Linalool Synthase Research Articles

Transcriptome Analyses Reveal Differences in the Metabolic Pathways of the Essential Oil Principal Components of Different Cinnamomum Chemotypes

The genus Cinnamomum exhibits a rich variety of chemotypes and is an economically important essential oil (EO)-producing plant belonging to the family Lauraceae. Here, we aimed to explore the potential differences in the terpenoid (the principal components of EOs) biosynthesis pathways of different chemotypes at the molecular level in four Cinnamomum species—C. camphora var. linaloolifera, C. kanehirae, C. longipaniculatum, and C. micranthum. Gas chromatography–mass spectrometry (GC-MS) was employed to elucidate the discrepancies in the chemical profiles and compositions of leaf EO terpenoids among the four Cinnamomum species. The results revealed significant variations in leaf EO yields. The main constituents of the leaf EOs from C. camphora var. linaloolifera and C. kanehirae were the acyclic monoterpene linalool, and those of C. longipaniculatum and C. micranthum were the monoterpene eucalyptol and the sesquiterpene β-caryophyllene, respectively. Furthermore, a comparative transcriptome analysis of the leaves from the four Cinnamomum species revealed that differentially expressed genes (DEGs) were significantly enriched in terpene-related entries. Specifically, 42 and 24 DEGs were significantly enriched to the mevalonate (MVA)/2-methylerythritol 4-phosphate (MEP) pathways and terpene synthase (TPS) activity, respectively. Most genes encoding proteins involved in the terpenoid precursor MVA and MEP pathways exhibited differential expression across the four species, which correlated with the distinct terpenoid profiles observed in their leaf EOs. Four acyclic monoterpene linalool synthase genes—Maker00024100, Maker00014813, Maker00014818, and Maker00018424—were highly expressed in C. camphora var. linaloolifera and C. kanehirae. A monoterpene eucalyptol synthesis gene, Maker00001509, was highly expressed in C. longipaniculatum, and a sesquiterpene β-stigmasterol synthesis gene, Maker00005791, was highly expressed in C. micranthum. These expression levels were subsequently validated through quantitative real-time polymerase chain reaction (qRT-PCR). In conclusion, the combined results of the GC-MS and transcriptome analyses revealed a strong correlation between the metabolite content of the EOs and gene expression. This research contributes to a better understanding of the differences in terpene accumulation in various chemotypes of Cinnamomum at the molecular and mechanistic levels, laying a solid foundation for the cultivation of an ideal Cinnamomum variety.

Engineering of Saccharomyces cerevisiae towards synthesis of linalool using linalool synthase from Magnolia champaca

Linalool is one of the commercially important fragrance molecule usually extracted from Lavandula angustifolia (lavender) and Ocimum basilicum (basil) plants. In the present study, efforts were made to produce this molecule in microbial system to meet demand-supply imbalance. Linalool synthase (LIS) gene from Magnolia champaca (Mc) and Coriandrum sativum (Cs) were successfully cloned and expressed in Saccharomyces cerevisiae CEN PK2–1 C. It was observed that expression of full-length LIS (fLIS) resulted in lesser linalool when compared to truncated LIS (tLIS) devoid of plastid signal for both Mc and Cs. In terms of linalool yield, MctLIS resulted in 1.27-fold higher linalool when compared to CstLIS. Later, when two more genes viz., TPI1 and ALD6 which presumably increase sterol pathway flux were overexpressed, actually resulted in lower linalool and increased acetate production. However, multicopy expression of MctLIS and tHMG1 in this strain has reversed the above phenomenon due to presumptive push-pull strategy. Finally, this engineered strain was cultivated in the 2 L bioreactor in fed-batch mode to obtain 10.85 µg/mL of linalool. Docking studies of homology model of MctLIS with geranyl pyrophosophate (GPP) revealed V387, Y361, T434, R427 and R249 as key interactions sites. The study reports the linalool production using LIS gene from Magnolia champaca for the first time and could be a potential chassis for further studies.

