Synthesis Of Terpenoids Research Articles

Transcriptional regulation modulates terpenoid biosynthesis of Pinus elliottii under drought stress

Global climate change has increased the frequency and severity of droughts, which may affect the biosynthesis of coniferous terpenes, but the regulatory mechanism is still unclear. We explored the mechanisms of terpenoid regulation in response to drought stress by resolving changes in terpenoid composition and gene expression under drought stress to better understand the expression profile of terpenoid synthesis in slash pine. We obtained three main results: (1) A total of 1393 differentially expressed genes (DEGs) were identified, mainly including MYB, AP2/ERF, WRKY and other transcription factors, which were reported to be associated with plant resistance. Real-time quantitative PCR (RT-qPCR) verified the reliability of transcriptomic data, and the analysis of its results shows a close correlation with the data obtained by RNA-seq. (2) A total of 39 DEGs encoding 7 types of the proteins that were identified in the terpene synthesis pathway; what’s more, TPS (e.g., PITA_24906, PITA_00655, PITA_09277) and CYP720B11 (PITA_22711) showed high expressions in diterpene synthesis. (3) A weighted coexpression network analysis (WGCNA) showed high correlations between levopimaric acid content and module genes expression. Network visualization of the 70 hub genes revealed this high degree of connectivity between PITA_24906 and other genes, suggesting that PITA_24906 might play a regulatory role in levopimaric acid under drought stress. Thus, the results of this study will lead to a better understanding of the defense mechanisms of slash pine and provide a basic reference for the management and genetic improvement of slash pine stands.

Understanding the Essential Metabolic Nodes in the Synthesis of 4-Acetylantroquinol B (4-AAQB) by Antrodia cinnamomea Using Transcriptomic Analysis

4-Acetylantroquinol B (4-AAQB) is a ubiquinone that has been shown to have multiple anticancer activities and is only found in the rare medicinal fungus A. cinnamomea in Taiwan. The large-scale production and application of 4-AAQB is thus limited due to the high host specificity, long production cycle, and low 4-AAQB content of A. cinnamomea. Additionally, the lack of molecular genetic studies on A. cinnamomea has hindered the study of the synthetic pathway of 4-AAQB. In this work, transcriptomic analysis was conducted to understand the essential metabolic nodes in the synthesis of 4-AAQB by A. cinnamomea based on the differences using glucose and fructose as carbon sources, respectively. The results showed that the glyoxylate and TCA cycle, terpenoid synthesis pathway, and the quinone ring modification pathway were clarified as the most significant factors associated with 4-AAQB synthesis. The enzymes ACS, ACU7, ACUE, GPS, PPT, P450, GEDA, YAT1, CAT2, and METXA in these pathways were the essential metabolic nodes in the synthesis of 4-AAQB. When fructose was used as the substrate, the expressions of these enzymes were upregulated, and the synthesis of some important intermediate metabolites was enhanced, thus promoting the accumulation of 4-AAQB. Our work understood the mechanism of fructose promoting the synthesis of 4-AAQB and identified the essential metabolic nodes which could provide the theoretical basis for the development of fermentation strategies to produce 4-AAQB by A. cinnamomea.

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803.

Cyanobacteria are a promising platform for the production of the triterpene squalene (C30), a precursor for all plant and animal sterols, and a highly attractive intermediate towards triterpenoids, a large group of secondary plant metabolites. Synechocystis sp. PCC 6803 natively produces squalene from CO2 through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native Synechocystis gene's impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δshc). Our in silico analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δshc mutant compared to the wildtype, including the pentose phosphate pathway, as well as lower glycolysis, while the tricarboxylic acid cycle predicted to be downregulated. Further, all enzymes of the MEP pathway and terpenoid synthesis, as well as enzymes from the central carbon metabolism, Gap2, Tpi and PyrK, were predicted to positively contribute to squalene production upon their overexpression. Each identified target gene was integrated into the genome of Synechocystis Δshc under the control of the rhamnose-inducible promoter Prha. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, ispH, ispE, and idi, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (sqs) in Synechocystis Δshc, which reached the highest production titer of 13.72 mg l-1 reported for squalene in Synechocystis sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Caste-specific expressions and diverse roles of takeout genes in the termite Reticulitermes speratus

