Mevalonic Acid Research Articles

Mevalonate Diphosphate Decarboxylase MVD/Erg19 Is Required for Ergosterol Biosynthesis, Growth, Sporulation and Stress Tolerance in Aspergillus oryzae.

Mevalonate diphosphate decarboxylase (MVD; EC 4.1.1.33) is a key enzyme of the mevalonic acid (MVA) pathway. In fungi, the MVA pathway functions as upstream of ergosterol biosynthesis, and MVD is also known as Erg19. Previously, we have identified Aoerg19 in Aspergillus oryzae using bioinformatic analysis. In this study, we showed that AoErg19 function is conserved using phylogenetic analysis and yeast complementation assay. Quantitative reverse transcription–PCR (qRT-PCR) indicated that Aoerg19 expression changed in different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg19 was located in the vacuole. Overexpression of Aoerg19 decreased the ergosterol content in A. oryzae, which may due to the feedback-mediated downregulation of Aoerg8. Consistent with the decrease in ergosterol content, both Aoerg19 overexpression and RNAi strains of A. oryzae are sensitive to abiotic stressors, including ergosterol biosynthesis inhibitor, temperature, salt and ethanol. Thus, we have identified the function of AoErg19 in A. oryzae, which may assist in genetic modification of MVA and the ergosterol biosynthesis pathway.

When neonatal inflammation does not mean infection: an early-onset mevalonate kinase deficiency with interstitial lung disease

Systemic inflammation in neonates is attributable to an infection in almost all cases. When inflammation persists, an autoinflammatory disease should be promptly suspected. We report here a case of mevalonate kinase deficiency (MKD) that presented at birth with mild symptoms and signs suggestive for a perinatal infection, together with the uncommon finding of interstitial lung disease. An extensive diagnostic work-up, performed after ineffective antibiotic treatment, demonstrated high levels of mevalonic acid in urine (7024 mM/M of creatinine, normal value <0.1). Next-generation sequencing showed a rare c.709A > T (p.T237S) homozygous mutation in the MVK gene, consistent with MKD. Treatment with anakinra led to a prompt resolution of symptoms and a sharp drop in serum inflammatory markers. The patient is now six months-old, currently undergoing evaluation for hematopoietic stem-cell transplantation. To our knowledge, this is the first case of MKD presenting within the first week of life with interstitial lung disease.

Transcriptome analysis identifies novel responses and potential regulatory genes involved in 12-deoxyphorbol-13-phenylacetate biosynthesis of Euphorbia resinifera

12-Deoxyphorbol-13-phenylacetate (DPP), found in Euphorbia resinifera, is a prototype of a new class of phorbol derivatives that function as protein kinase C activators with potent anti-tumor-promoting activity. To better understand the biosynthesis of DPP in E. resinifera and develop methods for its production, we used next-generation sequencing technologies to build a transcriptome dataset. We obtained a total of approximately 27,102,078 clean reads that were assembled into 90,448 unigenes. Of those, 129 and 59 unigenes were identified in the Terpenoid Backbone Biosynthesis (TBB) (KEGG map00900) and the Diterpenoid Biosynthesis (DB) (KEGG map00904) pathways, respectively. We investigated the expression patterns of 23 DPP biosynthesis-related genes and validated the results by quantitative real time PCR. The results showed that CMK (4-diphosphocytidyl-2-C-methyl-d-erythritol kinase), MECS (2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and IPI (Isopentenyl-diphosphate Delta-isomerase) of the methylerythritol 4-phosphate (MEP) pathway had a higher expression compared to other genes of TBB, whereas the expression levels of genes involved in the mevalonic acid (MVA) pathway were relatively lower and not significantly different from each other. Furthermore, the expression of the 23 genes in root, stem and young stem differed, and the statistical results indicated that HMGS (Hydroxymethylglutaryl-CoA synthase), MVK (mevalonic acid), MECT (2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase), MECS, HDS and IPI in the TBB pathway as well as KS in the DB pathway were significantly and positively correlated with the CS (casbene synthase) gene. These results suggested that both cytosol-localized MVA and plastid-localized MEP pathways may have a significant contribution in DPP accumulation, with MEP pathway playing a dominant role. To the best of our knowledge, this study is the first transcriptome analysis of E. resinifera and the results reported here will aid the identification of functional genes and better understanding of DPP biosynthesis.

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes

Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the β-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.

