This work was supported by a Norwich Research Park Studentship (JR), the joint Engineering and Physical Sciences Research Council/Biotechnological and Biological Sciences Research Council (BBSRC)-funded OpenPlant Synthetic Biology Research Centre grant (BB/L014130/1) (M.J.S., A.O.); a John Innes Centre Knowledge Exchange and Commercialization grant (BB/KEC1740/1) (B.B.); and the BBSRC Institute Strategic Programme Grant &#39;Molecules from Nature&#39; (BB/P012523/1) and the John Innes Foundation (A.O.). We would like to thank George Lomonossoff for providing the pEAQ vectors and Andrew Davis for photography.

<ol>
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The authors have nothing to disclose.

High-through-put vacuum infiltration allows this protocol to be used routinely in our laboratory for the preparative production of triterpene compounds of interest for various downstream applications. The vacuum infiltration apparatus described here is easily replicated. Addition of the vacuum reservoir is advisable to decrease the time required to pull the necessary vacuum, however it is possible to simply repurpose an unmodified vacuum oven as the infiltration chamber. Protecting the vacuum pump via addition of a suitable condenser is theoretically sensible, but in our laboratory we have found this to be unnecessary.
Infiltration coverage is compromised if the leaves are not fully immersed in the infiltration suspension. This problem is minimized by ensuring that the level of the suspension reaches the top surface of the plant holder, and that the plant holder is a tight fit for the infiltration bath. Note from Figure 2 that the top surface of the plant holder is recessed in to the infiltration bath when in place. This is achieved by panels on the middle edges of the holder which form the anchor point with the top of the infiltration bath. The infiltration suspension will require periodic additions to maintain the suspension level. This is best achieved by addition of excess infiltration suspension to prevent gradual reduction of the OD600 over the course of a large batch-wise infiltration. However, in our hands, substitution for water does not seem to have a noticeable effect on expected yields on a preparative scale, although this has not been quantitively investigated. How many plants can be infiltrated from one batch of infiltration suspension is still an open question. We routinely infiltrate between 100 and 200 plants with a single batch of infiltration suspension. In addition, it is normal for some soil to leach into the infiltration suspension over the course of the infiltration process. This has not been found to have a noticeable effect on infiltration efficacy.
During harvest it is advisable (but not critical) to harvest only the leaves that were infiltrated (some leaves will have grown post-infiltration). Selective harvest prevents dilution with unproductive tissue, which would otherwise increase the ratio of endogenous impurities relative to the compound of interest. This has a nominal impact on the ease of downstream purification, and increases the scale of these processes. When using tHMGR to boost precursor supply, a necrosed phenotype is often observed to develop over the post-infiltration growth period. This is normal and actually aids selective harvest and the downstream drying process. The dried leaf powder can usually be stored in a sealed container in a cool, dry, dark place, but ideally in a desiccator under vacuum. This depends on the stability of the compound of interested. Exercise caution if choosing to store in a -80 &#176;C freezer. Ensure that the dried leaves are in a waterproof container, and on removal from storage, allow this container to warm to room temperature prior to opening. Failure to do so will result in rewetting of the leaves, hindering down-stream processing.
The post-harvest steps in this protocol are provided for illustrative purposes, and to aid those researchers who may have limited practical chemistry experience. They can be substituted for many other natural product extraction/purification techniques. As detailed in the introduction, PSE has many advantages, however the capital cost of commercially available PSE apparatus may be prohibitive, and it is not essential. Basic ion-exchange resin treatment is a very convenient method to replace traditional saponification followed by liquid/liquid partitioning. However, it is not suitable for products containing carboxylic acid groups or functional groups that would be hydrolyzed under basic conditions, such as esters. These products would be expected to be retained on the basic ion exchange resin. However, there is potential to exploit this for a capture and release procedure (not documented here). Where traditional saponification would be appropriate, basic ion-exchange resin treatment serves as a convenient alternative. The chromatography method described in step 9, has been found to be generalizable for the compounds described in Figure 3. However, compounds of increased polarity may require an extended 100% ethyl acetate elution period. With reference to step 9.2, downstream fine purification is entirely compound specific, and is also dependent on scale. Repeated rounds of smaller-scale flash chromatography with optimized gradients, is usually sufficient to achieve a purity appropriate for NMR analysis. On larger scales, crystallization is often a convenient alternative. In difficult cases, preparative or semi-preparative high-performance liquid chromatography (HPLC) can be employed depending on the desired quantity of isolated compound. Examples of downstream purification processes for a range of representative compounds can be found elsewhere8.
