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Glioblastoma (GBM) is the deadliest adult brain cancer. Under the current standard of care, almost all patients succumb to the disease and novel treatments are urgently needed. Recognizing that GBMs are addicted to cholesterol, past clinical trials have repurposed statins against GBM but failed. The purpose of this study was to test whether treatments that upregulate the cholesterol biosynthesis pathway in GBM would generate a metabolic vulnerability that can be exploited using statins and to determine the underlying mechanisms.Effects of radiotherapy and temozolomide or dopamine receptor antagonists on the mevalonate pathway in GBM were assessed in vitro and in vivo. The impact of statins on self-renewal of glioma stem cells and median survival was studied. Branches of the mevalonate pathway were probed to identify relevant effector proteins.Cells surviving combination treatments that converge in activating the immediate early response, universally upregulated the mevalonate pathway and increased stemness of GBM cells through activation of the Rho-GTPase Rac-1. Activation of the mevalonate pathway and Rac-1 was inhibited by statins, which led to improved survival in mouse models of glioblastoma when combined with radiation and drugs that target the glioma stem cell pool and plasticity of glioma cells.We conclude that a combination of dopamine receptor antagonists and statins could potentially improve radiotherapy outcome and warrants further investigation.SignificanceCombination therapies that activate the mevalonate pathway in GBM cells after sublethal treatment enhance self-renewal and migratory capacity through Rac-1 activation, which creates a metabolic vulnerability that can be further potentially exploited using statins.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.

Les fièvres récurrentes de l'enfant sont de diagnostic étiologique difficile, car elles concernent des anomalies métaboliques ou génétiques souvent peu connues. Elles sont associées à des maladies dont les mécanismes physiopathologiques, et donc les marqueurs biologiques, commencent tout juste à être élucidés. Nous rapportons le cas d'un syndrome hyper-IgD, qui contrairement à sa dénomination, n'est pas toujours caractérisé par une élévation des IgD. La démarche diagnostique et les marqueurs associés sont décrits pour un diagnostic essentiel à la mise en place d'un traitement. The recurrent fevers of the child are of etiological diagnosis difficult because they concern metabolic or genetic abnormalities. They are associated with diseases whose pathophysiological mechanisms, and thus biological markers, are beginning to be elucidated. We report the case of a hyper IgD-syndrome, which unlike its name, is not always characterized by an elevation of IgD. The diagnostic procedure and the associated markers are described for an essential diagnosis for the implementation of a treatment. [ABSTRACT FROM AUTHOR]

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol's seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.

The mevalonate synthesis inhibitors, statins, are mainstay therapeutics for cholesterol management and cardiovascular health. Thirty years of research have uncovered supportive roles for the mevalonate pathway in numerous cellular processes that support oncogenesis, most recently macropinocytosis. Central to the diverse mechanisms of statin sensitivity is an acquired dependence on one mevalonate pathway output, protein geranylgeranylation. New chemical prenylation probes and the discovery of a novel geranylgeranyl transferase hold promise to deepen our understanding of statin mechanisms of action. Further, insights into statin selection and the counterproductive role of dietary geranylgeraniol highlight how we should assess statins in the clinic. Lastly, rational combination strategies preview how statins will enter the oncology toolbox.

Abstract Mevalonate kinase (MK) deficiency is a rare autosomal recessive metabolic disorder caused by pathogenic variants in the MVK gene with a broad phenotypic spectrum including autoinflammation, developmental delay and ataxia. Typically, neurological symptoms are considered to be part of the severe end of the phenotypical spectrum and are reported to be in addition to the autoinflammatory symptoms. Here, we describe a patient with MK deficiency with severe neurological symptoms but without autoinflammation and we found several similar patients in the literature. Possibly, the non‐inflammatory phenotype is related to a specific genotype: the MVK p.(His20Pro)/p.(Ala334Thr) variant. There is probably an underdetection of the neurological MK deficient phenotype without inflammatory symptoms as clinicians may not test for MK deficiency when patients present with only neurological symptoms. In conclusion, although rare, neurological symptoms without hyperinflammation might be more common than expected in MK deficiency. It seems relevant to consider MK deficiency in patients with psychomotor delay and ataxia, even if there are no inflammatory symptoms.