Acquisition of novel functions caused by gene duplication may be important for termite social evolution. To clarify this possibility, additional evidence is needed. An important example is takeout, encoding juvenile hormone binding protein. We identified 25 takeouts in the termite Reticulitermes speratus genome. RNA-seq revealed that many genes were highly expressed in specific castes. Two novel paralogs (RsTO1, RsTO2) were tandemly aligned in the same scaffold. Real-time qPCR indicated that RsTO1 and RsTO2 were highly expressed in queens and soldiers, respectively. Moreover, the highest RsTO1 expression was observed in alates during queen formation. These patterns were different from vitellogenins, encoding egg-yolk precursors, which were highly expressed in queens than alates. In situ hybridization showed that RsTO1 mRNA was localized in the alate-frontal gland, indicating that RsTO1 binds with secretions probably used for the defence during swarming flight. In contrast, increased RsTO2 expression was observed approximately 1 week after soldier differentiation. Expression patterns of geranylgeranyl diphosphate synthase, whose product functions in the terpenoid synthesis, were similar to RsTO2 expression. In situ hybridization indicated RsTO2-specific mRNA signals in the soldier-frontal gland. RsTO2 may interact with terpenoids, with a soldier-specific defensive function. It may provide additional evidence for functionalization after gene duplication in termites.

Methylierung nicht‐aktivierter Alkene mit modifizierten Methyltransferasen zur Diversifizierung von Terpenoiden

AbstractTerpenoide sind aus Isopren‐Bausteinen aufgebaut und haben zahlreiche biologische Funktionen. Eine selektive Modifizierung ihres Kohlenstoffgerüsts hat das Potenzial die biologischen Aktivitäten zu verändern oder zu optimieren. Die Synthese von Terpenoiden mit einem nicht‐natürlichen Kohlenstoffgerüst ist jedoch aufgrund der Komplexität dieser Moleküle oft ein schwieriges Unterfangen. In dieser Arbeit berichten wir über die Identifizierung und das Engineering von (S)‐Adenosyl‐l‐Methionin‐abhängigen Sterol‐Methyltransferasen für die selektive C‐Methylierung von linearen Terpenoiden. Das entwickelte Enzym katalysiert die selektive Methylierung von nicht‐aktivierten Alkenen in Mono‐, Sesqui‐ und Diterpenoiden zur Herstellung von C11‐, C16‐ und C21‐Derivaten. Die präparative Umsetzung und Isolation der Produkte zeigt, dass dieser Biokatalysator die Bildung von C−C‐Bindungen mit hoher Chemo‐ und Regioselektivität durchführt. Die Methylierung des Alkens erfolgt höchstwahrscheinlich über ein Carbokation‐Zwischenprodukt und eine regioselektive Deprotonierung. Diese Methode eröffnet neue Wege zur Modifizierung des Kohlenstoffgerüsts von Alkenen im Allgemeinen und Terpenoiden im Besonderen.

Methylation of Unactivated Alkenes with Engineered Methyltransferases To Generate Non-natural Terpenoids.

Terpenoids are built from isoprene building blocks and have numerous biological functions. Selective late-stage modification of their carbon scaffold has the potential to optimize or transform their biological activities. However, the synthesis of terpenoids with a non-natural carbon scaffold is often a challenging endeavor because of the complexity of these molecules. Herein we report the identification and engineering of (S)-adenosyl-l-methionine-dependent sterol methyltransferases for selective C-methylation of linear terpenoids. The engineered enzyme catalyzes selective methylation of unactivated alkenes in mono-, sesqui- and diterpenoids to produce C11 , C16 and C21 derivatives. Preparative conversion and product isolation reveals that this biocatalyst performs C-C bond formation with high chemo- and regioselectivity. The alkene methylation most likely proceeds via a carbocation intermediate and regioselective deprotonation. This method opens new avenues for modifying the carbon scaffold of alkenes in general and terpenoids in particular.

