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

Katherine  M. Murphy; Siwon Chung; Shruti Fogla; Hana  B. Minsky; Karen  Yong Zhu; Philipp Zerbe

doi:10.3791/59992

Biochemistry

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

Published: October 4, 2019 doi: 10.3791/59992

Katherine M. Murphy¹, Siwon Chung¹, Shruti Fogla¹, Hana B. Minsky¹, Karen Yong Zhu¹, Philipp Zerbe¹

¹Department of Plant Biology, University of California Davis

Summary

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.

Abstract

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.

Introduction

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.

Protocol

CAUTION: The protocols described here include the use of hazardous chemicals, sharp objects, electrical devices, hot objects, and other hazards that may result in injury. Appropriate personal protective equipment should be worn, and the appropriate safety procedures, including safety trainings, should be followed.

1. Preparation of materials and solutions

Prepare and autoclave lysogeny broth medium (LB) for 30 min: Per 1 L of medium, mix 10 g of tryptone, 5 g of bacterial yeast extract, and 10 g of NaCl and dissolve in 1 L of deionized (DI) water.
For 1 L co-expression cultures, prepare and autoclave Terrific Broth (TB) medium for 30 min: In a 2.8 L Erlenmeyer flask mix 12 g of tryptone, 24 g of bacterial yeast extract, and 40 mL of 10% (v/v) glycerol and dissolve in 860 mL of DI water. Prepare and autoclave 1.3 L of 10x phosphate buffer [1.3 L of DI water, 30.03 g of monopotassium phosphate (KH₂PO₄), and 163.02 g of dipotassium phosphate (K₂HPO₄)] and add to the above medium.
Prepare Super Optimal broth with Catabolite repression (SOC) media.
1. Prepare and autoclave for 30 min a solution containing 2% (w/v) tryptone, 0.5% (w/v) bacterial yeast extract, 8.5 mM NaCl, and 2.5 mM KCl.
2. Add sterile MgSO₄ and glucose both with a final concentration of 20 mM. Use 1 M NaOH to adjust to pH 7.0. Store the SOC media at -20 °C.
Prepare 1 L of 1 M sodium pyruvate. Autoclave for 15 min and store at 4 °C.
Prepare 20 mL of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) in sterile DI water. Store 1–2 mL aliquots at -20 °C.
Prepare infiltration buffer: 10 mM MES (1.952 g) and 10 mM MgCl₂ (2.033 g) dissolved in 1 L of DI water. Store at 4 °C.
Prepare and autoclave LB agar for 30 min: Per 100 mL of DI water, add 1 g of tryptone, 0.5 g of bacterial yeast extract, 1 g of NaCl, and 1.5 g of agar. Once LB agar is cool enough to handle the bottle, add antibiotics as required for the desired plasmid combinations. Pour agar plates using petri dishes and store at 4 °C.
1. See Table 1 for specifications regarding compound production in E. coli and N. benthamiana, respectively. Wrap plates containing rifampicin in foil to prevent degradation upon storage.

			Working concentration (µg/mL)
Antibiotic	Stock (mg/mL)	Solvent	1 plasmid	2 plasmids	3 or 4 plasmids
Carbenicillin	50	H₂O	50	25	20
Chloramphenicol	30	EtOH	30	20	20
Kanamycin	50	H₂O	50	25	20
Spectinomycin	30	H₂O	30	25	20
Gentamycin	50	H₂O	30
Rifampicin	10	MeOH	10

Table 1: Antibiotic concentrations for plasmid co-expression in E. coli or N. benthamiana.

2. Production of diterpenoid metabolites in E. coli

NOTE: The protocol described here for producing diterpenoid metabolites in E. coli has been adapted from a previously reported enzyme co-expression platform developed by the group of Dr. Reuben J. Peters (Iowa State University, IA, USA)¹³^,³².