Exploration of Thiamin thiazole synthase (THI4) Expression and Transcriptomes Involved in the Floral Volatiles of Caladium bicolor

4-methyl-5-vinylthiazole (MVT) is a significant volatile of caladium (Caladium bicolor) which produces a very high level of thiamin thiazole synthase (THI4) in male flowers. We explored transcriptomes upregulating MVT using RNA-seq during the six developmental stages of the male flower (Day−10 to Day0) in C. bicolor ‘Tapestry’. THI4 was the highest transcript throughout the male flower development. Additionally, the genes showing the high expression associated with floral volatiles of caladium on Day0 were trans-resveratrol di-O-methyltransferase (ROMT), chalcone synthase (CHS), 3-ketoacyl-CoA thiolase 2 (KAT2), and linalool synthase (TPS). These four genes correspond to the following elevated volatiles of caladium: 1,3,5-trimethoxybenzene, MVT, indole, methyl salicylate, and linalool on Day0 compared to Day−10. The upstream THI4 gene was cloned to drive a fluorescent gene (ZsGreen1) in transient and stable transgenic petunia and tobacco plants, showing the gene expression only in the male tissue. The tissue-specific expression of the caladium THI4 promoter could benefit crop production with minimal modification of plants. Investigating transcriptomes associated with caladium fragrance can help provide insight into understanding the regulatory mechanisms of floral volatiles of caladium.

FhMYB108 Regulates the Expression of Linalool Synthase Gene in Freesia hybrida and Arabidopsis.

Acting as the most abundant and widely distributed volatile secondary metabolites in plants, terpenoids play crucial roles in diverse physiological regulations and metabolic processes. Terpene synthases play a decisive role in determining the composition and diversity of terpenoids. Though the regulation of terpene synthases has been extensively investigated across various plant species, limited studies have focused on the upstream transcriptional regulation of terpene synthases. In this study, we have identified linalool as the predominant volatile compound that is released gradually from Freesia hybrida flowers throughout flower blooming. In the context of the transcriptome, a typical MYB transcription factor, FhMYB108, was screened based on homologous gene comparison. FhMYB108 is capable of regulating the expression of FhTPS1, and both their expression levels showed gradual increase during flower opening. Moreover, FhMYB108 exerts a stimulatory effect on the transcription of Arabidopsis thaliana AtTPS14, while no significant increase in AtTPS14 expression is observed upon the stabilization of FhMYB108 in A. thaliana. The highly expressed AtMYC2 in A. thaliana could interact with FhMYB108 to suppress the activation of AtTPS14 by FhMYB108. The present study not only elucidates the regulatory mechanism underlying linalool synthesis but also discovers the synergistic effect of MYB and bHLH transcription factors in governing the biosynthesis of volatile terpenoids.

Silver nanoparticles alleviate the impact of soil contamination and wastewater irrigation on rosemary plants: modulating of gene expression and secondary metabolites

A number of obstacles, including irrigated wastewater and soil contamination, arise in the growth of aromatic and medicinal plants. This study aimed to reduce the effects of contaminated soil and wastewater irrigation on rosemary (Rosmarinus officinalis L.) plants by using biosynthesized silver nanoparticles (AgNPs) produced by the ginger (Zingiber officinale) plant extract. The AgNPs were characterized using Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR). The experimental design involved three distinct groups of plants: one group was irrigated with regular tap water, another group was rooted in soil contaminated by sewage-wastewater and irrigated with processed wastewater, and the final group consisted of plants grown in wastewater-contaminated soil, irrigated with processed wastewater, and sprayed with 200 mM l−1 AgNPs. The study also examined the impact of different treatments on gene expression and secondary metabolite levels in rosemary plants. According to HPLC investigations, nineteen phenol compounds and flavonoids were identified in a methanolic extract of rosemary that was grown in contaminated soil, irrigated with wastewater, and sprayed with AgNPs. Plants treated with wastewater and nanoparticles produced quantities of secondary compounds, including resvertol, vanillic acid, and gallic acid with 1.11, 0.15, and 0.01 mg g−1 respectively, which are all regarded as significant antioxidants employed in the pharmaceutical industry. Hexokinase synthase (HK), geranyl diphosphate synthase (GPPS), and linalool synthase (LS) coding genes were found to have highly expressed expressions when plants grown in contaminated soil, wastewater-irrigated plants, and nanoparticle-sprayed plants, respectively, at a 23.2- and 5.54-fold level, where the HK gene was 8.7 times more strongly expressed. Conversely, plants grown in contaminated soil and irrigated with treated wastewater showed downregulation of these genes. Conclusively, using silver nanoparticles significantly reduced the influence of wastewater pollution on secondary metabolites in rosemary plants, which was increased by the gene expression results and was completely consistent with HPLC analysis.