Salicylic acid promotes terpenoid synthesis in the fungi Sanghuangporus baumii

Sanghuangporus baumii is a medicinal fungi with anti-inflammatory, liver protection and antitumour effects. Terpenoids are one of the main medicinal ingredients of S. baumii. However, terpenoid production by wild-type S. baumii cannot meet the market demand, which affects its application in medical care. Therefore, exploring how to increase terpenoid content in S. baumii is a promising path in this research field. Salicylic acid (SA) is a secondary metabolite. In this study, a concentration of 350 μmol/L SA was added into fungal cultivations for 2 and 4 days, and then the transcriptome and metabolome of untreated mycelia and treated with SA were analysed. The expression of some genes in the terpenoids biosynthesis pathway increased in SA-induced cultivations, and the content of isopentenyl pyrophosphate (IPP) and geranylgeranyl-PP (GGPP) increased significantly as well as the contents of triterpenoids, diterpenoids, sesquiterpenoids and carotenoids. The gene FPS was considered to be a key gene regulating terpenoid biosynthesis. Therefore, FPS was overexpressed in S. baumii by Agrobacterium tumefaciens-mediated genetic transformation. The gene FPS and its downstream gene (LS) expression levels were confirmed to be increased in the FPS overexpressing transformant, and terpenoid content was 36.98% higher than that of the wild-type strain in the evaluated cultivation conditions.

Identification and functional characterization of a new terpene synthase gene from Chrysanthemum indicum var. aromaticum

Terpene synthase (TPS) is a key enzyme in the synthesis of terpenoids in plants and plays an important role in the regulation of the synthesis of aromatic substances in plants. In this study, we treated Chrysanthemum indicum var. aromaticum as the experimental material to clone the CiTPS gene. The open reading frame of CiTPS is 1 818 bp, encoding a putative protein of 605 amino acids. There is a monoterpene synthase conserved domain and a highly conserved sequence, DDXXD, (N,D)D(L,I,V)X(S,T)XXXE and RRX8W, respectively, which belong to the monoterpene synthase family. The multiple sequence alignment and phylogenetic analysis showed that the CiTPS gene belongs to the TPS-b subfamily. The pBI121-TPS-GFP recombinant plasmid and the pBI121-GFP empty vector plasmid were introduced into Agrobacterium by electroporation and transferred into wild-type tobacco by the Agrobacterium-mediated method. Three transgenic CiTPS lines were screened, and the secretions of wild-type tobacco and transgenic tobacco T1 leaves in the vigorous growth period were analysed. The volatiles of the transgenic lines were similar to those of wild-type tobacco, but their monoterpenes increased significantly, and the sesquiterpenoids and diterpenoids decreased or remained unchanged, indicating that the CiTPS gene is related to the synthesis of the monoterpenes.

Disentangling the Potential Functions of miRNAs in the Synthesis of Terpenoids during the Development of Cinnamomum burmannii Leaves

The essential oil of Cinnamomum burmannii (Nees and T. Nees) Blume is rich in monoterpenes and sesquiterpenes. The post-transcriptional regulatory mechanisms controlling the expression of terpenoid-related genes have not yet been clarified in C. burmannii. Here, we conducted a metabolomic analysis of the leaves of C. burmannii across four developmental stages using gas chromatography–mass spectrometry. We also identified miRNAs and their target genes involved in terpenoid biosynthesis using small RNA sequencing. A total of 135 differentially expressed metabolites were detected, including 65 terpenoids, 15 aldehydes, and 13 benzenes. A total of 876 miRNAs from 148 families were detected, among which 434 miRNAs were differentially expressed, including three known miRNAs and 431 novel miRNAs. Four miRNAs (gma-miR5368, novel_miR_377, novel_miR_111, and novel_miR_251) were predicted to regulate the expression of four differential expressed genes involved in the monoterpenoid and sesquiterpenoid synthesis. miRNAs families miR396, miR5185, and miR9408 were predicted to play diverse regulatory roles in monoterpenoid and sesquiterpenoid synthesis during the leaf development of C. burmannii. The results of our study shed new light on the roles of regulatory genes in terpenoid biosynthesis. Our findings also have implications for the further promotion of essential oil production using the leaves of C. burmannii.