Transformation of competent cells with plasmid combinations.
1. Thaw chemically competent E. coli cells on ice (BL21DE3-C41 cells were used in this protocol).
2. Add 1 µL of a 100 ng/µL solution of each construct used for co-expression to 25 µL of competent cells in a 1.5 mL microtube. Do not vortex or mix by pipetting.
  NOTE: For optimal expression and activity of TPS and P450 enzymes, several sequence modifications need to be considered. For TPS, removal of the N-terminal plastidial transit peptide is often essential. Specifically, plastidial mono- and di-TPS typically require removal of the predicted transit peptide (using common prediction algorithms³⁷), whereas cytosolic sesqui-TPS can usually be used as full-length genes. With regards to P450s, codon optimization as well as removal or replacement with the leader sequence MAKKTSSKGK of the N-terminal transmembrane domain has proven effective in many cases³⁸^,³⁹. In addition, when co-expressing P450 enzymes a cytochrome P450 reductase (CPR) should be included to ensure sufficient P450 activity.
3. Incubate the mixture on ice for 30 min. Mix every 10 minutes by gently scraping the tube across a microtube rack.
4. Pre-incubate SOC media and LB agar plates at 37 °C containing antibiotics as required for the desired combination of constructs.
  NOTE: Each construct to be co-transformed must have a distinct antibiotic resistance, as well as distinct origins of replication to ensure optimal protein expression.
5. Heat shock the cell mixture at 42 °C for 1 minute, and then incubate on ice for at least 2 min.
6. Add 200 µL of warm SOC media.
7. Shake the cell mixture for 1 h at 37 °C and 200 rpm.
8. Add approximately 10 autoclaved glass beads to the warmed LB agar plate. Add 100 µL of the cell mixture and replace the lid. Shake plate horizontally with the lid on to distribute the cells evenly. Remove the glass beads by tapping them off into a waste container. Alternatively, use other preferred plating methods.
9. Incubate the LB agar plate at 37 °C overnight with the coated surface face down. The plate with transformed E. coli colonies can be used the following day or stored at 4 °C sealed in paraffin film for up to 2 weeks.
Preparation of inoculation cultures
1. On the following day, prepare a solution of LB medium with antibiotics required for the transformed plasmid combination using the concentrations provided in Table 1.
2. In a sterile hood, transfer 5 mL of LB medium to a 15 mL sterile glass test tube with a plastic breathable cap. Prepare one small culture tube for each desired large (1 L) culture.
3. Select individual E. coli colonies from the LB agar plate using a pipette tip. Inoculate each tube of LB with an E. coli colony by ejecting one pipette tip containing a selected colony into each tube.
4. Cap each inoculation culture test tube with a breathable plastic cap. Place the capped E. coli small cultures in a 37 °C shaking incubator for 12–24 h.
Preparation and induction of co-expression cultures
1. On the following day, add 100 mL of prepared 10x phosphate buffer to 900 mL of prepared TB for a final phosphate buffer concentration of 1x. Add necessary antibiotics with concentrations according to Table 1.
2. Shake at 140 rpm at 37 °C until warm (approximately 30 min).
3. Inoculate each flask of media for 1 L cultures with 5 mL of the inoculation culture. Retain the pipette tip used for inoculation culture inoculation in the inoculation culture tube so that, upon extraction with organic solvent in subsequent steps, there are no extracted plastic contaminants.
4. Incubate with shaking at 200 rpm until the optical density at 600 nm (OD₆₀₀) reaches 0.6, approximately 3 h. To measure the OD₆₀₀ with a spectrophotometer, use a mixture of sterile TB with phosphate buffer as a blank.
5. At the desired OD₆₀₀, set the incubator settings to 16 °C.
6. When co-expressing P450s, freshly prepare riboflavin and aminolevulinic acid, which are essential for sufficient P450 co-factor production. For every experiment, make 4 g/L riboflavin and 150 g/L aminolevulinic acid. Keep solution wrapped in foil until use, as riboflavin is light sensitive.
7. After the incubator has reached 16 °C (approximately 30 min), add 1 mL of 1 M IPTG, 1 mL of 4 g/L riboflavin, and 1 mL of 150 g/L aminolevulinic acid to each culture. For diterpenoid production, 25 mL of 1 M sodium pyruvate should be added to each culture to assure sufficient precursor formation.
  NOTE: All constructs used in this assay were under the same IPTG-inducible promoter. Different promoters can be used as desired.
8. Incubate at 16 °C and 140 rpm for 72 h. Add 25 mL of sodium pyruvate each subsequent day after induction if producing diterpenoids. Immediately use cultures are immediately used for separatory funnel extraction of metabolites; do not harvest or store cultures.

