Farnesyl Diphosphate Synthase Gene Research Articles

Farnesyl Diphosphate Synthase Gene Associated with Loss of Bone Mass Density and Alendronate Treatment Failure in Patients with Primary Osteoporosis

Farnesyl Diphosphate Synthase Gene Associated with Loss of Bone Mass Density and Alendronate Treatment Failure in Patients with Primary Osteoporosis

Carvacrol as a Stimulant of the Expression of Key Genes of the Ginsenoside Biosynthesis Pathway and Its Effect on the Production of Ginseng Saponins in Panax quinquefolium Hairy Root Cultures.

The accumulation of ginsenosides (triterpenic saponins) was determined in Panax quinquefolium hairy root cultures subjected to an elicitation process using carvacrol at 5, 10, 25, 50, 100, 250, and 500 μM concentrations during 24 and 72 h exposure. This study was the first one in which carvacrol was applied as an elicitor. The content of eight ginsenosides, Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Re, was determined using HPLC analysis. Moreover, the quantitative RT-PCR method was applied to assess the relative expression level of farnesyl diphosphate synthase, squalene synthase, and dammarenediol synthase genes in the studied cultures. The addition of carvacrol (100 μM) was an effective approach to increase the production of ginsenosides. The highest content and productivity of all detected saponins were, respectively, 20.01 mg∙g-1 d.w. and 5.74 mg∙L-1∙day-1 after 72 h elicitation. The production profile of individual metabolites in P. quinquefolium cultures changed under the influence of carvacrol. The biosynthesis of most examined protopanaxadiol derivatives was reduced under carvacrol treatment. In contrast, the levels of ginsenosides belonging to the Rg group increased. The strongest effect of carvacrol was noticed for Re metabolites, achieving a 7.72-fold increase in comparison to the control. Saponin Rg2, not detected in untreated samples, was accumulated after carvacrol stimulation, reaching its maximum concentration after 72 h exposure to 10 μM elicitor.

Boosting production of cembratriene-ol in Saccharomyces cerevisiae via systematic optimization.

Cembratriene-ol is a good biodegradable biopesticide ingredient with future potential applications in the field of sustainable agriculture. Cembratriene-ol is a monocyclic diterpenoid compound that is synthesized only in the trichome gland of Nicotiana plants. In this study, geranylgeranyl diphosphate synthase gene ggpps from Taxus canadensis and cbts*Δp were heterologously expressed in Saccharomyces cerevisiae W303-1A to successfully synthesize cembratriene-ol. The titer of cembratriene-ol was increased by 1.84-fold compared to the control by overexpressing the S. cerevisiae bifunctional (2E,6E)-farnesyl diphosphate synthase genes ERG20 and cbts*Δp under one promoter PGAP . The titer of cembratriene-ol in the engineered S. cerevisiae BY4741 was increased by 1.39-fold compared to the engineered S. cerevisiae W303-1A. The titer of cembratriene-ol in the engineered S. cerevisiae BY4741 was increased by 2.22-fold compared to the control by overexpressing ERG20 and cbts*Δp, respectively, using two promoters PGAP . Cembratriene-ol was found to be successfully synthesized via the integrated expression of cbts*Δp, ggpps and ERG20 on the genome of S. cerevisiae BY4741. The titer of cembratriene-ol in S. cerevisiae S25 was further increased by 1.80-fold compared to the control via dynamic control of the squalene synthase gene ERG9. Overexpression of the genes cbts*Δp and ggpps using pY26-GPD-TEF in S. cerevisiae S25 with their integration expression increased the titer of cembratriene-ol by 26.1-fold compared to S. cerevisiae S25. The titer of cembratriene-ol was significantly enhanced by mitochondrial compartmentalized expression of cbts*Δp and ggpps, which was 76.3-fold higher than that of the initial strain constructed. It was indicated that the systematic optimization has great potential in facilitating high-level production of cembratriene-ol production in S. cerevisiae.

Expression of PnSS Promotes Squalene and Oleanolic Acid (OA) Accumulation in Aralia elata via Methyl Jasmonate (MeJA) Induction.