Pitavastatin Selectively Kills PTEN Knock Out Cells and Cancer Organoids in Mouse Model via the Mevalonate Pathway

PTEN is one of the most frequently mutated tumor suppressors in human cancer. Loss of PTEN leads to hyperactivation of the PI3K–AKT signaling pathway, cell migration, and cell proliferation. Aiming to identify specific drugs that could target cancer cells, we screened 2650 FDA approved compounds and discovered that certain statins selectively kill Dictyostelium pten‐cells. This result carries over to mammalian cells: MCF10A cells lacking PTEN are more sensitive to statins than MCF10A wild type (WT) cells. By testing the differential sensitivity between MCF10A WT and pten−/− cells with multiple statins, we found that pitavastatin showed the best performance. Mevalonic acid and geranylgeranyl pyrophosphate (GGPP), but not farnesyl phyrophosphate or squalene, rescued pitavastatin treated MCF10A pten−/− cells. Pitavastatin induced an increase in RhoA expression suggesting that the requirement for geranylgeranylation of this GTPase may mediate the sensitivity of MCF10A pten−/− cells. The different sensitivity between MCF10A WT and pten−/− cells also carried over to three‐dimensional culture model (3D). Moreover, we found pitavastatin significantly decreased the growth and Twist1‐induced cell dissemination in 3D culture of murine mammary gland organoids. Finally, we tested the effects of pitavastatin on growth and survival of WT(FVB) and tumor organoids (MMTV‐PyMT (luminal breast cancer model), C31Tag (basal breast cancer model)) in 3D culture. Both MMTV‐PyMT and C31Tag were killed by 100 nM pitavastatin, while WT organoids survived. These findings implicate pitavastatin as a potential therapeutic candidate for tumors.Support or Funding InformationThis work was supported by NIH Grant R35 GM118177.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The mevalonate coordinates energy input and cell proliferation

The mevalonate pathway is known for the synthesis of cholesterol, but recent studies have reported that it also controls Hippo signaling, which is critical for the regulation of organ size and tumorigenesis. Here, we discover that the suppression of the mevalonate pathway inhibits the growth and proliferation of colon cancer cell lines. The results of transcriptomic and proteomic assays suggested that the mevalonate pathway controls multiple signaling pathways relevant to cell proliferation, and the results were further confirmed using western blot, PCR, and immunofluorescence assays. As cell proliferation is an energy-consuming process, we postulate that the mevalonate pathway may also control nutrient uptake to coordinate the processes of energy supply and cell proliferation. Here, we found that lovastatin, a mevalonate pathway inhibitor, suppresses glucose and amino acid uptake and lactate acid production. More importantly, mevalonic acid itself is sufficient to promote glucose uptake by colon cancer cells. In addition, we found that colon cancer tissues displayed a higher expression of mevalonate pathway enzymes, which may promote cell growth and stimulate energy uptake. Together, our findings establish the mevalonate pathway as a critical regulator in coordinating energy input and cell proliferation.

Constructing Yeast Chimeric Pathways To Boost Lipophilic Terpene Synthesis.

Synthetic chimeric biological system offers opportunities to illuminate principles of designing life, and a primary step is constructing synthetic chimeric pathways. Here, we constructed yeast chimeric pathways by transferring the genes from Saccharomyces cerevisiae pathways into another budding yeast Yarrowia lipolytica for in vivo assembly. We efficiently diversified gene option, combination, localization order, and copy number as expected. Convergence of two yeast pathways, especially mevalonic acid (MVA) pathways, remarkably enhanced synthesis of a lipophilic terpene, lycopene. In the selected champion strain with 50-fold of enhanced lycopene production, the chimeric MVA pathway gathered three S.cerevisiae genes with particular copies and locations. Amazingly, therein we discovered distinct transcriptional up-regulation of three significant pathways correlated with acetyl-CoA supply and tuning of cellular lipid amounts and composition. Modulating these pathways further improved lycopene production to 150-fold, a final 259 mg/L (approximately 80 mg/g DCW). We primarily showed the capacityof boosting the synthesis of lipophilic products with yeast chimeric pathways.