3. Separation and purification of metabolites

Separatory funnel extraction of metabolites
NOTE: It is important to use only glassware and glass pipettes when using organic solvents to prevent plasticizer contaminations.
1. In a fume hood, secure the separatory funnel onto a ring stand. Place a waste beaker underneath the separatory funnel.
2. Pour 500 mL of 50/50 (v/v) ethyl acetate/hexanes into the separatory funnel.
  NOTE: Solvent mixture should be adjusted based on the solubility and polarity of the targeted metabolites. Water-miscible solvents should be avoided to ensure suitable phase separation.
3. Add 500 mL of the E. coli culture to the separatory funnel and place on the glass stopper.
4. Shake the funnel to mix the culture with the extraction solvent, approximately 5–10x. Frequently de-gas the funnel by opening the spigot while the funnel is held upside down and pointed into the fume hood to release pressure. Repeat the shaking and de-gas procedure 2x.
5. Place the funnel upright in the ring stand and wait until the solvent layer (top) has separated from the aqueous (culture) layer (bottom), approximately 1 min.
  NOTE: When a large amount of bubbles is observed in the interphase, addition of a small volume of 5–10 mL EtOH can be added to improve phase separation.
6. Remove the stopper. Drain the E. coli layer into a waste beaker, retaining the solvent layer in the funnel.
7. Repeat the procedure using the remaining 500 mL of E. coli culture and the same 500 mL solvent used for the first extraction.
8. Drain the solvent containing the extracted metabolites into a clean flask. Avoid contamination with E. coli culture.
Rotary Evaporation Concentration
1. Prepare the rotary evaporation (rotovap) equipment: fill the water bath and set the temperature to 25 °C. For heat sensitive compounds, use a lower temperature setting or add ice to the water bath. Fill the condensing chamber with dry ice and set the rotating speed to 60–80 rpm.
2. Add approximately 700 mL of extracted metabolites to a 1 L evaporating flask, attach to the rotovap, and lower into the water bath. Turn the water bath heater on and set to 25 °C.
3. Start rotation of the evaporating flask, turn on the vacuum system, and gradually increase the suction to avoid rapid boiling of the metabolite solution into the waste flask. Evaporated solvent should begin condensing and dripping into the condensate-collecting (waste) flask.
4. When only a few mL of the metabolite solution remains in the evaporating flask, stop rotations and turn off the vacuum system. Raise the evaporating flask and depressurize by closing the vacuum line. Retain concentrated metabolite solution remaining in the evaporating flask. Dispose of the waste in the waste flask.
5. Continue rotary evaporation by adding up to 700 mL of additional extracted metabolite solution to the evaporating flask. Repeat process until all of the extracted metabolite solution has been concentrated.
6. Remove the concentrated metabolites from the evaporating flask by transferring with a glass pipette to new test tube. Rinse evaporating flask with 5 mL of 50/50 (v/v) ethyl acetate/hexanes or desired solvent mixture two times, transferring the rinsing solution to the test tube.
7. Store concentrated metabolites at -20 °C or -80 °C (depending on product stability) until further use.
