Diterpene Resin Acids Research Articles

Ultra High-Performance Supercritical Fluid Chromatography for the Quantitation of Diterpene Resin Acids in Norway Spruce Samples.

Picea abies (L.) H. Karst. (Pinaceae) is native to Northern, Central and Eastern Europe. The fast-growing tree reaches up to 50 m in height, has modest nutritional requirements and depends on sufficient water supply. The conifer, commonly called Norway spruce, produces exudates which are traditionally used to treat skin wounds in Northern European countries. Major bioactive constituents of the conifer oleoresin are diterpene resin acids (DRAs) of the abietane and the pimarane type. To assure consistent pharmaceutical quality of Norway spruce balm and commercial products thereof, an analytical method for the quantitation of DRAs is the prerequisite. However, high structural similarity among DRAs and their poor UV absorption makes chromatographic separation and detection challenging: Conventional liquid chromatography systems often fail to achieve sufficient separation, moreover, they are not sustainable. Gas chromatography on the other hand requires time-consuming derivatization prior to unacceptably long analyses (>60 min). These drawbacks prompted the development of the first validated supercritical fluid-based protocol for the separation and quantitation of eight DRAs, i.e., pimaric acid (1), sandaracopimaric acid (2), palustric acid (3), isopimaric acid (4), levopimaric acid (5), abietic acid (6), dehydroabietic acid (7), and neoabietic acid (8). By using an ultra high-performance supercritical fluid chromatography (UHPSFC) device hyphenated to a quadrupole mass detector, the DRAs were separated in less than 20 min on a Torus 2-Picolylamin (2-PIC) column (3.0 mm × 100 mm; 1.7 µm particle size) applying supercritical CO2 and ethanol as mobile phase. Regarding selectivity, accuracy (recovery rates: 87–108%), intermediate precision (between 6.6 and 11.1%), and linearity (R2 ≥ 0.99; linear between 0.75 μg/ml and 2.5 mg/ml), results were obtained in line with ICH guidelines. The lowest limit of detection (LOD) was at 0.75 μg/ml (7) and the lowest limit of quantitation (LOQ) at 2 μg/ml (8). As application examples, 22 Norway spruce balm samples and five commercial products were analyzed. The here presented protocol not only simplifies and shortens the analytical workflow, but also reduces the amount of organic solvent waste by about two thirds compared to conventional liquid chromatographic set-ups. These advantages qualify this fast and efficient method as an ideal tool for an environmentally friendly quality control of traditionally used wound-healing Norway spruce balm products.

Exudates of Picea abies, Pinus nigra, and Larix decidua: Chromatographic Comparison and Pro-Migratory Effects on Keratinocytes In Vitro.

Balms and resins of Picea abies, Larix decidua, and Pinus nigra are traditionally used to treat wounds. Three chromatographic techniques differing in separation capacity and technical demands were employed to distinguish among these plant exudates. A TLC method was established for fingerprint comparison, providing a quick overview of a large number of samples at low cost. HPLC-DAD (RP18) and UHPSFC-DAD (Torus 2-Picolylamin), hyphenated to ESI-MS, represented orthogonal chromatographic systems with high separation performance. The developed methods allow for the separation and detection of major and minor constituents belonging to different compound classes (phenyl carboxylic acids, lignans, diterpene resin acids). The qualitative compositions of the diterpene resin acids, the main compounds in the exudates, were comparable in all three genera. Differences were detected in the distribution of hydroxylated diterpene resin acids, pinoresinol, and hydroxycinnamic acids. The three tested chromatographic systems with varying demands on lab equipment offer appropriate tools for the quality assessment of Picea abies, Larix decidua, and Pinus nigra. The extracts were furthermore tested at three different concentrations (10 µg/mL, 3 µg/mL, and 1 µg/mL) for boosted re-epithelialization, a crucial step in the wound-healing process, in an in vitro HaCaT keratinocyte-based scratch assay. Lysophosphatidic acid (LPA, 10 µM) and extracts of several medicinal plants well known for their wound-healing properties (birch, marigold, St. John’s wort, manuka honey) were used as positive controls. Picea abies and Pinus nigra showed concentration dependency; significant activity was measured for Larix decidua at 3 µg/mL.

