CrJAT1 Regulates Endogenous JA Signaling for Modulating Monoterpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Many monoterpenoid indole alkaloids (MIAs) produced in Catharanthus roseus have demonstrated biological activities and clinical potential. However, their complex biosynthesis pathway in plants leads to low accumulation, limiting therapeutic applications. Efforts to elucidate the MIA biosynthetic regulatory mechanism have focused on improving accumulation levels. Previous studies revealed that jasmonic acid (JA), an important plant hormone, effectively promotes MIA accumulation by inducing the expression of MIA biosynthesis and transport genes. Nevertheless, excessive JA signaling can strongly inhibit plant growth, decreasing MIA productivity in C. roseus. Therefore, identifying key components balancing growth and MIA production in the JA signaling pathway is imperative for effective pharmaceutical production. Here, we identify a homolog of the jasmonate transporter 1, CrJAT1, through co-expression and phylogenetic analyses. Further investigation demonstrated that CrJAT1 can activate JA signaling to promote MIA accumulation without compromising growth. The potential role of CrJAT1 in redistributing intra/inter-cellular JA and JA-Ile may calibrate signaling to avoid inhibition, representing a promising molecular breeding target in C. roseus to optimize the balance between growth and specialized metabolism for improved MIA production.

Revelation of enzyme/transporter-mediated metabolic regulatory model for high-quality terpene accumulation in developing fruits of Lindera glauca.

Lindera glauca with rich resource and fruit terpene has emerged as potential material for utilization in China, but different germplasms show a variation for essential oil content and volatile profiling. This work aimed to determine key regulators (enzymes or transporters) and unravel mechanism of governing high production of essential oil of L. glauca fruit (EO-LGF). Temporal analysis of fruit growth and EO-LGF accumulation (yield, volatile compounds and contents) during development revealed a notable change in the contents of EO-LGF and its 45 compounds in developing fruits, and the major groups were monoterpene and sesquiterpene, showing good antioxidant and antimicrobial activities. To highlight molecular mechanism that govern such difference in terpene content and compound in developing fruits, Genome-wide assay was used to annotate 104 genes for terpene-synthesis pathway based on recent transcriptome data, and the comparative associations of terpene accumulative amount with gene transcriptional level were conducted on developing fruits to identify some crucial determinants (enzymes and transporters) with metabolic regulation model for high-quality terpene accumulation, involving in carbon allocation (sucrose cleavage, glycolysis and OPP pathway), metabolite transport, isoprene precursor production, C5-unit formation (MEP and MVA pathways), and mono-/sesqui-terpene synthesis. Our findings may present strategy for engineering terpene accumulation for utilization.

Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

The decline in cellular iron is crucial for differentiation in keratinocytes.

Iron is a vital metal for most biological functions in tissues, and its concentration is exquisitely regulated at the cellular level. During the process of differentiation, keratinocytes in the epidermis undergo a noticeable reduction in iron content. Conversely, psoriatic lesions, characterized by disruptions in epidermal differentiation, frequently reveal an excessive accumulation of iron within keratinocytes that have undergone differentiation. In this study, we clarified the significance of attenuated cellular iron content in the intricate course of epidermal differentiation. We illustrated this phenomenon through the utilization of hinokitiol, an iron chelator derived from the heartwood of Taiwanese hinoki, which forcibly delivers iron into cells independent of the intrinsic iron-regulation systems. While primary cultured keratinocytes readily succumbed to necrotic cell death by this iron chelator, mild administration of the hinokitiol-iron complex modestly disrupts the process of differentiation in these cells. Notably, keratinocyte model cells HaCaT and anaplastic skin rudiments exhibit remarkable resilience against the cytotoxic impact of hinokitiol, and the potent artificial influx of iron explains a suppressive effect selectively on epidermal differentiation. Moreover, the augmentation of iron content induced by the overexpression of divalent metal transporter 1 culminates in the inhibition of differentiation in HaCaT cells. Consequently, the diminution in cellular iron content emerges as an important determinant influencing the trajectory of keratinocyte differentiation.

Defensive glands in Stylotermitidae (Blattodea, Isoptera).

