Name: a customizable approach for the enzymatic production and purification of diterpenoid natural products
Uploaded: 2019-10-04T13:30:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products at JoVE.com

We gratefully acknowledge Dr. Reuben Peters (Iowa State University, USA) for providing the pIRS and pGGxZmAN2 constructs. Financial support for this work by the NSF Plant-Biotic Interactions Program (grant# 1758976 to P.Z.), the DOE Early Career Research Program (grant# DE-SC0019178 to P.Z.), the DOE Joint Genome Institute Community Science Program (grant# CSP2568 to P.Z.), the NSF Graduate Research Fellowship Program (to K.M.M.), and a UC Davis Dean&#8217;s Mentorship Award Fellowship (to K.M.M.) are gratefully acknowledged.

The authors declare they have no competing financial interests.

Broader investigation and application of diterpenoid natural products necessitates simple, inexpensive protocols to synthesize and purify sufficient quantities of desired compounds. The rapid increase in the number of available diterpenoid-metabolic enzymes from a broad range of species now provides an expansive inventory for the enzymatic production of diterpenoids using microbial and plant-based host systems. In addition, the modular architecture of many diterpenoid pathways enables the use of enzymes from the same or different species in &#8216;plug &#38; play&#8217; combinatorial engineering approaches to generate an array of natural and new nature-like diterpenoid natural products2,14,26,35.
E. coli is a preferred microbial host for natural product biosynthesis due to its robustness, ease of scalability, limited chemical complexity for reduced byproduct contamination, and the wealth of available tools for DNA assembly and expression optimization. In our experience, the platform described here is well-suited for producing product yields of up to several hundred mg of diterpene olefins and alcohols, which is suitable for many downstream applications including those proposed here. While not meeting industrial scale, the production platform described here can serve as a foundation for further pathway, host, and fermentation optimization as has been successfully demonstrated for related diterpenoids such as taxadiene and sclareol33,34. Over-expression of rate-limiting MVA or MEP pathway genes has been successfully established to overcome yield-limiting factors for diterpenoid biosynthesis, such as insufficient precursor supply and precursor flux into competing pathways13,32,33,39. Although proven successful in several studies, poor expression and catalytic activity of terpenoid-metabolic eukaryotic P450s and other membrane-bound enzymes in E. coli is a likely limiting factor33,39,48,49,50,51,52. Use of codon-optimized sequences and protein modifications, such as removal of the endoplasmic reticulum signal peptide or introduction of a plastidial signal peptide, have proven useful to increase soluble P450 expression14,38,49,50,53. Such modifications were also employed for the microbial co-expression of maize CYP71Z1839 used as an example pathway in this study. The protocols described are based on the use of plasmids carrying one or two genes per construct, all under the same inducible promoter. Where larger-scale gene combinations are desired, it is advisable to use various available multi-gene cassettes or gene stacking systems to mitigate reduced transformation efficiency and culture growth due to the use of multiple plasmids and antibiotics13.
With the broader availability of genetics and genomics resources, plant host systems also become increasingly suitable for the manufacture of natural products. Advantages include the ability of plants to produce the required natural precursors powered by photosynthesis, thus enabling product formation without the need to supplement precursor molecules54,55. N.&#160;benthamiana is already widely used for in vivo functional characterization and combinatorial expression of terpenoid and other natural product pathways14,35,36,40. Notable advantages of using N. benthamiana as a host system include the endogenous production of diterpenoid precursors, use of native gene sequences, simplified expression of eukaryotic P450s, ease of combinatorial gene transformation (as separate antibiotics are not required for transient co-transformation), and simple extraction of target products from leaf material. Where needed, diterpenoid production can be enhanced through co-expression of key MEP pathway genes to increase precursor supply36,41. Constraints for scalable diterpenoid production in N. benthamiana are more complex as compared to liquid microbial cultures due to the need for generating sufficient plant biomass, more labor-intensive product purification from chemically complex plant tissue, and possible undesired metabolization of target products through, for example, oxidation, glycosylation or dephosphorylation by endogenous enzymes36,43,44,45,46,47. However, this procedure can be scaled up to mg product quantities by increasing the number of plants used for agroinfiltration56.