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

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.

Unraveling the terpene synthase family and characterization of BsTPS2 contributing to (S)-( +)-linalool biosynthesis in Boswellia.

Boswellia tree bark exudes oleo-gum resin in response to wounding, which is rich in terpene volatiles. But, the molecular and biochemical basis of wound-induced formation of resin volatiles remains poorly understood. Here, we combined RNA-sequencing (RNA-seq) and metabolite analysis to unravel the terpene synthase (TPS) family contributing to wound-induced biosynthesis of resin volatiles in B. serrata, an economically-important Boswellia species. The analysis of large-scale RNA-seq data of bark and leaf samples representing more than 600 million sequencing reads led to the identification of 32 TPSs, which were classified based on phylogenetic relationship into various TPSs families found in angiosperm species such as TPS-a, b, c, e/f, and g. Moreover, RNA-seq analysis of bark samples collected at 0-24h post-wounding shortlisted 14 BsTPSs that showed wound-induced transcriptional upregulation in bark, suggesting their important role in wound-induced biosynthesis of resin volatiles. Biochemical characterization of a bark preferentially-expressed and wound-inducible TPS (BsTPS2) in vitro and in planta assays revealed its involvement in resin terpene biosynthesis. Bacterially-expressed recombinant BsTPS2 catalyzed the conversion of GPP and FPP into (S)-( +)-linalool and (E)-(-)-nerolidol, respectively, in vitro assays. However, BsTPS2 expression in Nicotiana benthamiana found that BsTPS2 is a plastidial linalool synthase. In contrast, cytosolic expression of BsTPS2 did not form any product. Overall, the present work unraveled a suite of TPSs that potentially contributed to the biosynthesis of resin volatiles in Boswellia and biochemically characterized BsTPS2, which is involved in wound-induced biosynthesis of (S)-( +)-linalool, a monoterpene resin volatile with a known role in plant defense.

Gene Expression of Monoterpene Synthases Is Affected Rhythmically during the Day in Lavandula angustifolia Flowers

Lavender essential oil (EO) is widely used for medicinal purposes. The significant monoterpenes’ abundance of linalool and linalool acetate accounts for more than 50% of lavender EO compounds. Monoterpenes synthesis differs throughout plant development as a result of the differential gene expression patterns in distinct cell types. Previously, we have reported that the chemical composition of Lavandula angustifolia cv. etherio EO was affected by diurnal harvest time. The aim of this was to evaluate if the gene expression of lavender monoterpenes synthases is altered during the day length and correlated with the accumulation of the major components of lavender EO. The relative expression of linalool synthase (LaLINS), limonene synthase (LaLIMS) and terpene synthase-like (LaTPS-l) was recorded in flowers at the 3rd to 5th stage every 3 h during two consecutive days using quantitative real-time PCR. The composition of the lavender EO was also monitored during the day length using GC-MS analysis. Our results indicate that the expression of genes involved in the synthesis of lavender EO, including linalool and limonene synthases, is accompanied by oscillations, picking at mid-day and leading to linalool acetate accumulation in the afternoon. In conclusion, the monoterpenes synthase expression in lavender flowers is rhythmically affected during the day, leading to a higher accumulation of EO compounds in the afternoon. These results will be helpful to monitor the biosynthesis of lavender EO to ensure a high-quality product. Furthermore, the outcome of this study will be useful for breeding programs in the lavender field to modulate the biosynthesis of linalool and linalool acetate during the flowering harvest period.