(-)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharmaceuticals. However, the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement. This study aimed to develop a sustainable strategy to enhance the biosynthesis of (-)-α-bisabolol by metabolic engineering. By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9, a de novo (-)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L (-)-α-bisabolol. Two key genes for (-)-α-bisabolol biosynthesis, ERG20 and MrBBS, were fused by a flexible linker (GGGS)3 under the GAL7 promoter control, and the titer was increased by 2.9-fold. Optimization of the mevalonic acid pathway and multi-copy integration further increased (-)-α-bisabolol production. To promote product efflux, overexpression of PDR15 led to an increase in extracellular production. Combined with the optimal strategy, (-)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L, which is the highest titer reported in yeast to date. This work provides a reference for the efficient production of (-)-α-bisabolol in yeast. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Isoprenoids are an essential and diverse class of molecules, present in all forms of life, that are synthesized from an essential common precursor derived from either the mevalonate pathway or the nonmevalonate pathway. Most bacteria have one pathway or the other, but the Gram-positive, facultative intracellular pathogen Listeria monocytogenes is unusual because it carries all the genes for both pathways. While the mevalonate pathway has previously been reported to be essential, here we demonstrate that the nonmevalonate pathway can support growth of strains 10403S and EGD-e, but only anaerobically. L. monocytogenes lacking the gene hmgR, encoding the rate-limiting enzyme of the mevalonate pathway, had a doubling time of 4 h under anaerobic conditions, in contrast to the 45 min doubling time of the wild type. In contrast, deleting hmgR in two clinical isolates resulted in mutants that grew significantly faster, doubling in approximately 2 h anaerobically, although they still failed to grow under aerobic conditions without mevalonate. The difference in anaerobic growth rate was traced to three amino acid changes in the nonmevalonate pathway enzyme GcpE, and these changes were sufficient to increase the growth rate of 10403S to the rate observed in the clinical isolates. Despite an increased growth rate, virulence was still dependent on the mevalonate pathway in 10403S strains expressing the more active GcpE allele.

Isoprenol (3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals. Biological production of isoprenol via the mevalonate pathway has been developed and optimized extensively in Escherichia coli, but high ATP requirements and isopentenyl diphosphate (IPP) toxicity have made it difficult to achieve high titer, yield, and large-scale production. To overcome these limitations, an IPP-bypass pathway was previously developed using the promiscuous activity of diphosphomevalonate decarboxylase, and enabled the production of isoprenol at a comparable yield and titer to the original pathway. In this study, we optimized this pathway, substantially improving isoprenol production. A titer of 3.7 g/L (0.14 g isoprenol per g glucose) was achieved in batch conditions using minimal medium by pathway optimization, and a further optimization of the fed-batch fermentation process enabled an isoprenol titer of 10.8 g/L (yield of 0.105 g/g and maximum productivity of 0.157 g L-1 h-1), which is the highest reported titer for this compound. The substantial increase in isoprenol titer via the IPP-bypass pathway in this study will facilitate progress toward commercialization.

Lepidoptera (butterflies and moths) make the six-carbon compounds homoisopentenyl pyrophosphate (HIPP) and homodimethylallyl pyrophosphate (HDMAPP) that are incorporated into 16, 17, and 18 carbon farnesyl pyrophosphate (FPP) analogues. In this work we heterologously expressed the lepidopteran modified mevalonate pathway, a propionyl-CoA ligase, and terpene cyclases in E. coli to produce several novel terpenes containing 16 carbons. Changing the terpene cyclase generated different novel terpene product profiles. To further validate the new compounds we confirmed 13C propionate was incorporated, and that the masses and fragmentation patterns were consistent with novel 16 carbon terpenes by GC-QTOF. On the basis of the available farnesyl pyrophosphate analogues lepidoptera produce, this approach should greatly expand the reachable biochemical space with applications in areas where terpenes have traditionally found uses.