The product extraction and purification protocols described here are compatible with E. coli and N. benthamiana, as well as S. cerevisiae and other plant or microbial host systems, and provide a cost-efficient approach that is easy to set up in both biology and chemistry laboratories and does not require expensive purification equipment. Metabolite extraction using a separatory funnel is well-suited for efficient extraction and phase separation prior to chromatographic purification. Funnel sizes can be readily adjusted to allow for larger culture volumes and reduce experimental time needed to extract from large cultures. We found the use of a hexane/ethyl acetate gradient to be ideal for extracting diterpenoids of different polarity as demonstrated here for the group of dolabralexins that comprise both hydrocarbon and oxygenated compounds (Figure 3). Depending on the properties of target products, other solvent mixtures may be advantageous. However, solvents must not be miscible with water to ensure successful extraction and phase separation using the separatory funnel technique. In addition, product loss through evaporation must be taken into account when using this approach for producing volatile organic compounds (VOCs), such as lower molecular weight mono- and sesqui-terpenoids and other VOCs. Chromatographic separation of diterpenoids of different levels of oxygenation using a larger-scale (~2 L) silica column has been advantageous in our experience, since it provides improved product separation and minimizes the need for iterative purification steps when using smaller column volumes. Column volumes and matrices can be adjusted as needed for the desired culture volume and the type of natural product. The purity of target products that can be achieved using this protocol is suitable for many downstream applications, such as bioactivity assays or for use in enzyme activity analyses. However, where higher purity levels are required, such as structural analyses via NMR, product purity can be efficiently enhanced by additional purification using (semi)-preparative HPLC.
This protocol described here has been optimized for the production of diterpenoid natural products, but can also be readily customized to related mono-, sesqui- and tri-terpenoids, as well as other natural product classes simply by generating the desired enzyme modules for combinatorial expression14,57. However, modifications of the procedures for product extraction and purification must be taken into consideration for compounds with higher volatility, such as mono- and sesqui-terpenoids, or higher polarity and functional modification as exemplified by glycosylation of many triterpenoids, phenylpropanoids, and other natural product classes.
Although industrial-scale platforms for the manufacture of natural products are available, the protocols described here offer an inexpensive, customizable tool that can be readily set up in most laboratories. As demonstrated by the production of maize dolabralexins here and elsewhere39, the product quantities and purity that can be achieved using this approach are typically sufficient to facilitate various downstream analysis and uses, including, but not limited to, various bioactivity studies, analysis of interactions between organisms, as well as for use as enzyme substrates or as starting material for semi-synthesis approaches.

Diterpenoids comprise a chemically diverse group of more than 12,000 predominantly polycyclic 20-carbon natural products that play critical roles in many organisms1. Fungi and plants produce the largest diversity of diterpenoids, but bacteria have also been shown to form bioactive diterpenoids (see reviews2,3,4,5). Rooted in their vast structural diversity, diterpenoids serve a multitude of biological functions. A few diterpenoids, such as gibberellin growth hormones, have essential functions in developmental processes5. However, the majority of diterpenoids serve as mediators of chemical defense and interorganismal interactions. Among these, diterpene resin acids in the pest and pathogen defense of coniferous trees and species-specific blends of antimicrobial diterpenoids in major food crops such as maize (Zea mays) and rice (Oryza sativa) have been most extensively studied6,7. These bioactivities provide a rich chemical repository for commercial applications, and select diterpenoids are used as important pharmaceuticals, food additives, adhesives, and other bioproducts of everyday modern life8,9,10. To advance research on the natural diversity and biological functions of diterpenoids and ultimately promote broader commercial applications, tools for the cost-efficient preparation of pure compounds are required. Large-scale isolation from plant material has been established for a few diterpenoid bioproducts, such as diterpene resin acids that are produced as a byproduct of the pulp and paper industry8. However, accumulation of diterpenoids in only specific tissues and under tight regulation by environmental stimuli often limits isolation of sufficient product amounts from the natural producer2. In addition, the structural complexity of diterpenoids hampers their production through chemical synthesis, although such approaches have been successful in several cases11,12. With the availability of advanced genomic and biochemical technologies, enzymatic production platforms have gained increasing attention for producing a range of diterpenoid compounds (see reviews13,14,15,16,17,18).
