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&quot; Tech Square Pot</td><td>McConkey Wholesale Grower&#039;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 &amp;beta;-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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M7506-500g</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</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>&lt;em&gt;Nicotiana benthamiana &lt;/em&gt;seed</td><td>USDA Germplasm Repository</td><td>Accession TW16</td><td>&lt;em&gt;N. benthamiana&lt;/em&gt;</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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JoVE Journal

Biochemistry

9.7K Views.  University of California Davis. Dit protocol is geschikt voor het produceren van gezuiverde diterpenoïden die kunnen worden gebruikt voor verschillende downstream-analyse en kan worden aangepast voor een reeks verschillende metabolietdoelen. Het belangrijkste voordeel van deze methode is het is een eenvoudige en goedkope methode om milligram hoeveelheden gespecialiseerde metabolieten te produceren. Door het uitwisselen van de genen en enzymen die worden gebruikt voor co-expressie, kan deze methode worden gewijzigd om ander...

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

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

Genetics

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Bioengineering

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

Chemistry

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

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

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

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Strategies for Expressing and Purifying Solute Carrier Transporters

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

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

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