraud et al., 2018) i.e. in a more organic acids, sugars and amino acids. Simultaneous accumulation of confined space that would demand storage at molar concentrations. such metabolites and depletion of water provides ideal conditions for These examples demonstrate that a wide range of plant species are able the formation of intracellular NADES phases for safe-storage of both to orchestrate the establishment of high-density distinct storage sites enzymes and natural products such as dhurrin. As stated above, where natural products with sparse water solubility are kept in soluble sorghum seedlings accumulate high amounts of dhurrin, and increased form at surprisingly and seemingly impossible high molar concentra- amounts of dhurrin are produced in the mature plant upon exposure to tions in confined cellular domains. In nature, specific plant species thus drought stress (O'Donnell et al., 2013). Dhurrin is an insect defense possess an inherent ability to biosynthesize, accumulate and store large compound that upon tissue disruption e.g. as caused by feeding insect quantities of bio-active natural products. The origin and physical larvae, is hydrolyzed by a β-glucosidase to release toxic hydrogen characteristics of these conspicuous storage structures remain con- cyanide gas (Gleadow and Møller, 2014; Morant et al., 2008; Poulton troversial. If the biological mechanisms orchestrating these abilities and Li, 1994; Sanchez-Perez et al., 2009; Swain et al., 1992). Dhurrin were known, they might be engineered into commercially grown plants also functions as a storage of nitrogen that can be remobilized by re- using synthetic biology approaches. cycling pathways, thus avoiding hydrogen cyanide formation and pro- Verpoorte and co-workers have proposed that Natural Deep Eutectic viding an important source of reduced nitrogen at specific develop- Solvents (NADESs) could be involved in establishing high-density bio- mental stages (Bjarnholt et al., 2018; Picmanova et al., 2015). Dhurrin condensates (Choi et al., 2011; Knudsen et al., 2018). The NADES is synthesized from the amino acid L-tyrosine (Fig. 1C). The enzymatic concept was coined based on nuclear magnetic resonance (NMR) conversion is catalyzed by a dynamic multi-component enzyme com- spectroscopy studies demonstrating the presence of sugars, protein plex, the dhurrin metabolon (Bassard et al., 2017; Jørgensen et al., amino acids, choline and organic acids in plant cells (Fig. 1A). A NADES 2005; Laursen et al., 2016). The metabolon is composed of a cyto- forms a highly viscous liquid - a liquid crystal - upon mixing specific chrome P450 oxidoreductase (POR) (Bavishi et al., 2018; Laursen et al., constituents in the right stoichiometric proportions, dissolving in water, 2014), two cytochromes P450 (CYP79A1 and CYP71E1) (Bak et al., and then lyophilizing until the mass remains constant. This applies for a 1998; Blomstedt et al., 2011; Kahn et al., 1999; Koch et al., 1995; mixture of glucose:tartaric acid in a 1:1 M ratio (approx. 8% water Sibbesen et al., 1995) and a UDP-glucosyltransferase (UGT85B1) remaining) (Fig. 1B), which has a significantly lowered melting point (Blomstedt et al., 2015; Jones et al., 1999). The membrane-bound compared to their individual components, resulting in the formation of CYP79A1 and CYP71E1 enzymes constitute less than 1% of the total a highly viscous syrup, as found in raisins (Grimplet et al., 2009). membrane protein in sorghum seedlings, yet catalyze the formation of Raisins maintain a liquid phase despite an almost complete removal of dhurrin up to 30% of the dry mass (Halkier and Møller, 1989; Sibbesen

Fig. 1. Formation of NADES derived from natural occurring metabolites in plants. A) Chemical structures of D-glucose, tartaric acid, malic acid, choline, glycerol and dhurrin. Mixtures of these metabolites were tested for their ability to form NADES and their potential role in stabilizing the dhurrin biosynthetic enzymes. B) Stoichiometric mixture of glucose and tartrate constitute a NADES with significantly lowered melting point compared tothe individual components. C) Biosynthetic pathway of the natural product dhurrin in S. bicolor.

