Linalool Biosynthesis Research Articles

Transcriptomic and free monoterpene analyses of aroma reveal that isopentenyl diphosphate isomerase inhibits monoterpene biosynthesis in grape (Vitis vinifera L.)

BackgroundMonoterpenes are among the most important volatile aromatic compounds contributing to the flavor and aroma of grapes and wine. However, the molecular basis of monoterpene biosynthesis has not yet been fully elucidated.ResultsIn our study, transcriptomics and gas chromatography-mass spectrometry (GC-MS) were used to mine candidate genes and transcription factors involved in monoterpene biosynthesis between high-monoterpene and zero-monoterpene table grape cultivars. We found that monoterpene biosynthesis was positively correlated by the expression of five genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (VvDXSs), one encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (VvHDR), three hydroxy-3-methylglutaryl-CoA synthases (VvHMGSs) and one mevalonate kinase (VvMVK), whereas the expression of one isopentenyl diphosphate isomerase (VvIDI) and one 3-hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) negatively correlated monoterpene biosynthesis. Of these genes, VvIDI was selected to validate its function in monoterpene accumulation through a transient overexpression experiment, and was shown to inhibit the biosynthesis of grape linalool and α-terpineol. Meanwhile, we found that a 64-amino acid extension sequence at the N-terminus can guide the VvIDI protein to target the chloroplast.ConclusionsThe findings of this study should help to guide future functional analysis of key genes as well as mining the potential regulatory mechanism of monoterpene biosynthesis in grapes and grape products.

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.

The genome assembly of Chimonanthus praecox var. concolor and comparative genomic analysis highlight the genetic basis underlying conserved and variable floral traits of wintersweet

Wintersweet (Chimonanthus praecox), a classical winter-blooming magnoliids species with great aesthetic value highlighting the on the brilliant yellow flower and pleasant floral aroma, possesses diverse varieties mainly classified in three groups (Concolor, Patens, and Rubrum group) with the most striking variation in the tepal color. The recent release of the genome in two wintersweet varieties belonging to Patens and Rubrum group successfully elucidated the molecular mechanisms underlying some ornamental traits highlighting the flowering time, floral scent and tepal color. In this study, the chromosome-scale genome of C. praecox var. concolor was assembled to further verify the placement of magnoliids and exploit the genetic variation underscoring the diverse flowering traits among different wintersweet groups. Phylogenomic analyses placed magnoliids including C. praecox var. concolor as the sister to eudicots. The C. praecox var. concolor’s genome experienced two rounds of whole genome duplication (WGD). Multiple gene families involved in floral development including ANR1 and PI genes were retained and expanded during these WGD events. The expansion and expression pattern of SVP and FUL genes suggest their pivotal role in unique character of winter-flowering. With the pleasant fragrance as the aesthetic characteristics, linalool and benzyl acetate were identified as the top abundant constituents in the floral scent of Concolor wintersweet. Integrating metabolic, genomic, and transcriptomic analyses with enzyme assays led to the identification of CpTPS3 (CpLIS) and CpBAHD2 (CpBEAT), which are responsible for the biosynthesis of linalool and benzyl acetate, respectively, in the Concolor variety. Taken together, the assembled genome of C. praecox var. concolor enables the comparative analysis of the genetic basis underlying both conserved and variable floral traits and provides a useful foundation for variety improvements by molecular breeding in wintersweet.

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.

The R2R3-MYB transcription factor OfMYB21 positively regulates linalool biosynthesis in Osmanthus fragrans flowers

Osmanthus fragrans is a well-known landscape ornamental tree species for its pleasing floral fragrance and abundance of flowers. Linalool, the core floral volatiles of O. fragrans, has tremendous economic value in the pharmaceuticals, cleaning products and cosmetics industries. However, the transcriptional regulatory network for the biosynthesis of linalool in O. fragrans remains unclear. Here, OfMYB21, a potential transcription factor regulating the linalool synthetase OfTPS2, was identified using RNA-seq data and qRT-PCR analysis. Yeast one-hybrid, dual-luciferase and EMSA showed that OfMYB21 directly binds to the promoter of OfTPS2 and activates its expression. Overexpression of OfMYB21 in the petals of O. fragrans led to up-regulation of OfTPS2 and increased accumulation of linalool, while silencing of OfMYB21 led to down-regulation of OfTPS2 and decreased biosynthesis of linalool. Subsequently, yeast two-hybrid, pull-down and BiFC experiments showed that OfMYB21 interacts with JA signaling factors OfJAZ2/3 and OfMYC2. Interestingly, the interaction between OfMYC2 and OfMYB21 further enhanced the transcription of OfTPS2, whereas OfJAZ3 attenuated this effect. Overall, our studies provided novel finding on the regulatory mechanisms responsible for the biosynthesis of the volatile monoterpenoid linalool in O. fragrans.