The large abundance of termites is partially achieved by their defensive abilities. Stylotermitidae represented by a single extant genus, Stylotermes, is a member of a termite group Neoisoptera that encompasses 83% of termite species and 94% of termite genera and is characterized by the presence of the frontal gland. Within Neoisoptera, Stylotermitidae represents a species-poor sister lineage of all other groups. We studied the structure of the frontal, labral and labial glands in soldiers and workers of Stylotermes faveolus, and the composition of the frontal gland secretion in S. faveolus and Stylotermes halumicus. We show that the frontal gland is a small active secretory organ in soldiers and workers. It produces a cocktail of monoterpenes in soldiers, and some of these monoterpenes and unidentified proteins in workers. The labral and labial glands are developed similarly to other termite species and contribute to defensive activities (labral in both castes, labial in soldiers) or to the production of digestive enzymes (labial in workers). Our results support the importance of the frontal gland in the evolution of Neoisoptera. Toxic, irritating and detectable monoterpenes play defensive and pheromonal functions and are likely critical novelties contributing to the ecological success of these termites.

LiNAC100 contributes to linalool biosynthesis by directly regulating LiLiS in Lilium 'Siberia'.

MAIN CONCLUSION: The transcription factor LiNAC100 has a novel function of regulating floral fragrance by directly regulating linalool synthase gene LiLiS. Lilium 'Siberia', an Oriental hybrid, is renowned as both a cut flower and garden plant, prized for its color and fragrance. The fragrance comprises volatile organic compounds (VOCs), primarily monoterpenes found in the plant. While the primary terpene synthases in Lilium 'Siberia' were identified, the transcriptional regulation of these terpene synthase (TPS) genes remains unclear. Thus, understanding the regulatory mechanisms of monoterpene biosynthesis is crucial for breeding flower fragrance, thereby improving ornamental and commercial values. In this study, we isolated a nuclear-localized LiNAC100 transcription factor from Lilium 'Siberia'. The virus-induced gene silencing (VIGS) of LiNAC100 was found to down-regulate the expression of linalool synthase gene (LiLiS) and significantly inhibit linalool synthesis. Conversely, transient overexpression of LiNAC100 produced opposite effects. Additionally, yeast one-hybrid and dual-luciferase assays confirmed that LiNAC100 directly activates LiLiS expression. Our findings reveal that LiNAC100 plays a key role in monoterpene biosynthesis in Lilium 'Siberia', promoting linalool synthesis through the activation of LiLiS expression. These results offer insights into the molecular mechanisms of terpene biosynthesis in Lilium 'Siberia' and open avenues for biotechnological enhancement of floral scent.

[Accumulation and biosynthesis mechanism of volatile oils in glandular scale of Agastache rugosa].

The volatile oils are the effective components of Agastache rugosa, which are stored in the glandular scale. The leaves of pulegone-type A. rugosa were used as materials to observe the leaf morphology of A. rugosa at different growth stages, and the components of volatile oils in gland scales were detected by GC-MS. At the same time, qRT-PCR was used to determine the relative expression of key enzyme genes in the biosynthesis pathway of monoterpenes in volatile oils. The results showed that the density of A. rugosa glandular scale decreased first and then tended to be stable. With the growth of leaves, the relative content of pulegone decreased from 79.26% to 3.94%(89.97%-41.44%), while that of isomenthone increased from 2.43% to 77.87%(0.74%-51.01%), and the changes of other components were relatively insignificant. The correlation analysis between the relative content of monoterpenes and the relative expression levels of their key enzyme genes showed that there was a significant correlation between the relative content of menthone and isomenthone and the relative expression levels of pulegone reductase(PR)(r>0.6, P<0.01). To sum up, this study revealed the accumulation rules of the main components of the contents of the glandular scale of A. rugosa and the expression rules of the key enzyme genes for biosynthesis, which provided a scientific basis and data support for determining the appropriate harvesting period and quality control of the medicinal herbs. This study also initially revealed the biosynthesis mechanism of the monoterpenes mainly composed of pulegone and isomenthone in A. rugosa, laying a foundation for further research on the molecular mechanism of synthesis and accumulation of monoterpenes in A. rugosa.

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development.

