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.

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This protocol affords convenient preparative-scaled access to triterpene compounds for use in downstream studies, such as structural characterization or the expiration of medicinally relevant biological activity. The protocol is quick, scalable, and allows combinations of enzymes to be utilized simply by the co-infiltration of different strains of Agrobacterium, negating the need to construct large multi-gene vectors. To begin, inoculate a selective LB agar plate with streaks of the desired Agrobacterium tumefaciens strains from glycerol stock cultures.

Incubate the plate overnight at 28 degrees Celsius in a standing incubator. The next day, individually inoculate 50 milliliters of selective LB media with a sample of each Agrobacterium tumefaciens strain. Incubate the culture overnight at 28 degrees Celsius in a shaking incubator at 200 rpm.

Individually transfer the 50-milliliter cultures to 1, 000 milliliters of selective LB media, and incubate overnight at 28 degrees Celsius in the shaking incubator at 200 rpm. The next day, individually pellet the Agrobacterium tumefaciens from the liquid cultures by centrifugation at 4, 000 times g. After discarding the supernatant, individually resuspend the pellets in 50 milliliters of freshly prepared MMA buffer.

Incubate the resuspended pellets at room temperature in the dark for one hour. Now, determine the appropriate volume of each Agrobacterium tumefaciens MMA suspension to produce a final OD 600 of at least 0.2 in a 10 liter total final volume. Combine the determined volumes of each suspension.

Now, add additional MMA buffer to the combined suspensions to a final volume of one liter, to be used immediately for vacuum infiltration. Transfer the one liter of infiltration suspension and the one liter of 10x strength MMA buffer to the infiltration bath, followed by an additional eight liters of water. Remove the detachable wings of the bespoke plant holder.

Insert four five-week-old plants that have been grown as described in the text protocol, and reattach the wings. Infiltration coverage is compromised if leaves are not fully submersed in infiltration suspension. This problem is minimized by ensuring that the level of the suspension reaches the top surface of the plant holder prior to vacuum treatment.

Invert the holder, and place it on top of the infiltration bath so as to submerge the leaves in the infiltration suspension. Transfer the infiltration bath with submerged plants to the infiltration chamber, and close the door. Ensure the air intake valve is closed on the infiltrator.

After turning on the vacuum pump, open the vacuum intake valve on the infiltration chamber. Once the pressure in the infiltration chamber has reduced by 880 millibar, shut the vacuum intake valve, and open the air intake valve. Upon returning to atmospheric pressure, open the door, and remove the infiltration bath.

Now, raise the plant holder until the plants are no longer submerged in the infiltration suspension. Then, gently shake the holder to allow excess suspension to run off the leaves and back into the infiltration bath. Finally, return the holder to the upright position, remove the detachable wings, and remove the plants.

Grow the infiltrated plants for five days at 25 degrees Celsius in 16 hours per day of light with daily watering. After five days of growth, harvest the leaves, and then dry them as described in the text protocol. Grind the dried leaves into a coarse powder via a convenient method, such as using a domestic food processor or manually crushing the leaves while contained within a bag.

Mix the leaf powder with quartz sand in a suitable container. This acts as a dispersant and improves the efficiency of the extraction process. Now, prepare four extraction cells for filling by first inverting them.

Then, insert the glass fiber filter, followed by the metal frit and holding plug. Return the extraction cells to the upright position, and add a one-centimeter-deep layer of quartz sand. Fill the extraction cells with the prepared leaf powder up to the fill line.

If needed, gently compress the powder during this process to facilitate addition of a greater amount in each extraction cell. Now, add a small layer of quartz sand to the top of the packed powder, and insert the top cellulose filter. Insert the packed extraction cells into the pressurized solvent extraction instrument, and run the desired method.

Once the method is complete, concentrate the combined extraction liquor to dryness via rotary evaporation under vacuum, as detailed in the text protocol. Check for the expected output of a dark green slurry. To remove the chlorophylls, using basic ion-exchange resin, first dissolve the crude extract in a minimal volume of ethanol.

Add 50 milliliters of basic ion-exchange resin beads to the resulting solution. Then, agitate at room temperature for 30 minutes. Add an additional 50 milliliters of basic ion-exchange resin beads every 30 minutes until the reaction has gone to completion.

At first, the basic ion-exchange resin beads are pink in color, and the liquid phase is green. As the reaction proceeds, the resin beads change to green and the liquid phase to orange or a murky brown color depending on the scale. Filter the reaction mixture through a short column of diatomaceous earth via a glass flash chromatography column, using hand bellows to apply pressure.

Rinse the collected resin beads with ethanol, followed by one-to-one ethanol to hexane, and finally with hexane. Use a rinse volume sufficient to resuspend the beads on top of the diatomaceous earth column. Finally, combine the filtrate, the rinses, and the concentrate to dryness by rotary evaporation under vacuum.

Shown here is an image of the leaves of one plant infiltrated with an expression construct for the fluorescent protein GFP after five days post-infiltration growth. This illustrates the extent of infiltration coverage that can be expected. The leaves are arranged from the top left to the bottom right of the image in descending order, with respect to their original height on the plant.

This image represents 0.8 grams of beta-amyrin produced using this protocol, which is greater than 98%pure. The experiment was conducted on a 459-plant scale and represents an isolated yield of 3.3 milligrams per gram of dry leaf material. While attempting this protocol, it is important to remember that only infiltrated leaves will be producing your compound of interest.

It is therefore advantageous to selectively harvest these leaves when possible to prevent dilution of your sample with unproductive tissue. The post-harvest steps in this protocol are provided for illustrative purposes. They can be substituted for other natural product extraction purification techniques depending on the availability of expertise and/or facilities at your institution.

We found that the basic ion-exchange resin treatment presented in this protocol is a convenient alternative to more traditional saponification followed by liquid/liquid partitioning for the removal of chlorophylls on larger scales. Before following this protocol, you should familiarize yourself with the material safety data for all substances used and take appropriate safety precautions. Local legislation concerning working with transgenic material should be checked and observed, including following appropriate containment procedures.

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Journal (Biochemistry)

Journal (Biochemistry)

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

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