Diterpene Synthase Research Articles

Functional kaurene-synthase-like diterpene synthases lacking a gamma domain are widely present in Oryza and related species.

Various diterpene synthases have been functionally identified in cultivated rice (Oryza sativa). These are the homologs of ent-copalyl diphosphate (ent-CDP) synthase and ent-kaurene synthase (KS) that are responsible for the biosynthesis of gibberellins, diterpenoid phytohormones. We isolated a cDNA encoding full-length OsKSL12, a previously uncharacterized KS like (KSL) enzyme that consists of a β-domain and an α-domain with an active center, but lacks an N-terminal γ-domain. Functional analysis using a bacterial expression system showed that recombinant OsKSL12 converted ent-CDP into ent-manool or ent-13-epi-manool. Comparative genomics revealed that functional OsKSL12 homologs exist in diverse wild species in the Oryzeae-Oryza nivara (Oryza rufipogon), Oryza coarctata, Oryza granulata, Leersia perrieri, and Leersia tisseranti. KSL12 homologs in O. granulata, L. perrieri, and L. tisseranti preferentially reacted with geranylgeranyl diphosphate rather than ent-CDP, resulting in geranyllinalool rather than ent-manool or ent-13-epi-manool as the main product, meaning that KSL12 functionally diversified during evolution in the Oryzeae.

Discovery and Functional Characterization of a Diverse Diterpene Synthase Family in the Medicinal Herb Isodon lophanthoides Var. gerardiana.

Isodon lophanthoides var. gerardiana (Lamiaceae), also named xihuangcao, is a traditional Chinese medicinal herb that exhibits a broad range of pharmacological activities. Abietane-type diterpenoids are the characteristic constituents of I. lophanthoides, yet their biosynthesis has not been elucidated. Although the aerial parts are the most commonly used organs of I. lophanthoides, metabolite profiling by gas chromatography-mass spectrometry showed the underground parts also contain large amounts of labdane diterpenoids including abietatriene, miltiradiene and ferruginol, which are distinct from the 13-hydroxy-8(14)-abietene detected in the aerial parts. Comparative transcriptome analysis of root and leaf samples identified a diverse diterpene synthase family including 6 copalyl diphosphate synthase (IlCPS1-6) and 5 kaurene synthase-like (IlKSL1-5). Here we report the functional characterization of six of these enzymes using yeast heterologous expression system. Both IlCPS1 and IlCPS3 synthesized (+)-copalyl diphosphate (CPP), in combination with IlKSL1 resulted in miltiradiene, precursor of abietane-type diterpenoids, while coupling with IlKSL5 led to the formation of hydroxylated diterpene scaffold nezukol. Expression profiling and phylogenetic analysis further support the distinct evolutionary relationship and spatial distribution of IlCPS1 and IlCPS3. IlCPS2 converted GGPP into labda-7,13E-dien-15-ol diphosphate. IlCPS6 was identified as ent-CPS, indicating a role in gibberellin metabolism. We further identified a single residue that determined the water addition of nezukol synthase IlKSL5. Substitution of alanine 513 with isoleucine completely altered the product outcome from hydroxylated nezukol to isopimara-7,15-diene. Together, these findings elucidated the early steps of bioactive abietane-type diterpenoid biosynthesis in I. lophanthoides and the catalytic mechanism of nezukol synthase.

Discovery and Biosynthesis of a Structurally Dynamic Antibacterial Diterpenoid.

A new bicyclic diterpenoid, benditerpenoic acid, was isolated from soil-dwelling Streptomyces sp. (CL12-4). We sequenced the bacterial genome, identified the responsible biosynthetic gene cluster, verified the function of the terpene synthase, and heterologously produced the core diterpene. Comparative bioinformatics indicated this Streptomyces strain is phylogenetically unique and possesses nine terpene synthases. The absolute configurations of the new trans-fused bicyclo[8.4.0]tetradecanes were achieved by extensive spectroscopic analyses, including Mosher's analysis, J-based coupling analysis, and computations based on sparse NMR-derived experimental restraints. Interestingly, benditerpenoic acid exists in two distinct ring-flipped bicyclic conformations with a rotational barrier of ≈16 kcal mol-1 in solution. The diterpenes exhibit moderate antibacterial activity against Gram-positive bacteria including methicillin and multi-drug resistant Staphylococcus aureus. This is a rare example of an eunicellane-type diterpenoid from bacteria and the first identification of a diterpene synthase and biosynthetic gene cluster responsible for the construction of the eunicellane scaffold.