The current protocol, as presented here, is used routinely in our laboratory, and has proven utility. However, there is still significant scope for further optimization of the expression platform. Work is ongoing in our laboratory to investigate how further pathway engineering and differential control of protein expression levels could improve triterpene production further, while mediating toxicity to host cells. There is also potential to investigate the manipulation of intracellular transport systems20,21, and the direction of enzymes to different cellular compartments22,23, avenues which could improve the efficiency of multiple oxidation events. Potential benefits to modifying the underlying morphology of key organelles could also be explored. For example, overexpression of the membrane domain of Arabidopsis thaliana HMGR has been observed to induce hypertrophy of the endoplasmic reticulum in plants24; a key site for CYP450 activity. This protocol is ideally suited for the production of milligram to gram-scale quantities of triterpene products, and can be employed in a research setting to access compounds for downstream study (e.g., the systematic exploration of structure-activity relationships, and preliminary investigations in to the pharmacodynamic and pharmacokinetic properties of lead compounds of clinical interest). However, the platform is linearly and reliably scalable simply by increasing the number of plants used, and the practicality of transient expression via agroinfiltration has been demonstrated on an industrial scale for the production of pharmaceutical proteins14, thus there is potential for this platform to be extended for commercial production of high-value triterpenes. Alternatively, it is also possible to envision the production of stable transformants carrying the finalized desire multigene biosynthetic pathway, allowing the mass cultivation and continued &#39;farming&#39; of transgenic N. benthamiana strains producing different compounds of commercial value.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Despite their immense diversity, all triterpene natural products are believed to be derived from the same linear precursor 2,3-oxidosqualene, a product of the mevalonate pathway in plants. Cyclization of 2,3-oxidosqualene is initiated and controlled by a family of enzymes termed oxidosqualene cyclases (OSCs). This cyclization step represents the first level of diversification, with hundreds of different triterpene scaffolds having been reported from nature1. These scaffolds are further diversified by tailoring enzymes including, but not limited to, cytochrome p450s (CYP450s)1,2. Such biosynthetic pathways can lead to immense complexity, sometimes resulting in final products which are barely recognizable from the parent triterpene. For example, the complex structure of the potent insecticidal and antifeedant limonoid azadirachtin is believed to derive from a tetracyclic triterpene of the tirucallane type3.
Many triterpene-derived natural products, even those with relatively unmodified parent scaffolds, have been shown to possess medicinally relevant biological activity4,5,6,7. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows.
Transient expression of triterpene biosynthetic enzymes from other plant species in N. benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products (Figure 1). This process can be used to functionally characterize candidate enzymes and reconstruct the biosynthetic pathways of important naturally-occurring metabolites. Equally, it can also be exploited in combinatorial biosynthetic approaches to produce novel triterpene products, a strategy that can result in libraries of structurally related analogs, allowing systematic exploration of the structure-activity relationships of biologically active lead compounds8,9.
Agroinfiltration is an efficient and simple means of achieving transient expression in N. benthamiana leaves. The process involves the infiltration of leaves with a suspension of A. tumefaciens carrying the binary expression construct(s) of interest. This is achieved via the application of pressure which forces the liquid through the stomata, displacing air in the intercellular space, and replacing it with the A. tumefaciens suspension. The bacteria transfer the respective T-DNAs to the interior of the plant cells, resulting in localized and transient protein expression in the infiltrated leaf tissue.
While any binary vector suitable for generating transgenic plants may be employed for transient expression, we utilize the Cowpea Mosaic Virus (CPMV)-derived Hypertranslational (HT) protein expression system10,11. In this system the gene of interest is flanked by untranslated regions (UTR) from the CPMV RNA-2. The 5&#39; UTR contains modifications resulting in very high levels of protein translation with no reliance on viral replication12. This technique has been developed into the Easy-And-Quick (pEAQ) binary vector series which includes site-specific recombination cloning protocol-compatible constructs (pEAQ-HT-DEST)10,11. Most pEAQ vectors also contain a tomato bushy stunt virus-derived P19 silencing suppressor gene13 within the T-DNA portion of the expression cassette, which circumvents the need to coinfiltrate a separate P19-carrying strain and affords very high-level protein expression in the host plant cell10,11.
Use of N. benthamiana as an expression host has particular advantages when working with plant biosynthetic pathways. The cell architecture intrinsically supports appropriate mRNA and protein processing, and proper compartmentalization, in addition to possessing the necessary co-enzymes, reductases (for CYP450s), and metabolic precursors. The carbon source is photosynthesis; thus, plants can simply be grown in good quality compost, requiring only water, CO2 (from the air), and sunlight as inputs. The platform is also extremely convenient for co-expression of different combinations of proteins, as this can be achieved facilely by the co-infiltration of different strains of A. tumefaciens, negating the need to build large multigene expression cassettes. Furthermore, the process can be linearly and reliably scaled simply by increasing the number of plants used in the experiment.