2 C. Knudsen, et al. Phytochemistry 170 (2020) 112214 et al., 1994). different NADES constituents were included in the assays to assess their The micro-environment surrounding the dhurrin metabolon possible effect on the biosynthetic enzymes. Maximal activity ofthe including the lipid membrane is important for high flux through the membrane associated metabolon, namely POR2B, CYP79A1 and metabolon, increasing the conversion of L-tyrosine to the product CYP71E1, catalyzing the conversion of L-tyrosine to p-hydro- dhurrin (Laursen et al., 2016). Likewise, the cytosolic micro-environ- xymandelonitrile, was observed at a 5% concentration of NADESs ment has been proposed to serve as a stabilizing solvent for dhurrin and composed of glucose:tartrate and glucose:malate as monitored by the prevent auto-hydrolysis at cytosolic pH (Knudsen et al., 2018; Møller conversion of tyrosine (Fig. 2B). The NADES concentration of 5% is et al., 2016). With the hypothesis that dhurrin is being stored in within the macromolecule concentration range of 5%–40% assumed to cytosolic or apoplastic high-density bio-condensates formed in a be present in the cytosol of healthy hydrated cells (Ellis and Minton, NADES-based environment, we aimed to examine whether the enzymes 2003). Enzymatic activity was strongly decreased at higher of the dhurrin metabolon would be active in such a hypothetical concentrations of these NADESs, such as 25% NADESs corresponding to cellular environment. Additionally, we investigated the stability of the 1 M of glucose:tartrate and 1.2 M glucose:malate. The reduced activity dhurrin metabolon in NADES, thereby mimicking conditions resulting might be attributed to the high viscosity of the medium, which would from abiotic stresses such as dessication and heat. This study adds to the restrict domain motion related to catalytic function (Laursen et al., increasing evidence that a NADES-based cellular environment might 2011) and diffusion of substrates. The presence of choline chloride was represent a third intracellular phase that complements the aqueous and found to have a stronger inhibitory effect on microsomal activity with lipid phases in plant cells as suggested by Verpoorte and coworkers total inhibition at and above 20%, corresponding to 0.5 M glucose and (2011). 1 M choline in the 1:2 NADES (Fig. 2B). The focus was therefore shifted to NADESs composed of an organic acid and a sugar. In these NADES systems, salts of simple organic acids such as tartaric acid, malic acid, 2. Results maleic acid and malonic acid are common. We chose to proceed with a NADES composed of glucose as the sugar and tartaric acid as the 2.1. Enzyme activity in the presence of NADES organic acid since these two constituents displayed minimal effect on the enzyme activity when present at 12%, corresponding to 0.45 M of As stated in the introduction, NADESs are composed of common glucose:tartrate and 0.58 M glucose:malate, as required for studying metabolites present in living organisms including plants. These include the recovery of enzymatic activity upon storage in high NADES sugars, protein amino acids, choline and organic acids (Choi et al., concentrations followed by dilution. 2011). In general, NADES consists of a hydrogen-bonding salt and a neutral species present in an eutectic molar ratio with a melting point that is much lower than those of each constituent (Dai et al., 2013b; 2.2. NADES-based stabilization of the dhurrin biosynthetic enzymes Hammond et al., 2017). The most common eutectic mixtures are composed of an organic acid and a sugar, an organic acid and choline In order to investigate the ability of the glucose:tartrate NADES to chloride, or choline chloride and sugar. preserve the dhurrin synthesizing enzymes during heat and desiccation To select an appropriate NADES for the experimental work, initial stress, we expressed the genes encoding CYP79A1 and CYP71E1 in biosynthetic experiments were performed using microsomes prepared Saccharomyces cerevisiae and UGT85B1 and POR2B in Escherichia coli, from etiolated sorghum seedlings (Fig. 2A). Previous studies have de- isolated the proteins formed and reconstituted CYP79A1, CYP71E1 and monstrated that the microsomes catalyze the conversion of L-tyrosine to POR2B in proteoliposomes composed of native lipids extracted from p-hydroxymandelonitrile i.e. all the