Promoter characterization of a citrus linalool synthase gene mediating interspecific variation in resistance to a bacterial pathogen

BackgroundTerpenoids play essential roles in plant defense against biotic stresses. In Citrus species, the monoterpene linalool mediates resistance against citrus canker disease caused by the gram-negative bacteria Xanthomonas citri subsp. citri (Xcc). Previous work had associated linalool contents with resistance; here we characterize transcriptional responses of linalool synthase genes.ResultsLeaf linalool contents are highly variable among different Citrus species. “Dongfang” tangerine (Citrus reticulata), a species with high linalool levels was more resistant to Xcc than “Shatian” pummelo (C. grandis) which accumulates only small amounts of linalool. The coding sequences of the major leaf-expressed linalool synthase gene (STS4) are highly conserved, while transcript levels differ between the two Citrus species. To understand this apparent differential transcription, we isolated the promoters of STS4 from the two species, fused them to a GUS reporter and expressed them in Arabidopsis. This reporter system revealed that the two promoters have different constitutive activities, mainly in trichomes. Interestingly, both linalool contents and STS4 transcript levels are insensitive to Xcc infestation in citrus plants, but in these transgenic Arabidopsis plants, the promoters are activated by challenge of a bacterial pathogen Pseudomonas syringae, as well as wounding and external jasmonic acid treatment.ConclusionsOur study reveals variation in linalool and resistance to Xcc in citrus plants, which may be mediated by different promoter activities of a terpene synthase gene in different Citrus species.

Metabolic engineering of Escherichia coli for efficient production of linalool from biodiesel-derived glycerol by targeting cofactors regeneration and reducing acetate accumulation

Linalool is a valuable monoterpenoid compound commercially used in cosmetics and personal care industry as well as a ‘drop-in’ biofuel. Direct plant extraction or chemical synthesis of linalool suffers from insufficient abundance in plant and inefficient purification procedures. Herein, a powerful green cell factory has been developed to meet the increasing demands for more sustainable and efficient production of this terpene alcohol. The de novo production of linalool from biodiesel-derived glycerol was achieved by engineered Escherichia coli harboring the exogenous mevalonate pathway followed by coupling with geranyl diphosphate synthase and linalool synthase (LIS). Then, the Mentha aquatica LIS showing the most efficient linalool biosynthesis was identified, and subsequently tuned by ribosomal binding site (RBS) engineering and site-directed mutagenesis to facilitate linalool production. A further engineered strain equipped with an engineered NADP+-dependent pyruvate dehydrogenase complex (PDH) and phosphoketolase bypass, remarkably decreasing the by-products formation together with boosting substrate utilization efficiency. A final titer of 1.07 g/L linalool with a yield of 0.10 g/g glycerol was gained in flask culture and further increase to 4.16 g/L in 2-L fed-batch fermentation, which is the highest reported linalool titer and yield in E. coli to the best of our knowledge. In addition, the engineering strategies established here not only lay a potential production platform for linalool and its derivatives, but also provide references to minimize by-products secretion and improve carbon use efficiency.

Boosting Geranyl Diphosphate Synthesis for Linalool Production in Engineered Yarrowia lipolytica.

Linalool is a pleasant-smelling monoterpenoid widely found in the essential oils of most flowers. Due to its biologically active properties, linalool has considerable commercial potential, especially in the food and perfume industries. In this study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce linalool de novo. The (S)-linalool synthase (LIS) gene from Actinidia argute was overexpressed to convert geranyl diphosphate (GPP) into linalool. Flux was diverted from farnesyl diphosphate (FPP) synthesis to GPP by introducing a mutated copy of the native ERG20F88W-N119W gene, and CrGPPS gene from Catharanthus roseus on its own and as part of a fusion with LIS. Disruption of native diacylglycerol kinase enzyme, DGK1, by oligo-mediated CRISPR-Cas9 inactivation further increased linalool production. The resulting strain accumulated 109.6mg/L of linalool during cultivation in shake flasks with sucrose as a carbon source. CrGPPS expression in Yarrowia lipolytica increased linalool accumulation more efficiently than the ERG20F88W-N119W expression, suggesting that the increase in linalool production was predominantly influenced by the level of GPP precursor supply.