Identification of key genes involved in flavonoid and terpenoid biosynthesis and the pathway of triterpenoid biosynthesis in Passiflora edulis

Passion fruit (Passiflora edulis Sims) is a vine of the Passiflora genus in the Passifloraceae family. The extracted components include flavonoids and terpenoids, which have good anti-anxiety and anti-inflammatory effects in humans. In this study, we analyzed the transcriptomes of four tissues of the ‘Zixiang’ cultivar using RNA-Seq, which provided a dataset for functional gene mining. The de novo assembly of these reads generated 96 883 unigenes, among which 61 022 unigenes were annotated (62.99% yield). In addition to its edible value, another important application of passion fruit is its medicinal value. The flavonoids and terpenoids are mainly derivatives of luteolin, apigenin, cycloartane triterpenoid saponins and other active substances in leaf extracts. A series of candidate unigenes in the transcriptome data that are potentially involved in the flavonoid and terpenoid synthesis pathways were screened using homology-based BLAST and phylogenetic analysis. The results showed that the biosynthesis of triterpenoids in passion fruit comes from the branches of the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathways, which is different from the MVA pathway that is used in other fruit trees. Most of the candidate genes were found to be highly expressed in the leaves and/or flowers. Quantitative real-time PCR (qRT-PCR) verification was carried out and confirmed the reliability of the RNA-Seq data. Further amplification and functional analysis of these putative unigenes will provide additional insight into the biosynthesis of flavonoids and terpenoids in passion fruit.

Transcription Factors ZjMYB39 and ZjMYB4 Regulate Farnesyl Diphosphate Synthase- and Squalene Synthase-Mediated Triterpenoid Biosynthesis in Jujube

Jujube (Ziziphus jujuba Mill.) is rich in valuable bioactive triterpenoids. However, the regulatory mechanism underlying triterpenoid biosynthesis in jujube remains poorly studied. Here, we characterized the triterpenoid content in wild jujube and cultivated jujube. The triterpenoid content was higher in wild jujube than in cultivated jujube, triterpenoids were most abundant in young leaves, buds, and later stages of development. The transcriptome analysis and correlation analysis showed that differentially expressed genes (DEGs) were enriched in the terpenoid synthesis pathways, and triterpenoids content was strongly correlated with farnesyl diphosphate synthase (ZjFPS), squalene synthase (ZjSQS), and transcription factors ZjMYB39 and ZjMYB4 expression. Gene overexpression and silencing analysis indicated that ZjFPS and ZjSQS were key genes in triterpenoid biosynthesis and transcription factors ZjMYB39 and ZjMYB4 regulated triterpenoid biosynthesis. Subcellular localization experiments showed that ZjFPS and ZjSQS were localized to the nucleus and endoplasmic reticulum and ZjMYB39 and ZjMYB4 were localized to the nucleus. Yeast one-hybrid, glucuronidase activity, and dual-luciferase activity assays suggested that ZjMYB39 and ZjMYB4 regulate triterpenoid biosynthesis by directly binding and activating the promoters of ZjFPS and ZjSQS. These findings provide insights into the underlying regulatory network of triterpenoids metabolism in jujube and lay theoretical and practical foundation for molecular breeding.