Previous work in our laboratory has demonstrated the utility of this platform for preparative scale experiments. This included the preparation of novel triterpenes for use in bioactivity assays and scale up to achieve gram quantities of isolated product. Furthermore, accumulation of heterogenous triterpene products can be increased several fold by co-expression of an N-terminal-truncated, feedback-insensitive form of 3-hydroxy, 3-methyglutary-coenzyme A reductase (tHMGR), a rate-limiting upstream enzyme in the mevalonate pathway8.
Key to such preparative-scale experiments is the ability to conveniently up-scale the infiltration process. In a typical experiment, tens to hundreds of plants may be required to achieve the target quantity of isolated product. Infiltrating individual leaves by hand (using a needless syringe) is operationally demanding, and often prohibitively time-consuming, rendering this method impractical for routine scale-up. Vacuum infiltration offers advantages over hand infiltration, as it is not dependent on the skill level of the operator and allows the infiltration of a greater area of the leaf surface. This procedure is used commercially for the large-scale production of pharmaceutical proteins14. This protocol utilizes an easily replicable vacuum infiltration apparatus, which can be constructed from commercially available parts. This allows the simultaneous infiltration of up to 4 plants, affording rapid and practical batch-wise infiltration of hundreds of plants in a short period of time (Figure 2a - 2b). The vacuum infiltration apparatus consists of a vacuum oven which forms the infiltration chamber (Figure 2f). The oven is connected to a pump via a vacuum reservoir. This greatly reduces the time required to achieve the desired vacuum in the infiltration chamber. Plants are secured in a bespoke holder and inverted into a 10 L stainless-steel water bath filled with A. tumefaciens suspension (Figure 2a - 2c). Complete immersion of the aerial parts of the plants is important for efficient infiltration. The water bath is then placed within the infiltration chamber (Figure 2d - 2e), and the vacuum applied to draw the air out of the leaf interstitial spaces. Once the pressure has been reduced by 880 mbar (a process which takes approximately 1 min) the infiltration chamber is brought quickly back to atmospheric pressure over 20 - 30 s by opening the oven inlet valve, whereupon infiltration is complete.
Five days after infiltration the plant material is ready for harvesting and subsequent extraction and isolation of the desired product(s). From this point the process is simply one of natural product extraction and purification from leaf material, a workflow that is familiar to any natural product chemist. Many different methods for initial extraction and subsequent purification exist15. The most appropriate choice of methods and the exact conditions used is highly dependent on the particular chemical properties of the compound of interest, in addition to the availability of skills and/or equipment. It is not possible to include in this protocol a fully generalizable, step-by-step method for the downstream processing of harvested plant leaf material to isolated product that could be followed blindly for any triterpene product of interest, nor would it be appropriate to attempt to do so. However, this protocol will provide an overview of the basic workflow used in our laboratory and some methods for the early stages of the process, which in our experience have proved generalizable for most oxygenated aglycone triterpene products. This includes two relatively uncommon techniques, namely, Pressurized Solvent Extraction (PSE), and a convenient heterogenous phase method for chlorophyll removal using a strongly basic ion exchange resin.
PSE is a highly efficient technique for the extraction of small organic molecules from solid matrixes. Extractions are performed under pressure (ca. 100 - 200 bar), the main advantage being the ability to use alleviated temperatures which exceed the boiling point of the extraction solvent. This can significantly reduce the time and amount of solvent required to achieve an exhaustive extraction, when compared to other hot solvent techniques such as simple refluxing or Soxhlet extractions16. Commercial bench-top PSE instruments are available, which utilize interchangeable extraction cells, and automated solvent handling, heating, and monitoring. This makes this technique extremely convenient. It is also arguably less hazardous, particularly for operators with limited practical chemistry experience.
Saponification of crude leaf extracts under reflux followed by liquid/liquid partitioning is a common technique for the bulk removal of chlorophylls prior to subsequent purification or analysis. However, this can often be operationally demanding on larger scales. Furthermore, detection of the interface, or product loss due to the formation of emulsions can be problematic. The use of strongly basic ion-exchange resins to perform heterogenous phase hydrolysis can serve as a convenient alternative. The pigmented portion of the hydrolyzed chlorophyll remains adhered to the resin and can be simply removed by filtration. This protocol utilizes a preparative scale adaptation of a previously reported analytic procedure17 that employs a commercially available basic ion-exchange resin.
Below we describe a detailed and rapid protocol for the preparative scale production of triterpenes utilizing this plant-based platform. This protocol is used routinely in our laboratory to prepare tens to hundreds of milligrams of isolated triterpene product for applications such a structural characterization by nuclear magnetic resonance (NMR) spectroscopy, and/or further study in functional assays.