The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses of cardiomyocytes to 7KCh. A 7KCh treatment inhibited the growth of cardiac cells and their mitochondrial oxygen consumption. It was accompanied by a compensatory increase in mitochondrial mass and adaptive metabolic remodeling. The application of [U-13C] glucose labeling revealed an increased production of malonyl-CoA but a decreased formation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) in the 7KCh-treated cells. The flux of the tricarboxylic acid (TCA) cycle decreased, while that of anaplerotic reaction increased, suggesting a net conversion of pyruvate to malonyl-CoA. The accumulation of malonyl-CoA inhibited the carnitine palmitoyltransferase-1 (CPT-1) activity, probably accounting for the 7-KCh-induced suppression of β-oxidation. We further examined the physiological roles of malonyl-CoA accumulation. Treatment with the inhibitor of malonyl-CoA decarboxylase, which increased the intracellular malonyl-CoA level, mitigated the growth inhibitory effect of 7KCh, whereas the treatment with the inhibitor of acetyl-CoA carboxylase, which reduced malonyl-CoA content, aggravated such a growth inhibitory effect. Knockout of malonyl-CoA decarboxylase gene (Mlycd−/−) alleviated the growth inhibitory effect of 7KCh. It was accompanied by improvement of the mitochondrial functions. These findings suggest that the formation of malonyl-CoA may represent a compensatory cytoprotective mechanism to sustain the growth of 7KCh-treated cells. [ABSTRACT FROM AUTHOR]

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Clear cell renal cell carcinoma (CC-RCC) is a devastating disease with limited therapeutic options available for advanced stages. The objective of this study was to investigate HMG-CoA reductase inhibitors, also known as statins, as potential therapeutics for CC-RCC. Importantly, treatment with statins was found to be synthetically lethal with the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in 90% of CC-RCC driving the disease. This effect has been confirmed in three different CC-RCC cell lines with three different lipophilic statins. Inhibition of mevalonate synthesis by statins causes a profound cytostatic effect at nanomolar concentrations and becomes cytotoxic at low micromolar concentrations in VHL-deficient CC-RCC. The synthetic lethal effect can be fully rescued by both mevalonate and geranylgeranylpyrophosphate, but not by squalene, indicating that the effect is due to disruption of small GTPase isoprenylation and not the inhibition of cholesterol synthesis. Inhibition of Rho and Rho kinase (ROCK) signaling contributes to the synthetic lethality effect, and overactivation of hypoxia-inducible factor signaling resulting from VHL loss is required. Finally, statin treatment is able to inhibit both tumor initiation and progression of subcutaneous 786-OT1-based CC-RCC tumors in mice. Thus, statins represent potential therapeutics for the treatment of VHL-deficient CC-RCC. Mol Cancer Ther; 17(8); 1781-92. ©2018 AACR.

IntroductionAsthma manifests as chronic airflow obstruction with persistent inflammation and airway hyperresponsiveness. The immunomodulatory and anti-inflammatory properties of the HMG-CoA reductase (HMGCR) inhibitors (a.k.a. statins), suggest a therapeutic role in chronic inflammatory lung diseases. However, despite positive laboratory investigations and promising epidemiological data, clinical trials using statins for the treatment of asthma have yielded conflicting results. Inadequate statin levels in the airway compartment could explain these findings. Areas covered: HMGCR is in the mevalonate (MA) pathway and MA signaling is fundamental to lung biology and asthma. This article will discuss clinical trials of oral statins in asthma, review lab investigations relevant to the systemic versus inhaled administration of statins, address the advantages and disadvantages of inhaled statins, and answer the question: is there a role for inhaled statins in the treatment of asthma? Expert commentary: If ongoing investigations show that oral administration of statins has no clear clinical benefits, then repurposing statins for delivery via inhalation is a logical next step. Inhalation of statins bypasses first-pass metabolism by the liver, and therefore, allows for delivery of significantly lower doses to the airways at greater potency. Statins could become the next major class of novel inhalers for the treatment of asthma.

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an "IPP-bypass" isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either kcat or Ki of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.

BackgroundWith increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints.ResultsWe characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate.ConclusionBy combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.