Silica Column Purification
1. Prepare glassware by rinsing a 1 L beaker, glass funnel, 50 mL test tubes, and 3.2 L chromatography column (equipped with a glass fret) once with hexane and once with ethyl acetate. Label the 50 mL test tubes, which will be used to collect fractions.
2. Add 2 L of silica gel (230–400 mesh, grade 60) to a 3.2 L chromatography column with a 2 L reservoir capacity and a fritted disk, then load sand to form a 5 cm layer at the top of the column.
3. Prepare the column for chromatography by flushing it thoroughly with 2 L of hexane. At all times, there should be a thin (~0.5 cm) layer of the solvent liquid above the sand layer of the column to ensure the column does not dry out or acquire air pockets. A glass inlet adaptor connected to an air hose can be used to gently increase the flow rate through the column rather than gravity alone.
4. Load the concentrated metabolite extract (see section 3.2) onto the column. Rinse the bottle that contained the sample 3x with hexane and add to the column to ensure all of the sample has been transferred.
5. Using the following gradient, load 100 mL at a time and collect 50 mL fractions in labeled test tubes: 100% hexanes 3x, 10% (v/v) ethyl acetate in hexanes 3x, 12.5% (v/v) ethyl acetate in hexanes 3x, 15% (v/v) ethyl acetate in hexanes 3x, 20% (v/v) ethyl acetate in hexanes 3x, 40% (v/v) ethyl acetate in hexanes 3x, 60% (v/v) ethyl acetate in hexanes 3x, and 100% ethyl acetate 4x.
  NOTE: Gradient should be adjusted based on compound size and polarities and desired separation.
6. Using a glass pipette, transfer 1 mL from each fraction into a labeled GC vial. Analyze each sample via GC-MS to determine which fractions contain the desired metabolites and their level of purity.
  NOTE: The GC-MS method suitable for the metabolites produced in this method has been described in Mafu et al. 2018³⁹. In brief, all analyses were performed on an GC with an XL MS detector using an HP-5MS column (see Table of Materials), a sample volume of 1 µL, and oven temperature ramp from 50 °C to 300 °C at 20 °C min^-1.
7. After determining which fractions contain the compound(s) of interest, combine all fractions that contain the same compound. Properly dispose of fractions that do not contain any compounds into a waste container. Repeat the rotary evaporation procedure if necessary to concentrate the purified metabolites.
8. If additional purification is necessary, use (semi-)preparative HPLC to improve product purity. HPLC protocols should be adapted based on individual equipment specifications and compounds of interest.
  1. Resuspend dried silica chromatography samples in 1 mL hexane (diterpene hydrocarbons) or acetonitrile (oxygenated diterpenoids), and filter through a filter syringe to prevent contamination with small particles.
  2. For non-polar diterpenoids, use a CN column (see Table of Materials) with a recommended flow rate of 1 mL/min and a hexane:ethyl acetate gradient, with fractions collected every 1 minute.
  3. For polar diterpenoids, use a C18 column (see Table of Materials) with a recommended flow rate of 3 mL/min and an acetonitrile:water gradient, with fractions collected every 1 minute.
  4. Dry all HPLC purified fractions under a nitrogen stream and resuspend in 1 mL hexane before GC-MS analysis to assess product abundance and purity.