Identification and Tissue-Specific Expression Analysis of CYP720B Subfamily Genes in Slash Pine and Loblolly Pine

Diterpene resin acids (DRAs) are major components of pine oleoresin that can effectively resist the invasion of insects and pathogenic microorganisms. The subfamily of cytochrome P450s, CYP720B, catalyzes diterpene products into DRAs. Identifying CYP720B subfamily members and revealing the characteristics of tissue-specific expression would help understand diterpene-rich structures and diverse types. Slash pine and loblolly pine are important pines that provide oleoresin products. In this study, we identified CYP720B candidate genes based on the Pinus taeda V2.0 genome and full-length transcriptome of slash pine by PacBio. A total of 17 genes in slash pine and 19 in loblolly pine were identified and classified into four main clades by phylogenetic analysis. An analysis of cis-acting elements showed that CYP720B genes were closely related to adversity resistance. The gene expression of these candidates in different tissues was quantified by real-time quantitative PCR (RT–qPCR) analysis. Most of the genes showed relatively higher expression levels in roots and stems than in the other tissues, corresponding with the results of DRA component detection by gas chromatography–mass spectrometry (GC–MS), which indicated that stems and roots might be important tissues in oleoresin biosynthesis. These results provide a valuable resource for a better understanding of the biological role of individual CYP720Bs in slash pine and loblolly pine.

Mountain Pine Beetle Epidemic: An Interplay of Terpenoids in Host Defense and Insect Pheromones.

The mountain pine beetle epidemic has highlighted the complex interactions of bark beetles with conifer host defenses. In these interactions, oleoresin terpenoids and volatiles, produced and released by the host tree, can be both harmful and beneficial to the beetle's success in colonizing a tree and completing its life cycle. The insect spends almost its entire life, from egg to adult, within the bark and phloem of a pine host, exposed to large quantities of complex mixtures of oleoresin terpenoids. Conifer oleoresin comprises mostly monoterpenes and diterpene resin acids as well as many different sesquiterpenes. It functions as a major chemical and physical defense system. However, the insect has evolved host colonization behavior and enzymes for terpenoid metabolism and detoxification that allow it to overcome some of the terpenoid defenses and, importantly, to co-opt pine monoterpenes as cues for host search and as a precursor for its own pheromone system. The insect-associated microbiome also plays a role in the metabolism of conifer terpenoids.

Diterpene Resin Acids and Olefins in Calabrian Pine (Pinus nigra subsp. laricio (Poiret) Maire) Oleoresin: GC-MS Profiling of Major Diterpenoids in Different Plant Organs, Molecular Identification and Expression Analysis of Diterpene Synthase Genes

A quali-quantitative analysis of diterpenoid composition in tissues obtained from different organs of Pinus nigra subsp. laricio (Poiret) Maire (Calabrian pine) was carried out. Diterpene resin acids were the most abundant diterpenoids across all the examined tissues. The same nine diterpene resin acids were always found, with the abietane type prevailing on the pimarane type, although their quantitative distribution was found to be remarkably tissue-specific. The scrutiny of the available literature revealed species specificity as well. A phylogeny-based approach allowed us to isolate four cDNAs coding for diterpene synthases in Calabrian pine, each of which belonging to one of the four groups into which the d3 clade of the plants’ terpene synthases family can be divided. The deduced amino acid sequences allowed predicting that both monofunctional and bifunctional diterpene synthases are involved in the biosynthesis of diterpene resin acids in Calabrian pine. Transcript profiling revealed differential expression across the different tissues and was found to be consistent with the corresponding diterpenoid profiles. The isolation of the complete genomic sequences and the determination of their exon/intron structures allowed us to place the diterpene synthase genes from Calabrian pine on the background of current ideas on the functional evolution of diterpene synthases in Gymnosperms.

Identification of the diterpenoid biosynthesis genes and their expression status in relation to oleoresin yield of masson pine