Aralia elata is an important herb due to the abundance of pentacyclic triterpenoid saponins whose important precursors are squalene and OA. Here, we found that MeJA treatment promoted both precursors accumulation, especially the latter, in transgenic A. elata, overexpressing a squalene synthase gene from Panax notoginseng(PnSS). In this study, Rhizobium-mediated transformation was used to express the PnSS gene. Gene expression analysis and high-performance liquid chromatography (HPLC) were used to identify the effect of MeJA on squalene and OA accumulation. The PnSS gene was isolated and expressed in A. elata. Transgenic lines showed a very high expression of the PnSS gene and farnesyl diphosphate synthase gene (AeFPS) and a slightly higher squalene content than the wild-type, but endogenous squalene synthase (AeSS), squalene epoxidase (AeSE), and β-amyrin synthase (Aeβ-AS) gene were decreased as well as OA content. Following one day of MeJA treatment, the expression levels of PeSS, AeSS, and AeSE genes increased significantly. On day 3, the maximum content of both products reached 17.34 and 0.70 mg·g-1, which increased 1.39- and 4.90-fold than in the same lines without treatment. Transgenic lines expressing PnSS gene had a limited capability to promote squalene and OA accumulation. MeJA strongly activated their biosynthesis pathways, leading to enhance yield.

A new sesquiterpene synthase catalyzing the formation of (R)-β-bisabolene from medicinal plant Colquhounia coccinea var. mollis and its anti-adipogenic and antibacterial activities

The sesquiterpene β-bisabolene possessing R and S configurations is commonly found in plant essential oils with antimicrobial and antioxidant activities. Here, we report the cloning and functional characterization of a (R)-β-bisabolene synthase gene (CcTPS2) from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. The biochemical function of CcTPS2 catalyzing the cyclization of farnesyl diphosphate to form a single product (R)-β-bisabolene was characterized through an engineered Escherichia coli producing diverse polyprenyl diphosphate precursors and in vitro enzyme assay, indicating that CcTPS2 was a high-fidelity (R)-β-bisabolene synthase. The production of (R)-β-bisabolene in an engineered E. coli strain harboring the exogenous mevalonate pathway, farnesyl diphosphate synthase and CcTPS1 genes was 17 mg/L under shaking flask conditions. Ultimately, 120 mg of purified (R)-β-bisabolene was obtained from the engineered E. coli, and its structure was elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, and specific rotation). Four chimeric enzymes were constructed through domain swapping, which altered the product outcome, indicating the region important for substrate and product specificity. In addition, (R)-β-bisabolene exhibited anti-adipogenic activity in the model organism Caenorhabditis elegans and antibacterial activity selectively against Gram-positive bacteria.

Functional characterization and transcriptional activity analysis of Dryopteris fragrans farnesyl diphosphate synthase genes.

Farnesyl diphosphate synthase (FPS), a key enzyme of the terpene metabolic pathway, catalyzes the precursor of sesquiterpene compounds farnesyl diphosphate (FPP) synthesis, and plays an important role in regulating plant growth and development. Dryopteris fragrans is a medicinal plant rich terpenoids. In this study, the function of the gene was verified in vitro and in vivo, the promoter of the gene was amplified and its transcriptional activity was analyzed. In the present study, we report the molecular cloning and functional characterization of DfFPS1 and DfFPS2, two FPS genes from D. fragrans. We found that the two genes were evolutionarily conserved. Both DfFPS genes were highly expressed in the gametophyte and mature sporophyte leaves, and their expression levels increased in response to methyl jasmonate (MeJA) and high temperature. Both DfFPS proteins were localized in the cytoplasm and could catalyze FPP synthesis in vitro. We also found that the overexpression of DfFPS genes in tobacco plants promoted secondary metabolite accumulation but exhibited negligible effect on plant growth and development. However, the transgenic plants exhibited tolerance to high temperature and drought. The promoters of the two genes were amplified using fusion primer and nested integrated polymerase chain reaction (FPNI-PCR). The promoter sequences were truncated and their activity was examined using the β-glucuronidase (GUS) gene reporter system in tobacco leaves, and we found that both genes were expressed in the stomata. The transcriptional activity of the promoters was found to be similar to the expression pattern of the genes, and the transcriptional core regions of the two genes were mainly between -943 bp and -740 bp of proDfFPS1. Therefore, we present a preliminary study on the function and transcriptional activity of the FPS genes of D. fragrans and provide a basis for the regulation of terpene metabolism in D. fragrans. The results also provide a novel basis for the elucidation of terpene metabolic pathways in ferns.