Abstract Neem is a medicinal plant used as antimalarial, antibacterial, antiviral, insecticide, and antimicrobial drug. This study aimed to investigate and predict the effect of yeast extract and sampling time on cell growth, secondary metabolites synthesis, SQS1 and MOF1 genes expression by response surface methodology. The highest fresh and dry cell weights were 580.25 g/L and 21.01 g/L, respectively obtained 6 days after using 100 mg/L yeast extract. The highest azadirachtin accumulation and production were 16.08 mg/g DW and 219.78 mg/L obtained 2 and 4 days, respectively after using 25 mg/L yeast extract. Maximum mevalonic acid accumulation (1.75 mg/g DW) and production (23.77 mg/L) were observed 2 days after application of 50 mg/L yeast extract. The highest amount of squalene accumulation (0.22 mg/g DW) and production (4.53 mg/L) were achieved 4 days after using 50 mg/L yeast extract. Prediction results exhibited the highest azadirachtin accumulation (13.61 mg/g DW) and production (190.50 mg/L), mevalonic acid accumulation (0.50 mg/g DW) and production (5.57 mg/L), and squalene accumulation (0.30 mg/g DW) by using 245 mg/L yeast extract for 2 days, 71 mg/L yeast extract for 2 days, 200 mg/L yeast extract for 4.96 days, without yeast extract for 6.54 days and 4 days, respectively. Also, it was predicted that the highest squalene production is achieved by long-term exposure to high concentrations of yeast extract. The qRT-PCR analysis displayed the maximum relative gene expression of SQS1 and MOF1 by using 150 and 25 mg/L yeast extract for 4 and 2 days treatment.

The major oxidized product of cholesterol, 7-Ketocholesterol (7KCh), causes cellular oxidative damage. In the present study, we investigated the physiological responses of cardiomyocytes to 7KCh. A 7KCh treatment inhibited the growth of cardiac cells and their mitochondrial oxygen consumption. It was accompanied by a compensatory increase in mitochondrial mass and adaptive metabolic remodeling. The application of [U-13C] glucose labeling revealed an increased production of malonyl-CoA but a decreased formation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) in the 7KCh-treated cells. The flux of the tricarboxylic acid (TCA) cycle decreased, while that of anaplerotic reaction increased, suggesting a net conversion of pyruvate to malonyl-CoA. The accumulation of malonyl-CoA inhibited the carnitine palmitoyltransferase-1 (CPT-1) activity, probably accounting for the 7-KCh-induced suppression of β-oxidation. We further examined the physiological roles of malonyl-CoA accumulation. Treatment with the inhibitor of malonyl-CoA decarboxylase, which increased the intracellular malonyl-CoA level, mitigated the growth inhibitory effect of 7KCh, whereas the treatment with the inhibitor of acetyl-CoA carboxylase, which reduced malonyl-CoA content, aggravated such a growth inhibitory effect. Knockout of malonyl-CoA decarboxylase gene (Mlycd−/−) alleviated the growth inhibitory effect of 7KCh. It was accompanied by improvement of the mitochondrial functions. These findings suggest that the formation of malonyl-CoA may represent a compensatory cytoprotective mechanism to sustain the growth of 7KCh-treated cells.

Branched C5 alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C5 alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C5 alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete "decoupling" of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C5 alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

BackgroundConsiderable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemical scaffolds and include many potent antibiotic and cytotoxic agents.ResultsOne hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the 'MAR4' clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites.ConclusionsWe found that marine-derived MAR4 streptomycetes possess a relatively high genetic potential for HI biosynthesis. The combination of horizontal gene transfer, duplication, and rearrangement indicate that complex evolutionary processes account for the high level of HI gene cluster diversity in these bacteria, the products of which may provide a yet to be defined adaptation to the marine environment.