Comparative proteome profiling of fusarium chlamydosporum and elucidation of pigment biosynthetic pathway under nitrogen stress

The main aim of this study was to understand the protein expression of F. chlamydosporum in two different medium composition in varying concentrations of nitrogen. The interesting phenomenon of producing diverse pigments by a single strain in different concentrations of Nitrogen made us further to explore the difference in the protein expression of the fungus when grown in these two media. For this, we had adopted non-gel-based method of protein separation by LC-MS/MS analysis followed by label free identification of proteins by SWATH analysis. The molecular and biological functions of each protein and their Gene Ontology annotations were analyzed by UniProt KB and KEGG pathway; the secondary metabolite pathways and the carbohydrate metabolic pathways were analyzed by DAVID bioinformatics tool. The positively regulated proteins biologically functioned for the secondary metabolite production in optimized medium were Diphosphomevalonate decarboxylase (terpenoid backbone biosynthesis), Phytoene synthase (carotenoid biosynthesis), 6,7-dimethyl-8-ribityllumazine synthase (riboflavin biosynthesis). The main characteristic change observed was that proteins related to carotenoid biosynthesis and terpenoid synthesis were not regulated in nitrogen limited medium. Except for the protein 6,7-dimethyl-8-ribityllumazine synthase, all the enzymes related to fatty acid biosynthesis and polyketide chain elongation were up regulated. Apart from the proteins related to secondary metabolite production, two novel proteins were found to be up regulated in Nitrogen Limited Medium; C-fem protein responsible for fungal pathogenesis and DAO domain containing protein which functions as a neuromodulator and catalyzes the synthesis of dopamine. SignificanceThis particular strain of F. chlamydosporum of immense genetic and biochemical diversity represents an interesting example of a microorganism which can produce a variety of bioactive compounds and this can be exploited in various industries. The production of carotenoids and polyketides by this fungus when grown in the same media with different concentrations of Nitrogen has been published by us following which we analyzed the proteome sequence of the fungus in varying Nutrient conditions. Following the proteome analysis and expression, we could derive the pathway leading to the biosynthesis of varying secondary metabolites by the fungus which has not been published or studied so far.

Genome, transcriptome, and metabolome analyses provide new insights into the resource development in an edible fungus Dictyophora indusiata.

Dictyophora indusiata (Vent. Ex Pers.) Fisch. (DI) is an edible and medicinal fungus widely used in East Asian countries. However, during DI cultivation, the formation of fruiting bodies cannot be regulated, which leads to yield and quality losses. The present study performed a combined genome, transcriptome, and metabolome analysis of DI. Using Nanopore and Illumina sequencing approaches, we created the DI reference genome, which was 67.32 Mb long with 323 contigs. We identified 19,909 coding genes on this genome, of which 46 gene clusters were related to terpenoid synthesis. Subsequent transcriptome sequencing using five DI tissues (cap, indusia, mycelia, stipe, and volva) showed high expression levels of genes in the cap, indicating the tissue's importance in regulating the fruiting body formation. Meanwhile, the metabolome analysis identified 728 metabolites from the five tissues. Mycelium was rich in choline, while volva was rich in dendronobilin; stipe had monosaccharides as the primary component, and the cap was the main source of indole acetic acid (IAA) synthesis. We confirmed the importance of tryptophan metabolism for DI fruiting body differentiation based on KEGG pathway analysis. Finally, the combined multiomics analysis identified three new genes related to IAA synthesis of the tryptophan metabolic pathway in the cap, which may regulate DI fruiting body synthesis and improve DI quality. Thus, the study's findings expand our understanding of resource development and the molecular mechanisms underlying DI development and differentiation. However, the current genome is still a rough draft that needs to be strengthened.

Systematic identification of TPS genes in Gossypium and their characteristics in response to flooding stress.

Terpene synthases (TPS) is a key enzyme in the synthesis of plant terpenoids. Studies on TPSs have not been reported in Gossypium barbadense and Gossypium arboreum. 260 TPSs were identified in Gossypium, including 71 in Gossypium hirsutum, 75 in Gossypium. barbadense, 60 in Gossypium. arboreum, and 54 in Gossypium raimondii. We systematically analyzed the TPS gene family of Gossypium from three aspects: gene structure, evolutionary process and gene function. (1) Gene structure: Based on the protein structure of two conserved domains (PF01397 and PF03936), the TPS gene family is divided into five clades: TPS -a, -b, -c, -e/f and -g. (2) Evolution: Whole genome duplication and segmental duplication are the main modes of TPS gene amplification. (3) Function: The abundance of cis-acting elements may reveal the functional diversity of TPSs in cotton. TPS gene has tissue specific expression in cotton. The hypomethylation of the exon of TPSs may help to enhance the adaptability of cotton to flooding stress. In conclusion, this study can broaden the understanding of structure-evolution-function of the TPS gene family, and provide reference for the mining and verification of new genes.

The chromosome-scale assembly of the Salvia rosmarinus genome provides insight into carnosic acid biosynthesis.

Rosemary (Salvia rosmarinus) is considered a sacred plant because of its special fragrance and is commonly used in cooking and traditional medicine. Here, we report a high-quality chromosome-level assembly of the S. rosmarinus genome of 1.11Gb in size; the genome has a scaffold N50 value of 95.5Mb and contains 40 701 protein-coding genes. In contrast to other diploid Labiataceae, an independent whole-genome duplication event occurred in S. rosmarinus at approximately 15 million years ago. Transcriptomic comparison of two S. rosmarinus cultivars with contrasting carnosic acid (CA) content revealed 842 genes significantly positively associated with CA biosynthesis in S. rosmarinus. Many of these genes have been reported to be involved in CA biosynthesis previously, such as genes involved in the mevalonate/methylerythritol phosphate pathways and CYP71-coding genes. Based on the genomes and these genes, we propose a model of CA biosynthesis in S. rosmarinus. Further, comparative genome analysis of the congeneric species revealed the species-specific evolution of CA biosynthesis genes. The genes encoding diterpene synthase and the cytochrome P450 (CYP450) family of CA synthesis-associated genes form a biosynthetic gene cluster (CPSs-KSLs-CYP76AHs) responsible for the synthesis of leaf and root diterpenoids, which are located on S. rosmarinus chromosomes 1 and 2, respectively. Such clustering is also observed in other sage (Salvia) plants, thus suggesting that genes involved in diterpenoid synthesis are conserved in the Labiataceae family. These findings provide new insights into the synthesis of aromatic terpenoids and their regulation.

Transcriptome Analysis Reveals Genes Involved in Responses of Eucalyptus to Gall Wasp Infestation

Leptocybe invasa is a gall wasp causing significant damage to Eucalyptus species. This study used RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) associated with early L. invasa infestation in eucalyptus parents and their F1-progenies. A total of 14,648 significant DEGs were identified from U22-tolerant and C153-susceptible parents, and extremely tolerant and susceptible pools of their F1-progenies. A total of 324/632 and 182/205 DEGs specific for the tolerant group and the susceptible group were up-regulated, respectively. Expression analysis by qRT-PCR of the selected DEGs was comparable with the results of RNA-seq. Expression analysis of the selected genes using the top five progenies from each pool was consistent with that in the parents. Three genes (RCA, SUI1, GCN5) were up-regulated after infestation in all tested tolerant plants, suggesting their important roles in the tolerant phenotype. Using expression and STRING analysis, our results suggest that early response at three days after gall wasp infestation increased protein and terpenoid synthesis, and increased transportation of these molecules. In addition, wounding also increased photosynthesis and glycolysis. These processes involved the interaction of several plant hormones, such as JA, Auxin, and ABA. The information obtained from this study could be useful for future Eucalyptus breeding programs.

Full-length transcriptome sequencing reveals the molecular mechanism of monoterpene and sesquiterpene biosynthesis in Cinnamomum burmannii.