Conifer oleoresin is a highly precious raw material for industrial sectors and a precursor for biofuels. Diterpenoid, particularly diterpenoid resin acids (DRAs), is one of the most abundant oleoresin components. Masson pine (Pinus massoniana L.) is one of the most important native species for oleoresin yield in China. To better understand the biosynthesis of diterpenoid in high oleoresin-yield masson pine and develop methods for its production in needle and trunk xylem, we used the combination of SMRT full-length transcriptome and Illumina RNA sequencing technologies to establish a transcriptome dataset. Approximately 20,560 and 17,950 full-length non-redundant transcripts were assembled in trunk xylem and needle, respectively. Of those, 29 and 20 DEGs were identified involving in the Terpenoid Backbone Biosynthesis pathway (TBB) (KO 00900) and the Diterpenoid Biosynthesis pathway (DB) (KO 00904) in trunk xylem, as well as 35 and 12 DEGs in the TBB and the DB in the needle, respectively. Compared with the low oleoresin-yield germplasm, 13 up-regulated DEGs with complete open reading frame (ORF) sequence were obtained by Venn analysis in trunk xylem and needle of a high oleoresin-yield clone. Subsequently, the expression patterns of the 16 diterpenoid biosynthesis-related genes (including three types of DXS genes) were validated by quantitative real-time PCR in both 12-year-old and 1-year-old high and low oleoresin-yield masson pine clones. The results showed that the 13 genes in the higher yield oleoresin trees were proved to be higher expression in comparison with the lower ones irrespectively of the tree age; as well as MCS (2-C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase), and IDI (Isopentenyl-diphosphate Delta-isomerase) of the methylerythritol 4-phosphate (MEP) pathway demonstrated a higher expression compared to that of other TBB genes. Both cytosol-localized MVA and plastid-localized MEP pathways gave significant contributions to diterpenoid accumulation, and the latter played a more dominant role. To clarify the potential relationships between oleoresin yield and the expression patterns of 16 genes from the TBB and DB pathways, the Pearson correlation analysis was performed using three tissues (young needle, old needle, and trunk xylem) from the 60-year-old tree. Remarkably, HMGR (Hydroxy methylglutaryl-CoA reductase) and ent-CDS (Ent-copalyl diphosphate synthase) were positive correlation with oleoresin yield in the new needle, whereas FPPS (Farnesyl pyrophosphate synthase) was a negative correlation with that in the old needle; furthermore, HMGS (Hydroxy methylglutaryl-CoA synthase), IDI and CDS (Copalyl diphosphate synthase) were positively correlated with oleoresin synthesis in the trunk xylem. Moreover, the Pearson correlation analysis was also carried out to determine the potential relationships between the 13 genes of the TBB pathways and ent-CDS, CDS, and LDS ((13E)-labda-7 13-dien-15-ol synthase) from DB pathways within three tissues of the 60-year-old masson pine. Furthermore, the diterpenoid and diterpene resin acids (DRAs) demonstrated highly positive correlations with oleoresin yield, and DRAs were detected as the most abundant diterpenoids in oleoresin. Promisingly, HMGR, MCS, HDS, IDI, ent-CDS, and CDS may be used as the key target genes of the molecular-assisted selection for high-oleoresin-yielding germplasm in this species.

Primary and Secondary Metabolite Profiles of Lodgepole Pine Trees Change with Elevation, but Not with Latitude.

Climate change has a large influence on plant functional and phenotypic traits including plant primary and secondary metabolites. One well-established approach to investigating the variation in plant metabolites involves studying plant populations along elevation and latitude gradients. We considered how two space-for-time climate change gradients (elevation and latitude) influence carbohydrate reserves (soluble sugars, starches) and secondary metabolites (monoterpenes, diterpene resin acids) of lodgepole pine trees in western Canada. We were particularly interested in the relationship of terpenes and carbohydrates with a wide range of tree, site, and climatic factors. We found that only elevation had a strong influence on the expression of both terpenes and carbohydrates of trees. Specifically, as elevation increased, concentrations of monoterpenes and diterpenes generally increased and soluble sugars (glucose, sucrose, total sugars) decreased. In contrast, latitude had no impact on either of terpenes or carbohydrates. Furthermore, we found a positive relationship between concentrations of starch and total terpenes and diterpenes in the elevation study; whereas neither starches nor sugars were correlated to terpenes in the latitude study. Similarly, both terpenes and carbohydrates had a much greater number of significant correlations to site characteristics such as slope, basal area index, and sand basal area, in the elevational than in the latitude study. Overall, these results support the conclusion that both biotic and abiotic factors likely drive the patterns of primary and secondary metabolite profiles of lodgepole pine along geographical gradients. Also, presence of a positive relationship between terpenes and starches suggests an interaction between primary ad secondary metabolites of lodgepole pine trees.