<table><tbody><tr><td>GC-MS instrument</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Thermocycler suitable for performing PCR reactions</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Gel electrophoresis apparatus</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Adjustable air displacement micropipettes (Sizes : 1000 &amp;micro;L, 200 &amp;micro;L, 100 &amp;micro;L, 20 &amp;micro;L, 10 &amp;micro;L, and 2 &amp;micro;L )</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Wide top liquid nitrogen carrier (size: 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Benchtop microcentrifuge (Capacity: 24 x 1.5/2mL, Max. speed: =/&gt; 15000 rpm)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Benchtop centrifuge (Capacity: 20 x 50 mL, Max. speed: =/&gt; 4000 rpm)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Large Floor-standing centrifuge (Capacity: 4 x 1L, Max. speed: =/&gt; 4000 rpm)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Standing incubator</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Shaking incubator (Flask Clamp sizes: 10 mL, 50 mL, 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Centrifuge bottles (1 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Vacuum infiltration apparatus</td><td>bespoke</td><td>n/a</td><td></td></tr><tr><td>9 x 9 cm plant pots</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Freeze drying apparatus (=/&gt; 8 litre condenser capacity)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Buchi SpeedExtractor 914 with 120 mL extraction cells (PSE instrument)</td><td>Buchi</td><td>SpeedExtractor E-914</td><td></td></tr><tr><td>Rotary evaporator apparatus</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Evaporation flasks (Sizes: 100 mL, 500 mL, 1 L, 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Duran bottles (Sizes: 1 L, 10 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Conical flasks (Sizes 100 mL, 2 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Spatulas (assorted sizes)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Analytical balance (5 figures)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Balance (2 figures)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Measuring cylinders (10 mL, 100 mL, 1 L)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>A -80 freezer</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Biotage Isolera instrument with 250 mL collection rack and bottles (automated flash chromatography instrument</td><td>Biotage</td><td>ISO-1EW</td><td></td></tr><tr><td>Safety glasses</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Lab coat</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Autoclave</td><td>any</td><td>n/a</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micropipette tips (Sizes : 1000 &amp;micro;L, 200 &amp;micro;L, 100 &amp;micro;L, 20 &amp;micro;L, 10 &amp;micro;L, and 2 &amp;micro;L )</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Microcentrifuge tubes (Size: 2 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Centrifuge tubes (Sizes: 15 mL, 50 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Disposable Transfer pipettes (size 2 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Petri dishes (Size: 100 mL)</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Biotage&amp;reg; SNAP KP-SIL 100 g flash cartridges</td><td>Biotage</td><td>FSK0-1107-0100</td><td></td></tr><tr><td>HPLC vials (1 mL), vial inserts and caps</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Disposable purple nitrile gloves</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Top filter for E-914, cellulose</td><td>Buchi</td><td>51249</td><td></td></tr><tr><td>Bottom filter for E-916 / 914, glass fiber</td><td>Buchi</td><td>11055932</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Quartz