Essential oil of Cinnamomum burmannii is rich in monoterpenes and sesquiterpenes and is widely used in cosmetics and medicines. Knowledge about the enzymes that catalyze the formation of monoterpenes and sesquiterpenes in C. burmannii is insufficient. Therefore, anatomy observation of C. burmannii at the four developmental stages (7days, CBS1; 14days, CBS2; 21days, CBS3, and 28days, CBS4) were conducted to elucidate the origins of essential oil production. Twelve full-length transcriptomes of C. burmannii leaves at the four stages were generated using Oxford Nanopore Technologies. GC-MS analysis revealed 15 monoterpene and sesquiterpenes dramatically increased from CBS1 to CBS4. A weighted correlation network analysis (WGCNA) in association and differentially expressed genes across four developmental stages were performed. A total of 44 differentially expressed genes (DEGs) were involved in terpenoid syntheses during leaf development. Among them, the DEGs of the mevalonate acid (MVA) pathway were predominantly expressed at CBS1, while those of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway showed increased expression from CBS2 to CBS4. Besides, fourteen genes were associated with monoterpene synthesis and nine with sesquiterpene synthesis. Functions of these DEGs were further predicted with regard to gene expression profile and phylogenetic relationship with those characterized in previous studies. In addition, 922 long noncoding RNAs (lncRNAs) were detected, of which twelve were predicted to regulate monoterpene and sesquiterpene biosynthesis. The present study provided new insights the molecular mechanisms of monoterpenoid and sesquiterpenoid syntheses of C. burmannii.

Integrated transcriptomic and metabolomic analyses revealed the molecular mechanism of terpenoid formation for salicylic acid resistance in Pulsatilla chinensis callus.

As a kind of traditional Chinese medicine, Pulsatilla chinensis (Bunge) Regel is well known for its anti-inflammation and anti-cancer activities, which are attributed to its active components including total saponins and monomers. To clarify the synthesis and metabolism mechanisms of class components in callus terpenes of P. chinensis, a certain concentration of salicylic acid (SA) hormone elicitor was added to the callus before being analysed by transcriptomic and metabolomic techniques. Results showed that the content of Pulsatilla saponin B4 in the callus suspension culture was significantly increased up to 1.99% with the addition of SA. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the differentially expressed genes were mainly enriched in 122 metabolic pathways, such as terpenoid metabolism-related pathways: terpenoid skeleton synthesis pathway, monoterpenoid biosynthesis pathways, diterpenoid biosynthesis pathways, and ubiquinone and other terpenoid-quinone biosynthesis pathways. A total of 31 differentially accumulated metabolites were obtained from four differential groups. Amongst 21 kinds of known chemical components in P. chinensis, deoxyloganic acid was the only monoterpenoid; the others are triterpenoids. In summary, this study found that SA elicitors can affect the metabolic changes of terpenoids in P. chinensis callus, which provided a basis for analysing the genetic regulation of terpenoid components of leucons.

Optimization of mevalonate production using acetate by precursor balancing and flux redistribution in Escherichia coli

Mevalonate is an important organic acid that can be used to synthesize bioplastics and is utilized as a key precursor for the synthesis of terpenoids and biofuels. To develop economically feasible microbial fermentation, the use of acetate, which is an inexpensive and non-edible feedstock, as the substrate is advantageous. However, a previous study that used acetate as a carbon source reported low levels of mevalonate production, which was caused by a precursor imbalance and insufficient acetyl-CoA. Mevalonate production was substantially improved by simultaneously regulating the expression levels of mvaE and mvaS to balance the precursors. Then, the excess carbon flux for cell growth was redirected to mevalonate by repressing gltA, which encodes citrate synthase, resulting in efficient production. The optimized strain produced a yield of 0.41 g mevalonate/g acetate (72% of the theoretical maximum yield). Finally, repeated feeding resulted in the highest reported mevalonate titer (7.9 g/L) among all reported studies using acetate. These results show that precise regulation of the expression levels of mvaE, mvaS, and gltA is a powerful strategy for mevalonate production. Moreover, this strategy can be widely applied to the production of mevalonate using other substrates.