Abstract P6-12-02: isomiR-140-3p-regulated mevalonic acid pathway as a potiental target for prevention of triple negative breast cancer

Abstract Background: Prevention of triple negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis. Methods: In order to identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represents early stages of TNBC development. We identified direct gene targets of an isomiRNA and using cell based and in vivo model systems we demonstrate the utility of targeting downstream pathways for prevention of TNBC. Results: These analyses showed that 5'isomiRNA of miR-140-3p (miR-140-3p-1) were deregulated in the normal-to- preneoplastic transition. We also identified novel direct gene targets of miR140-3p-1, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, and found that these too are deregulated in the normal to preneoplastic transition, resulting in activation of the cholesterol synthesis pathway. Upregulation in the cholesterol pathway creates a metabolic vulnerability that can be targeted with the statin class of inhibitors. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin inhibits growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin treatment induced HMGCR transcript levels in the explanted xenografts, with higher transcript induction directly correlated with the degree of histological progression of lesions, indicating an adaptive resistance mechanism that circumvents statin targeting of HMGCR. Therefore, we hypothesized that dual targeting of HMGCR and the feedback loops are necessary to overcome this adaptive resistance. To test this hypothesis, we generated MCF10AT1 cells resistant to fluvastatin. We then treated these resistance AT1 cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin effectively abrogated the statin-induced upregulation of HMGCR and sensitized these resistant cells to fluvastatin. Conclusions: Our preclinical data suggests that the statin treatment induced homeostatic upregulation of the cholesterol pathway is a barrier to effective chemoprevention with statins. However, our results also suggest that this adaptive resistance mechanism can be abrogated by combined treatment with statin and AMPK activators such as aspirin. Clinical studies of this combined regimen should be considered in high risk patients. Citation Format: Bhardwaj A, Singh H, Trinidad CM, Albarracin C, Hunt KK, Bedrosian I. isomiR-140-3p-regulated mevalonic acid pathway as a potiental target for prevention of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-12-02.

Ubiquinone Ameliorates Simvastatin Induced Respiratory Chain Complex Function Impairment in High‐Fat High‐Cholesterol Diet Fed Mice

Long‐term statins therapy is the primary treatment of hyperlipidemia. A ubiquinone (Co‐enzyme Q10, CoQ10) supplement ameliorates the statins side effects, including skeletal muscle and liver damage, whereas the mechanism involving respiratory chain complex function in a hyperlipidemia animal model remains unknown. Herein, it is reported that the administration of CoQ10 increases plasma CoQ10 level significantly without affecting the lipid lowering effects of statins in high‐fat high cholesterol diet fed mice. Compared with thr statins group, the combination therapy of CoQ10 and statin (Simva + CoQ10) reduces the activity enhancement of plasma CK, ALT, and AST, and restores the activity of complex I in skeletal muscle (2.7‐fold increased) and liver (42% increased). Moreover, the activities of complex II are increased in the muscle (29% increased) and liver (5.8‐folds increased) significantly. Statins decrease the total CoQ10 concentration in mitochondria, while Simva + CoQ10 restore the total CoQ10 concentration in mitochondria from muscle (41% increased) and liver (1.1‐folds increased), respectively. Therefore, administration of CoQ10 ameliorates the function loss of mitochondria respiratory chain complex induced by statins in muscle and liver are concluded.Practical Applications: Statins are widely used in clinical treatment of hypercholesterolemia and atherosclerotic diseases via inhibiting the synthesis of mevalonic acid to achieve the purpose of reducing endogenous cholesterol loading, to display anti‐inflammatory effects, and to stabilize the atherosclerotic plaques. However, the shortage of MVA may cause the low level of ubiquinone which subsequently leads to increasing various side effects, including muscle and liver damage. This study introduces a novel combined administration which could ameliorate the statins induced muscle and liver damage for hypercholesterolemia individuals. Moreover, the method shows a beneficial effect on mitochondrion integrity and functions in skeletal muscle and liver.Statin administration inhibits the 3‐hydroxy‐3‐methyl glutaryl coenzyme A reductase (HMGCoA‐R), which subsequently decreases the level of endogenous Co enzyme Q10 (CoQ). CoQ supplementation increases the level of CoQ in mitochondria, and ameliorates the function loss of mitochondrial respiratory chain complex induced by statins.

Expression patterns of some genes involved in tanshinone biosynthesis in Salvia miltiorrhiza roots