Identification and functional study of oleoresin terpenoid biosynthesis-related genes in masson pine (Pinus massoniana L.) based on transcriptome analysis

Masson pine (Pinus massoniana L.) is one of the most important species for oleoresin production worldwide. However, the mechanism of oleoresin synthesis in masson pine remains poorly studied. We revealed the key differentially expressed genes associated with terpenoid biosynthesis in trees with different yields of oleoresin in different tissues (trunk xylem and needle) during different seasons (spring and autumn) based on the combined application of the SMRT full-length transcriptome and Illumina RNA sequencing under field operations. A total of 76,754 full-length non-redundant transcripts were obtained in the study. Transcriptome data were annotated with KEGG and GO, and they have been shown to be of high quality by qRT-PCR. Chloroplastic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway and cytosolic mevalonate (MVA) pathway genes have different preferences under different conditions. Moreover, the phylogenetic analysis results revealed three classes of DXS genes with different functions. Among the tissue-specific genes, AACT1 was expressed in the trunk xylem at the highest levels, while MCS (1, 4) and HDR2 were significantly upregulated in the needle. Among stage-specific genes, DXS6 and HDS6 were expressed in the trunk xylem in August (autumn) at the highest levels. Furthermore, all of the terpene synthase (TPS) differentially expressed genes (DEGs) were clustered into the gymnosperm-specific TPS-d subfamily, including sesquiterpene synthase and diterpene synthase genes. According to the protein sequence alignment, diTPS1 and diTPS5 with three conserved active sites catalysed the synthesis of diterpene resin acids. In the low-yield oleoresin clone, diTPS (1, 5) and GGPPS (4, 6) were upregulated in the trunk xylem. The integration of the transcriptomic data indicated that the AACT, MCS, HDR, DXS, HDS, and TPS (with two conserved active sites) may play critical roles in the terpenoids biosynthesis of masson pine. The results of our study can be useful for developing an understanding of the coniferous genome and to significantly accelerate confer breeding.

Molecular underpinnings of methyl jasmonate-induced resistance in Norway spruce.

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm.

Norway Spruce Balm: Phytochemical Composition and Ability to Enhance Re-epithelialization In Vitro.

The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids ( 1: - 3: ), the lignan pinoresinol ( 4: ), four hydroxylated derivatives of dehydroabietic acid (DHAA) ( 5: - 8: ), and dehydroabietic acid ( 9: ) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids-pimaric acid ( 10: ) and isopimaric acid ( 11: )-and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

Spatial variation in soil available water holding capacity alters carbon mobilization and allocation to chemical defenses along jack pine stems

Carbohydrate metabolism in trees has received considerable attention due to its role in global forest decline. However, mobilization, storage, and allocation of carbohydrates show large variation along tree stems due to various source-sink relationships. Currently, we have a limited understanding of the causes or consequences of carbohydrate variation on plant chemical defenses. We measured non-structural carbohydrates (NSC), monoterpenes, and diterpene resin acids along jack pine (Pinus banksiana) stems before inoculating with a phytopathogenic fungus in multiple sites with different soil available water holding capacities (AWHC). We then assessed induced defense responses in trees by measuring lesion areas (necrotic tissues), NSC mobilization, and terpene concentrations along different stem heights. Before inoculation, NSC concentrations increased with stem height, diterpene resin acid concentrations decreased, and monoterpene concentrations did not differ. However, with increasing AWHC, NSC concentrations decreased, monoterpene concentrations increased, and diterpene resin acid concentrations did not differ. After inoculation, trees on the high AWHC sites developed smaller lesions, mobilized local NSC, and induced higher monoterpene concentrations in the phloem and lesions. Diterpene resin acid concentrations did not differ with AWHC but differed at each stem height. Inoculation density showed no difference. We conclude that gradients in soil AWHC can affect stem vulnerability in jack pine trees by influencing carbon mobilization, allocation, and utilization.

Embedding MIL-100(Fe) with magnetically recyclable Fe3O4 nanoparticles for highly efficient esterification of diterpene resin acids and the associated kinetics

Abstract MIL-100(Fe) embedded with magnetic Fe3O4 nanoparticles (Fe3O4/MIL-100(Fe) composite) is directly used as an effective and recyclable heterogeneous catalyst for the rosin esterification. The Fe3O4/MIL-100(Fe) composite shows unexpectedly high rosin conversion of 94.8% at 240 °C within 2.5 h, which is higher than that obtained using a homogenous ZnO catalyst. Characterizations reveals that the Fe3O4/MIL-100(Fe) composite exhibits apparent catalytic activity due to the interaction of MIL-100(Fe) with Fe3O4. The kinetics of rosin esterification using Fe3O4/MIL-100(Fe) composite and ZnO is studied. The effect of glycerol can be considered as constant due to the excess supplies. For Fe3O4/MIL-100(Fe) composite, a first order kinetic model is obtained, but for ZnO catalyst, a second order kinetic model is obtained, affording the activation energies of 25.794–28.808 and 33.048–34.358 kJ mol−1, respectively, indicating that the presence of Fe3O4/MIL-100(Fe) composite reduces the energy barier that is required in the esterification, thus making it easier to perform. Further, the Fe3O4/MIL-100(Fe) composite shows acceptable stability and reusability for six cycles.

Functions of mountain pine beetle cytochromes P450 CYP6DJ1, CYP6BW1 and CYP6BW3 in the oxidation of pine monoterpenes and diterpene resin acids.

The mountain pine beetle (MPB; Dendroctonus ponderosae) is a forest insect pest that attacks several different pine (Pinus) species in its native range of distribution in western North America. MPB are exposed for most of their life cycle to the chemical defenses of their hosts. These defenses are dominated by oleoresin secretions containing mostly various monoterpenes and diterpene resin acids (DRAs). Cytochrome P450 enzymes (P450s) of the MPB are thought to be involved in the metabolism of at least some of these defense compounds. Here we describe the cloning and characterization of three MPB P450s, CYP6DJ1, CYP6BW1 and CYP6BW3, and their functions in the oxidation of various monoterpenes and diterpene resin acids. CYP6DJ1 oxidizes the monoterpenes (+)-(4R)-limonene, (–)-(4S)-limonene and terpinolene and produces (4R,8R)-limonene-8,9-epoxide, (4R,8S)-limonene-8,9-epoxide, (4S,8S)-limonene-8,9-epoxide, (4S,8R)-limonene-8,9-epoxide, perilla alcohol and several unidentified oxidized compounds. These products of CYP6DJ1 were also identified in extracts of MPB treated with the same monoterpenes. CYP6BW1 and CYP6BW3 both oxidize the DRAs abietic acid, dehydroabietic acid, neoabietic acid, levopimaric acid, palustric acid, and isopimaric acid, producing hydroxylated and epoxidized DRAs. CYP6DJ1, CYP6BW1 and CYP6BW3 appear to contribute to the metabolism of oleoresin terpenes as part of the MPB’s ability to cope with host defenses.

Dehydroabietic Acid Suppresses Inflammatory Response Via Suppression of Src-, Syk-, and TAK1-Mediated Pathways.

Dehydroabietic acid (DAA) is a naturally occurring diterpene resin acid derived from coniferous plants such as Pinus and Picea. Various bioactive effects of DAA have been studied including antibacterial, antifungal, and anticancer activities. However, the anti-inflammatory mechanism of DAA remains unclear. We evaluated the anti-inflammatory effect of DAA in macrophage cell lines. Dehydroabietic acid clearly reduced nitric oxide (NO) production and inflammatory gene expression decreased according to RT-PCR results. Dehydroabietic acid displayed anti-inflammatory activity at the transcriptional level in results from NF-κB- or AP-1-mediated luciferase assays. To identify the DAA target protein, we investigated NF-κB and AP-1 pathways by Western blotting analysis. Dehydroabietic acid suppressed the activity of proto-oncogene tyrosine protein kinase (Src) and spleen tyrosine kinase (Syk) in the NF-κB cascade and transforming growth factor beta-activated kinase 1 (TAK1) in the AP-1 cascade. Using overexpression strategies, we confirmed that DAA targeted these kinases. Our findings demonstrate the anti-inflammatory effects and molecular mechanism of DAA. This suggests that DAA has potential as a drug or supplement to ameliorate inflammation.

The cytochrome P450 CYP6DE1 catalyzes the conversion of \u03b1-pinene into the mountain pine beetle aggregation pheromone trans-verbenol

The recent outbreak of the mountain pine beetle (Dendroctonus ponderosae; MPB) has affected over 20 M hectares of pine forests in western North America. During the colonization of host trees, female MPB release the aggregation pheromone (−)-trans-verbenol. (−)-trans-Verbenol is thought to be produced from the pine defense compound (−)-α-pinene by cytochrome P450 (P450) dependent hydroxylation. MPB may also use P450s for the detoxification of other monoterpenes of the pine defense system. Here we describe the functional characterization of MPB CYP6DE1. CYP6DE1, but not the closely related CYP6DE2, used the bicyclic monoterpenes (−)-α-pinene, (+)-α-pinene, (−)-β-pinene, (+)-β-pinene and (+)-3-carene as substrates. CYP6DE1 was not active with other monoterpenes or diterpene resin acids that were tested as substrates. trans-Verbenol is the major product of CYP6DE1 activity with (−)-α-pinene or (+)-α-pinene as substrates. When tested with blends of different ratios of (−)-α-pinene and (+)-α-pinene, CYP6DE1 produced trans-verbenol with an enantiomeric profile that was similar to that produced by female MPB exposed to the α-pinene enantiomers.