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.

Genome-wide analyzation and functional characterization on the TPS family provide insight into the biosynthesis of mono-terpenes in the camphor tree

Terpene synthase (TPS) plays an important role in terpenoids biosynthesis. Cinnamomum camphora (camphor tree) contains dozens of terpenoids with medicinal value, especially borneol, which has been widely used since ancient times. However, limited information is available regarding the genome-wide identification and characterization of the TPS family in the C. camphora. In this study, 82 CcTPS genes were identified from the camphor tree genome (CTG). Gene cluster and sequence syntenic analysis suggested that tandem duplication occurred within the TPS family of the CTG, especially for the TPS-b subfamily. The chemotype-specific gene expression analysis showed significantly differential expression patterns among six chemotypes. It is worth noting that three genes (CcTPS26, CcTPS49 and CcTPS72) exhibited relatively high expression in the borneol-type camphor tree, compared to the other five chemotypes. Further functional characterization of them indicated that they were all bornyl diphosphate synthases (BPPSs), which function in catalyzing GPP into BPP and then undergoes dephosphorylation to yield borneol. This is the first report that multiple BPPSs exist within a single species. Intriguingly, CcTPS49 and CcTPS72 lead to the generation of dextral-borneol, while CcTPS26 contributes to the biosynthesis of levo-borneol. In addition, the functional characterization of another six CcTPSs suggested that they are responsible for the biosynthesis of linalool, eucalyptol and several other monoterpenes in camphor tree. In conclusion, these novel results provide a foundation for further exploration of the role of the CcTPS gene family and shed light on a better understanding of the biosynthesis and accumulation of monoterpenes in camphor tree.

Biogenesis of flavor-related linalool is diverged and genetically conserved in tree peony (Paeonia × suffruticosa).

Floral scent is an important and genetically complex trait in horticultural plants. Tree peony (Paeonia × suffruticosa) originates in the Pan-Himalaya and has nine wild species divided into two subsections, Delavayanae and Vaginatae. Their flowers are beloved worldwide for their sweet floral fragrance, yet the flavor-related volatiles and underlying biosynthetic pathways remain unknown. Here, we characterized the volatile blends of all wild tree peony species and found that the flavor-related volatiles were highly divergent, but linalool was a unique monoterpene in subsect. Delavayanae. Further detection of volatiles in 97 cultivars with various genetic backgrounds showed that linalool was also the characteristic aroma component in Paeonia delavayi hybrid progenies, suggesting that linalool was conserved and dominant within subsect. Delavayanae and its hybrids, instead of species and cultivars from subsect. Vaginatae. Global transcriptome analysis of all wild tree peony species and 60 cultivars revealed five candidate genes that may be involved in key steps of linalool biosynthesis; especially the expressions of three TPS genes, PdTPS1, PdTPS2, and PdTPS4, were significantly positively correlated with linalool emissions across tree peony cultivars. Further biochemical evidence demonstrated that PdTPS1 and PdTPS4 were the pivotal genes determining the species-specific and cultivar-specific emission of linalool. This study revealed a new insight into floral scent divergence in tree peony and would greatly facilitate our understanding of the phylogeny and evolution of Paeonia.

Methyl Jasmonate Induces Genes Involved in Linalool Accumulation and Increases the Content of Phenolics in Two Iranian Coriander (Coriandrum sativum L.) Ecotypes.