sand, 0.3 - 0.9 mm</td><td>Buchi</td><td>37689</td><td></td></tr><tr><td>Celite 545 (diatomaceous earth)</td><td>Simga-Alrich</td><td>22140-1KG-F</td><td></td></tr><tr><td>Silica gel 60 (0.040 -0.0063 mm)</td><td>Merck</td><td>1.09385.5000</td><td></td></tr><tr><td>2-(N-morpholino)-ethanesulphonic acid</td><td>Melford</td><td>B2002</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Simga-Alrich</td><td>208337-100G</td><td></td></tr><tr><td>KOH</td><td>Simga-Alrich</td><td>60377-1KG</td><td></td></tr><tr><td>3&amp;rsquo;5&amp;rsquo;-dimethoxy-4&amp;rsquo;-acetophenone (acetosyringone)</td><td>Simga-Alrich</td><td>D134406-1G</td><td></td></tr><tr><td>Rifampicin</td><td>Alfa Aesar</td><td>J60836</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Gibco</td><td>11815-032</td><td></td></tr><tr><td>Streptomycin</td><td>Simga-Alrich</td><td>S6501-100G</td><td></td></tr><tr><td>Gentamycin</td><td>MP biomedicals</td><td>190057</td><td></td></tr><tr><td>Agarose LE gel</td><td>Melford</td><td>MB1203</td><td></td></tr><tr><td>LB broth Millar</td><td>Simga-Alrich</td><td>L3522-250G</td><td></td></tr><tr><td>Agar</td><td>Simga-Alrich</td><td>A5306-250G</td><td></td></tr><tr><td>NaCl</td><td>Simga-Alrich</td><td>S5886-500G</td><td></td></tr><tr><td>KCl</td><td>Simga-Alrich</td><td>P9541-500G</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Alrich</td><td>M2773-500G</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Alrich</td><td>G7021-100G</td><td></td></tr><tr><td>Ambersep&amp;reg;&amp;nbsp;900 hydroxide (basic ion exchange resin)</td><td>Sigma-Alrich</td><td>06476-1KG</td><td></td></tr><tr><td>Hexane</td><td>Fisher</td><td>H/0406/PB17</td><td></td></tr><tr><td>Ethyl acetate</td><td>Fisher</td><td>E/0900/PB17</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher</td><td>D/1852/PB17</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>M/4056/PB17</td><td></td></tr><tr><td>Ethidium bromide</td><td>Sigma-Alrich</td><td>E7637-1G</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>20821-330</td><td></td></tr><tr><td>Milli-Q water</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Levington F1 Compost</td><td>any</td><td>n/a</td><td></td></tr><tr><td>Levington F2 Compost</td><td>any</td><td>n/a</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (Plasmid purification kit).</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>NEB 5-alpha Competent E. coli</td><td>Supplier New England Biolabs Ltd</td><td>C2987I</td><td></td></tr><tr><td>pEAQ-HT-DEST1 vector</td><td>Lomonossoff laboratory John Innes Center</td><td>n/a</td><td></td></tr><tr><td>pDONR207</td><td>Thermo-Fisher</td><td>pDONR207</td><td></td></tr><tr><td>A. tumefaciens LBA4404</td><td>Thermo-Fisher</td><td>18313015</td><td></td></tr><tr><td>GATEWAY LR CLONASE(tm) II ENZYME MIX (site-specific recombination cloning protocol)</td><td>Thermo-Fisher</td><td>11791020</td><td></td></tr><tr><td>GATEWAY BP CLONASE (tm) II ENZYME MIX (site-specific recombination cloning protocol)</td><td>Thermo-Fisher</td><td>11789020</td><td></td></tr><tr><td>GO TAQ G2 GREEN MASTER MIX</td><td>Promega UK Ltd</td><td>M7822</td><td></td></tr><tr><td>PHUSION HIGH FIDELITY DNA POLYMERASE</td><td>New England Biolabs Ltd</td><td>M0530S</td><td></td></tr><tr><td>dNTP SET 100mm</td><td>Thermo-Fisher</td><td>10297018</td><td></td></tr><tr><td>RNEASY PLANT MINI KIT</td><td>Qiagen Ltd</td><td>74904</td><td></td></tr><tr><td>RQ1 RNASE-FREE DNASE</td><td>Promega UK Ltd</td><td>M6101</td><td></td></tr><tr><td>Invitrogen SuperScript III First-Strand Synthesis System</td><td>Fisher</td><td>10308632</td><td></td></tr><tr><td>Trytone</td><td>Sigma-Alrich</td><td>T7293-250G</td><td></td></tr><tr><td>Nicotiana benthamiana seeds</td><td>any reputable supplier</td><td>n/a</td><td></td></tr></tbody></table>

