Implicating the red body of Nannochloropsis in forming the recalcitrant cell wall polymer algaenan.

Stramenopile algae contribute significantly to global primary productivity, and one class, Eustigmatophyceae, is increasingly studied for applications in high-value lipid production. Yet much about their basic biology remains unknown, including the nature of an enigmatic, pigmented globule found in vegetative cells. Here, we present an in-depth examination of this "red body," focusing on Nannochloropsis oceanica. During the cell cycle, the red body forms adjacent to the plastid, but unexpectedly it is secreted and released with the autosporangial wall following cell division. Shed red bodies contain antioxidant ketocarotenoids, and overexpression of a beta-carotene ketolase results in enlarged red bodies. Infrared spectroscopy indicates long-chain, aliphatic lipids in shed red bodies and cell walls, and UHPLC-HRMS detects a C32 alkyl diol, a potential precursor of algaenan, a recalcitrant cell wall polymer. We propose that the red body transports algaenan precursors from plastid to apoplast to be incorporated into daughter cell walls.

Biosynthesis of chlorophyll c in a dinoflagellate and heterologous production in planta.

Chlorophyll c is a key photosynthetic pigment that has been used historically to classify eukaryotic algae. Despite its importance in global photosynthetic productivity, the pathway for its biosynthesis has remained elusive. Here we define the CHLOROPHYLL C SYNTHASE (CHLCS) discovered through investigation of a dinoflagellate mutant deficient in chlorophyll c. CHLCSs are proteins with chlorophyll a/b binding and 2-oxoglutarate-Fe(II) dioxygenase (2OGD) domains found in peridinin-containing dinoflagellates; other chlorophyll c-containing algae utilize enzymes with only the 2OGD domain or an unknown synthase to produce chlorophyll c. 2OGD-containing synthases across dinoflagellate, diatom, cryptophyte, and haptophyte lineages form a monophyletic group, 8 members of which were also shown to produce chlorophyll c. Chlorophyll c1 to c2 ratios in marine algae are dictated in part by chlorophyll c synthases. CHLCS heterologously expressed in planta results in the accumulation of chlorophyll c1 and c2, demonstrating a path to augment plant pigment composition with algal counterparts.

Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide.

The diterpenoid triepoxides triptolide and triptonide from Tripterygium wilfordii (thunder god wine) exhibit unique bioactivities with potential uses in disease treatment and as a non-hormonal male contraceptives. Here, we show that cytochrome P450s (CYPs) from the CYP71BE subfamily catalyze an unprecedented 18(4→3) methyl shift required for biosynthesis of the abeo-abietane core structure present in diterpenoid triepoxides and in several other plant diterpenoids. In combination with two CYPs of the CYP82D subfamily, four CYPs from T. wilfordii are shown to constitute the minimal set of biosynthetic genes that enables triptonide biosynthesis using Nicotiana benthamiana and Saccharomyces cerevisiae as heterologous hosts. In addition, co-expression of a specific T. wilfordii cytochrome b5 (Twcytb5-A) increases triptonide output more than 9-fold in S. cerevisiae and affords isolation and structure elucidation by NMR spectroscopic analyses of 18 diterpenoids, providing insights into the biosynthesis of diterpenoid triepoxides. Our findings pave the way for diterpenoid triepoxide production via fermentation.

Circular biomanufacturing through harvesting solar energy and CO2.

Using synthetic biology, it is now time to expand the biosynthetic repertoire of plants and microalgae by utilizing the chloroplast to augment the production of desired high-value compounds and of oil-, carbohydrate-, or protein-enriched biomass based on direct harvesting of solar energy and the consumption of CO2. Multistream product lines based on separate commercialization of the isolated high-value compounds and of the improved bulk products increase the economic potential of the light-driven production system and accelerate commercial scale up. Here we outline the scientific basis for the establishment of such green circular biomanufacturing systems and highlight recent results that make this a realistic option based on cross-disciplinary basic and applied research to advance long-term solutions.

Synthesis of C-Glucosylated Octaketide Anthraquinones in Nicotiana benthamiana by Using a Multispecies-Based Biosynthetic Pathway.

Carminic acid is a C-glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane-bound enzymes.

Localization and in-Vivo Characterization of Thapsia garganica CYP76AE2 Indicates a Role in Thapsigargin Biosynthesis.

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily.

Tripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis.

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.

Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme.

BACKGROUND: Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4. RESULTS: We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45-55 µg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes. CONCLUSIONS: It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.

Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide.

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana.

To respond to the rapidly growing number of genes putatively involved in terpenoid metabolism, a robust high-throughput platform for functional testing is needed. An in planta expression system offers several advantages such as the capacity to produce correctly folded and active enzymes localized to the native compartments, unlike microbial or prokaryotic expression systems. Two inherent drawbacks of plant-based expression systems, time-consuming generation of transgenic plant lines and challenging gene-stacking, can be circumvented by transient expression in Nicotiana benthamiana. In this chapter we describe an expression platform for rapid testing of candidate terpenoid biosynthetic genes based on Agrobacterium mediated gene expression in N. benthamiana leaves. Simultaneous expression of multiple genes is facilitated by co-infiltration of leaves with several engineered Agrobacterium strains, possibly making this the fastest and most convenient system for the assembly of plant terpenoid biosynthetic routes. Tools for cloning of expression plasmids, N. benthamiana culturing, Agrobacterium preparation, leaf infiltration, metabolite extraction, and automated GC-MS data mining are provided. With all steps optimized for high throughput, this in planta expression platform is particularly suited for testing large panels of candidate genes in all possible permutations.

Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii.

Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.