The medicinal herb coriander (Coriandrum sativum L.), with a high linalool (LIN) content, is widely recognized for its therapeutic benefits. As a novel report, the goals of this study were to determine how methyl jasmonate (MeJA) affects total phenolic content (TPC), LIN content, flavonoid content (TFC), and changes in gene expression involved in the linalool biosynthesis pathway (CsγTRPS and CsLINS). Our findings showed that, in comparison to the control samples, MeJA treatment substantially enhanced the TPC, LIN, and TFC content in both ecotypes. Additionally, for both Iranian coriander ecotypes, treatment-induced increases in CsγTRPS and CsLINS expression were connected to LIN accumulation in all treatments. A 24 h treatment with 150 µM MeJA substantially increased the LIN content in the Mashhad and Zanjan ecotypes, which was between 1.48 and 1.69 times greater than that in untreated plants, according to gas chromatography–mass spectrometry (GC-MS) analysis. Our findings demonstrated that MeJA significantly affects the accumulation of LIN, TPC, and TFC in Iranian C. sativum treated with MeJA, which is likely the consequence of gene activation from the monoterpene biosynthesis pathway. Our discoveries have improved the understanding of the molecular mechanisms behind LIN synthesis in coriander plants.

Study on the Composition of Hop Terpenes and Terpenoids by Steam Distillation and Effects on Beer Flavor by an Ale Yeast

Model systems of adding three distillates (hop oil, water-soluble aqueous, and residual aqueous) in model wort (buffer) and wort were used to produce model beers and special beers, respectively (with an ale yeast and fermentation at 18 °C). The attributes of hoppy flavor and organoleptic quality were comprised of sustained, surged, generated, or diminished oxygenated compounds and hydrocarbons by yeast biosynthesis in hop essential oil and non-volatile precursors. The matrix of content and instrumental composition of oxygenated terpenoids and hydrocarbons (terpenes and sesquiterpenes) and sensorial profile of model systems was directionally correlated to the matrix and sensory of the early hopped, late hopped, and dry hopped beers. The occurrence of hop aroma profile of the residual aqueous is reminiscent to traditional early kettle hopped beer. The water-soluble volatile components added beer were remarkably close to the distinctive odor profile of late hopped beer. However, dry hopped beer was seemingly a coupling of the hop oil and the water soluble aqueous added beers, but its perception was closer to the latter. The mechanism of the growth of nerol, citronellol, and methyl geranate via a reversed biosynthesis of free linalool by ale yeast and fermentation at 18 °C is contrary to a pathway via geraniol from the hop oil or the water-soluble aqueous. Free linalool was sustained after yeast hydrolysis of linalyl glycoside and related to metabolites in the residual aqueous due to sluggish yeast activity in the model wort (buffer) but not in the wort.

Differential gene expression associated with a floral scent polymorphism in the evening primrose Oenothera harringtonii (Onagraceae)

BackgroundPlant volatiles play an important role in both plant-pollinator and plant-herbivore interactions. Intraspecific polymorphisms in volatile production are ubiquitous, but studies that explore underlying differential gene expression are rare. Oenothera harringtonii populations are polymorphic in floral emission of the monoterpene (R)-(−)-linalool; some plants emit (R)-(−)-linalool (linalool+ plants) while others do not (linalool- plants). However, the genes associated with differential production of this floral volatile in Oenothera are unknown. We used RNA-Seq to broadly characterize differential gene expression involved in (R)-(−)-linalool biosynthesis. To identify genes that may be associated with the polymorphism for this trait, we used RNA-Seq to compare gene expression in six different Oenothera harringtonii tissues from each of three linalool+ and linalool- plants.ResultsThree clusters of differentially expressed genes were enriched for terpene synthase activity: two were characterized by tissue-specific upregulation and one by upregulation only in plants with flowers that produce (R)-(−)-linalool. A molecular phylogeny of all terpene synthases identified two putative (R)-(−)-linalool synthase transcripts in Oenothera harringtonii, a single allele of which is found exclusively in linalool+ plants.ConclusionsBy using a naturally occurring polymorphism and comparing different tissues, we were able to identify candidate genes putatively involved in the biosynthesis of (R)-(−)-linalool. Expression of these genes in linalool- plants, while low, suggests a regulatory polymorphism, rather than a population-specific loss-of-function allele. Additional terpene biosynthesis-related genes that are up-regulated in plants that emit (R)-(−)-linalool may be associated with herbivore defense, suggesting a potential economy of scale between plant reproduction and defense.

Putative regulatory role of hexokinase and fructokinase in terpenoid aroma biosynthesis in Lilium ‘Siberia’

Lily is one of the most economically important flowers worldwide due to its elegant appearance and appealing scent, which is mainly composed of monoterpene ocimene, linalool and benzenoids. Sugars are the primary products of plants, with fructose and hexose sugars being the substrate material for most organic compounds and metabolic pathways in plants. Herein, we isolated and functionally characterized hexokinase (LoHXK) and fructokinase (LoFRK) from Lilium ‘Siberia’ flower, which indicated their potential roles in floral aroma production. Real-time PCR analysis showed that LoHXK and LoFRK were highly expressed in the flower filament. Overexpression and virus-induced gene silencing (VIGS) assays revealed that LoHXK and LoFRK significantly modified the emission of β-ocimene and linalool contents via regulation of expression of key structural volatile synthesis genes (LoTPS1 and LoTPS3). Under exogenous glucose and fructose application, the volatile contents of β-ocimene and linalool were increased and the expression levels of key structural genes were upregulated. The emission of β-ocimene and linalool followed a diurnal circadian rhythm. Determination of carbon fluxes via 13C-labeled glucose and 13C-labeled fructose experiments showed that the mass spectra of ocimene and linalool significantly increased, however, the m/z ratio of ethyl benzoate did not change. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that LoFRK interacted with LoMYB1 and LoMYB2 proteins. Together, these results suggest that hexokinase and fructokinase may play significant roles in the regulation of ocimene and linalool biosynthesis in Lilium ‘Siberia’.

The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.

Bioinformatics-aided identification, characterization and applications of mushroom linalool synthases

Enzymes empower chemical industries and are the keystone for metabolic engineering. For example, linalool synthases are indispensable for the biosynthesis of linalool, an important fragrance used in 60–80% cosmetic and personal care products. However, plant linalool synthases have low activities while expressed in microbes. Aided by bioinformatics analysis, four linalool/nerolidol synthases (LNSs) from various Agaricomycetes were accurately predicted and validated experimentally. Furthermore, we discovered a linalool synthase (Ap.LS) with exceptionally high levels of selectivity and activity from Agrocybe pediades, ideal for linalool bioproduction. It effectively converted glucose into enantiopure (R)-linalool in Escherichia coli, 44-fold and 287-fold more efficient than its bacterial and plant counterparts, respectively. Phylogenetic analysis indicated the divergent evolution paths for plant, bacterial and fungal linalool synthases. More critically, structural comparison provided catalytic insights into Ap.LS superior specificity and activity, and mutational experiments validated the key residues responsible for the specificity.

Seasonal emission of monoterpenes from four chemotypes of Cinnamomum camphora

Plant terpenes are the main functional agents in forest bathing and have great value in several industries. To uncover the seasonal variation of Cinnamomum camphora monoterpene emission and the difference among the chemotypes for promoting the development of forest bathing and exploitation of monoterpenes using the species, the emission of monoterpenes and related gene expression in the camphor chemotype, eucalyptol chemotype, linalool chemotype and borneol chemotype of C. camphora were analyzed in May (spring), July (summer) and November (autumn), and the stored monoterpenes and storage structure were investigated. Eleocytes were the storage structure of monoterpenes in C. camphora, which widely existed in the leaf palisade tissue and spongy tissue, as well as around the glands and vascular bundles. The 4 chemotypes of C. camphora released different monoterpene components which mainly derived from the immediate emission after synthesis. Camphor, eucalyptol, linalool and endo-borneol showed the uppermost emission rate and were the typical monoterpenes in the corresponding chemotype, respectively, due to the high expression of the genes encoding bornyl diphosphate synthase (E5.5.1.8), linalool synthase (TSP14) and (+)-neomenthol dehydrogenase (E1.1.1.208) for the biosynthesis of camphor, linalool and endo-borneol, respectively, and only expression of the gene encoding 1,8-cineole synthase (TSP-cin) for eucalyptol biosynthesis. Among the 4 chemotypes, the linalool chemotype showed the highest emission rate of monoterpenes in the same month (without significant difference with the eucalyptol chemotype in November), due to the high expression of the genes (dxs, dxr, ispF, gcpE, and GGPS) in methylerythritol-4-phosphate (MEP) pathway, α-terpineol synthase gene (E4.2.3.111) and TSP14. Among the 3 months, temperature was the remarkably varied meteorological condition. The highest monoterpene emission rate from the 4 chemotypes of C. camphora was detected in July, which should result from the up-regulated expression of related genes in monoterpene biosynthesis and improved vaporization and diffusion of stored monoterpenes under high temperature, with the former providing a major contribution.

Genome-Wide Identification and Expression Profile of TPS Gene Family in Dendrobium officinale and the Role of DoTPS10 in Linalool Biosynthesis.

Terpene synthase (TPS) is a critical enzyme responsible for the biosynthesis of terpenes, which possess diverse roles in plant growth and development. Although many terpenes have been reported in orchids, limited information is available regarding the genome-wide identification and characterization of the TPS family in the orchid, Dendrobium officinale. By integrating the D. officinale genome and transcriptional data, 34 TPS genes were found in D. officinale. These were divided into four subfamilies (TPS-a, TPS-b, TPS-c, and TPS-e/f). Distinct tempospatial expression profiles of DoTPS genes were observed in 10 organs of D. officinale. Most DoTPS genes were predominantly expressed in flowers, followed by roots and stems. Expression of the majority of DoTPS genes was enhanced following exposure to cold and osmotic stresses. Recombinant DoTPS10 protein, located in chloroplasts, uniquely converted geranyl diphosphate to linalool in vitro. The DoTPS10 gene, which resulted in linalool formation, was highly expressed during all flower developmental stages. Methyl jasmonate significantly up-regulated DoTPS10 expression and linalool accumulation. These results simultaneously provide valuable insight into understanding the roles of the TPS family and lay a basis for further studies on the regulation of terpenoid biosynthesis by DoTPS in D. officinale.

Efficient Biosynthesis of R-(-)-Linalool through Adjusting the Expression Strategy and Increasing GPP Supply in Escherichia coli.

R-(-)-linalool is widely used in pharmaceutical, agrochemical, and fragrance industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method is urgently needed. To induce the R-(-)-linalool biosynthesis pathway in Escherichia coli, we expressed several heterologous (3R)-linalool synthases (LISs) and then chose a suitable LIS from Streptomyces clavuligerus (bLIS) for further study. The bLIS expression was markedly elevated by using optimized ribosomal binding sites and protein fusion tags. To increase the geranyl diphosphate content, we tested various alterations in prenyltransferases and their mutants. The final strain accumulated 100.1 and 1027.3 mg L-1 R-(-)-linalool under shake flask and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date. This work could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.

De novo biosynthesis of linalool from glucose in engineered Escherichia coli

Linalool is an important terpenoids of floral scents and has wide applications. In the past, several groups reported on a strategy to establish biosynthesis of linalool in yeast based on co-expression of Saccharomyces cerevisiae farnesyl diphosphate synthase ERG20 and Actinidia arguta linalool synthase LIS. However, ERG20 has both geranyl diphosphate synthase and farnesyl diphosphate synthase activities, which can lead to metabolic flow to farnesyl diphosphate. In this study, a heterologous linalool biosynthesis pathway was constructed in Escherichia coli and showed that using Abies grandis geranyl diphosphate synthase GPPS2 instead of ERG20 can effectively improve linalool biosynthesis. Subsequently, we further improved the biosynthesis of linalool by overexpression of isopentenyl diphosphate isomerase Idi.

Improved linalool production in Saccharomyces cerevisiae by combining directed evolution of linalool synthase and overexpression of the complete mevalonate pathway

With special fragrance and various biological properties, linalool has wide applications in food, pharmaceuticals and cosmetics industries. Microbial biosynthesis has become a promising approach to linalool production, however its efficiency is limited by the limited precursor supply and poor efficiency of linalool synthase. With the goal of enhancing heterologous linalool production in Saccharomyces cerevisiae, protein engineering and metabolic engineering were simultaneously employed to solve these bottlenecks. Linalool-producing yeast was first constructed by introduction of t67OMcLIS selected out from a collection of linalool synthases. The efficiency of linalool biosynthesis was then improved by strengthening the supply of geranyl pyrophosphate (GPP) as precursor via overexpression of the complete mevalonate (MVA) pathway and a GPP synthase variant. To accelerate the conversion of the accumulated GPP to linalool, t67OMcLIS was engineered by means of directed evolution after development of a competition-based color-indicated high-throughput screening method. Expression of the t67OMcLIS variant in the engineered S. cerevisiae with enhanced precursor supply yielded 53.14 mg/L of linalool in biphasic shake-flask culture. This study delivered an efficient linalool-producing yeast cell factory and meanwhile provided efficient strategies for biosynthesis of other valuable monoterpenes.