Tanshinones are the major bioactive components of Salvia miltiorrhiza, and synthesized through the cytosol-localized mevalonic acid (MVA) and plastid-localized methylerythritol 4-phosphate (MEP) pathways. To reveal correlations between gene expression and tanshinone accumulation, transcript-level variations in five candidate genes involved in tanshinone biosynthesis in the roots of S. miltiorrhiza were investigated at different developmental stages. Additionally, the accumulation of tanshinone I, tanshinone IIA, and cryptotanshinone were analyzed by liquid chromatography tandem mass spectrometry. Compared with the other genes examined, SmCMK (encoding the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase) expression was significantly positively correlated with tanshinone production, and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) likely functions as a non-rate-limiting enzyme in the tanshinone-biosynthesis pathways of S. miltiorrhiza during different developmental stages, with significant correlations observed between SmDXR and SmCMK expression. Furthermore, no significant correlations were observed between the expression of SmAACT (encoding the enzyme acetyl-CoA acyltransferase), SmHMGR2 (encoding the enzyme 3-hydroxy-3-methylglutaryl-COA reductase), and SmFPPS (encoding the enzyme farnesyl diphosphate synthase), which are involved in the MVA pathway, and tanshinone production during the developmental stages examined, suggesting that the MVA pathway might contribute less to tanshinone accumulation as compared with the MEP pathway. The results in this study indicated that SmCMK might play an important role in their biosynthesis and accumulation. Overall, this study contributes to a better understanding of tanshinone biosynthesis during the different developmental stages of S. miltiorrhiza.

Synthesizing ginsenoside Rh2 in Saccharomyces cerevisiae cell factory at high-efficiency

Synthetic biology approach has been frequently applied to produce plant rare bioactive compounds in microbial cell factories by fermentation. However, to reach an ideal manufactural efficiency, it is necessary to optimize the microbial cell factories systemically by boosting sufficient carbon flux to the precursor synthesis and tuning the expression level and efficiency of key bioparts related to the synthetic pathway. We previously developed a yeast cell factory to produce ginsenoside Rh2 from glucose. However, the ginsenoside Rh2 yield was too low for commercialization due to the low supply of the ginsenoside aglycone protopanaxadiol (PPD) and poor performance of the key UDP-glycosyltransferase (UGT) (biopart UGTPg45) in the final step of the biosynthetic pathway. In the present study, we constructed a PPD-producing chassis via modular engineering of the mevalonic acid pathway and optimization of P450 expression levels. The new yeast chassis could produce 529.0 mg/L of PPD in shake flasks and 11.02 g/L in 10 L fed-batch fermentation. Based on this high PPD-producing chassis, we established a series of cell factories to produce ginsenoside Rh2, which we optimized by improving the C3–OH glycosylation efficiency. We increased the copy number of UGTPg45, and engineered its promoter to increase expression levels. In addition, we screened for more efficient and compatible UGT bioparts from other plant species and mutants originating from the direct evolution of UGTPg45. Combining all engineered strategies, we built a yeast cell factory with the greatest ginsenoside Rh2 production reported to date, 179.3 mg/L in shake flasks and 2.25 g/L in 10 L fed-batch fermentation. The results set up a successful example for improving yeast cell factories to produce plant rare natural products, especially the glycosylated ones.

Ligand-based virtual screening and molecular docking of two cytotoxic compounds isolated from Papaver lacerum

This study revealed that the Papaver lacerum extract strongly inhibited HeLa cell proliferation, resulting in 13% cell viability. As a result of phytochemical studies, one known compound, Tyrosol-1-O-β-xylopyranosyl-(1→6)-O-β-glucopyranoside) (I), and one new compound, 5-O-(6-O-α-rhamnopyronosyl-β-glucopyronosyl) mevalonic acid (II), were isolated. Compounds I and II were found to possess a moderate cytotoxic effect with an IC50 of 66.4 μM (p < 0.0001) and 54 μM (p < 0.0001), respectively. The ligand-based virtual screening technique was used to reveal the possible molecular target of compounds I and II. The molecular target was identified as protein-tyrosine kinase Syk for compound I, and aldo-keto reductase family-1 for compound II. Molecular docking was used to assess the binding affinity of the compounds with the targets obtained from ligand-based virtual screening.

Ent-homocyclopiamine B, a Prenylated Indole Alkaloid of Biogenetic Interest from the Endophytic Fungus Penicillium concentricum.

Ent-homocyclopiamine B (1), a new prenylated indole alkaloid bearing an alicyclic nitro group along with 2-methylbutane-1,2,4-triol (2) were isolated from an endophytic fungus Penicillium concentricum of the liverwort Trichocolea tomentella (Trichocoleaceae). The structure of 1 was elucidated through extensive spectroscopic analyses and comparison with data reported for a structurally related nitro-bearing Penicillium metabolite, clopiamine C (3), which contain an indolizidine ring instead of the quinolizine ring in 1. The new compound, ent-homocyclopiamine B, exhibited slight growth inhibition against Gram-positive bacteria. Based on the reported biosynthesis of related compounds and the isolation of the mevalonic acid derived compound 2-methyl-1,2,4-butanetriol (2), we proposed that ent-homocylopiamine B (1) was biosynthesized from lysine and prenyl group-producing mevalonic pathway.