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2-regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

Spatiotemporal Regulation and Transport Engineering for Sustainable Production of Geraniol in Candida glycerinogenes.

Geraniol is an attractive natural monoterpene with significant industrial and commercial value in the fields of pharmaceuticals, condiments, cosmetics, and bioenergy. The biosynthesis of monoterpenes suffers from the availability of key intermediates and enzyme-to-substrate accessibility. Here, we addressed these challenges in Candida glycerinogenes by a plasma membrane-anchoring strategy and achieved sustainable biosynthesis of geraniol using bagasse hydrolysate as substrate. On this basis, a remarkable 2.4-fold improvement in geraniol titer was achieved by combining spatial and temporal modulation strategies. In addition, enhanced geraniol transport and modulation of membrane lipid-associated metabolism effectively promoted the exocytosis of toxic monoterpenes, significantly improved the resistance of the engineered strain to monoterpenes and improved the growth of the strains, resulting in geraniol yield up to 1207.4 mg L-1 at shake flask level. Finally, 1835.2 mg L-1 geraniol was obtained in a 5 L bioreactor using undetoxified bagasse hydrolysate. Overall, our study has provided valuable insights into the plasma membrane engineering of C. glycerinogenes for the sustainable and green production of valuable compounds.

Atmospheric Degradation of Ecologically Important Biogenic Volatiles: Investigating the Ozonolysis of (E)-ß-Ocimene, Isomers of α and ß-Farnesene, α-Terpinene and 6-Methyl-5-Hepten-2-One, and Their Gas-Phase Products.

Biogenic volatile organic compounds (bVOCs), synthesised by plants, are important mediators of ecological interactions that can also undergo a series of reactions in the atmosphere. Ground-level ozone is a secondary pollutant generated through sunlight-driven reactions between nitrogen oxides (NOx) and VOCs. Its levels have increased since the industrial revolution and reactions involving ozone drive many chemical processes in the troposphere. While ozone precursors often originate in urban areas, winds may carry these hundreds of kilometres, causing ozone formation to also occur in less populated rural regions. Under elevated ozone conditions, ozonolysis of bVOCs can result in quantitative and qualitative changes in the gas phase, reducing the concentrations of certain bVOCs and resulting in the formation of other compounds. Such changes can result in disruption of bVOC-mediated behavioural or ecological interactions. Through a series of gas-phase experiments using Gas Chromatography Mass Spectrometry (GC-MS) and Proton Transfer Reaction Mass Spectrometry (PTR-MS), we investigated the products and their yields from the ozonolysis of a range of ubiquitous bVOCs, which were selected because of their importance in mediating ecological interactions such as pollinator and natural enemy attraction and plant-to-plant communication, namely: (E)-ß-ocimene, isomers of α and ß-farnesene, α-terpinene and 6-methyl-5-hepten-2-one. New products from the ozonolysis of these compounds were identified, and the formation of these compounds is consistent with terpene-ozone oxidation mechanisms. We present the degradation mechanism of our model bVOCs and identify their reaction products. We discuss the potential ecological implications of the degradation of each bVOC and of the formation of reaction products.

Cloning and Functional Characterization of 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase (LiMCT) Gene in Oriental Lily (Lilium 'Sorbonne').

2-C-methyl-D-erythritol-phosphate cytidylyltransferase (MCT) is a key enzyme in the MEP pathway of monoterpene synthesis, catalyzing the generation of 4- (5'-pyrophosphate cytidine)-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol-4-phosphate. We used homologous cloning strategy to clone gene, LiMCT, in the MEP pathway that may be involved in the regulation of floral fragrance synthesis in the Lilium oriental hybrid 'Sorbonne.' The full-length ORF sequence was 837 bp, encoding 278 amino acids. Bioinformatics analysis showed that the relative molecular weight of LiMCT protein is 68.56 kD and the isoelectric point (pI) is 5.12. The expression pattern of LiMCT gene was found to be consistent with the accumulation sites and emission patterns of floral fragrance monoterpenes in transcriptome data (unpublished). Subcellular localization indicated that the LiMCT protein is located in chloroplasts, which is consistent with the location of MEP pathway genes functioning in plastids to produce isoprene precursors. Overexpression of LiMCT in Arabidopsis thaliana affected the expression levels of MEP and MVA pathway genes, suggesting that overexpression of the LiMCT in A. thaliana affected the metabolic flow of C5 precursors of two different terpene synthesis pathways. The expression of the monoterpene synthase AtTPS14 was elevated nearly fourfold in transgenic A. thaliana compared with the control, and the levels of carotenoids and chlorophylls, the end products of the MEP pathway, were significantly increased in the leaves at full bloom, indicating that LiMCT plays an important role in regulating monoterpene synthesis and in the synthesis of other isoprene-like precursors in transgenic A. thaliana flowers. However, the specific mechanism of LiMCT in promoting the accumulation of isoprene products of the MEP pathway and the biosynthesis of floral monoterpene volatile components needs further investigation.