All terpenoids, including diterpenoids, are derived from two isomeric isoprenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)19 that, in turn, are formed through the mevalonate (MVA) or the methylerythritol-5-phosphate (MEP) pathway. Terpenoid biosynthesis proceeds through the MEP pathway in bacteria and the MVA pathway in fungi, whereas plants possess a cytosolic MVA and a plastidial MEP pathway, with the latter being the primary route toward diterpenoid formation20. Condensation of IPP and DMAPP by prenyl transferases yields the central 20-carbon precursor to all diterpenoids, geranylgeranyl diphosphate (GGPP)20. Downstream of GGPP formation, two enzyme families, terpene synthases (TPSs) and cytochrome P450 monooxygenases (P450s) largely control the formation of the vast chemical diversity of terpenoid metabolism21,22. Diterpene synthases (diTPSs) catalyze the committed carbocation-driven cyclization and rearrangement of GGPP to form various stereospecific bi-, poly-, or macro-cyclic diterpene scaffolds1,3,23,24. Oxygenation and further functional decoration of these scaffolds is then facilitated by P450 enzymes and select other enzyme families22,25. TPSs and P450s commonly exist as species-specific, multi-gene families that can form modular biosynthetic networks, where combining different enzyme modules along a common blueprint enables the formation of a broad range of compounds2,26. The rapid discovery of functionally distinct enzymes operating in modular terpenoid pathways in recent years has provided expanding opportunities for their use as a versatile parts list for metabolic engineering of partial or complete pathways in both microbial and plant-based production platforms. For example, yeast (Saccharomyces cerevisiae) has been applied successfully to engineer multi-enzyme pathways for the manufacture of terpenoid bioproducts, such as the antimalarial drug artemisinin27, the sesquiterpenoid biofuels bisabolene and farnesene28, but also select diterpenoids29,30,31. Likewise, engineered Escherichia coli platforms for the industrial-scale manufacture have been established for a few diterpenoid metabolites, including the Taxol precursor taxadiene used as an anti-cancer drug and the diterpene alcohol, sclareol, used in the fragrance industry13,32,33,34. Advances in genetic engineering and transformation technologies also have made plant host systems increasingly viable for producing plant natural products9,14,35,36. In particular, the close tobacco relative, Nicotiana benthamiana, has become a widely used chassis for terpenoid pathway analysis and engineering, due to the ease of Agrobacterium-mediated transformation of multiple gene combinations, efficient biosynthesis of endogenous precursors, and high biomass14,35,36.
Drawing on these established platforms for terpenoid biosynthesis, we describe here easy-to-use and cost-efficient methods for the enzymatic production of diterpenoids and the purification of single compounds. The presented protocols illustrate how E. coli and N. benthamiana platforms engineered for enhanced diterpenoid precursor biosynthesis can be utilized for the combinatorial expression of different diTPSs and P450 enzymes to generate desired diterpenoid compounds. Application of this protocol to produce and purify structurally different diterpenoids is shown by example of specialized diterpenoids from maize (Zea mays), termed dolabralexins, endogenous biosynthesis of which recruits two diTPS and one P450 enzyme. Purification of different dolabralexins ranging from olefins to oxygenated derivatives is then achieved by combining separatory funnel extraction with large-scale silica column chromatography and preparative high-pressure liquid chromatography (HPLC). The described protocols are optimized for the production of diterpenoids, but can also be readily adapted for related terpenoid classes, as well as other natural products for which enzyme resources are available. Compounds produced using this approach are suitable for various downstream applications, including but not limited to, structural characterization via nuclear magnetic resonance (NMR) analysis, use as substrates for enzyme functional studies, and a range of bioactivity assays.