membrane-bound steps of the etiolated sorghum seedlings (Laursen et al., 2016)(Fig. 3A). The dhurrin pathway catalyzed by CYP79A1, CYP71E1 and POR2B. These stability of the enzymes in the proteoliposomes was tested by incuba- analyses utilized classical tricine or phosphate buffers in the presence of tion in high NADES concentrations (72%) followed by dilution to 12%. NADPH and oxygen (Bak et al., 2000; Jones et al., 1999; Kahn et al., Upon dilution of the NADES solution, 82% of the initial tyrosine con- 1999; Laursen et al., 2011). Because the enzymes in the version rate was recovered (Fig. 3B). Glycerol at high concentrations dhurrin pathway are organized within a metabolon, no accumulation was included as a control for crowding effects (Ellis and Minton, 2003) of intermediates such as p-hydroxyphenylacetaldoxime and p- and enzymes in glycerol displayed 61% recovery of activity upon di- hydroxyphenylacetonitrile is observed. The p-hydroxymandelonitrile lution from 72% (7.8 M) to 12% (1.3 M). The UGT85B1 recovered intermediate is labile and dissociates into p-hydroxybenzaldehyde and approximately 70% of the initial activity upon dilution of the NADES as hydrogen cyanide. Product formation from 14C-L-tyrosine was monitored following administration of p-hydroxymandelonitrile and monitored by autoradiography following separation using thin layer radiolabeled UDP-glucose (Fig. 3B). This demonstrates that NADES may chromatography (TLC) (Kahn et al., 1999). In the present study, provide an inert environment protecting against irreversible

Fig. 2. Dhurrin biosynthesis in the presence of different NADESs. A) Etiolated sorghum seedlings used for preparation of microsomes. B) Tyrosine conversion assay in microsomes at different NADES concentrations indicates an optimum at 5% NADES for both glucose:tartrate and glucose:malate. Values are mean of three technical replicates ± SD.

3 C. Knudsen, et al. Phytochemistry 170 (2020) 112214

Fig. 3. NADES-based stabilization of the dhurrin biosynthetic enzymes. A) Illustration of proteoliposomes comprising the POR2B, CYP79A1, CYP71E1 and UGT85B1 reconstituted in liposomes composed of phospholipids extracted from etiolated sorghum seedlings (Metabolon). B) Recovery of activity upon storage of enzymes in NADES and glycerol compared to buffer upon dilution displayed as relative conversion of tyrosine forthe Metabolon samples and conversion of cyanohydrin to dhurrin for the UGT85B1 samples. Values are mean of three technical replicates ± SD. C) Stability of dhurrin biosynthetic enzymes stored at room temperature in aqueous buffer, NADES and glycerol. Samples were diluted in buffer prior to activity assay. Values are mean of three technical replicates ± SDandfittedtoadouble exponential decay. D) Bar plot showing relative activity of the enzymes following incubation at various temperatures for 30 min in aqueous buffer, NADES and glycerol. Samples were diluted in buffer prior to activity assay. All values are mean of three independent technical replicates ±SD.

4 C. Knudsen, et al. Phytochemistry 170 (2020) 112214 denaturation or inhibition of proteins. tyrosine into dhurrin was negligible. However, the activity of the To further test the ability of NADES to stabilize enzyme activity pathway enzymes was recovered by dilution of the NADES (Figs. 2 and during long-term storage, the dhurrin biosynthetic enzymes recon- 3). This demonstrates that the inactivation of the biosynthetic enzymes stituted in proteoliposomes were kept at room temperature in the at the high concentrations of the glucose:tartrate NADES was reversible, concentrated glucose:tartrate NADES, glycerol or in aqueous buffer despite almost complete removal of water from the system. Substitution prior to dilution and activity assays. The activity of all proteins followed of the water shell by sugars upon desiccation preserved the biosynthetic a double exponential decay indicating two phases of protein dena- machinery. Combined with the increased heat stability of each of these turation (Fig. 3C). CYP79A1 and CYP71E1 maintained activity for more enzymes in the glucose:tartrate NADES when compared with aqueous than 30 d when stored in glucose:tartrate NADES compared to 5 d in buffer (Fig. 3C and D), this provides further evidence that NADES are aqueous buffer and 15 d in glycerol. Assays were performed byad- capable