4. Production of diterpenoid metabolites using N. benthamiana

NOTE: The protocol described here for producing diterpenoid metabolites in N. benthamiana has been adapted from previously reported studies³⁵^,³⁶^,⁴⁰^,⁴¹. The below protocol is specific to syringe-infiltration of N. benthamiana leaves. Other infiltration methods, such as vacuum infiltration are equally suitable. Binary T-DNA vector systems, such as pCAMBIA130035Su (pLIFE33) or pEAQ-HT⁴⁰^,⁴¹^,⁴², that enable propagation in E. coli and A. tumefaciens and gene expression in plant hosts are suitable for this protocol.

Planting Nicotiana benthamiana
1. Fill one 750 mL pot with potting soil and water the pot. Add ~20 tobacco seed and gently tap with finger so that they are in the soil. Do not cover with soil.
  NOTE: Seeds can be generated through self-pollination for seed propagation. Seeds for this experiment were obtained from the UC Davis Department of Plant Biology, and are publicly available through USDA germplasm repository (https://npgsweb.ars-grin.gov/gringlobal/accessiondetail.aspx?id=1450450).
2. Leave pot in a growth chamber with the following conditions, watering every other day to ensure the soil does not dry out. Grow plants at 26 °C and 60% humidity with 16:8 h day:night cycle for all steps in this protocol.
3. After 1 week, fill ~20 pots with potting soil and water the pots. Using forceps, grasp a tobacco seedling by the stem and gently remove from the source pot, placing one seedling in each new pot and carefully burring the roots. Do not damage the leaves or roots.
4. Water the plants every other day by placing water in the tray the pots are in (also called “bottom watering”). Every fourth watering, include generic fertilizer in the water according to package instructions.
Freeze-thaw transformation of Agrobacterium tumefaciens strain GV3101 competent cells
1. Thaw Agrobacterium tumefaciens strain GV3101 (or other preferred strain) competent cells on ice (~1 h). Pre-chill 0.2 mL microcentrifuge tubes on ice. Warm SOC media at 28 °C.
2. Gently mix cells with the pipet tip (do NOT pipet up and down) and aliquot 15 µL of cells for each transformation into the chilled 1.5 mL microtubes.
3. Add 1-5 µL (~400 ng) of DNA to each tube of competent cells and mix gently by scraping tube along a tube rack.
  NOTE: Desired constructs need not have different antibiotic resistances because they are each being transformed separately and will be mixed before infiltration.
4. Place microtubes in liquid nitrogen for 5 min.
5. Remove microtubes from liquid nitrogen and place the tubes on the room-temp rack. Thaw tubes for 5 min in 37 °C incubator.
6. Add 250 µL of the pre-warmed (28 °C) SOC to each microtube. Shake at 28–30 °C, 225 rpm, for 3 h.
7. Plate 50 µL of transformed cells onto LB agar plates containing the necessary antibiotics described in Table 1 using sterile glass beads, as described in step 2.1.8.
8. Incubate plates inverted at 28–30 °C for 48 h. Growth within the 1^st day of incubation may be a sign of contamination. Transformed A. tumifaciens can be used following the 2-day incubation or stored at 4 °C sealed in paraffin film for up to 2 weeks.
Agrobacterium-mediated transient enzyme co-expression in Nicotiana benthamiana
1. Prune 4-week-old Nicotiana benthamiana plants 2 days before infiltration by removing lower leaves. Leave 4 uppermost leaves. Water the plants. Do not water the plants 24 h before infiltration in order to allow for open stomata and easier infiltration.