Production of santalenes and bergamotene in Nicotiana tabacum plants.

Terpenes play an important role in plant–insect relationships, and these relationships can potentially be modified by altering the profile of terpenes emitted from plants using metabolic engineering methods. Transgenic plants generated by employing such methods offer the prospect of low-cost sustainable pest management; in this regard, we used chloroplast targeting and cytosolic mevalonic acid pathway enhancement in this study to investigate the interaction of santalenes and bergamotene with insects. The santalene- and bergamotene-emitting transgenic tobacco plants thus generated were utilized to study host preference in the green peach aphid (Myzus persicae (Sulzer)). The results showed that co-expression of either 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) or truncated HMGR with santalene synthase led to the production of higher amounts of santalenes and bergamotene in transgenic tobacco plants, and that these santalene- and bergamotene-emitting plants were attractive to green peach aphids. We accordingly propose that such transgenic plants may have potential application in pest management as a trap crop to prevent green peach aphid infestation of wild-type tobacco plants.

Unique evolution of vitamin A as an external pigment in tropical starlings.

Pigments are largely responsible for the appearance of organisms. Most biological pigments derive from the metabolism of shikimic acid (melanins), mevalonic acid (carotenoids) or levulinic acid (porphyrins), which thus generate the observed diversity of external phenotypes. Starlings are generally dark birds despite iridescence in feathers, but 10% of species have evolved plumage pigmentation comprising bright colors that are known to be produced only by carotenoids. However, using micro-Raman spectroscopy, we have discovered that the bright yellow plumage coloration of one of these species, the Afrotropical golden-breasted starling Cosmopsarus regius, is not produced by carotenoids, but by vitamin A (all-trans-retinol). This is the first organism reported to deposit significant amounts of vitamin A in its integument and use it as a body pigment. Phylogenetic reconstructions reveal that the retinol-based pigmentation of the golden-breasted starling has independently appeared in the starling family from dark ancestors. Our study thus unveils a unique evolution of a new class of external pigments consisting of retinoids.

The isomiR-140-3p-regulated mevalonic acid pathway as a potential target for prevention of triple negative breast cancer

BackgroundPrevention of triple-negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis.MethodsTo identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represent early stages of TNBC development. We have identified direct gene targets of isomiRNA-140-3p and by using cell-based and in vivo model systems we have demonstrated the utility of targeting downstream pathways for prevention of TNBC.ResultsThese analyses showed that 5’isomiRNA of miR-140-3p (miR-140-3p-1) and its novel direct gene targets, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, were deregulated in the normal-to-preneoplastic transition. Upregulation in the cholesterol pathway creates metabolic vulnerability that can be targeted. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin to inhibit growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin-induced HMGCR transcript levels to be directly correlated with the degree of histological progression of lesions, indicating that the extent of cholesterol pathway suppression directly correlates with abrogation of the tumorigenic process. To block the HMGCR feedback response to statins, we treated resistant preneoplastic cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin and metformin effectively abrogated the statin-induced aberrant upregulation of HMGCR and sensitized these resistant cells to fluvastatin.ConclusionsThese results suggest the potential use of combined treatment with statin and aspirin for prevention of TNBC.

Terpenes and isoprenoids: a wealth of compounds for global use.

Role of terpenes and isoprenoids has been pivotal in the survival and evolution of higher plants in various ecoregions. These products find application in the pharmaceutical, flavor fragrance, and biofuel industries. Fitness of plants in a wide range of environmental conditions entailed (i) evolution of secondary metabolic pathways enabling utilization of photosynthate for the synthesis of a variety of biomolecules, thereby facilitating diverse eco-interactive functions, and (ii) evolution of structural features for the sequestration of such compounds away from the mainstream primary metabolism to prevent autotoxicity. This review summarizes features and applications of terpene and isoprenoid compounds, comprising the largest class of secondary metabolites. Many of these terpene and isoprenoid biomolecules happen to be high-value bioproducts. They are essential components of all living organisms that are chemically highly variant. They are constituents of primary (quinones, chlorophylls, carotenoids, steroids) as well as secondary metabolism compounds with roles in signal transduction, reproduction, communication, climatic acclimation, defense mechanisms and more. They comprise single to several hundreds of repetitive five-carbon units of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, there are two pathways that lead to the synthesis of terpene and isoprenoid precursors, the cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. The diversity of terpenoids can be attributed to differential enzyme and substrate specificities and to secondary modifications acquired by terpene synthases. The biological role of secondary metabolites has been recognized as pivotal in the survival and evolution of higher plants. Terpenes and isoprenoids find application in pharmaceutical, nutraceutical, synthetic chemistry, flavor fragrance, and possibly biofuel industries.