Bacterial diterpene synthases prenylate small molecules.

The biosynthesis of terpenoid natural products begins with a carbocation-based cyclization or prenylation reaction. While these reactions are mechanistically similar, there are several families of enzymes, namely terpene synthases and prenyltransferases, that have evolved to specifically catalyze terpene cyclization or prenylation reactions. Here, we report that bacterial diterpene synthases, enzymes that are traditionally considered to be specific for cyclization, are capable of efficiently catalyzing both diterpene cyclization and the prenylation of small molecules. We investigated this unique dual reactivity of terpene synthases through a series of kinetic, biocatalytic, structural, and bioinformatics studies. Overall, this study unveils the ability of terpene synthases to catalyze C-, N-, O-, and S-prenylation on small molecules, proposes a substrate decoy mechanism for prenylation by terpene synthases, supports the physiological relevance of terpene synthase-catalyzed prenylation in vivo, and addresses questions regarding the evolution of prenylation function and its potential role in natural products biosynthesis.

Discovery and modulation of diterpenoid metabolism improves glandular trichome formation, artemisinin production and stress resilience in Artemisia annua.

Plants synthesize diverse diterpenoids with numerous functions in organ development and stress resistance. However, the role of diterpenoids in glandular trichome (GT) development and GT-localized biosynthesis in plants remains unknown. Here, the identification of 10 diterpene synthases (diTPSs) revealed the diversity of diterpenoid biosynthesis in Artemisia annua. Protein-protein interactions (PPIs) between AaKSL1 and AaCPS2 in the plastids highlighted their potential functions in modulating metabolic flux to gibberellins (GAs) or ent-isopimara-7,15-diene-derived metabolites (IDMs) through metabolic engineering. A phenotypic analysis of transgenic plants suggested a complex repertoire of diterpenoids in Artemisia annua with important roles in GT formation, artemisinin accumulation and stress resilience. Metabolic engineering of diterpenoids simultaneously increased the artemisinin yield and stress resistance. Transcriptome and metabolic profiling suggested that bioactive GA4 /GA1 promote GT formation. Collectively, these results expand our knowledge of diterpenoids and show the potential of diterpenoids to simultaneously improve both the GT-localized metabolite yield and stress resistance, in planta.

Isolation and Functional Characterization of the Promoters of Miltiradiene Synthase Genes, TwTPS27a and TwTPS27b, and Interaction Analysis with the Transcription Factor TwTGA1 from Tripterygium wilfordii.

Miltiradiene synthase (MS) genes, TwTPS27a and TwTPS27b, are the key diterpene synthase genes in the biosynthesis of triptolide, which is an important medicinally active diterpenoid in Tripterygium wilfordii. However, the mechanism underlying the regulation of key genes TwTPS27a/b in triptolide biosynthesis remains unclear. In this study, the promoters of TwTPS27a (1496 bp) and TwTPS27b (1862 bp) were isolated and analyzed. Some hormone-/stress-responsive elements and transcription factor (TF) binding sites were predicted in both promoters, which might be responsible for the regulation mechanism of TwTPS27a/b. The β-glucuronidase (GUS) activity analysis in promoter deletion assays under normal and methyl jasmonate (MeJA) conditions showed that the sequence of −921 to −391 bp is the potential core region of the TwTPS27b promoter. And the TGACG-motif, a MeJA-responsive element found in this core region, might be responsible for MeJA-mediated stress induction of GUS activity. Moreover, the TGACG-motif is also known as the TGA TF-binding site. Yeast one-hybrid and GUS transactivation assays confirmed the interaction between the TwTPS27a/b promoters and the TwTGA1 TF (a MeJA-inducible TGA TF upregulating triptolide biosynthesis in T. wilfordii), indicating that TwTPS27a/b are two target genes regulated by TwTGA1. In conclusion, our results provide important information for elucidating the regulatory mechanism of MS genes, TwTPS27a and TwTPS27b, as two target genes of TwTGA1, in jasmonic acid (JA)-inducible triptolide biosynthesis.