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.

Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Isolation and characterization of a gene encoding farnesyl diphosphate synthase from \(\textit{Panax vietnamensis}\) Ha et Grushv

Panax vietnamensis Ha et Grushv. is a species of the genus Panax native to Central Vietnam, containing a family of triterpene saponins named ginsenosides. This group of biomolecules possesses valuable therapeutic properties against cancer, hepatitis, diabetes, inflammation as well as stress and anxiety. Farnesyl diphosphate synthase (FPS) is a key enzyme participating in the ginsenoside biosynthesis pathway. In this study, a FPS gene from P. vietnamensis (PvFPS) was isolated and characterized. The PvFPS cDNA contained an open reading frame of 1032 bp, encoding a polypeptide chain of 342 amino acid residues. Nucleotide sequence comparison showed that FPS was highly conserved among most species, with two Aspartate-rich motifs responsible for product chain length determination strongly sustained. PvFPS was closely related to those of the same genera and order and differed from those from other kingdoms. PvFPS expression was detected at a greater level in root tissues than in leaves in all ages. Our findings provided information concerning the properties of a crucial gene in the ginsenoside biosynthesis, thus enhancing our understanding of this important pathway.

The relationship between EuSRPP1 gene expression and rubber biosynthesis in Eucommia ulmoides Oliver (Du-zhong)

Because of the unique properties, trans-polyisoprene rubber (TPI) has gradually realized that it has great development potential. Eucommia ulmoides Oliver (E. ulmoides Oliver), also named Du-zhong, is one of the few woody plants producing TPI. The significant expansion of small rubber particle protein and rubber elongation factor (SRPP/REF) genes in E. ulmoides Oliver may be associated with the producing of TPI. To understand the relationship between EuSRPP1 gene expression and rubber biosynthesis in E. ulmoides Oliver, in this study, an overexpression transgenic (OT) vector of EuSRPP1 driven by the act promoter and an RNA interference (RNAi) expression vector of EuSRPP1 were constructed. Then, Agrobacterium-mediated genetic transformation of E. ulmoides Oliver was conducted to obtain the OT and RNAi seedlings. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) showed that the EuSRPP1 expression levels in OT seedlings significantly increased, and were 2.53 × and 3.68 × higher in OT-1 and OT-2 seedlings, respectively, than the wild-type (WT) seedlings, Meanwhile, the expression levels of E. ulmoides Isopentenyl Diphosphate Isomerase (EuIPPS) and E. ulmoides Farnesyl Diphosphate Synthase (EuFPS) genes in OT plants, which are related to rubber biosynthesis synthesis, were also up-regulated. To the contrary, EuSRPP1 gene expression levels in the RNAi-1 and RNAi-2 seedlings significantly decreased, which were 3.92% and 42.38% of the WT seedlings, respectively. EuIPPS and EuFPS genes expression levels in the RNAi seedlings were also down-regulated. The TPI content analysis revealed that the TPI content in the overexpression EuSRPP1 callus increased to 1.85 × that of the WT callus, whereas that in the RNAi callus decreased to only 52% of the WT callus. Molecular weights of rubber were significantly different in OT callus (2.31 × 106 Da) and RNAi callus (0.36 × 106 Da). Observation of the rubber particles revealed that the rubber particle electron density was higher in OT seedlings, and the immunogold localization results showed that EuSRPP1 was localized to rubber particles. These results indicate that the EuSRPP1 expression level influences the chain elongation, particle development, and content of rubber in E. ulmoides Oliver.

High-Level Patchoulol Biosynthesis in Artemisia annua L.