2. Add 10 mL of LB with working concentrations of the appropriate antibiotics for chosen constructs and Agrobacterium strain (described in Table 1) to a 50 mL sterile glass test tube with a foil lid.
3. Inoculate using a pipette tip to swab a single colony of transformed Agrobacterium and eject the tip into the LB media. Each Agrobacterium transformant should have at least 2 small cultures.
4. Incubate overnight at 28 °C and 220 rpm.
5. Measure optical density at 600 nm (OD₆₀₀) of the overnight cultures using a spectrophotometer. Dilute the overnight cultures to an OD₆₀₀of 1.
  NOTE: Optimal OD₆₀₀ values may vary when using other Agrobacterium strains.
6. Distribute 10 mL of diluted overnight culture into 50 mL conical tubes.
7. Harvest the bacteria by centrifugation at 3500 rpm for 15 min at room temp. Pour off and discard the supernatant.
8. Re-suspend the cultures in 10 mL infiltration buffer by gently shaking the tube and rolling on a tube rack. The OD₆₀₀should equal 1 for each construct.
9. Generate the combinations desired for infiltrations by combining equal volumes of each transformed cell line. The OD₆₀₀should equal 1 for each infiltration. Estimate 5 mL of infiltration solution per leaf, 2 leaves per plant.
10. Attach tubes horizontally to a rocker and rock gently for 2 h at room temperature.
11. Infiltrate ~2 leaves per N. benthamiana plant using approximately 5 mL of infiltration mixture per leaf. Infiltrate healthy leaves with a needleless syringe on the underside of the leaves while exerting a counter-pressure with a finger on the top side of the leaf to ensure infiltration solution suffuses the leaf tissue.
12. Mark all infiltrated leaves with a black marker or other indicator. Place infiltrated plants in the growth chamber for 5 days. Keep plants well-watered.
  NOTE: An incubation time of five days has proven sufficient for reaching diterpenoid levels sufficient for most downstream analyses, while maintaining time-efficiency of product formation. Longer incubation periods can be tested, where higher product amounts are required.
Metabolite extraction and purification from transformed Nicotiana benthamiana
1. Harvest infiltrated leaves from plants by clipping them from the plant. Add ~100 mL of liquid nitrogen and a single leaf to a mortar, then grind using a mortar and pestle or tissue mill until obtaining a fine powder.
2. Add powdered tissue to a GC-MS vial to the 500 µL demarcation. Add 1.5 mL of 50/50 (v/v) ethyl acetate/hexane or desired solvent mixture to the vial and cap tightly.
  1. For expressions that have been tested to provide the desired products, pool ground tissue into a larger flask or test tube, then add ~2x the amount of solvent than tissue volume and shake overnight. Proceed to step 4.4.5 for purification.
3. Place all vials in a microtube rack and tape down tightly to secure. Extract under vigorous shaking overnight at room temperature.
4. Transfer 400 µL of extract and 600 µL of hexane into a fresh GC-MS vial. Do not aliquot any leaf tissue. Analyze samples using GC-MS using the method described in step 3.3.6.1.
5. After analysis via GC-MS for presence of desired metabolites, pool leaf extracts together and proceed through rotary evaporation, silica column chromatography, and HPLC to obtain pure compounds as described in sections 3.2 and 3.3.