<table>
	<tbody>
		<tr>
			<td>1020 Trays</td>
			<td>Greenhouse Megastore</td>
			<td>CN-FLHD</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(N-morpholino)ethanesulfonic acid</td>
			<td>Sigma</td>
			<td>M8250-500g</td>
			<td>MES</td>
		</tr>
		<tr>
			<td>4&#34; Tech Square Pot</td>
			<td>McConkey Wholesale Grower&#39;s Supply</td>
			<td>JMCTS4</td>
			<td></td>
		</tr>
		<tr>
			<td>5977 Extractor XL MS</td>
			<td>Agilent</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>7890B GC</td>
			<td>Agilent</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>271004</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial yeast extract</td>
			<td>Fisher</td>
			<td>BP9727-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>CTechGlass</td>
			<td>BK-2001-015B</td>
			<td></td>
		</tr>
		<tr>
			<td>Cap, 9 mm blue screw, PFTE</td>
			<td>Agilent</td>
			<td>5185-5820</td>
			<td>GC vial cap</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Genesee</td>
			<td>25-532</td>
			<td>Carb</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Fisher</td>
			<td>50247423</td>
			<td>Chlor</td>
		</tr>
		<tr>
			<td>Chromatography column</td>
			<td>CTechGlass</td>
			<td>CL-0015-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear humidity dome</td>
			<td>Greenhouse Megastore</td>
			<td>CN-DOME</td>
			<td></td>
		</tr>
		<tr>
			<td>ColiRollers Plating Beads</td>
			<td>Sigma</td>
			<td>71013</td>
			<td>Glass beads</td>
		</tr>
		<tr>
			<td>CoorsTek Porcelain Mortars</td>
			<td>Fisher</td>
			<td>12-961A</td>
			<td>mortar</td>
		</tr>
		<tr>
			<td>CoorsTek Porcelain Pestles</td>
			<td>Fisher</td>
			<td>12-961-5A</td>
			<td>pestle</td>
		</tr>
		<tr>
			<td>Delta-Aminolevulinic acid hydrochloride</td>
			<td>Sigma</td>
			<td>50981039</td>
			<td>Aminoleuvolinic acid</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>A962-4</td>
			<td>EtOH</td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Fisher</td>
			<td>E1454</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>14-432-22</td>
			<td>Falcon tubes</td>
		</tr>
		<tr>
			<td>Fisherbrand Disposable Cuvettes</td>
			<td>Fisher</td>
			<td>14-955-127</td>
			<td>cuvette</td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid</td>
			<td>Fisher</td>
			<td>FB0875713</td>
			<td>petri dish</td>
		</tr>
		<tr>
			<td>Fisherbrand Polypropylene Microtube Storage Racks</td>
			<td>Fisher</td>
			<td>05-541</td>
			<td>microtube rack</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>Fisher</td>
			<td>H292-4 (CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>HP-5MS</td>
			<td>Agilent</td>
			<td>19091S-433</td>
			<td>GC column</td>
		</tr>
		<tr>
			<td>Inlet adapter</td>
			<td>CTechGlass</td>
			<td>AD-0006-003</td>
			<td>glass inlet adapter</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Fisher</td>
			<td>BP1755-100</td>
			<td>IPTG</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher</td>
			<td>BP9065</td>
			<td>Kan</td>
		</tr>
		<tr>
			<td>KIM-KAP Caps, Disposable, Polypropylene, Kimble Chase</td>
			<td>VWR</td>
			<td>60825-798</td>
			<td>breathable test tube lids</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Acros</td>
			<td>223210010</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma</td>
			<td>M7506-500g</td>
			<td>MgSO4</td>
		</tr>
		<tr>
			<td>Miracle-Gro Water Soluble All Purpose Plant Food</td>