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

Stereospecific linalool production utilizing two-phase cultivation system in Pantoea ananatis

Linalool is a monoterpene alcohol, which imparts floral scents to a variety of plants and is extensively used in various kinds of products, such as processed foods and beverages for fragrances and flavors. However, linalool from natural resources is racemate, and production of linalool enantiomers is difficult. To produce stereospecific linalool, we evaluated linalool synthase genes (LINS) from plants, such as Actinidia arguta (AaLINS) and Coriandrum sativum (CsLINS) for (S)-specific LINS and a gram-positive bacterium Streptomyces clavuligerus (ScLINS) for (R)-specific LINS, with Pantoea ananatis strain as the host. Among the 16 LINS examined, AaLINS and ScLINS showed the best (S)-linalool production and (R)-linalool production, respectively, with 100 % enantio excess. Co-expression of the mutated farnesyl diphosphate synthase gene, ispA* (S80 F), from Escherichia coli along with the LINS genes also improved linalool production. In order to prevent volatilization and cell toxicity of linalool, two-phase cultivation with isopropyl myristate was done, which had positive effects on linalool production. The carbon flux to the MVA pathway from glucose was increased by inactivating a membrane-bound glucose dehydrogenase. Overall, 5.60 g/L (S)-linalool and 3.71 g/L (R)-linalool were produced from 60.0 g/L glucose by introduction of AaLINS-ispA* and ScLINS-ispA* in P. ananatis, respectively.

Effects of Exogenous Elicitors on Triterpenoids Accumulation and Expression of Farnesyl Diphosphate Synthase Gene in Inonotus obliquus

Exogenous elicitors can influence accumulation of triterpenoids by regulating FPS (farnesyl pyrophosphate synthase) in Inonotus obliquus, such as birch bark (BB), birch rhizosphere soil (BS), Frankia alni (FA), and Rhizobium indigoferae (RI). Among them, the highest yield of biomass (15.6 mg/mL) could be detected by treatment with RI at 2 µg/mL for 10 d, about 2.1-fold of the control. Results showed the significant effect of elicitors on accumulation of triterpenoids by regulating FPS expression level. RI stimulated mycelium to achieve the highest accumulation of triterpenoids about 48.2 mg/g, which was about over 4-fold of the control (9.5 mg/g). Its effect on FPS expression level was greater than that of others, which was about over 10-fold of the control. However, accumulation of triterpenoids and FPS expression level were clearly down-regulated under treatment with BS. In addition, two endogenous factors (H2O2 and NO) in I. obliquus could affect accumulation of triterpenoids by regulating FPS expression level. Effect of RI on H2O2 and NO contents were higher than that of others, about 3.5-fold of the control. The minimum value of H2O2 content was detected by treatment with BS for 10 d, which was about 0.5-fold of the control. RI that assist with CAT (catalase) and SNP (sodium nitroprusside dehydrate) could stimulate triterpenoid contents to achieve highest accumulation, which was about 5.5-fold of the control. Fluctuation of H2O2 and NO content seems to play a pivotal role in accumulation of triterpenoids by regulating FPS expression level.

Production of glutamate and stereospecific flavors, (S)-linalool and (+)-valencene, by Synechocystis sp. PCC6803

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3g/L in 143h (34°C, 100μmolm-2s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4mg/L (S)-linalool in 160h (30°C, 50μmolm-2s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E.coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6mg/L (S)-linalool in 160h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E.coli were introduced, and the resultant strain produced 9.6mg/L of (+)-valencene in 166h (30°C, 50μmolm-2s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.

Production of the Inaccessible Sesquiterpene (-)-5-Epieremophilene by Metabolically Engineered Escherichia coli.

(-)-5-Epieremophilene, an epimer of the versatile sesquiterpene (+)-valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1-3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (-)-5-epieremophilene production with yields of 42.4-76.0 mg/L in shake-flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1-FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (-)-5-epieremophilene were achieved. Biological assay indicated that (-)-5-epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC50 =1.25 μg/cm2 ), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (-)-5-epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.