Abstract Background Climate plays an essential role in forest health, and climate change may increase forest productivity losses due to abiotic and biotic stress. Increased temperature leads to the increased formation of ozone (O3). Ozone is formed by the interaction of sunlight, molecular oxygen and by the reactions of chemicals commonly found in industrial and automobile emissions such as nitrogen oxides and volatile organic compounds. Although it is well known that productivity of Northern red oak (Quercus rubra) (NRO), an ecologically and economically important species in the forests of eastern North America, is reduced by exposure to O3, limited information is available on its responses to exogenous stimuli at the level of gene expression. Results RNA sequencing yielded more than 323 million high-quality raw sequence reads. De novo assembly generated 52,662 unigenes, of which more than 42,000 sequences could be annotated through homology-based searches. A total of 4140 differential expressed genes (DEGs) were detected in response to O3 stress, as compared to their respective controls. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the O3-response DEGs revealed perturbation of several biological pathways including energy, lipid, amino acid, carbohydrate and terpenoid metabolism as well as plant-pathogen interaction. Conclusion This study provides the first reference transcriptome for NRO and initial insights into the genomic responses of NRO to O3. Gene expression profiling reveals altered primary and secondary metabolism of NRO seedlings, including known defense responses such as terpenoid biosynthesis.

Abstract Background The fuel ethanol industry has made tremendous progress in the last decades. Ethanol can be obtained by fermentation using a variety of biomass materials as the feedstocks. However, few studies have been conducted on ethanol utilization by microorganisms. The price of petroleum-derived ethanol, easily made by the hydrolysis of ethylene, is even lower than that of bioethanol. If ethanol can be metabolized by microorganisms to produce value-added chemicals, it will open a new door for the utilization of inexpensive ethanol resources. Results We constructed an engineered Escherichia coli strain which could utilize ethanol as the sole carbon source. The alcohol dehydrogenase and aldehyde dehydrogenase from Aspergillus nidulans was introduced into E. coli and the recombinant strain acquired the ability to grow on ethanol. Cell growth continued when ethanol was supplied after glucose starvation and 2.24 g L−1 of ethanol was further consumed during the shake-flasks fermentation process. Then ethanol was further used for the production of mevalonic acid by heterologously expressing its biosynthetic pathway. Deuterium-labeled ethanol-D6 as the feedstock confirmed that mevalonic acid was synthesized from ethanol. Conclusions This study demonstrated the possibility of using ethanol as the carbon source by engineered E. coli strains. It can serve as the basis for the construction of more robust strains in the future though the catabolic capacity of ethanol should be further improved.

Azadirachta indica is used to insects repellent, control diabetes, and combat with cancer. In this study, the effect of different concentrations of chitosan and sampling times on cell suspension culture and SQS1 and MOF1 genes expression were studied and response surface methodology was used to construct the prediction models for azadirachtin, mevalonic acid and squalene accumulation and production. The highest fresh and dry cell weight and azadirachtin accumulation were 726.21 g/L, 20.76 g/L, and 11.74 mg/g DW obtained 6 days after using 50 mg/L chitosan, respectively. The highest azadirachtin production was 165.50 mg/L achieved by using 50 mg/L chitosan for 8 days. Maximum mevalonic acid accumulation (1.87 mg/g DW) and production (22.50 mg/L) were observed 2 and 4 days after the addition of 50 mg/L chitosan and control condition, respectively. The highest amount of squalene accumulation (0.319 mg/g DW) and production (3.56 mg/L) were obtained 10 days after using 50 mg/L chitosan. Also, the prediction results showed the highest azadirachtin accumulation (10.76 mg/g DW) and production (136.83 mg/L), mevalonic acid accumulation (1.869 mg/g DW) and production (20.576 mg/L) and squalene accumulation (0.217 mg/g DW) and production (2.23 mg/L) by using 71.50 mg/L chitosan for 5.24 days, 66.25 mg/L chitosan for 9.1 days, 2.5 mg/L chitosan for 2 days, without chitosan for 2 days, 98.50 mg/L chitosan for 2 days and 80.25 mg/L chitosan for 6.08 days, respectively. The qRT-PCR analysis indicated the maximum relative expression of SQS1 and MOF1 genes by using 25 and 50 mg/L chitosan for 6 days. Key message: For the first time, chitosan concentrations and sampling times optimized by response surface methodology for azadirachtin, mevalonic acid and squalene accumulation and production and investigated SQS1 and MOF1 genes expression. [ABSTRACT FROM AUTHOR]

In animals, cholesterol is made from 5-carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)-mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate-3-kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)-mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation.