			<td>Miracle-Gro</td>
			<td>2756810</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 200</td>
			<td>Retsch</td>
			<td>20.746.0001</td>
			<td>tissue mill</td>
		</tr>
		<tr>
			<td>Nalgene Fernbach culture flask</td>
			<td>Sigma</td>
			<td>Z360236</td>
			<td>2.8 L flask</td>
		</tr>
		<tr>
			<td>New Brunswick I26</td>
			<td>Eppendorf</td>
			<td>M1324-0000</td>
			<td>Shaking incubator</td>
		</tr>
		<tr>
			<td>Nicotiana benthamiana seed</td>
			<td>USDA Germplasm Repository</td>
			<td>Accession TW16</td>
			<td>N. benthamiana</td>
		</tr>
		<tr>
			<td>OverExpress C41(DE3) Chemically Competent Cells</td>
			<td>Lucigen</td>
			<td>60442</td>
			<td>C41-DE3 cells</td>
		</tr>
		<tr>
			<td>Parafilm M wrapping film</td>
			<td>Fisher</td>
			<td>S37440</td>
			<td>Parafilm</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P-9541</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic anhydrous</td>
			<td>Fisher</td>
			<td>P288-3</td>
			<td>Dipotassium phosphate</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td></td>
			<td></td>
			<td>Monopotassium phosphate</td>
		</tr>
		<tr>
			<td>Pyrex disposable culture tubes, rimless</td>
			<td>Sigma</td>
			<td>CLS9944516</td>
			<td>test tubes</td>
		</tr>
		<tr>
			<td>Pyruvate Acid Sodium Salt</td>
			<td>Fisher</td>
			<td>501368477</td>
			<td>Sodium pyruvate</td>
		</tr>
		<tr>
			<td>Retort Ring Stands</td>
			<td>CTechGlass</td>
			<td>ST00</td>
			<td>ring stand</td>
		</tr>
		<tr>
			<td>Riboflavin</td>
			<td>Amresco</td>
			<td>0744-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Sigma</td>
			<td>R7382</td>
			<td>Rif</td>
		</tr>
		<tr>
			<td>Rotovap</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sand, 50-70 mesh particle size</td>
			<td>Sigma</td>
			<td>274739-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica</td>
			<td>Fisher</td>
			<td>AC241660010</td>
			<td>silica gel</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>5271-3</td>
			<td>NaCl</td>
		</tr>
		<tr>
			<td>Sodum hydroxide</td>
			<td>Fisher</td>
			<td>SS266-1</td>
			<td>NaOH</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Fisher</td>
			<td>501368607</td>
			<td>Spec</td>
		</tr>
		<tr>
			<td>Squibb Separatory Funnel</td>
			<td>CTechGlass</td>
			<td>FN-1060-006</td>
			<td>Separatory funnel</td>
		</tr>
		<tr>
			<td>Sunshine Mix #1</td>
			<td>Sungro Horticulture</td>
			<td></td>
			<td>Potting soil</td>
		</tr>
		<tr>
			<td>Thermo Scientific Snap Cap Low Retention Microcentrifuge Tubes</td>
			<td>Fisher</td>
			<td>21-402-902</td>
			<td>microtube</td>
		</tr>
		<tr>
			<td>Triangle funnel</td>
			<td>CTechGlass</td>
			<td>FN-0035</td>
			<td>funnel</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>BP14212</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial, screw, 2 mL, amber, WrtOn</td>
			<td>Agilent</td>
			<td>5182-0716</td>
			<td>GC vial</td>
		</tr>
		<tr>
			<td>visible spectrophotometer, V-1200</td>
			<td>VWR</td>
			<td>634-6000P</td>
			<td>spectrophotometer</td>
		</tr>
		<tr>
			<td>ZORBAX Eclipse XDB-C18</td>
			<td>Agilent</td>
			<td>990967-202</td>
			<td>HPLC column</td>
		</tr>
		<tr>
			<td>ZORBAX Eclipse XDB-CN</td>
			<td>Agilent</td>
			<td>990967-905</td>
			<td>HPLC column</td>
		</tr>
	</tbody>
</table>

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.