Development of Corynebacterium glutamicum as a monoterpene production platform.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Geranyl diphosphate synthase large subunits OfLSU1/2 interact with small subunit OfSSUII and are involved in aromatic monoterpenes production in Osmanthus fragrans.

Osmanthus fragrans is a famous ornamental tree species for its pleasing floral fragrance. Monoterpenoids are the core floral volatiles of O. fragrans flowers, which have tremendous commercial value. Geranyl diphosphate synthase (GPPS) is a key enzyme that catalyzes the formation of GPP, the precursor of monoterpenoids. However, there are no reports of GPPSs in O. fragrans. Here, we performed RNA sequencing on the O. fragrans flowers and identified three GPPSs. Phylogenetic tree analysis showed that OfLSU1/2 belonged to the GPPS.LSU branch, while the OfSSUII belonged to the GPPS.SSU branch. OfLSU1, OfLSU2 and OfSSUII were all localized in chloroplasts. Y2H and pull-down assays showed that OfLSU1 or OfLSU2 interacted with OfSSUII to form heteromeric GPPSs. Site mutation experiments revealed that the conserved CXXXC motifs of OfLSU1/2 and OfSSUII were essential for the interaction between OfLSU1/2 and OfSSUII. Transient expression experiments showed that OfLSU1, OfLSU2 and OfSSUII co-expressed with monoterpene synthase genes OfTPS1 or OfTPS2 improved the biosynthesis of monoterpenoids (E)-ß-ocimene and linalool. The heteromeric GPPSs formed by OfLSU1/2 interacting with OfSSUII further improves the biosynthesis of monoterpenoids. Overall, these preliminary results suggested that the GPPSs play a key role in regulating the production of aromatic monoterpenes in O. fragrans.

Synthesis of Trifluoromethylated Monoterpenes by an Engineered Cytochrome P450.

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature's repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.

CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus.

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

Adjusting function of camphor on primary metabolism in Cinnamomum camphora stressed by high temperature.

Cinnamomum camphora has great economic value for its wide utilization in traditional medicine and furniture material, and releases lots of monoterpenes to tolerate high temperature. To uncover the adjusting function of monoterpenes on primary metabolism and promoting their utilization as anti-high temperature agents, the photosynthetic capacities, primary metabolite levels, cell ultrastructure and associated gene expression were surveyed in C. camphora when it was blocked monoterpene biosynthesis with fosmidomycin (Fos) and fumigated with camphor (a typical monoterpene in the plant) under high temperature (Fos+38 °C+camphor). Compared with the control (28 °C), high temperature at 38 °C decreased the starch content and starch grain size, and increased the fructose, glucose, sucrose and soluble sugar content. Meanwhile, high temperature also raised the lipid content, with the increase of lipid droplet size and numbers. These variations were further intensified in Fos+ 38 °C treatment. Compared with Fos+ 38 °C treatment, Fos+ 38 °C+camphor treatment improved the starch accumulation by promoting 4 gene expression in starch biosynthesis, and lowered the sugar content by suppressing 3 gene expression in pentose phosphate pathway and promoting 15 gene expression in glycolysis and tricarboxylic acid cycle. Meanwhile, Fos+ 38 °C+camphor treatment also lowered the lipid content, which may be caused by the down-regulation of 2 genes in fatty acid formation and up-regulation of 4 genes in fatty acid decomposition. Although Fos+ 38 °C+camphor treatment improved the photosynthetic capacities in contrast to Fos+ 38 °C treatment, it cannot explain the variations of these primary metabolite levels. Therefore, camphor should adjust related gene expression to maintain the primary metabolism in C. camphora tolerating high temperature.