Combining Protein and Organelle Engineering for Linalool Overproduction in Saccharomyces cerevisiae.

Linalool, a plant-derived high-value monoterpene, is widely used in the perfume, cosmetic, and pharmaceutical industries. Recently, engineering microbes to produce linalool has become an attractive alternative to plant extraction or chemical synthesis approaches. However, the low catalytic activity of linalool synthase and the shortage of precursor pools have been considered as two key factors for low yields of linalool. In this study, we rationally engineered the entrance of the substrate-binding pocket of linalool synthase (t67OMcLISM) and successfully increased the catalytic efficiency of this enzyme toward geranyl pyrophosphate. Specifically, F447E and F447A, with decreased entrance hydrophobicity and steric hindrance, increased linalool production by 2.2 and 1.9 folds, respectively. Subsequently, cytoplasm and peroxisomes were harnessed to boost linalool synthesis in Saccharomyces cerevisiae, achieving a high titer of linalool (219.1 mg/L) in shake-flask cultivation. Finally, the engineered diploid strain produced 2.6 g/L of linalool by 5 L fed-batch fermentation, which was the highest production in yeast to date. The protein engineering and biosynthetic pathway compartmentalization in the peroxisome provide references for the microbial production of other monoterpenes.

Volatile Composition and Classification of Paeonia lactiflora Flower Aroma Types and Identification of the Fragrance-Related Genes.

Flower scent is one of the main ornamental characteristics of herbaceous peony, and the improvement of flower fragrance is a vital objective of herbaceous peony breeding. In this study, 87 herbaceous peony cultivars were divided into three groups (no/light fragrance, medium fragrance, and strong fragrance) based on their sensory evaluation scores, and 16 strong fragrance cultivars and one no fragrance cultivar were selected for subsequent analysis. Sixty-eight volatile components were detected in these 17 cultivars based on solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS), and 26 types were identified as important scent components. They were composed of terpenoids, benzenoids/phenylpropanoids, and fatty acid derivatives. According to the content and odor threshold of these main aroma components, the characteristic aroma substances of herbaceous peony were identified, including linalool, geraniol, citronellol, and phenylethyl alcohol (2-PE). The cultivars of strong scented herbaceous peony were divided into three types: rose scent, lily scent, and mixed scent. We explored the possible key genes of characteristic aroma substances in herbaceous peony petals with different odors through the qRT-PCR. The key genes encoding monoterpene biosynthesis were found to be PlDXS2, PlDXR1, PlMDS1, PlHDR1, PlGPPS3, and PlGPPS4. In addition, the linalool synthase (LIS) gene and the geraniol synthase (GES) gene were also found. PlAADC1, PlPAR1, and PlMAO1, related to the biosynthesis of 2-PE were detected, and the synthetic pathway of 2-PE was speculated. In conclusion, these findings revealed that the difference in gene expression of monoterpene and 2-PE synthesis pathway was related to the difference in the fragrance of herbaceous peony. This study explored the releasing pathway of herbaceous peony characteristic aroma substances and provided key genetic resources for fragrance improvement.

Structural Understanding of Fungal Terpene Synthases for the Formation of Linear or Cyclic Terpene Products.