Schematic workflow for diterpenoid production using E. coli 
Figure 1 illustrates the described workflow for diterpenoid production. The protocol outlined here has been adapted from a previously described E. coli platform for diterpenoid biosynthesis13,32 for use of larger-volume cultures and purification of desired diterpenoid products via silica chromatography. To demonstrate the use of this protocol, we used a recently identified dolabralexin pathway from maize that comprises two diTPSs, ZmAN2 (Zm00001d029648) and ZmKSL4 (Zm00001d032858), a multi-functional P450 (CYP71Z18, Zm00001d014134), and a cytochrome P450 reductase (ZmCPR2, Zm00001d026483) (Figure 2). In brief, E. coli BL21DE3-C41 competent cells were pre-transformed with the pCDFDuet:IRS and pACYC-Duet:GGPPS/ZmAN2 plasmids13,32. The pCDFDuet:IRS plasmid contains key enzymes for diterpenoid precursor production, including 1-deoxy-D-xylulose-5-phosphate synthase (dxs), 1-deoxy-D-xylulose-5-phosphate reductase (dxr), and isopentenyl diphosphate isomerase (idi), and was shown to increase diterpenoid formation in E. coli13. The pACYC-Duet:GGPPS/ZmAN2 plasmid contains the maize ent-copalyl diphosphate synthase ZmAN2 and a GGPP synthase from Abies grandis. Enzymes catalyzing the committed reactions in dolabralexin biosynthesis were then co-transformed as plasmids pET28b:ZmKSL4 and pETDUET:ZmCPR2/ZmCYP71Z18. For details on sequences and plasmid constructs see Mafu et al. 201839.
A GC-MS chromatogram of the extracted enzyme products is shown in Figure 3A, illustrating the formation of three dolabralexin compounds, namely dolabradiene (1.2 &#177; 0.25 mg/L culture), epoxydolabrene (0.65 &#177; 0.2 mg/L culture), and epoxydolabranol (11.4 &#177; 1.1 mg/L culture) as quantified based on a standard curve using the diterpenoid sclareol. Sclareol was used as a reference standard, due to its similar structure and chemical properties as compared to dolabralexins. Typically observed minor byproducts include chloramphenicol, the indole derivatives oxindole and indole-5-aldehyde, and the precursor geranylgeranyl diphosphate (GGPP) (Figure 3). Indole commonly represents the primary byproduct, but is not shown here, due to its retention time shorter than the set solvent delay of 7 min to preserve the integrity of the GC-MS instrument.
Schematic workflow of diterpenoid production using N. benthamiana 
Figure 4 depicts an overview of the expression of the dolabralexin pathway in N. benthamiana. For the products described here, the following constructs were transformed separately into A. tumefaciens strain GV3101: pLife33:p19 (expressing the p19 gene silencing suppressor protein), pLife33:ZmCYP71Z18, pLife33:ZmAN2, pLife33:ZmKSL4. Full-length native sequences of the maize dolabralexin pathway genes were used in the binary T-DNA vector pLife3341 with kanamycin resistance for propagation in E. coli and A. tumefaciens. Co-expression of upstream terpenoid pathway genes is optional, since the precursor geranylgeranyl diphosphate is endogenously formed in N. benthamiana. However, several studies have successfully employed such approaches to increase diterpenoid formation in N. benthamiana14,36,41. As illustrated in Figure 3, co-expression successfully produced dolabradiene and 15,16-epoxydolabrene. Unlike enzyme co-expression in E. coli, 15,16-epoxydolabranol was not detected in metabolite extracts.