Clinical, Diagnostic and Prognostic Significance of Farnesyl Diphosphate Synthase Gene Polymorphism in Patients with Osteoarthritis: Decreased Bone Density and Overweight

A comprehensive study of 96 patients with osteoarthritis (OA) revealed that AA homozygotes prevail, the frequency of which is significantly higher than heterozygotes or homozygotes with the SS genotype (53.1 ± 5.1 %, 41.7 ± 5.0 % and 5, 2 ± 2.3 %, respectively; p <0.05). The frequency of homozygotes with the AA genotype significantly (almost 8–10 times; p <0.001) prevails over the frequency of SS homozygotes. The generalized WOMAC index in the patient groups was 48.8 ± 1.8 % and was large in AA homozygotes with a tendency to prevail in the severity structure of the manifestations of stiffness. Among homozygotes, AA have an increased body mass (BM) or obesity of 83.3 ± 4.8 %, among heterozygotes - 75.0 ± 6.8 %, among homozygotes of SS - 80.0 ± 19.1 % of people. The apelin content in the blood plasma of patients with OA depending on the BM and the variant polymorphism of the FDPS gene is characterized by the fact that with homozygosity SS, in the case of an increase in BM, the apelin content decreases, while with AA homozygosity it does not change significantly. The relationship between the FDPS gene polymorphism and the OA stage is characterized by the prevalence of more severe radiological signs of articular cartilage degradation among homozygous AA alleles.

Features of the Structural and Functional State of Bone Tissue in Patients with Osteoarthritis: Analysis of the Relationship with the Plasma Content of Apelin and Polymorphism of the Farnesyl Diphosphate Synthase Gene

The objective: to study the effect of the level of plasma apelin in relation to the polymorphism of the farnesyl diphosphate synthase gene and the structural and functional state of bone tissue (SPSCT) in patients with osteoarthritis (OA).Materials and methods. In a comprehensive study of 96 patients with OA, it was found that among all patients who took part in the study, patients with homozygous AA genotype (according to FDPS) prevailed, the frequency of occurrence of which was significantly higher than in patients with heterozygous AS genotype and homozygous SS: 53,1 ± 5.1%, 41.7 ± 5.0% and 5.2 ± 2.3%, respectively (p <0.05).Results. The frequency of patients with a homozygous genotype AA is significantly (almost 8–10 times; p <0.001) higher than the frequency of occurrence of patients with a homozygous SS genotype. In patients with a homozygous SS genotype among patients with OA comorbid with osteoporosis, the level of plasma apelin was significantly higher than in patients with OA with osteopenia: 72.49 ± 3.84 pg/cm3 and 42.97 ± 2.15 pg/cm3 respectively (p <0.05). As a result of the study, the effect of FDPS gene polymorphism on the levels of variability of the concentration of plasma apelin was proved. It should be noted that in patients with OA with the AA genotype without violations of SPSCT, plasma apelin levels differed from patients with osteopenia and significantly exceeded the same indicator in patients of the genotyped group for osteoporosis (p <0.05).Conclusion. The effect of the gene on the frequency and nature of violations of the structural and functional state of bone tissue in patients with osteoarthritis (OA) has been proved: the homozygous AA variant is the most prognostically unfavorable, in the presence of which the formation of combined osteopenia in patients with OA is associated with an increase in the level of plasma apelin, and osteoporosis with its decrease . In this case, an increase in the concentration of apelin in osteopenia can be assessed as transient and used as a prognostic marker.

Identification and Characterization of trans-Isopentenyl Diphosphate Synthases Involved in Herbivory-Induced Volatile Terpene Formation in Populus trichocarpa.