An Enzyme-Mediated Aza-Michael Addition Is Involved in the Biosynthesis of an Imidazoyl Hybrid Product of Conidiogenone B.

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,β-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.

A pair of threonines mark ent-kaurene synthases for phytohormone biosynthesis

All land plants (embryophytes) must contain an ent-kaurene synthase (KS), as the ability to produce this olefin from ent-copalyl diphosphate (ent-CPP) is required for phytohormone biosynthesis. These KSs have frequently given rise to other class I diterpene synthases that catalyze distinct reactions for more specialized plant metabolism. Indeed, the prevalence of such gene duplication and neofunctionalization has obscured phylogenetic assignment of function. Here a pair of threonines is found to be conserved in all land plant KS involved in phytohormone biosynthesis, and their role in enzyme function investigated. Surprisingly, these threonines are not required, nor even particularly important for efficient production of ent-kaurene from ent-CPP. In addition, these threonines do not seem to affect protein structure or stability. Moreover, the absence of codon bias and positioning within an intron do not support a role in transcription or translation either. Despite their lack of apparent function, this pair of threonines are nevertheless completely conserved in all embryophyte KS from phytohormone biosynthesis. Thus, regardless of exact role, this serves as a diagnostic mark for such KS, enabling more confident distinction of these critical enzymes.

Identification and functional characterization of <i>ent</i>-kaurene synthase gene in <i>Ilex latifolia</i>

Diterpenes are the most complex and abundant plant metabolites, some of which play significant roles in both primary and secondary metabolism. Ent-kaurene synthase is the key enzyme for gibberellins (GAs) biosynthesis, which may act as a catalyst in the formation of ent-kaurene, the precursor for GAs, by cyclization or rearrangement of the substrate ent-copalyl diphosphate (ent-CPP). Ilex latifolia Thunb (Aquifoliaceae) is a widely distributed Chinese plant whose leaves are used to process a popular Chinese bitter tea named 'Kudingcha', which has anti-microbial, anti-oxidant and anti-inflammatory properties. Here, we isolated a diterpene synthase gene from the leaf transcriptome of I. latifolia, and further identified its biosynthesis activity by in vitro enzymatic testing. The heterologous expressed ent-kaurene synthase of I. latifolia (IlKS) in E. coli could catalyze ent-copalyl diphosphate (ent-CPP) to form ent-kaurene. Tissue-specific expression indicated that IlKS had the highest transcript level in roots which is maybe the major location of ent-kaurene biosynthesis. This study would help us to determine diterpenoid metabolism and GAs biosynthesis in I. latifolia, to better understand the regulation function of GAs in growth and development.

Diterpene Biosynthesis in Catenulispora acidiphila: On the Mechanism of Catenul-14-en-6-ol Synthase.

A new diterpene synthase from the actinomycete Catenulispora acidiphila was identified and the structures of its products were elucidated, including the absolute configurations by an enantioselective deuteration approach. The mechanism of the cationic terpene cyclisation cascade was deeply studied through the use of isotopically labelled substrates and of substrate analogues with partially blocked reactivity, resulting in derailment products that gave further insights into the intermediates along the cascade. Their chemistry was studied, leading to the biomimetic synthesis of a diterpenoid analogue of a brominated sesquiterpene known from the red seaweed Laurencia microcladia.

Discovery and characterization of diterpene synthases in Chamaecyparis formosensis Matsum. which participated in an unprecedented diterpenoid biosynthesis route in conifer

Chamaecyparis formosensis Matsum. is an endemic and precious coniferous species of Taiwan, and is known for a high abundance of specialized metabolites, which contributes to the excellent timber durability. Several terpenoids were identified and isolated from C. formosensis wood and needles, and exhibit anti-fungal and anti-bacterial bioactivities, which may participate in plant defense against pathogens. In various identified compounds, not only cadinene and ferruginol, were identified in C. formosensis extracts but also unique diterpenoids, which include pisferal, totarol, and derivates of isoabienol. To understand the biosynthesis of these specific diterpenoids, we conducted a series of functional characterization of the C. formosensis diterpene synthases (CfdiTPSs), which participate in skeleton formation and differentiation of diterpenes. In this study, we identified eight diTPSs from C. formosensis transcriptome, and they all contain either class I or class II motif, which indicates they are all monofunctional enzymes. These candidates consist of three class II diTPSs and five class I diTPSs, and after conducting in vivo and in vitro assays, class II diTPS CfCPS1 was characterized as a (+)-copalyl diphosphate synthase ((+)-CPS), and class I diTPSs CfKSL1 could further convert (+)-copalyl diphosphate ((+)-CPP) to levopimaradiene. Meanwhile, CfKSL1 also accepted labda-13-en-8-ol diphosphate (LPP) as substrate and formed monoyl oxide. Another class I diTPS, CfKSL4, exhibits a strong enzymatic ability of isoabienol synthase, which is firstly reported in conifer. This finding provides potential participants in the biosynthesis of unique diterpenoids, and with this knowledge, we can further expand our understanding of diterpenoid metabolism in Cupressaceae and their potential role in plant defense.