of fulfilling the role of mediating heat, dehydration ministration of tyrosine as substrate. Hence, the dramatic decrease of and drought tolerance. Interestingly, the soluble enzyme UGT85B1 CYP71E1 activity is the cumulative effect of stability and the coupling catalyzing the final step in the dhurrin biosynthetic pathway was only between CYP79A1 and CYP71E1. UGT85B1 was more stable in glycerol marginally influenced by the high concentrations of glucose inthe compared to both the NADES and aqueous buffer, maintaining activity glucose:tartrate NADES (Fig. 3B). This demonstrates that glucose when for more than 200 d (Fig. 3C). The temperature stability of the dhurrin present in NADES does not compete with the UDP-glucose binding site pathway enzymes was monitored in the glucose:tartrate NADES, in of UGT85B1. Regardless of enzyme function, the presence of NADES as glycerol and in aqueous buffer to assess whether the NADES would also a third phase during desiccation is capable to prevent precipitation of protect against heat stress. All enzymes showed increased stability in enzymes, metabolites and polymers and can preserve enzyme function the NADES at higher temperatures, resulting in at least 50% activity until the water balance is restored. preservation following exposure to 60 °C for 30 min (Fig. 3D). 3.3. NADES phases assembled by intracellular liquid-liquid phase 3. Discussion separation

3.1. Formation of NADES in plants and possible applications NADESs form a “liquid crystal” through formation of inter- and intramolecular hydrogen bonds. This results in a high melting point In the present study we demonstrated that a glucose:tartrate NADES depression causing the solids to liquify and, in many cases, to remain serves as an excellent in vitro stabilizing solvent for the dhurrin fluid at room temperature. The unparalleled solubilization capacity of metabolon and possibly dhurrin. In the plant tissue, storage of dhurrin NADESs would suggest that certain combinations of NADES con- in high-density bio-condensates composed of a glucose:tartrate or stituents also possess the ability to establish hydrogen bonds to specific similar based NADESs is thus a possibility. NADESs can be formed from bio-active natural products. The water molecules present in NADESs are general metabolites such as sugars, amino acids and organic acids, all of highly coordinated by solvophobic sequestration, forming nanos- which are present in considerable amounts in the cells of all living tructured domains and worm-like structures orchestrated by the eu- organisms (Choi et al., 2011). Sugars serve the purpose of energy sto- tectic pair. The ionic clusters are part of a complex and structurally rage, but the reason that many of the other simple metabolites are disordered hydrogen-bonding network. In this way, the NADESs may present in high amounts has remained unresolved. Small organic mo- function as super-molecules mimicking the polymers known to stabilize lecules such as proline, trehalose and sucrose are known to contribute liquid-liquid phase separation in animal cells (Banani et al., 2017; to freezing tolerance in wheat (Kovacs et al., 2011) and a number of Brangwynne et al., 2009; Gall, 2003; Pederson, 2011). Analyses using a sugars and amino acids have been implicated in drought tolerance range of different scattering techniques demonstrated that NADESs (Hoekstra et al., 2001). Based on the pioneering research from the formation is not based on classical phase separation (Hammond et al., groups of Robert Verpoorte and Young Hae Choi at Leiden University, 2017). Hence the properties of NADESs are retained by dilution with up subsets of many additional organic metabolites have been suggested to to 40–50 mass % H2O. Upon further addition of water, the unique function as NADES providing, a third liquid phase for storage of high properties of the NADES are gradually abolished. local concentrations of natural products in plant cells (Choi et al., 2011; Dolgin, 2018). Cellular constituents uncovered by NMR-based meta- 3.4. Biological relevance of intracellular NADES phases bolomic analyses were found to be able to possibly form more than 30 combinations of liquid NADESs (Choi et al., 2011). Bio-production of Our data support that NADESs may have an in vivo function in plants high value natural products in engineered cells