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.


Introduction
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 nature 1 .
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 type 3 .
Many triterpene-derived natural products, even those with relatively unmodified parent scaffolds, have been shown to possess medicinally relevant biological activity 4,5,6,7 . 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 compounds 8,9 .
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 . In this system the gene of interest is flanked by untranslated regions (UTR) from the CPMV RNA-2. The 5' UTR contains modifications resulting in very high levels of protein translation with no reliance on viral replication 12 . 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 gene 13 within the T-DNA portion of the expression cassette, which circumvents the need to coinfiltrate a separate P19carrying strain and affords very high-level protein expression in the host plant cell 10,11 .
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 coenzymes, reductases (for CYP450s), and metabolic precursors. The carbon source is photosynthesis; thus, plants can simply be grown in good quality compost, requiring only water, CO 2 (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 3hydroxy, 3-methyglutary-coenzyme A reductase (tHMGR), a rate-limiting upstream enzyme in the mevalonate pathway 8 .
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 proteins 14 . 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 exist 15 . 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 extractions 16 . 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 procedure 17 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.

Protocol
Note: Before following this protocol, become familiar with the material safety data for all substances used, and take appropriate safety precautions. These include but are not limited to: wearing safety glasses when working with glass under vacuum, and handling dry silica gel in a fume cupboard. It is also essential that local legislation concerning working with transgenic material is checked and observed, including following appropriate containment procedures.

Generate A. tumefaciens Strains for Infiltration
Note: We provide here a simplified description of the steps required to generate suitable A. tumefaciens strains for transient expression. Further details of these steps are described by Sainsbury et al. 8 .
1. Amplify by PCR or synthesize the protein coding region of the gene of interest flanked by 5' and 3' attB sequences suitable for generation of an entry clone for use with a site-specific recombination cloning protocol 18,19 .  18,19 to generate an entry clone using any non-kanamycin-based entry vector (we use pDONR207 which is commercially available). Note: The integrity of the entry clone should be confirmed by sequencing, before using to generate the pEAQ-HT-DEST1 expression construct. The pEAQ-HT-DEST1 is available on request from the Lomonossoff laboratory at the John Innes Centre in Norwich, UK. 4. Isolate the pEAQ-HT-DEST1 vector from the expression clone generated in step 1.1.3, and store at -20 °C prior to use in step 1.3.
Note: Any commercially available convenient spin column-based plasmid purification kit designed for extracting plasmids from cloning strains of E. coli is suitable. Follow the specific instructions of the chosen plasmid purification kit (see Table of Materials). Note: This culture can be revived for future experiments by streaking onto LB agar plates with appropriate antibiotics (described below).

Harvest the Infiltrated Leaves
1. After 5 days of growth, harvest the leaves. 2. Autoclave waste plant material, pots, and soil prior to disposal.

Dry the Harvested Leaves
Note: The exact lyophilization procedure described below depends on the nature of the available lyophilization apparatus available.

Pressurized Solvent Extraction
Note: The proceeding steps refer to use of a commercially available PSE instrument (see Table of Materials). Please refer to the manufacturer's literature for more detailed information on operation. The instrument carries out 4 extractions in parallel.
1. Grind the dried leaves into a course powder via a convenient method (e.g., for most triterpene compounds of interest use a domestic food processor, or manually crush the leaves while contained within a bag). Note: Grinding with a pestle and mortar under liquid nitrogen is not usually necessary. 1. Mix the leaf powder with quartz sand (0.3 -0.9 mm, 20% by volume) in a suitable container; this acts as a dispersant and improves the efficiency of the extraction process.

Basic Ion Exchange Resin Treatment for Removal of Chlorophylls
Note: The quantities quoted in the following steps assume an original working scale of 100 -150 plants.
Step 8 is not suitable for products containing carboxylic acid groups, or functional groups that would be hydrolyzed under basic conditions such as esters.
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 OD 600 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 °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 elsewhere 8 .
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 systems 20,21 , and the direction of enzymes to different cellular compartments 22,23 , 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 plants 24 ; 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 proteins 14 , 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 'farming' of transgenic N. benthamiana strains producing different compounds of commercial value.

Disclosures
The authors have nothing to disclose.