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol.

Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution. Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9-en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π-cation, dipole-cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.

Crystal Structure and Mechanistic Molecular Modeling Studies of Mycobacterium tuberculosis Diterpene Cyclase Rv3377c.

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.

Revision of the Cyclisation Mechanism for the Diterpene Spiroviolene and Investigations of Its Mass Spectrometric Fragmentation.

The diterpene spiroviolene, its diterpene synthase from Streptomyces violens and the experimentally determined terpene cyclisation mechanism were reported in 2017. Recently, the structure of spiroviolene was revised based on a total synthesis, with consequences for the cyclisation mechanism. Herein, a reinvestigation of the terpene cyclisation to spiroviolene and the mass spectrometric fragmentation mechanism investigated by 13C‐labelling experiments are presented.

Improved cis-Abienol production through increasing precursor supply in Escherichia coli

cis-Abienol, a natural diterpene-diol isolated from balsam fir (Abies balsamea), can be employed as precursors for the semi-synthesis of amber compounds, which are sustainable replacement for ambergris and widely used in the fragmented industry. This study combinatorially co-expressed geranyl diphosphate synthase, geranylgeranyl diphosphate synthase, Labda-13-en-8-ol diphosphate synthase and diterpene synthase, with the best combination achieving ~ 0.3 mg/L of cis-abienol. An additional enhancement of cis-abienol production (up to 8.6 mg/L) was achieved by introducing an exogenous mevalonate pathway which was divided into the upper pathway containing acetyl-CoA acetyltransferase/HMG-CoA reductase and HMG-CoA synthase and the lower pathway containing mevalonate kinase, phosphomevalonate kinase, pyrophosphate mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. The genetically modified strain carrying chromosomal copy of low genes of the mevalonate with the trc promoter accumulated cis-abienol up to 9.2 mg/L in shake flask. Finally, cis-abienol titers of ~ 220 mg/L could be achieved directly from glucose using this de novo cis-abienol-producing E. coli in high-cell-density fermentation. This study demonstrates a microbial process to apply the E. coli cell factory in the biosynthesis of cis-abienol.

Divergent Evolution of the Diterpene Biosynthesis Pathway in Tea Plants (Camellia sinensis) Caused by Single Amino Acid Variation of ent-Kaurene Synthase.

Most plant terpenoids are classified as secondary metabolites. A small portion of them are products of primary metabolism biosynthesized by relatively conserved pathways. Gibberellins (GAs), which are essential for plant growth and development, are diterpenoid phytohormones. (E,E,E)-Geranylgeranyl diphosphate (GGPP) is the precursor for both GAs and other diterpenoids of secondary metabolism. ent-Kaurene biosynthesis from GGPP is a key step of GA formation, which is catalyzed by two sequential and dedicated diterpene synthases (diTPSs): ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) of the terpene synthase gene family. Sharing a common evolutionary origin, CPS and KS belong to different TPS subfamilies. Tea plant (Camellia sinensis), the subject of this study, is a leaf-based economic crop. Budbreak mainly manipulated by GAs is a primary factor for targeted tea breeding. The key genes for gibberellin biosynthesis are known; however, they have not yet been characterized in tea plants. Here, we identified and functionally characterized three diterpene biosynthesis-related genes, including one CPS and two highly similar KSs in tea plants. These genes were initially identified through transcriptome sequencing. The functional characterization determined by enzymatic activity assay indicated that CsCPS could catalyze GGPP to form ent-copalyl diphosphate (ent-CPP), which was further used as the substrate by CsKS1 to produce ent-kaurene or by CsKS2 to produce 16α-hydroxy-ent-kaurane with ent-kaurene as a minor product, respectively. We demonstrated that the divergent evolution of diterpene biosynthesis in tea plants resulted from gene duplication of KSs, followed by functional divergence caused by single amino acid variation. This study would provide an insight into the diterpenoid metabolism and GA biosynthesis in tea plants to further understand leaf bud development or insect resistance and to provide a genetic basis for tea plant breeding.

