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.
This protocol is suitable for producing purified diterpenoids that can be used for different downstream analysis and can be customized for a range of different metabolite targets. The main advantage of this method is it's a simple and inexpensive method to produce milligram quantities of specialized metabolites. By exchanging the genes and enzymes used for co-expression, this method can be modified to produce other terpenoids, including monoterpenoids and sesquiterpenoids, as well as other metabolite classes such as flavonoids.
For diterpenoid metabolic production and transformation, begin by warming chemically competent E.coli cells on ice. When the culture has thawed, add 25 microliters of competent cells per construct to a chilled 1.5 milliliter microtube and add 1 microliter of a 100 nanograms per microliter solution of each construct used for co-expression. Incubate the mixtures on ice for 30 minutes with gentle mixing every 10 minutes by scraping the tubes across a microtube rack.
At the end of the incubation, heat shock the cell mixtures at 42 degrees celsius for one minute before placing the cells back on ice for at least two more minutes. Next, add 200 microliters of 37 degrees celsius SOC medium to the tubes. And incubate the cultures for one hour at 37 degrees celsius and 200 rotations per minute.
At the end of the incubation, add approximately 10 autoclaved glass beads and 100 microliters of cells to one 37 degrees celsius warmed lysogeny broth agar plate with the appropriate antibiotics. And shake the plate horizontally with the lid in place to evenly distribute the cells. Then, gently tap the glass beads into a waste container and incubate the plate at 37 degrees celsius overnight, coated surface side down.
The next day, remove the plate from the incubator. Add 5 milliliters of freshly prepared lysogeny broth medium with the appropriate antibiotics to one 15 milliliter sterile glass tube with a plastic breathable cap per planned 1 liter culture, and use a pipet tip to pick individual transformed E.Coli colonies from the agar plate. Add one picked colony to each of the prepared 15 milliliter tubes and cap each tube with the accompanying breathable plastic cap.
Then, place the capped E.coli small cultures in a 37 degrees celsius shaking incubator for 12 to 24 hours. The next day add 100 milliliters of 10x PBS to 900 milliliters of terrific broth in a 1 liter flask per small culture with the appropriate antibiotics. And place the flasks at 37 degrees celsius and 140 rotations per minute for approximately 30 minutes.
When the terrific broth is warm, add the entire 5 milliliter volume of each inoculation culture to one 1 liter culture flask per culture. And place the flasks in the shaking incubator at 140 rotations per minute for approximately 3 hours. When the optical density at 600 nanometers reaches 0.6, reduce the temperature of the incubator to 16 degrees celsius.
Once the incubator has cooled, add 1 milliliter of one molar IPTG, 1 milliliter of freshly prepared 4 grams per liter riboflavin and 1 milliliter of freshly prepared 150 grams per liter aminolevulinic acid to each culture. For diterpenoid production, add 25 milliliters of one molar sodium pyruvate to each culture to ensure a sufficient precursor formation. Then, incubate the cultures at 16 degrees celsius and 140 rotations per minute for 72 hours, adding 25 milliliters of sodium pyruvate daily to the appropriate cultures.
For metabolite separation and purification, secure a separatory funnel onto a ring stand in a fume hood. And place a waste container under the funnel. Pour 500 milliliters of a 50/50 solution of ethyl acetate and hexanes into the separatory funnel.
And add 500 milliliters of the transformed E.coli co-expression culture of interest into the funnel. Place the glass stopper on the funnel and shake the funnel 5 to 10 times to mix the culture with the extraction solvent, frequently opening the spigot while the funnel is upside down and point it into the fume hood to degas the funnel. After a second round of shaking and degassing as just demonstrated, place the funnel upright in the ring stand for approximately one minute.
When the top solvent layer has separated from the aqueous culture layer, remove the stopper and drain the E.coli layer into a waste beaker, retaining the solvent layer within the funnel. Then, repeat the extraction with the remaining 500 milliliters of the culture using the same 500 milliliters of solvent used for the first extraction, before draining the solvent containing the extracted metabolites into a clean flask. Here, our representative gas chromatography-mass spectrometry chromatogram of typical extracted enzyme products is shown.
Commonly observed minor byproducts include chloramphenicol and the indole derivatives oxindole and indole pivalaldehyde. After diterpenoid purification via silica column chromatography, three dolabralexin compounds can be obtained, as quantified based on a standard curve using the diterpenoid sclareol. Silica chromatography is ideal for achieving a high purity of the target compounds.
Since it enables the simple preparation of diterpene olefins and oxygenated derivatives and readily removes the major contaminate oxindole, which is retained on the silica matrix. Further, the co-expression of the terpenoid pathway genes can be used to produce diterpenoids in N.Benthamiana, resulting in the production of dolabradiene and 15, 16 epoxydolabrene. The solvents used during the extraction should be optimized for the physical chemical properties of the metabolites of interest and must not be mixable with water to maintain two distinct layers.
Following this procedure, purified metabolites can be used for downstream analysis, including structural analysis and antibiotic acids. This technique enabled the discovery of novel diterpenoids, including those shown here, allowing these metabolites to be analyzed for their chemical structure and new biactivies. Take precautions when using organic solvents in a separatory funnel.
Always be sure to dispose of biological waste according to your lab safety standards.