transient expression in nicotiana benthamiana leaves for triterpene production at a preparative scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Typical isolated yields are dependent on the enzyme/enzyme combination under investigation, the chemical stability of the target compound, and how challenging the purification process was. We have previously reported isolated yields of &#946;-amyrin derivatives from our combinatorial biosynthesis experiments ranging from 0.12 to 3.87 mg per gram of dry N. benthamiana leaf powder. These compounds ranged widely in the level of oxidation, and included unnatural combinations of enzymes (Figure 3)8. This protocol is used routinely in our laboratory to produce tens or hundreds of milligrams of purified product for structural characterization by NMR spectroscopy and downstream functional assays. We have also demonstrated the preparative utility of this platform by producing 0.8 g of the triterpene scaffold &#946;-amyrin at a purity of greater than 98%. This experiment used 459 plants, and represents an isolated yield of 3.3 mg per gram of dry N. benthamiana leaf powder8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58169/58169fig1.jpg" /> 
Figure 1: Schematic summary. Schematic representation of the process exploiting transient expression of biosynthetic enzymes in N. benthamiana leaf to divert endogenous supplies of 2,3-oxidosqualene towards the preparative production of new high-value triterpene products. <a href="https://www.jove.com/files/ftp_upload/58169/58169fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58169/58169fig2.jpg" /> 
Figure 2: Vacuum infiltrator construction and process. a) Bespoke plant holder one wing detached. b) Bespoke plant holder wings attached. c) Inversion and submersion of plants. d) Insertion of the infiltration bath. e) Infiltration bath in place within the infiltration chamber. f) Complete vacuum infiltrator: (1) Vacuum pump (2) Connection from vacuum pump to vacuum reservoir (3) Vacuum reservoir isolation valve (4) Connection between vacuum reservoir and the infiltration chamber (5) Pressure gauge (6) Air intake valve (7) Infiltration chamber (8) Vacuum reservoir. g) Representative leaves from one plant infiltrated with an expression construct for green fluorescent protein (GFP) after five days post-infiltration growth. <a href="https://www.jove.com/files/ftp_upload/58169/58169fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58169/58169fig3.jpg" /> 
Figure 3: Previously reported representative results for the preparative production of &#946;-amyrin derivatives using this protocol8. This table is ordered by isolated yields. Columns are conditionally formatted with color indication of relative value. Red = high, Green = low (though intermediate yellow). <a href="https://www.jove.com/files/ftp_upload/58169/58169fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale at JoVE.com

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

transient-expression-nicotiana-benthamiana-leaves-for-triterpene

Research

JoVE Journal

Biochemistry

17.2K Views.  John Innes Centre. Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

Video: Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Environment

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Biology

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing&#160;strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment&#39;s purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing ...

Efficient Extraction and Purification of GFP-His from N. benthamiana Leaves

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

Author Spotlight: Optimizing Apoplast Protein Extraction for Efficient Recovery of Recombinant Proteins from Plant Cells

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

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