of cyanobacteria, algae and other organisms, by establishing structurally confined high-density or plants is currently challenging, being hampered by bottleneck issues NADES-based bio-condensates, in which the biosynthetic enzymes and such as low production levels of the desired bio-active natural product, bio-active natural products are stored in a soluble form. The inherent autotoxicity and storage capacity issues. Storage within NADES-based properties of NADESs could enhance plant robustness because a compartments might overcome some of these bottlenecks and facilitate NADES has almost no vapor pressure and thus retains water during the path to the biological production of high value molecules based on desiccation caused by drought stress and heat waves. That is, in in- solar energy and carbon dioxide from the atmosphere as the sole carbon stances where dehydration of cells would result in protein precipitation source. or denaturation, the formation of NADESs and presence of sequestered water molecules may provide an environment in which proteins can 3.2. In vitro stabilization of the dhurrin biosynthetic enzymes in NADES maintain solubility and intramolecular structures until rehydration occurs. Using the enzymes in the biosynthetic pathway for the cyanogenic Independent of whether a NADES phase is membrane encapsulated glucoside dhurrin as model enzymes in natural product metabolism or not, NADES-based liquid phase separation would prevent osmotic (Fig. 1C), we investigated the ability of equimolar glucose:tartrate issues related to accumulation of molar concentrations of natural NADESs to stabilize the two cytochromes P450 CYP79A1 and CYP71E1, products and thus prevent disruption of cell homeostasis (Aguilera- the UDP-glucosyltransferase UGT85B1 and the cytochrome P450 Gomez and Rabouille, 2017; Courchaine et al., 2016; Kuchler et al., oxidoreductase POR2B (Fig. 3). Optimum enzyme activity was found at 2016). In addition to providing storage capacity for bio-active 5% NADESs (Fig. 2B), which suggests that the cellular interior may compounds, NADES-based assemblies may also serve to mediate and represent a dilute NADES environment (Ellis and Minton, 2003). At fine-tune numerous biological processes. A higher order of metabolic high concentration of the glucose:tartrate NADESs, conversion of and catalytic control may be achieved by limiting or improving enzyme

5 C. Knudsen, et al. Phytochemistry 170 (2020) 112214 access and activity toward their substrates or by embedding entire were germinated between two layers of gauze for 3–4 d at 28 °C in the biosynthetic pathways in NADES-based assemblies. The high solubi- dark. Seedlings were harvested with a razor blade, avoiding collecting lizing capacity of the NADES would imply that the biosynthesis of some the seed coats. Seedlings were homogenized using a mortar and pestle natural products sparsely soluble in water could occur in NADESs, in in 2 ×(w/v) homogenization buffer (250 mM sucrose, 100 mM Tris (pH which both substrate and enzymes are dissolved. Typically, Km values 7.9), 50 mM NaCl, 2 mM EDTA, 2 mM DTT) and 10% (w/w) of poly- are determined in dilute aqueous buffers. Such values may not be very vinylpolypyrrolidone (PVPP). The homogenate was filtered through relevant for enzymes exhibiting their catalytic functions in a NADES. two layers of 22 μm nylon cloth, following which cell debris were Dehydration of bio-active natural products and/or enzymes stored removed by centrifugation (10,000 g, 4 °C, 10 min). The microsomal in NADESs might prevent some enzymatic conversions. NADESs may membranes were isolated from the supernatant obtained by thus have multiple roles in the regulation, production and storage of ultracentrifugation (150,000 g, 4 °C, 1 h) and resuspended in re- natural products in addition to their ability to function as exquisite suspension buffer (50 mM Tris (pH 7.9), 100 mM NaCl, 2 mM DTT,DTT solvents. Formation of phase-separated NADES droplets in plant cells added just before use) using a marten hair soft brush and homogenized may thus contribute to the biosynthesis and compartmentalized storage using a Potter-Elvehjelm homogenizer. of natural products. 4.4. Expression and purification of CYP79A1, CYP71E1, POR2B and 3.5. Applications of NADES inspired by nature UGT85B1