Terpene synthases (TPSs), known gatekeepers of terpenoid diversity, are the main targets for enzyme engineering attempts. To this end, we have determined the crystal structure of Agrocybe pediades linalool synthase (Ap.LS), which has been recently reported to be 44-fold and 287-fold more efficient than bacterial and plant counterparts, respectively. Structure-based molecular modeling followed by in vivo as well as in vitro tests confirmed that the region of 60-69aa and Tyr299 (adjacent to the motif "WxxxxxRY") are essential for maintaining Ap.LS specificity toward a short-chain (C10) acyclic product. Ap.LS Y299 mutants (Y299A, Y299C, Y299G, Y299Q, and Y299S) yielded long-chain (C15) linear or cyclic products. Molecular modeling based on the Ap.LS crystal structure indicated that farnesyl pyrophosphate in the binding pocket of Ap.LS Y299A has less torsion strain energy compared to the wild-type Ap.LS, which can be partially attributed to the larger space in Ap.LS Y299A for better accommodation of the longer chain (C15). Linalool/nerolidol synthase Y298 and humulene synthase Y302 mutations also produced C15 cyclic products similar to Ap.LS Y299 mutants. Beyond the three enzymes, our analysis confirmed that most microbial TPSs have asparagine at the position and produce mainly cyclized products (δ-cadinene, 1,8-cineole, epi-cubebol, germacrene D, β-barbatene, etc.). In contrast, those producing linear products (linalool and nerolidol) typically have a bulky tyrosine. The structural and functional analysis of an exceptionally selective linalool synthase, Ap.LS, presented in this work provides insights into factors that govern chain length (C10 or C15), water incorporation, and cyclization (cyclic vs acyclic) of terpenoid biosynthesis.

Functional characterization of a bark-specific monoterpene synthase potentially involved in wounding- and methyl jasmonate-induced linalool emission in rubber (Hevea brasiliensis)

Rubber (Hevea brasiliensis) is a latex-producing plant that often encounters mechanical wounding, as well as pathogen and pest attacks through wound sites during and after tapping. Terpenoids play an important role in the ecological interactions of many plant species, and their diversity is mainly generated by enzymes known as terpene synthases (TPS). In this study, one cDNA sequence encoding a putative terpene synthase, HbTPS20, was obtained from the bark tissues of H. brasiliensis. The encoded protein contains 610 amino acids with a putative N-terminal plastid transit peptide of approximately 70 residues. It belongs to the TPS-b subfamily. Further phylogenetic analysis showed that HbTPS20 formed a separate branch that diverged from the progenitor of all other potentially functional terpene synthases of the rubber TPS-b subfamily. The truncated HbTPS20 without the signal peptide coding sequence was successfully expressed in E. coli and in vitro enzymatic assays with geranyl diphosphate (GPP) or neryl diphosphate (NPP) as a substrate defined HbTPS20 as an active linalool synthase (HbLIS) with the ability to produce linalool as the principal product. RT-qPCR analysis showed that the highest transcript levels of HbTPS20 were found in barks, and this gene was expressed at 2.26- and 250-fold greater levels in the bark tissues of wounded and MeJA-treated plants, respectively, than in those of the control plants. This indicates that this gene may be involved in the induced stress responses of rubber.

Combinatorial transient gene expression strategies to enhance terpenoid production in plants.

The monoterpenoid linalool and sesquiterpenoid costunolide are ubiquitous plant components that have been economically exploited for their respective essential oils and pharmaceutical benefits. In general, monoterpenes and sesquiterpenes are produced by the plastid 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytosolic mevalonate (MVA) pathways, respectively. Herein, we investigated the individual and combinatorial potential of MEP and MVA pathway genes in increasing linalool and costunolide production in Nicotiana benthamiana. First, six genes from the MEP (1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, geranyl pyrophosphate synthase, and linalool synthase) and MVA (acetoacetyl-CoA-thiolase, hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, germacrene A synthase, germacrene A oxidase, and costunolide synthase) pathways were separately cloned into the modular cloning (MoClo) golden gateway cassette. Second, the cassettes were transformed individually or in combination into the leaves of N. benthamiana by agroinfiltration. Five days post infiltration (DPI), all selected genes were transiently 5- to 94-fold overexpressed. Quantification using gas chromatography-Q-orbitrap-mass spectrometry (GC-Q-Orbitrap-MS) determined that the individual and combinatorial expression of MEP genes increased linalool production up to 50-90ng.mg-1 fresh leaf weight. Likewise, MVA genes increased costunolide production up to 70-90ng.mg-1 fresh leaf weight. Our findings highlight that the transient expression of MEP and MVA pathway genes (individually or in combination) enhances linalool and costunolide production in plants.

Enhancement of linalool production in Saccharomyces cerevisiae by utilizing isopentenol utilization pathway

BackgroundLinalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites.ResultsIn this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20F96W−N127W (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20F96W−N127W genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg2+ addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation.ConclusionsThe results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.