Heterologous production of levopimaric acid in Saccharomyces cerevisiae

BackgroundLevopimaric acid (LA), a type of diterpene resin acid produced by plants, is a significant industrial intermediate that is mainly produced via phytoextraction. This work aimed to apply synthetic biology to produce LA in yeast strains from a simple carbon source.ResultsLevopimaradiene (LP), the precursor of LA, was produced via LP synthase (LPS) expression in yeast. LPS was then modified by N-terminal truncating and site-directed mutagenesis. The strain containing t79LPSMM (79 N-terminal amino acid truncating and M593I/Y700F mutation) produced 6.92 mg/L of LP, which were 23-fold higher than the strain containing LPS. Next, t79LPSMM was expressed in a new metabolically engineered chassis, and the final LP production increased 164-folds to 49.21 mg/L. Three cytochrome P450 reductases (CPRs) were co-expressed with CYP720B1 (the enzyme responsible for LA production from LP) in yeast to evaluate their LA producing abilities, and the CPR from Taxus cuspidata (TcCPR) was found to be the best (achieved 23.13 mg/L of LA production). CYP720B1 and TcCPR genes overexpression in the multi-copy site of the S.cerevisiae genome led to a 1.9-fold increase in LA production to 45.24 mg/L in a shake-flask culture. Finally, LA production was improved to 400.31 mg/L via fed-batch fermentation in a 5-L bioreactor.ConclusionsThis is the first report to produce LA in a yeast cell factory and the highest titer of LA is achieved.

Conifer Diterpene Resin Acids Disrupt Juvenile Hormone-Mediated Endocrine Regulation in the Indian Meal Moth Plodia interpunctella.

Diterpene resin acids (DRAs) are important components of oleoresin and greatly contribute to the defense strategies of conifers against herbivorous insects. In the present study, we determined that DRAs function as insect juvenile hormone (JH) antagonists that interfere with the juvenile hormone-mediated binding of the JH receptor Methoprene-tolerant (Met) and steroid receptor coactivator (SRC). Using a yeast two-hybrid system transformed with Met and SRC from the Indian meal moth Plodia interpunctella, we tested the interfering activity of 3704 plant extracts against JH III-mediated Met-SRC binding. Plant extracts from conifers, especially members of the Pinaceae, exhibited strong interfering activity, and four active interfering DRAs (7α-dehydroabietic acid, 7-oxodehydroabietic acid, dehydroabietic acid, and sandaracopimaric acid) were isolated from roots of the Japanese pine Pinus densiflora. The four isolated DRAs, along with abietic acid, disrupted the juvenile hormone-mediated binding of P. interpunctella Met and SRC, although only 7-oxodehydroabietic acid disrupted larval development. These results demonstrate that DRAs may play a defensive role against herbivorous insects via insect endocrine-disrupting activity.

Signaling function of dehydroabietinal in plant defense and development

Plants provide the bulk of the nearly 12,000 diterpenoid natural products, which include diterpene resin acids that have important medicinal and industrial applications. However, the biological function of these terpenoids in plants is poorly understood. Here, we highlight recent work that implicates a signaling function in angiosperms for some of these diterpenoids. In particular, this review will focus on the involvement of an abietane diterpenoid, dehydroabietinal, in the activation of systemic acquired resistance, and draw attention to new evidence that supports a role for dehydroabietinal in the transition from the vegetative phase of growth to reproductive development in the crucifer plant Arabidopsis thaliana.

Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.

Isolation a P450 gene in Pinus armandi and its expression after inoculation of Leptographium qinlingensis and treatment with methyl jasmonate

In conifers, the cytochrome P450 monooxygenase of the CYP720B family plays an important role in the synthesis of diterpene resin acids, which are specialised metabolites of the oleoresin defence. We isolated CYP720B19 from Pinus armandi and found that it belongs to the CYP720B family. The CYP720B19 open reading frame of 1467 bp encodes a protein of 488 amino acid residues with high similarity to abietadienol/abietadienal oxidase. Differential expression of the CYP720B19 gene significantly changed when plants were inoculated with the fungus Leptographium qinlingensis, mechanically wounded, or treated with methyl jasmonate (MeJa) and Tween at 4 days and 8 days. Increased expression of the CYP720B19 gene after these treatments suggested that the gene was involved in pine defence against inoculation by L. qinlingensis, MeJa treatment and mechanical injury.