In parallel to the implications of our studies, specific NADESs have CYP79A1 and CYP71E1 were expressed in the yeast (Saccharomyces been demonstrated to function as excellent storage media for human cerevisiae) strain BY4741 and purified as described elsewhere (Laursen interferon-alpha 2a, an important therapeutic protein. Enzyme activity, et al., 2013, 2016; Pompon et al., 1996). POR2B was expressed in Es- long-term storage as well as resistance to denaturation at high tem- cherichia coli cell strain BL21 (DE3) and purified as described elsewhere peratures were improved (Lee et al., 2018). Likewise, lipases were (Laursen et al., 2016; Wädsater et al., 2012). UGT85B1 was expressed shown to possess dramatically enhanced activities in a NADES as a C-terminal fusion protein with green fluorescent protein (GFP) (Elgharbawy et al., 2018). NADESs based on trehalose and glycerol are interspaced with a tobacco etch virus (TEV) protease cleavage site and being used as cryoprotective agents based on their strong effects on the including a His-tag as described elsewhere (Laursen et al., 2016). water crystallization/freezing and melting process, resulting in the formation of a reduced number of ice crystals and hence reduced ice 4.5. Preparation of proteoliposomes crystal damage in cells; a crucial parameter for their survival following freezing and thawing (Castro et al., 2018). The three-subunit mem- Native sorghum lipids were extracted with chloroform and me- brane-spanning photosynthetic reaction center from Rhodobacter thanol directly from microsomes prepared from etiolated sorghum sphaeroides was shown to be stabilized in choline-based NADESs seedlings (Bligh and Dyer, 1959). The concentration of phospholipids (Milano et al., 2017). The photosynthetic reaction center complex was determined by the method of Rouser et al. (1966). retained its activity upon illumination to carry out charge separation Liposomes were prepared with CYP79A1, CYP71E1 and POR2B and reduce a quinone acceptor using cytochrome c as the electron reconstituted in native sorghum lipids as follows; Phospholipids donor. (1000 nmol) were solubilized in buffer (50 mM Tris-HCl, 100 mM NaCl, In conclusion, our data on the ability of the glucose:tartrate NADES 50 mM cholate, pH 7.5) and mixed with 1.3 nmol CYP79A1, 4 nmol to stabilize long-term activity and heat-tolerance of the membrane- CYP71E1 and 4 nmol POR2B in a total volume of 1000 μL and in- bound and soluble enzymes in dhurrin biosynthesis adds to the pro- cubated for 1 h at 4 °C. Detergent was removed by addition of an equal posed role and importance of NADESs in biological systems. volume of Bio-Beads SM-2 adsorbent (Biorad, Denmark) and incubation for 3 h at 4 °C. Bio-Beads were removed by centrifugation (1000 g for 4. Experimental 20 min) and the supernatant was collected for further studies. POR2B proteoliposomes were prepared in the same proportions as 4.1. Chemicals described above except without CYP79A1 and CYP71E1 and using 25% DLPG/75% DLPC (DLPG, 1,2-dilauroyl-sn-glycero-3-phosphoglycerol; All chemicals were of analytical grade and purchased from Sigma DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine (Avanti Polar lipids, Aldrich (Denmark) unless stated otherwise. Dhurrin was chemically USA). synthesized as previously described (Møller et al., 2016). 4.6. Setups for enzyme activity and stability assays 4.2. Preparation of NADES 4.6.1. NADES optimum on sorghum microsomes The three different NADESs, composed of 1:1 D-glucose and dipo- The conversion of tyrosine catalyzed by the membrane-anchored tassium tartrate (glu:tart), 1:1 D-glucose and sodium malate (glu:mal) POR2B, CYP79A1 and CYP71E1 to p-hydroxymandelonitrile was as- and 1:2 D-glucose and choline chloride (glu:chol), were mixed with 20% sayed in the presence of 0, 4, 8, 12, 16, 20, 24, 28, 36, 44 and 48% w/w H2O at 70 °C under magnetic stirring until a clear solution was NADES or glycerol. Assay conditions are described below. obtained. The resulting stock solutions named 80% NADES contained the following concentrations glu:tart (3.0 M D-glucose and 3.0 M 4.6.2. Time course on proteoliposomes dipotassium tartrate), glu:mal (3.8 M D-glucose and 3.8 M sodium Proteoliposomes containing POR2B (4.0 μM), CYP79A1 (1.3 μM) malate), glu:chol (2.1 M D-glucose and 4.2 M choline chloride), with and CYP71E1 (4.0 μM) as described above and UGT85B1 (18.2 μM) glycerol (8.7 M