In response to insect herbivory, poplar releases a blend of volatiles that plays important roles in plant defense. Although the volatile bouquet is highly complex and comprises several classes of compounds, it is dominated by mono- and sesquiterpenes. The most common precursors for mono- and sesquiterpenes, geranyl diphosphate (GPP) and (E,E)-farnesyl diphosphate (FPP), respectively, are in general produced by homodimeric or heterodimeric trans-isopentenyl diphosphate synthases (trans-IDSs) that belong to the family of prenyltransferases. To understand the molecular basis of herbivory-induced terpene formation in poplar, we investigated the trans-IDS gene family in the western balsam poplar Populus trichocarpa. Sequence comparisons suggested that this species possesses a single FPP synthase gene (PtFPPS1) and four genes encoding two large subunits (PtGPPS1.LSU and PtGPPS2.LSU) and two small subunits (PtGPPS.SSU1 and PtGPPS.SSU2) of GPP synthases. Transcript accumulation of PtGPPS1.LSU and PtGPPS.SSU1 was significantly upregulated upon leaf herbivory, while the expression of PtFPPS1, PtGPPS2.LSU, and PtGPPS.SSU2 was not influenced by the herbivore treatment. Heterologous expression and biochemical characterization of recombinant PtFPPS1, PtGPPS1.LSU, and PtGPPS2.LSU confirmed their respective IDS activities. Recombinant PtGPPS.SSU1 and PtGPPS.SSU2, however, had no enzymatic activity on their own, but PtGPPS.SSU1 enhanced the GPP synthase activities of PtGPPS1.LSU and PtGPPS2.LSU in vitro. Altogether, our data suggest that PtGPPS1.LSU and PtGPPS2.LSU in combination with PtGPPS.SSU1 may provide the substrate for herbivory-induced monoterpene formation in P. trichocarpa. The sole FPP synthase PtFPPS1 likely produces FPP for both primary and specialized metabolism in this plant species.

Increased production of ganoderic acids by overexpression of homologous farnesyl diphosphate synthase and kinetic modeling of ganoderic acid production in Ganoderma lucidum

BackgroundGanoderic acids (GAs), derived from the medicinal mushroom Ganoderma lucidum, possess anticancer and other important pharmacological activities. To improve production of GAs, a homologous farnesyl diphosphate synthase (FPS) gene was overexpressed in G. lucidum. Moreover, the influence of FPS gene overexpression on GA production was investigated by developing the corresponding mathematical models.ResultsThe maximum levels of total GAs and individual GAs (GA-T, GA-S, and GA-Me) in the transgenic strain were 2.76 mg/100 mg dry weight (DW), 41 ± 2, 21 ± 5, and 28 ± 1 μg/100 mg DW, respectively, which were increased by 2.28-, 2.27-, 2.62-, and 2.80-folds compared with those in the control. Transcription levels of squalene synthase (SQS) and lanosterol synthase (LS) genes during GA biosynthesis were upregulated by 2.28- and 1.73-folds, respectively, in the transgenic G. lucidum. In addition, the developed unstructured models had a satisfactory fit for the process of GA production in submerged cultures of G. lucidum. Analysis of the kinetic process showed that FPS gene overexpression had a stronger positive impact on GA production compared with its influence on cell growth. Also, FPS gene overexpression led to a higher non-growth-associated-constant β (1.151) over the growth-associated-constant α (0.026) in the developed models.ConclusionsFPS gene overexpression is an effective strategy to improve the production of GAs in G. lucidum. The developed mathematical models are useful for developing a better GA production process in future large-scale bioreactors.

Production of alarm pheromone starts at embryo stage and is modulated by rearing conditions and farnesyl diphosphate synthase genes in the bird cherry-oat aphid Rhopalosiphum padi.

The major component of aphid alarm pheromone is (E)-β-farnesene (EβF), but the molecular mechanisms of EβF synthesis are poorly understood. Here we established a biological model to study the modulation of EβF synthesis in the bird cherry-oat aphid Rhopalosiphum padi by using quantitative polymerase chain reaction, gas chromatography/mass spectrometry and RNA interference. Our results showed that the rearing conditions significantly affected the weight of adult and modulated EβF synthesis in a transgenerational manner. Specifically, the quantity of EβF per milligram of aphid was significantly reduced in the individually reared adult or 1st-instar nymphs derived from 1-day-old adult reared individually, but EβF in the nymph derived from 2-day-old adult that experienced collective conditions returned to normal. Further study revealed that the production of EβF started in embryo and was extended to early nymphal stage, which was modulated by farnesyl diphosphate synthase genes (RpFPPS1 and RpFPPS2) and rearing conditions. Knockdown of RpFPPS1 and RpFPPS2 confirmed the role played by FPPS in the biosynthesis of aphid alarm pheromone. Our results suggested that the production of EβF starts at the embryo stage and is modulated by FPPS and rearing conditions in R. padi, which sheds lights on the modulatory mechanisms of EβF in the aphid.