Representative Results

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 ± 0.25 mg/L culture), epoxydolabrene (0.65 ± 0.2 mg/L culture), and epoxydolabranol (11.4 ± 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).

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. Please click here to view a larger version of this figure.

Figure 2: Dolabralexin biosynthetic pathway and gene constructs used in this study. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

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 ‘plug & play’ 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. 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.

Disclosures

The authors declare they have no competing financial interests.

Acknowledgments

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’s Mentorship Award Fellowship (to K.M.M.) are gratefully acknowledged.

Materials

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

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References

Peters, R. J. Two rings in them all: the labdane-related diterpenoids. Natural Product Reports. 27 (11), 1521-1530 (2010).
Zerbe, P., Bohlmann, J. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering. Trends in Biotechnology. 33 (7), 419-428 (2015).
Toyomasu, T. Recent advances regarding diterpene cyclase genes in higher plants and fungi. Bioscience, Biotechnology, and Biochemistry. 72 (5), 1168-1175 (2008).
Smanski, M. J., Peterson, R. M., Huang, S. X., Shen, B. Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis. Current Opinion in Chemical Biology. 16 (1-2), 132-141 (2012).
Salazar-Cerezo, S., Martinez-Montiel, N., Garcia-Sanchez, J., Perez, Y. T. R., Martinez-Contreras, R. D. Gibberellin biosynthesis and metabolism: A convergent route for plants, fungi and bacteria. Microbiological Research. 208, 85-98 (2018).
Keeling, C. I., Bohlmann, J. Diterpene resin acids in conifers. Phytochemistry. 67 (22), 2415-2423 (2006).
Schmelz, E. A., et al. Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins. Plant Journal. 79 (4), 659-678 (2014).
Bohlmann, J., Keeling, C. I. Terpenoid biomaterials. Plant Journal. 54 (4), 656-669 (2008).
Mafu, S., Zerbe, P. Plant diterpenoid metabolism for manufacturing the biopharmaceuticals of tomorrow: prospects and challenges. Phytochemistry Reviews. 17 (1), 113-130 (2017).
Hillwig, M. L., Mann, F. M., Peters, R. J. Diterpenoid biopolymers: new directions for renewable materials engineering. Biopolymers. 95 (2), 71-76 (2011).
Jorgensen, L., et al. 14-step synthesis of (+)-ingenol from (+)-3-carene. Science. 341 (6148), 878-882 (2013).
Line, N. J., Burns, A. C., Butler, S. C., Casbohm, J., Forsyth, C. J. Total Synthesis of (-)-Salvinorin A. Chemistry. 22 (50), 17983-17986 (2016).
Morrone, D., et al. Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Applied Microbioly and Biotechnology. 85 (6), 1893-1906 (2010).
Kitaoka, N., Lu, X., Yang, B., Peters, R. J. The application of synthetic biology to elucidation of plant mono-, sesqui-, and diterpenoid metabolism. Molecular Plant. 8 (1), 6-16 (2015).
George, K. W., et al. Metabolic engineering for the high-yield production of isoprenoid-based C(5) alcohols in E. coli. Scientific Reports. 5, 11128 (2015).
Paddon, C. J., Keasling, J. D. Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nature Reviews Microbiology. 12 (5), 355-367 (2014).
Keasling, J. D. Synthetic biology and the development of tools for metabolic engineering. Metabolic Engineering. 14 (3), 189-195 (2012).
Kampranis, S. C., Makris, A. M. Developing a yeast cell factory for the production of terpenoids. Computational and Structural Biotechnology Journal. 3 (4), e201210006 (2012).
Tholl, D. Biosynthesis and biological functions of terpenoids in plants. Advances in Biochemical Engineering/Biotechnology. 148, 63-106 (2015).
McGarvey, D. J., Croteau, R. Terpenoid metabolism. The Plant Cell. 7 (7), 1015-1026 (1995).
Chen, F., Tholl, D., Bohlmann, J., Pichersky, E. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. The Plant Journal. 66 (1), 212-229 (2011).
Pateraki, I., Heskes, A. M., Hamberger, B. Cytochromes P450 for terpene functionalisation and metabolic engineering. Advances in Biochemical Engineering/Biotechnology. 148, 107-139 (2015).
Cao, R., et al. Diterpene cyclases and the nature of the isoprene fold. Proteins. 78 (11), 2417-2432 (2010).
Reiling, K. K., et al. Mono and diterpene production in Escherichia coli. Biotechnology and Bioengineering. 87 (2), 200-212 (2004).
Hamberger, B., Plant Bak, S. P450s as versatile drivers for evolution of species-specific chemical diversity. Philosophical Transactions of the Royal Society B: Biological Sciences. 368 (1612), 20120426 (2013).
Jia, M., Potter, K. C., Peters, R. J. Extreme promiscuity of a bacterial and a plant diterpene synthase enables combinatorial biosynthesis. Metabolic Engineering. 37, 24-34 (2016).