Presence of 15,16-epoxydolabrene in leaf extracts demonstrated the activity of CYP71Z18 in N. benthamiana. As 15,16-epoxydolabranol was shown to be stable after extraction from microbial cultures (Figure 3) as well as after isolation from maize root tissues in previous studies39, it appears plausible that the hydroxylated product is glycosylated by endogenous glycosyltransferases and subsequently sequestered in the vacuole, rendering it inaccessible to extraction with the organic solvent mixtures used here for extraction36,43,44,45,46. Similar undesired product modifications in the context of pathway engineering in N. benthamiana have been reported in previous studies47. As shown for co-expression in E. coli, transient expression in N. benthamiana results in the extraction of several byproducts, including linear alkanes of different chain length as based on comparison to reference mass spectra databases. Compound titers extracted from leaf material were found to be on average 2.4 +/- 0.5 mg dolabradiene and 0.9 +/- 0.3 mg 15,16-epoxydolabrene per g dry leaf tissue. These titers cannot be compared directly to the E. coli co-expression system given the different experimental set ups.
Diterpenoid purification 
Diterpenoid purification was achieved using silica column chromatography and subsequent semi-preparative HPLC. Metabolite extracts from 12 L of pooled E. coli cultures were purified using silica column chromatography to separate the three focal dolabralexin compounds (Figure 3A). Silica chromatography is ideal for achieving high purity of the target compounds, since it enables simple separation of diterpene olefins and oxygenated derivatives, and readily removes the major contaminant, oxindole, which is retained on the silica matrix (Figure 3A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59992/59992fig1.jpg" /> 
Figure 1: Workflow for diterpenoid production in E. coli and metabolite purification from liquid bacterial cultures. Dashed boxes depict optional steps where additional purification is required. (A) Representative image of extracted E. coli culture using a separatory funnel. (B) Representative image of metabolite extract purification using silica chromatography. &#160; <a href="https://www.jove.com/files/ftp_upload/59992/59992fig1large.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/59992/59992fig2v2.jpg" /> 
Figure 2: Dolabralexin biosynthetic pathway and gene constructs used in this study. <a href="https://www.jove.com/files/ftp_upload/59992/59992fig2large.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/59992/59992fig3v2.jpg" /> 
Figure 3: GC-MS results. Shown are representative GC-MS chromatograms of purified diterpenoid products obtained using enzyme co-expression assays in (A) E. coli and (B) N. benthamiana. Product identifications are based on comparisons to authentic standards and reference mass spectra of the National Institute of Standards and Technology (NIST) mass spectral library. 1, oxindole; 2, indole-5-aldehyde; 3, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-; 4, 6-O-Acetyl-1-[[4-bromophenyl]thio]-a-d-glucoside S,S-dioxide; 5, dolabradiene; 6, 15,16-epoxydolabrene; 7, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)-; 8, 15,16-epoxydolabranol; 9 and 12, unknown; 10, chloramphenicol; 11, 3,7,11,15-tetramethyl-2-hexadecen-1-ol; 13-16, alkanes. <a href="https://www.jove.com/files/ftp_upload/59992/59992fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59992/59992fig4.jpg" /> 
Figure 4: Diterpenoid production in N. benthamiana. (A) Workflow of diterpenoid production in N. benthamiana and metabolite purification from leaf material. (B) Representative image of N. benthamiana plants ready for infiltration experiments, before pruning. (C) Representative images of N. benthamiana plants after pruning. (D) Image of syringe-infiltrated leaves. Darker areas have been infiltrated. <a href="https://www.jove.com/files/ftp_upload/59992/59992fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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