Silver and copper-oxide nanoparticles prepared with GA3 induced defense in rice plants and caused mortalities to the brown planthopper, Nilaparvata lugens (Stål)

BackgroundNanoparticles have been employed as nanopesticides for pest control in agriculture. However, the harmful effects of their chemical synthesis on human and environmental health have resulted in increased use of green synthetic approaches, including the use of plant extracts. The brown planthopper, Nilaparvata lugens (Stål) (BPH), is a severe pest of rice plants (Oryza sativa L.), especially in Asia. It is usually controlled chemically but has developed resistance against many insecticides. ResultsIn this study, we synthesized metallic silver (Ag-NPs) and copper-oxide (CuO-NPs) nanoparticles using the exogenous phytohormone, gibberellic acid (GA3), as a reducing agent. We then sprayed them separately on rice plants and BPH together and evaluated their effects on the plants and insects. SEM and TEM images showed that the synthesis was successful, indicated by the sizes (25–60 nm), uniform shape and spherical and cubical structures of Ag-NPs, as well as by the rugby sheet-like of CuO-NPs with lateral sizes of 150–340 nm and thickness of 30–70 nm. Independent applications of the nanoparticles and GA3 on rice plants induced different volatile profiles, of which the highest number emitted was under Ag-NPs, including the highest emission of linalool. Transcriptome analysis showed that Ag-NPs-treated rice plants showed different transcriptome profiles compared to the control, 24 h after treatment, including the upregulation of the linalool synthase gene, genes of plants transcription factors such as WRKY, bHLH and NAC and other genes involved in plant defense responses. In all treatments, the mortality rate of BPH increased with an increase in NPs concentrations over time but was prominent under Ag-NPs treatment. The LC50 values for Ag-NPs and CuO-NPs decreased with an increase in time. Also, the nanoparticles increased the activities of protective enzymes (POD, SOD and CAT), inhibited that of detoxification enzymes (A-CHE, ACP and AKP), and reduced total protein concentrations in the BPH. ConclusionsThese results show that synthesizing nanoparticles using phytohormones may be a safer and environmentally friendly option, which also holds promise for controlling the BPH in rice production.

Volatile metabolomics and coexpression network analyses provide insight into the formation of the characteristic cultivar aroma of oolong tea (Camellia sinensis)

Finished oolong tea products with distinctly characteristic cultivar aromas generally have higher economic value. To advance our understanding of the aromatic differences between Camellia sinensis cv. Tieguanyin (TGY) and Jinguanyin (JGY) cultivars and their underlying formation mechanism, volatile metabolomics analysis, electronic nose (EN) analysis, sensory evaluation, and RNA sequencing were performed during this study. The EN and sensorial analysis showed a significant difference in odour characteristics between the two cultivars, which featured sweet floral and fruity aromas and green floral aromas, respectively. A metabolomics analysis of tea products showed that linalool (floral, OAV = 50.6) and geraniol (rose-like and sweet, OAV = 1.9) may contribute to the characteristic aroma. The volatile determination at each stage during the oolong tea manufacturing process emphasized that the significant difference in linalool and geraniol contents in fresh leaves from the two cultivars and the increase rate were potential reasons for their characteristic aroma and suggested that the “Tanqing” treatment clarified and intensified the characteristic cultivar aroma. A linalool synthase CsLIN with the expected catalytic activity was identified by coexpression network analyses. Collectively, our work pinpoints two key aroma substances within two cultivars and elaborates on the potential cause of characteristic cultivar aroma formation.

Phycocyanin Fusion Constructs for Heterologous Protein Expression Accumulate as Functional Heterohexameric Complexes in Cyanobacteria.

Overexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein "P" of interest is expressed as a fusion with the abundant CpcB β-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous "P" protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (α,β*P)3CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, β*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (α,β*P)3CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per β-subunit. (iii) The (α,β*P)3CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,β*P)3CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon α-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (α,β*P)3CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,β)3 compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest.