D-limonene Synthase Research Articles

Identification and functional analysis of ZmDLS associated with the response to biotic stress in maize.

Terpenes are the main class of secondary metabolites produced in response to pest and germ attacks. In maize (Zea mays L.), they are the essential components of the herbivore-induced plant volatile mixture, which functioned as a direct or indirect defense against pest and germ attacks. In this study, 43 maize terpene synthase gene (ZmTPS) family members were systematically identified and analyzed through the whole genomes of maize. Nine genes, including Zm00001d032230, Zm00001d045054, Zm00001d024486, Zm00001d004279, Zm00001d002351, Zm00001d002350, Zm00001d053916, Zm00001d015053, and Zm00001d015054, were isolated for their differential expression pattern in leaves after corn borer (Ostrinia nubilalis) bite. Additionally, six genes (Zm00001d045054, Zm00001d024486, Zm00001d002351, Zm00001d002350, Zm00001d015053, and Zm00001d015054) were significantly upregulated in response to corn borer bite. Among them, Zm00001d045054 was cloned. Heterologous expression and enzyme activity assays revealed that Zm00001d045054 functioned as d-limonene synthase. It was renamed ZmDLS. Further analysis demonstrated that its expression was upregulated in response to corn borer bites and Fusarium graminearum attacks. The mutant of ZmDLS downregulated the expressions of Zm00001d024486, Zm00001d002351, Zm00001d002350, Zm00001d015053, and Zm00001d015054. It was more attractive to corn borer bites and more susceptible to F. graminearum infection. The yeast one-hybrid assay and dual-luciferase assay showed that ZmMYB76 and ZmMYB101 could upregulate the expression of ZmDLS by binding to the promoter region. This study may provide a theoretical basis for the functional analysis and transcriptional regulation of terpene synthase genes in crops.

Improve the production of D-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation.

D-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of D-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and D-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase D-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved D-limonene production, while the best strain yielded 62.31mg/L D-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding D-limonene yeasts and the parental strain under the stress of different D-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to D-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport D-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to D-limonene. This study provided a valuable platform for other monoterpenes' biosynthesis in yeast during alcoholic beverage fermentation.

Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation.

d-Limonene, a cyclic monoterpene, possessescitrus-like olfactory property and multi-physiologicalfunctions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W-N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W-N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 fusion protein, and the best strain yielded 9.8mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

Genetic diversity of limonene synthase genes in Rongan kumquat (Fortunella crassifolia).

D-limonene is the main component of citrus essential oils. In the monoterpene biosynthetic pathway, geranyl diphosphate reacts with monoterpenes to form the prenyl-carbocation intermediate to produce d-limonene. In this study, d-limonene synthase (FcLS) genes were first isolated from Rongan kumquat (Fortunella crassifolia Swingle). Sequencing analysis revealed that the open reading frames of 18 FcLS genes contain 12 single nucleotide polymorphisms, which resulted in the variation of FcLS proteins, indicating that the limonene synthase genes are a large family in F. crassifolia. This phenomenon has not been reported in Citrus. The predicted FcLS proteins showed a high amino acid sequence identity with other Citrus limonene synthases and also had the typical structures of limonene synthase protein. FcLS1 was validated to be a functional d-limonene synthase by prokaryotic expression.

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

BackgroundLimonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly.ResultsIn this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization.ConclusionsThis study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

Engineering d-limonene synthase down-regulation in orange fruit induces resistance against the fungus Phyllosticta citricarpa through enhanced accumulation of monoterpene alcohols and activation of defence.

Terpene volatiles play an important role in the interactions between specialized pathogens and fruits. Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, is associated with crop losses in different citrus-growing areas worldwide. The pathogen may infect the fruit for 20-24weeks after petal fall, but the typical hard spot symptoms appear when the fruit have almost reached maturity, caused by fungal colonization and the induction of cell lysis around essential oil cavities. d-Limonene represents approximately 95% of the total oil gland content in mature orange fruit. Herein, we investigated whether orange fruit with reduced d-limonene content in peel oil glands via an antisense (AS) approach may affect fruit interaction with P.citricarpa relative to empty vector (EV) controls. AS fruit showed enhanced resistance to the fungus relative to EV fruit. Because of the reduced d-limonene content, an over-accumulation of linalool and other monoterpene alcohols was found in AS relative to EV fruit. A global gene expression analysis at 2h and 8days after inoculation with P.citricarpa revealed the activation of defence responses in AS fruit via the up-regulation of different pathogenesis-related (PR) protein genes, probably as a result of enhanced constitutive accumulation of linalool and other alcohols. When assayed invitro and invivo, monoterpene alcohols at the concentrations present in AS fruit showed strong antifungal activity. We show here that terpene engineering in fruit peels could be a promising method for the development of new strategies to obtain resistance to fruit diseases.

Impact of d-limonene synthase up- or down-regulation on sweet orange fruit and juice odor perception

Citrus fruits are characterized by a complex mixture of volatiles making up their characteristic aromas, being the d-limonene the most abundant one. However, its role on citrus fruit and juice odor is controversial. Transgenic oranges engineered for alterations in the presence or concentration of few related chemical groups enable asking precise questions about their contribution to overall odor, either positive or negative, as perceived by the human nose. Here, either down- or up-regulation of a d-limonene synthase allowed us to infer that a decrease of as much as 51 times in d-limonene and an increase of as much as 3.2 times in linalool in juice were neutral for odor perception while an increase of only 3 times in ethyl esters stimulated the preference of 66% of the judges. The ability to address these questions presents exciting opportunities to understand the basic principles of selection of food.

Resistance to pathogens in terpene down-regulated orange fruits inversely correlates with the accumulation of D-limonene in peel oil glands

Volatile organic compounds (VOCs) are secondary metabolites acting as a language for the communication of plants with the environment. In orange fruits, the monoterpene D-limonene accumulates at very high levels in oil glands from the peel. Drastic down-regulation of D-limonene synthase gene expression in the peel of transgenic oranges harboring a D-limonene synthase transgene in antisense (AS) configuration altered the monoterpene profile in oil glands, mainly resulting in reduced accumulation of D-limonene. This led to fruit resistance against Penicillium digitatum (Pd), Xanthomonas citri subsp. citri (Xcc) and other specialized pathogens. Here, we analyze resistance to pathogens in independent AS and empty vector (EV) lines, which have low, medium or high D-limonene concentrations and show that the level of resistance is inversely related to the accumulation of D-limonene in orange peels, thus explaining the need of high D-limonene accumulation in mature oranges in nature for the efficient attraction of specialized microorganism frugivores.

Presence of monoterpene synthase in four Labiatae species and Solid-Phase Microextraction- Gas chromatography-Mass Spectroscopy analysis of their aroma profiles.

Background:The family Lamiaceae (Labiatae) has included some medicinal plants. some monoterpene synthases, including linalool and limonene synthases, have been cloned and functionally characterized from several plants of Labiatae family.Materials and Methods:In this study, presence of linalool and limonene synthases, in four species of Labiatae family including Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea has been determined by molecular biological techniques together with the Head space SPME – GC-MS analysis of the aroma profile of these species.Results:Indicated that none of the plant species produced distinguishable bands with primer pairs related to d-limonene synthase. Distinguishable bands around 1800 bp in cDNA samples of L. angustifolia, H. officinalis and S. sclarea were observed regarding to the presence of linalool synthase. Head space SPME-GC-MS analysis of the aroma profiles of the above-mentioned plants showed that linalool (31.0%), linalyl acetate (18.2%), were found as the major compounds of L. angustifolia, while geraniol (5.5%), nerol (34.0%) and α- citral (52.0%) were identified as the main compounds of the N. cataria. The major components of H. officinalis and S. sclarea oils were determined as cis-pinocamphone (57.3%), and linalool (19.0%), linalyl acetate (51.5%), respectively.Conclusion:H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above-mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene. The profiles of the main components of these plants are in agreement with molecular assays.

Terpene Down-Regulation Triggers Defense Responses in Transgenic Orange Leading to Resistance against Fungal Pathogens

Terpenoid volatiles are isoprene compounds that are emitted by plants to communicate with the environment. In addition to their function in repelling herbivores and attracting carnivorous predators in green tissues, the presumed primary function of terpenoid volatiles released from mature fruits is the attraction of seed-dispersing animals. Mature oranges (Citrus sinensis) primarily accumulate terpenes in peel oil glands, with d-limonene accounting for approximately 97% of the total volatile terpenes. In a previous report, we showed that down-regulation of a d-limonene synthase gene alters monoterpene levels in orange antisense (AS) fruits, leading to resistance against Penicillium digitatum infection. A global gene expression analysis of AS versus empty vector (EV) transgenic fruits revealed that the down-regulation of d-limonene up-regulated genes involved in the innate immune response. Basal levels of jasmonic acid were substantially higher in the EV compared with AS oranges. Upon fungal challenge, salicylic acid levels were triggered in EV samples, while jasmonic acid metabolism and signaling were drastically increased in AS orange peels. In nature, d-limonene levels increase in orange fruit once the seeds are fully viable. The inverse correlation between the increase in d-limonene content and the decrease in the defense response suggests that d-limonene promotes infection by microorganisms that are likely involved in facilitating access to the pulp for seed-dispersing frugivores.

Production of the monoterpene limonene and modulation of apoptosis‐related proteins in embryonic‐mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from Japanese catnip (Schizonepeta tenuifolia)

The monoterpene D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo. Unlike plants, animals are unable to synthesize limonene de novo and obtain limonene through dietary sources. In the present study we established transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D-limonene synthase gene obtained from Japanese catnip (Schizonepeta tenuifolia). Apoptosis was not observed in the limonene-producing cells. A concomitant increase in the level of apoptosis-related protein Bcl-2 (B-cell lymphoma protein 2) and decreases in the levels of Bad (Bcl-2 antagonist of cell death) and phosphorylated JNK (c-Jun N-terminal kinase) were observed in limonene-producing cells. Limonene-producing cells may provide a useful new system to investigate the in vivo function of this monoterpene.

Characterization of the 5' Flanking Region of the Citrus d-Limonene Synthase Gene, Which Shows a Quantitatively Preferential Expression in Peel

To obtain a peel-specific promoter useful for citrus transgenic research, the 5' flanking region of a citrus d-limonene synthase gene (CitMTSE2), which showed a high degree of expression in the peel of mature fruits, was isolated from the satsuma mandarin (Citrus unshiu Marc.) Bacterial Artificial Chromosome (BAC) library. Promoter activity was characterized by particle bombardment, agroinjection and Arabidopsis transformation analyses. Approximately 5 kbps in the 5' upstream region (PCuMTSE2) contained several cis-motifs relating to guard cell-specific and stress-related genes. PCuMTSE2 fused to the uidA gene was directly incorporated into tissues from citrus and its relatives using particle bombardment. The results showed that PCuMTSE2 conferred β-glucronidase (GUS) activity in fruits rather than in seed and leaf tissues. Agroinjection analysis using citrus fruits showed PCuMTSE2 has promoter activity in peel but not in juice sacs. In a transgenic Arabidopsis-incorporated PCuMTSE2::uidA construct, GUS activity was detected in the joint between the stalk and silique. These results suggest that PCuMTSE2 could be utilized as a promoter regulating the quantitatively preferential expression in peel, and is useful for studies of manipulation by genetic engineering in citrus.

Isolation and characterization of a new d-limonene synthase gene with a different expression pattern in Citrus unshiu Marc

We isolated a new d-limonene synthase gene (CitMTSE2) from Satsuma mandarin ( Citrus unshiu Marc.) and compared its mRNA expression pattern with that of the previously isolated CitMTSE1. The predicted protein of CitMTSE2 showed 84.2% identity to that of CitMTSE1 at the amino acid level and possessed the typical structure and chemical features of general monoterpene synthases, such as transit peptides in the N-terminus, sequence motifs of RR and DDXXD, and cation-dependent reactions. A functional test demonstrated that CitMTSE2 is a d-limonene synthase gene. Under different expression regulations in response to the fruit developmental stage, the mRNA expression of CitMTSE2 indicated a specificity to fruit tissue and was different from that of CitMTSE1. Occurring mainly in peel at an early stage of fruit development, the mRNA expression profile of CitMTSE2 was similar to that of d-limonene biosynthesis in C. unshiu.

Molecular cloning and functional characterization of four monoterpene synthase genes from Citrus unshiu Marc.

We isolated four novel cDNA clones for monoterpene synthase genes, CitMTSE1, CitMTS3, CitMTS61, and CitMTS62, from Satsuma mandarin ( Citrus unshiu Marc.). Functional tests of in vitro translation demonstrated that CitMTSE1, CitMTS3, CitMTS61, and CitMTS62 coded d-limonene synthase, gamma-terpinene synthase, gamma-terpinene synthase, and beta-pinene synthase, respectively. Their predicted proteins possessed typical characteristics for monoterpene synthases, such as transit peptides in the N-terminus, RR sequences, and DDXXD motifs. In citrus monoterpene synthase genes, the enzymatic product-specific interval length variations among conservative domains were conserved among interspecies, and they were conspicuous between limonene synthase genes and other cyclic monoterpene synthase genes, although the total amino acid length of each clone was similar. A phylogenetic tree indicated that differentiation in citrus monoterpene synthases might occur before the divergence of Citrus species. Besides, the mRNA expressions of most clones were active in the peel at an early stage of fruit development. CitMTSE1 and CitMTS3 were also powerfully expressed in the flower at anthesis, suggesting a different regulation mechanism.

Molecular cloning, functional expression and characterization of d-limonene synthase from Agastache rugosa.

We cloned the gene of d-limonene synthase (ArLMS) from Agastache rugosa (Labiatae). The function of ArLMS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. ArLMS consisted of 2077 nucleotides including 1839 bp of coding sequence that encodes a protein of 613 amino acids. This protein has a 60 kDa molecular weight, which is identical to that of d-limonene synthase from Schizonepeta tenuifolia (Labiatae). The deduced amino acid sequence of ArLMS shows high homology with the known d- and l-limonene synthases from Labiatae plants. Here, we discussed the amino acid residues responsible for the stereochemical regulation in limonene biosynthesis.

Molecular cloning, functional expression and characterization of d-limonene synthase from Schizonepeta tenuifolia.

Limonene is one of the most simple cyclic monoterpenes, and two enantiomers, d- and l-limonene occur due to the chiral carbon at 4-position. Cyclization of GPP into limonene is catalyzed by the limonene synthase, and some l-limonene synthase cDNAs have already been cloned from several species of plants, mainly from Labiatae family. However, the d-limonene synthase gene has not yet been obtained, therefore, no information is available on the molecular mechanism of stereochemical regulation in limonene formation. To resolve this, we cloned the d-limonene synthase gene (dLMS) from Schizonepeta tenuifolia (Labiatae) by a reverse genetic approach, and we found that both d- and l-limonene synthase share similar features such as a transit peptide, an arginine rich domain, and a metal cation binding site in their structures. Here, we report on the cloning of dLMS, and the putative stereochemical regulation mechanism is discussed based on the comparison of the deduced amino acid sequence of dLMS with those of known l-limonene synthases.