Paddon, C. J., et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 496 (7446), 528-532 (2013).
Peralta-Yahya, P. P., Zhang, F., del Cardayre, S. B., Keasling, J. D. Microbial engineering for the production of advanced biofuels. Nature. 488 (7411), 320-328 (2012).
Pateraki, I., et al. Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. Elife. 6, e23001 (2017).
Ignea, C., et al. Carnosic acid biosynthesis elucidated by a synthetic biology platform. Proceedings of the National Academy of Sciences of the United States of America. 113 (13), 3681-3686 (2016).
Ignea, C., et al. Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast. Metabolic Engineering. 28, 91-103 (2015).
Cyr, A., Wilderman, P. R., Determan, M., Peters, R. J. A modular approach for facile biosynthesis of labdane-related diterpenes. Journal of the American Chemical Society. 129 (21), 6684-6685 (2007).
Ajikumar, P. K., et al. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science. 330 (6000), 70-74 (2010).
Schalk, M., et al. Toward a biosynthetic route to sclareol and amber odorants. Journal of the American Chemical Society. 134 (46), 18900-18903 (2012).
Andersen-Ranberg, J., et al. Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis. Angewandte Chemie International Edition in English. 55 (6), 2142-2146 (2016).
Bruckner, K., Tissier, A. High-level diterpene production by transient expression in Nicotiana benthamiana. Plant Methods. 9 (1), 46 (2013).
Emanuelsson, O., Brunak, S., von Heijne, G., Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nature Protocols. 2 (4), 953-971 (2007).
Hausjell, J., Halbwirth, H., Spadiut, O. Recombinant production of eukaryotic cytochrome P450s in microbial cell factories. Bioscience Reports. 38 (2), BSR20171290 (2018).
Mafu, S., et al. Discovery, biosynthesis and stress-related accumulation of dolabradiene-derived defenses in maize. Plant Physiology. 176 (4), 2677-2690 (2018).
Zerbe, P., et al. Gene discovery of modular diterpene metabolism in nonmodel systems. Plant Physiology. 162 (2), 1073-1091 (2013).
Bach, S. S., et al. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. Methods in Molecular Biology. , 245-255 (2014).
Sainsbury, F., Thuenemann, E. C., Lomonossoff, G. P. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnology Journal. 7 (7), 682-693 (2009).
Wang, B., et al. Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua. Metabolic Engineering. 38, 159-169 (2016).
Reed, J., Osbourn, A. Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Reports. 37 (10), 1431-1441 (2018).
Dong, L., Jongedijk, E., Bouwmeester, H., Der Krol, A. V. an Monoterpene biosynthesis potential of plant subcellular compartments. New Phytologist. 209 (2), 679-690 (2016).
Liu, Q., et al. Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana. PLoS One. 6 (8), e23255 (2011).
Karunanithi, P. S., et al. Functional characterization of the cytochrome P450 monooxygenase CYP71AU87 indicates a role in marrubiin biosynthesis in the medicinal plant Marrubium vulgare. BMC Plant Biology. 19 (1), 114 (2019).
Pelot, K., et al. Functional diversity of diterpene synthases in the biofuel crop switchgrass. Plant Physiology. 178 (1), 54-71 (2018).
Wang, Q., Hillwig, M. L., Wu, Y., Peters, R. J. CYP701A8: a rice ent-kaurene oxidase paralog diverted to more specialized diterpenoid metabolism. Plant Physiology. 158 (3), 1418-1425 (2012).
Wang, Q., Hillwig, M. L., Peters, R. J. CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice. The Plant Journal. 65 (1), 87-95 (2011).
Morrone, D., Chen, X., Coates, R. M., Peters, R. J. Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis. Biochemical Journal. 431 (3), 337-344 (2010).
Swaminathan, S., Morrone, D., Wang, Q., Fulton, D. B., Peters, R. J. CYP76M7 is an ent-cassadiene C11alpha-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice. The Plant Cell. 21 (10), 3315-3325 (2009).
Gnanasekaran, T., et al. Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme. Journal of Biological Engineering. 9 (1), 24 (2015).
De Luca, V., Salim, V., Atsumi, S. M., Yu, F. Mining the biodiversity of plants: a revolution in the making. Science. 336 (6089), 1658-1661 (2012).
Wurtzel, E. T., Kutchan, T. M. Plant metabolism, the diverse chemistry set of the future. Science. 353 (6305), 1232-1236 (2016).
Menon, A., et al. Mobile-based insulin dose adjustment for type 2 diabetes in community and rural populations: study protocol for a pilot randomized controlled trial. Therapeutic Advances in Endocrinology and Metabolism. 10, 2042018819836647 (2019).
Moses, T., et al. OSC2 and CYP716A14v2 catalyze the biosynthesis of triterpenoids for the cuticle of aerial organs of Artemisia annua. The Plant Cell. 27 (1), 286-301 (2015).

Biochemistry