Evolution of Labdane-Related Diterpene Synthases in Cereals.

Gibberellins (GAs) are labdane-related diterpenoid phytohormones that regulate various aspects of higher plant growth. A biosynthetic intermediate of GAs is ent-kaurene, a tetra-cyclic diterpene that is produced through successive cyclization of geranylgeranyl diphosphate catalyzed by the two distinct monofunctional diterpene synthases-ent-copalyl diphosphate synthase (ent-CPS) and ent-kaurene synthase (KS). Various homologous genes of the two diterpene synthases have been identified in cereals, including rice (Oryza sativa), wheat (Triticum aestivum) and maize (Zea mays), and are believed to have been derived from GA biosynthetic ent-CPS and KS genes through duplication and neofunctionalization. They play roles in specialized metabolism, giving rise to diverse labdane-related diterpenoids for defense because a variety of diterpene synthases generate diverse carbon-skeleton structures. This review mainly describes the diterpene synthase homologs that have been identified and characterized in rice, wheat and maize and shows the evolutionary history of various homologs in rice inferred by comparative genomics studies using wild rice species, such as Oryza rufipogon and Oryza brachyantha. In addition, we introduce labdane-related diterpene synthases in bryophytes and gymnosperms to illuminate the macroscopic evolutionary history of diterpene synthases in the plant kingdom-bifunctional enzymes possessing both CPS and KS activities are present in bryophytes; gymnosperms possess monofunctional CPS and KS responsible for GA biosynthesis and also possess bifunctional diterpene synthases facilitating specialized metabolism for defense.

Identification of Three Monofunctional Diterpene Synthases with Specific Enzyme Activities Expressed during Heartwood Formation in Western Redcedar (Thuja plicata) Trees.

Upon harvest, Western redcedar (WRC; Thuja plicata) trees have a high incidence and extent of heartwood rot. While monoterpenoids and lignans have been linked to rot resistance in this species, other specialized metabolites, such as diterpenes, are likely to contribute to rot resistance. Here we report the cloning and functional assessment of three putative diterpene synthase (TpdiTPS) genes expressed during heartwood formation in WRC. The predicted proteins of the three genes lack either of the two catalytically independent active sites typical of most diTPS, indicating monofunctional rather than bifunctional activity. To identify potential catalytic activities of these proteins, we expressed them in genetically engineered Escherichia coli strains that produce four potential substrates, geranylgeranyl diphosphate (GGDP), ent, syn, and normal stereoisomers of copalyl diphosphate (CDP). We found that TpdiTPS3 used GGDP to produce CDP. TpdiTPS2 used normal CDP to produce levopimaradiene. TpdiTPS1 showed stereoselectivity as it used normal CDP to produce sandaracopimaradiene and syn-CDP to produce syn-stemod-13(17)-ene. These genes and protein enzymatic activities have not been previously reported in WRC and provide an opportunity to assess their potential roles in heartwood rot resistance in this economically important species.

A Fungal Diterpene Synthase Is Responsible for Sterol Biosynthesis for Growth.

A conserved open reading frame, dps, is described in Pestalotiopsis microspora, sharing a remarkable similarity with fungal diterpene synthases whose function is less studied. Loss-of-function approach manifested that dps was necessary for the growth and the development of the fungus. A deletion strain, dpsΔ, showed a fundamental retardation in growth, which could deliberately be restored by the addition of exogenous sterols to the media. Gas chromatography–mass spectrometry analysis confirmed the loss of the ability to produce certain sterols. Thus, the tolerance and the resistance of dpsΔ to several stress conditions were impaired. Secondary metabolites, such as the polyketide derivative dibenzodioxocinones, were significantly diminished. At the molecular level, the deletion of dps even affected the expression of genes in the mevalonate pathway. This report adds knowledge about fungal diterpene synthases in Pestalitiopsis microspora.

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.