glycerol) as a control. were suspended in 10 vol buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5), glycerol (28% H2O) or NADES (28% H2O). Samples were prepared 4.3. Growth of etiolated sorghum seedlings and preparation of microsomal in triplicates and stored at RT. For each time point, 10 μl was diluted fractions 6× prior to activity assay (12% final concentration of glycerol and NADES). Microsomes were prepared from Sorghum bicolor (L.) Moench (Poaceae) as described elsewhere (Halkier and Møller, 1989). Briefly, 4.6.3. Heat treatment on proteoliposomes seeds of S. bicolor BTx-623 (Seedtek Pty Ltd, Toowoomba, Australia) Samples were solubilized in buffer (100 mM NaCl, 50 mM Tris-HCl,

6 C. Knudsen, et al. Phytochemistry 170 (2020) 112214

pH 7.5), glycerol (28% H2O) or NADES (28% H2O) in a volume of and Typhoon scanner control software version 5.0. Product formation 100 μl before heat treatment for 30 min at either 30 °C, 40 °C, 50 °C, was quantified using the software ImageQuant TL ver. 7.0 and theuse 14 60 °C, 70 °C, 80 °C and 90 °C followed by cooling on ice for 30 min prior of a standard curve of 5–100 pmol UDP-[ C]-D-glucose. to activity assay. Author contribution 4.7. Activity assays C.K, B.L.M and T.L. designed the research project; C.K, K.B, K.M.V. Microsomes, proteoliposomes and recombinant UGT85B1 were in- and T.L. performed in vitro experiments; M.S.M. performed chemical cubated in 72% NADES for all experiments related to activity recovery, synthesis of dhurrin and non-commercial intermediates; H.T.S, D.P.D, time course and heat treatment. Samples were diluted 6 × in buffer B.L.M. and T.L. initiated and supervised the research; C.K, D.P.D, B.L.M. (100 mM NaCl, 50 mM Tris-HCl, pH 7.5) in the assay reactions. and T.L. wrote the manuscript. All authors approved the final version of the manuscript. 4.7.1. Cytochrome P450 oxidoreductase, POR Reductase activity was measured using cytochrome c (cyt c) as Declaration of competing interest electron acceptor at different concentrations of NADES (containing

100-28% H2O) in buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.5). The The authors declare no conflict of interest. assay mixtures (total volume: 1 ml) contained 0.12 pmol POR protein in 25% DLPG/75% DLPC liposomes, 1 mM NADPH and 50 μM Acknowledgement cyt c. Reduction of cyt c was monitored at 550 nm using a PerkinElmer Lambda 650 UV/Vis spectrophotometer (extinction This research was supported by the VILLUM Research Center “Plant coefficient = 21.2 mM−1 cm−1). The slope of the curve in the linear Plasticity” (Grant no. VKR023054); by the ‘bioSYNergy’ program of region was used to determine the catalytic activity (Guengerich et al., Center for Synthetic Biology (University of Copenhagen Excellence 2009). For determination of reductase activity after heat Program for Interdisciplinary Research); by an ERC Advanced Grant to treatment, POR samples were diluted to 6 × corresponding to a final B.L.M. (ERC-2012-ADG_20120314, Project No: 323034); and by concentration of 88% H2O in both NADES or glycerol samples prior to Innovation Fund Denmark (12–131834). T.L. was supported by a fel- performing the activity assay. lowship awarded by the VILLUM Foundation (95-300-73023), by a fellowship awarded by the Novo Nordisk Foundation 4.7.2. Sorghum microsomes and recombinant cytochromes P450 enzymes (NNF17OC0024886) and a Sapere Aude Starting Grant awarded by the (CYPs) Independent Research Fund Denmark (7026-00041A). K.B. was sup- CYP activity was measured in assay mixtures (total volume: 30 μl) ported by P4FIFTY Marie Curie Actions (FP7, REA grant agreement containing either microsomes (1.5 μg/μL total protein) or CYP79A1 number 289217). D.P.D. received funding from the European Union (0.22 pmol) and CYP71E1 (0.67 pmol) in sorghum lipid proteolipo- Seventh Framework Programme (FP7/2007–2013) under grant agree- 14 somes, 17 μM [UL- C]-L-tyrosine (specific activity: 391 μCi/μmol, ment 275422, which supported a Marie Curie International Outgoing Larodan Fine Chemical AB, Malmö, Sweden), 100 μM L-tyrosine, 1 mM Fellowship. The Danish Strategic Research Council Grant “SPOTLight” NADPH, 100 mM NaCl and 50 mM Tris-HCl (pH 7.5). Incubation (30 °C, supported the work of H.T.S. 300 rpm, 30 min) was terminated by direct application of aliquots