Inhibition of Adenosine Monophosphate-Activated Protein Kinase-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Signaling Leads to Hypercholesterolemia and Promotes Hepatic Steatosis and Insulin Resistance.

Adenosine monophosphate–activated protein kinase (AMPK) regulates multiple signaling pathways involved in glucose and lipid metabolism in response to changes in hormonal and nutrient status. Cell culture studies have shown that AMPK phosphorylation and inhibition of the rate‐limiting enzyme in the mevalonate pathway 3‐hydroxy‐3‐methylglutaryl (HMG) coenzyme A (CoA) reductase (HMGCR) at serine‐871 (Ser871; human HMGCR Ser872) suppresses cholesterol synthesis. In order to evaluate the role of AMPK‐HMGCR signaling in vivo, we generated mice with a Ser871‐alanine (Ala) knock‐in mutation (HMGCR KI). Cholesterol synthesis was significantly suppressed in wild‐type (WT) but not in HMGCR KI hepatocytes in response to AMPK activators. Liver cholesterol synthesis and cholesterol levels were significantly up‐regulated in HMGCR KI mice. When fed a high‐carbohydrate diet, HMGCR KI mice had enhanced triglyceride synthesis and liver steatosis, resulting in impaired glucose homeostasis. Conclusion: AMPK‐HMGCR signaling alone is sufficient to regulate both cholesterol and triglyceride synthesis under conditions of a high‐carbohydrate diet. Our findings highlight the tight coupling between the mevalonate and fatty acid synthesis pathways as well as revealing a role of AMPK in suppressing the deleterious effects of a high‐carbohydrate diet.

Metabolomic Markers of Phthalate Exposure in Plasma and Urine of Pregnant Women.

Phthalates are known endocrine disruptors and found in almost all people with several associated adverse health outcomes reported in humans and animal models. Limited data are available on the relationship between exposure to endocrine disrupting chemicals and the human metabolome. We examined the relationship of metabolomic profiles in plasma and urine of 115 pregnant women with eleven urine phthalate metabolites measured at 26 weeks of gestation to identify potential biomarkers and relevant pathways. Targeted metabolomics was performed by selected reaction monitoring liquid chromatography and triple quadrupole mass spectrometry to measure 415 metabolites in plasma and 151 metabolites in urine samples. We have chosen metabolites with the best defined peaks for more detailed analysis (138 in plasma and 40 in urine). Relationship between urine phthalate metabolites and concurrent metabolomic markers in plasma and urine suggested potential involvement of diverse pathways including lipid, steroid, and nucleic acid metabolism and enhanced inflammatory response. Most of the correlations were positive for both urine and plasma, and further confirmed by regression and PCA analysis. However, after the FDR adjustment for multiple comparisons, only 9 urine associations remained statistically significant (q-values 0.0001–0.0451), including Nicotinamide mononucleotide, Cysteine T2, Cystine, and L-Aspartic acid. Additionally, we found negative associations of maternal pre-pregnancy body mass index (BMI) with more than 20 metabolomic markers related to lipid and amino-acid metabolism and inflammation pathways in plasma (p = 0.01–0.0004), while Mevalonic acid was positively associated (p = 0.009). Nicotinic acid, the only significant metabolite in urine, had a positive association with maternal BMI (p = 0.002). In summary, when evaluated in the context of metabolic pathways, the findings suggest enhanced lipid biogenesis, inflammation and altered nucleic acid metabolism in association with higher phthalate levels. These results provide new insights into the relationship between phthalates, common in most human populations, and metabolomics, a novel approach to exposure and health biomonitoring.

An in Vitro Biosynthesis of Sesquiterpenes Starting from Acetic Acid.

The enzymatic synthesis of terpenes was investigated by using a cascade based on the mevalonic acid pathway. Suitable enzymes from all kingdoms of life were identified and combined to give rise to geosmin and patchoulol as representative compounds. The pathway was studied in three separate segments, which were subsequently combined in a ten-step cascade plus added cofactor regeneration systems. The cascade delivers farnesyl pyrophosphate with >40 % conversion and cyclises it to sesquiterpenes with >90 % conversion.