Escherichia coli was used as a microbial system for the heterologous synthesis of β-phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-phellandrene synthase (PHLS), alone or in combination with Picea abies geranyl-diphosphate synthase in E. coli, resulted in no β-phellandrene accumulation, in sharp contrast to observations with PHLS-transformed cyanobacteria. Lack of β-phellandrene biosynthesis in E. coli was attributed to the limited endogenous carbon partitioning through the native 2-C-methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic (Synechocystis) and non-photosynthetic bacteria (E. coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems.

BackgroundThe increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects, including a reduction in cancer risk.MethodsWe investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations.ResultsIn vitro studies showed that cerivastatin, pitavastatin, and fluvastatin were the most potent anti-proliferative, autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently, pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment, tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells.ConclusionsOur study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies.

Isoprenoids make up a remarkably diverse class of more than 25000 biomolecules that include familiar compounds such as cholesterol, chlorophyll, vitamin A, ubiquinone, and natural rubber. The two essential building blocks of all isoprenoids, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are ubiquitous in the three domains of life. In most eukaryotes and archaea, IPP and DMAPP are generated through the mevalonate pathway. We have identified two novel enzymes, mevalonate-3-kinase and mevalonate-3-phosphate-5-kinase from Thermoplasma acidophilum, which act sequentially in a putative alternate mevalonate pathway. We propose that a yet unidentified ATP-independent decarboxylase acts upon mevalonate 3,5-bisphosphate, yielding isopentenyl phosphate, which is subsequently phosphorylated by the known isopentenyl phosphate kinase from T. acidophilum to generate the universal isoprenoid precursor, IPP.

The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs ('statins') directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD)), and cancer.

The high yields required for the economical production of chemicals and fuels using microbes can be difficult to achieve due to the complexities of cellular metabolism. An alternative to performing biochemical transformations in microbes is to build biochemical pathways in vitro, an approach we call synthetic biochemistry. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chemical isoprene. We construct an in vitro synthetic biochemical pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl-CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl-CoA derived isoprenoids from glucose in vitro.

Heterologous expression of the isoprene synthase gene in the cyanobacterium Synechocystis PCC 6803 conferred upon these microorganisms the property of photosynthetic isoprene (C₅H₈) hydrocarbons production. Continuous production of isoprene from CO₂ and H₂O was achieved in the light, occurring via the endogenous methylerythritol-phosphate (MEP) pathway, in tandem with the growth of Synechocystis. This work addressed the issue of photosynthetic carbon partitioning between isoprene and biomass in Synechocystis. Evidence is presented to show heterologous genomic integration and cellular expression of the mevalonic acid (MVA) pathway genes in Synechocystis endowing a non-native pathway for carbon flux amplification to isopentenyl-diphosphate (IPP) and dimethylallyl-diphosphate (DMAPP) precursors of isoprene. Heterologous expression of the isoprene synthase in combination with the MVA pathway enzymes resulted in photosynthetic isoprene yield improvement by approximately 2.5-fold, compared with that measured in cyanobacteria transformed with the isoprene synthase gene only. These results suggest that the MVA pathway introduces a bypass in the flux of endogenous cellular substrate in Synechocystis to IPP and DMAPP, overcoming flux limitations of the native MEP pathway. The work employed a novel chromosomal integration and expression of synthetic gene operons in Synechocystis, comprising up to four genes under the control of a single promoter, and expressing three operons simultaneously. This is the first time an entire biosynthetic pathway with seven recombinant enzymes has been heterologously expressed in a photosynthetic microorganism. It constitutes contribution to the genetic engineering toolkit of photosynthetic microorganisms and a paradigm in the pursuit of photosynthetic approaches for the renewable generation of high-impact products.