(10 μl) onto TLC plates (Silica gel 60 F254, Merck). Substrates and References intermediates were separated using a mobile phase of toluene: ethyl acetate: methanol (30:8:1 by volume). Authentic standards (40 pmol) Aguilera-Gomez, A., Rabouille, C., 2017. Membrane-bound organelles versus membrane- were spotted on the TLC plates and visualized by UV luminescence. less compartments and their control of anabolic pathways in Drosophila. Dev. Biol. 428, 310–317. Radiolabeled reaction products were visualized by O/N exposure of the Bak, S., Kahn, R.A., Nielsen, H.L., Møller, B.L., Halkier, B.A., 1998. Cloning of three TLC plates to Phosphoimager screens (Molecular Dynamics) and de- A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) veloped using a Typhoon TRIOTM (GE Healthcare) and Typhoon Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic scanner control software version 5.0 (Amersham Biosciences). Product glucoside dhurrin. Plant Mol. Biol. 36, 393–405. formation was quantified using the software ImageQuant TL ver. 7.0 Bak, S., Olsen, C.E., Halkier, B.A., Møller, B.L., 2000. Transgenic tobacco and Arabidopsis (GE Health Care) and the use of a standard curve of 12.8–256 pmol plants expressing the two multifunctional sorghum cytochrome P450 enzymes, 14 CYP79A1 and CYP71E1, are cyanogenic and accumulate metabolites derived from [UL- C]-L-tyrosine. For NADES optimum concentration determination intermediates in dhurrin biosynthesis. Plant Physiol. 123, 1437–1448. assays were performed as described above and addition of various Banani, S.F., Lee, H.O., Hyman, A.A., Rosen, M.K., 2017. Biomolecular condensates: or- concentrations of NADES i.e. 0, 4, 8, 12, 16, 20, 40, 60 and 72% ganizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298. NADES. Bassard, J.E., Møller, B.L., Laursen, T., 2017. Assembly of dynamic P450-mediated metabolons-order versus chaos. Curr Mol Biol Rep 3, 37–51. Bavishi, K., Li, D., Eiersholt, S., Hooley, E., Petersen, T., Møller, B.L., Hatzakis, N., 4.7.3. UDP-glucosyltransferase (UGT) Laursen, T., 2018. Direct observation of multiple conformational states in UGT85B1 catalytic activity was measured in assay mixtures (total Cytochrome P450 oxidoreductase and their modulation by membrane environment and ionic strength. Sci Rep-Uk 8, 6817. volume: 30 μl) containing recombinant UGT85B1 (1.2 pmol), 3.33 μM Bjarnholt, N., Neilson, E.H.J., Crocoll, C., Jorgensen, K., Motawia, M.S., Olsen, C.E., 14 UDP-[UL- C]-D-glucose (specific activity: 250 μCi/μl, PerkinElmer), Dixon, D.P., Edwards, R., Møller, B.L., 2018. Glutathione transferases catalyze re- 83.3 μM unlabeled UDP-D-glucose, 10 mM p-hydroxymandelonitrile cycling of auto-toxic cyanogenic glucosides in sorghum. Plant J. 94, 1109–1125. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. (TCI Europe N.V.), 100 mM NaCl and 50 mM Tris-HCl (pH 7.5). Can. J. Biochem. Physiol. 37, 911–917. Incubation (30 °C, 300 rpm, 30 min) was terminated by direct appli- Blomstedt, C.K., O’Donnell, N.H., Bjarnholt, N., Neale, A.D., Hamill, J.D., Møller, B.L., Gleadow, R.M., 2015. Metabolic consequences of knocking out UGT85B1, the gene cation of aliquots (20 μl) onto TLC plates (Silica gel 60 F254, Merck). encoding the glucosyltransferase required for synthesis of dhurrin in Sorghum bicolor. Substrates and intermediates were separated using a mobile phase of Plant Cell Physiol. 57, 373–386. freshly mixed ethyl acetate: acetone: dichloromethane: methanol: water Blomstedt, C.K., Gleadow, R.M., O'Donnell, N., Naur, P., Jensen, K., Laursen, T., Olsen, (40:30:12:10:8 by volume). For determination of the migration of the C.E., Stuart, P., Hamill, J.D., Møller, B.L., Neale, A.D., 2011. A combined biochemical dhurrin, an authentic standard (40 pmol) was spotted on the TLC plates screen and TILLING approach identifies mutations in Sorghum bicolor L. Moench resulting in acyanogenic forage production. Plant Biotechnol. J 10, 54–66. and visualized by UV luminescence. Radiolabeled reaction products Brangwynne, C.P., Eckmann, C.R., Courson, D.S., Rybarska, A., Hoege, C., Gharakhani, J., were visualized by O/N exposure of the TLC plates to Phosphoimager Julicher, F., Hyman, A.A., 2009. Germline P granules are liquid droplets that localize screens (Molecular Dynamics) and developed using a Typhoon TRIO™ by controlled dissolution/condensation. Science 324, 1729–1732.

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