Identification and Characterization of a Nerol Synthase in Fungi.

Nerol, a linear monoterpenoid, is naturally found in essential oils of various plants and is widely used in the fragrance, food, and cosmetic industries. Nerol synthase, essential for nerol biosynthesis, has previously been identified only in plants that use NPP as the precursor. In this study, a novel fungal nerol synthase, named PgfB, was cloned and characterized from Penicillium griseofulvum. In vitro enzymatic assays showed that PgfB could directly convert the substrate GPP into nerol. Furthermore, the successful expression of PgfB and its homologous protein in Saccharomyces cerevisiae resulted in the heterologous production of nerol. Finally, crucial amino acid residues for PgfB's catalytic activity were identified through site-directed mutagenesis. This research broadens our understanding of fungal monoterpene synthases and presents precious gene resources for the industrial production of nerol.

Combined in†vitro/in silico approaches, molecular dynamics simulations and safety assessment of the multifunctional properties of thymol and carvacrol: A comparative insight.

Bioactive compounds derived from medicinal plants have acquired immense attentiveness in drug discovery and development. The present study investigated in vitro and predicted in silico the antibacterial, antifungal, and antiviral properties of thymol and carvacrol, and assessed their safety. The performed microbiological assays against Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica Typhimurium revealed that the minimal inhibitory concentration values ranged from (0.078 to 0.312 mg/mL) and the minimal fungicidal concentration against Candida albicans was 0.625 mg/mL. Molecular docking simulations, stipulated that these compounds could inhibit bacterial replication and transcription functions by targeting DNA and RNA polymerases receptors with docking scores varying between (-5.1 to -6.9 kcal/mol). Studied hydroxylated monoterpenes could hinder C. albicans growth by impeding lanosterol 14α-demethylase enzyme and showed a (ΔG=-6.2 and -6.3 kcal/mol). Computational studies revealed that thymol and carvacrol could target the SARS-Cov-2 spike protein of the Omicron variant RBD domain. Molecular dynamics simulations disclosed that these compounds have a stable dynamic behavior over 100 ns as compared to remdesivir. Chemo-computational toxicity prediction using Protox II webserver indicated that thymol and carvacrol could be safely and effectively used as drug candidates to tackle bacterial, fungal, and viral infections as compared to chemical medication.

Maize terpene synthase 1 impacts insect behavior via the production of monoterpene volatiles ß-myrcene and linalool.

Plant-derived volatiles are important mediators of plant-insect interactions as they can provide cues for host location and quality, or act as direct or indirect defense molecules. The volatiles produced by Zea mays (maize) include a range of terpenes, likely produced by several of the terpene synthases (TPS) present in maize. Determining the roles of specific terpene volatiles and individual TPSs in maize-insect interactions is challenging due to the promiscuous nature of TPSs in vitro and their potential for functional redundancy. In this study, we used metabolite GWAS of a sweetcorn diversity panel infested with Spodoptera frugiperda (fall armyworm) to identify genetic correlations between TPSs and individual volatiles. This analysis revealed a correlation between maize terpene synthase 1 (ZmTPS1) and emission of the monoterpene volatiles linalool and ß-myrcene. Electroantennogram assays showed gravid S. frugiperda could detect both linalool and ß-myrcene. Quantification of headspace volatiles in a maize tps1 loss-of-function mutant confirmed that ZmTPS1 is an important contributor to linalool and ß-myrcene emission in maize. Furthermore, pairwise choice assays between tps1 mutant and wild-type plants showed that ZmTPS1, and by extension its volatile products, aid host location in the chewing insect S. frugiperda, yet repel the sap-sucking pest, Rhopalosiphum maidis (corn leaf aphid). On the other hand, ZmTPS1 had no impact on indirect defense via the recruitment of the parasitoid Cotesia marginiventris. ZmTPS1 is therefore an important mediator of the interactions between maize and its insect pests.