Neem is a medicinal plant used as antimalarial, antibacterial, antiviral, insecticide, and antimicrobial drug. This study aimed to investigate and predict the effect of yeast extract and sampling time on cell growth, secondary metabolites synthesis, SQS1 and MOF1 genes expression by response surface methodology. The highest fresh and dry cell weights were 580.25 g/L and 21.01 g/L, respectively obtained 6 days after using 100 mg/L yeast extract. The highest azadirachtin accumulation and production were 16.08 mg/g DW and 219.78 mg/L obtained 2 and 4 days, respectively after using 25 mg/L yeast extract. Maximum mevalonic acid accumulation (1.75 mg/g DW) and production (23.77 mg/L) were observed 2 days after application of 50 mg/L yeast extract. The highest amount of squalene accumulation (0.22 mg/g DW) and production (4.53 mg/L) were achieved 4 days after using 50 mg/L yeast extract. Prediction results exhibited the highest azadirachtin accumulation (13.61 mg/g DW) and production (190.50 mg/L), mevalonic acid accumulation (0.50 mg/g DW) and production (5.57 mg/L), and squalene accumulation (0.30 mg/g DW) by using 245 mg/L yeast extract for 2 days, 71 mg/L yeast extract for 2 days, 200 mg/L yeast extract for 4.96 days, without yeast extract for 6.54 days and 4 days, respectively. Also, it was predicted that the highest squalene production is achieved by long-term exposure to high concentrations of yeast extract. The qRT-PCR analysis displayed the maximum relative gene expression of SQS1 and MOF1 by using 150 and 25 mg/L yeast extract for 4 and 2 days treatment. [ABSTRACT FROM AUTHOR]

Squalene epoxidase (SQLE) is a key enzyme in the mevalonate-cholesterol pathway that plays a critical role in cellular physiological processes. It converts squalene to 2,3-epoxysqualene and catalyzes the first oxygenation step in the pathway. Recently, intensive efforts have been made to extend the current knowledge of SQLE in cancers through functional and mechanistic studies. However, the underlying mechanisms and the role of SQLE in cancers have not been fully elucidated yet. In this review, we retrospected current knowledge of SQLE as a rate-limiting enzyme in the mevalonate-cholesterol pathway, while shedding light on its potential as a diagnostic and prognostic marker, and revealed its therapeutic values in cancers. We showed that SQLE is regulated at different levels and is involved in the crosstalk with iron-dependent cell death. Particularly, we systemically reviewed the research findings on the role of SQLE in different cancers. Finally, we discussed the therapeutic implications of SQLE inhibitors and summarized their potential clinical values. Overall, this review discussed the multifaceted mechanisms that involve SQLE to present a vivid panorama of SQLE in cancers.

Background: Here, we investigated the underlying transcriptional-level evidence behind phytochemical differences between two metabolically extreme genotypes of Thymus daenensis. The genotypes 'Zagheh-11' (thymol/carvacrol type, poor in essential oil [EO] [2.9%] but rich in triterpenic acids) and 'Malayer-21' (thymol type and rich in EO [3.8%]) were selected from an ongoing breeding program and then clonally propagated for further experimental use., Materials and Methods: GC-MS, GC-FID, and HPLC-PDA were utilized to monitor the fluctuation of secondary metabolites at four phenological stages (vegetative, bud burst, early, and full-flowering stages). The highest phytochemical divergence was observed at early flowering stage. Both genotypes were subjected to mRNA sequencing (approximately 100 million paired reads) at the aforementioned stage. The expression patterns of four key genes involved in the biosynthesis of terpenoids were also validated using qRT-PCR., Results: Carvacrol content in 'Zagheh-11' (26.13%) was approximately 23 times higher than 'Malayer-21' (1.12%). Reciprocally, about 10% higher thymol was found in 'Malayer-21' (62.15%). Moreover, the concentrations of three major triterpenic acids in 'Zagheh-11' were approximately as twice as those found in 'Malayer-21'. Transcriptome analysis revealed a total of 1840 unigenes that were differentially expressed, including terpene synthases, cytochrome P450, and terpenoid backbone genes. Several differentially expressed transcription factors (such as MYB, bZIP, HB-HD-ZIP, and WRKY families) were also identified. These results suggest that an active cytosolic mevalonate (MVA) pathway may be linked to higher levels of sesquiterpenes, triterpenic acids, and carvacrol in 'Zagheh-11'. The chloroplastic pathway of methyl erythritol phosphate (MEP) may have also contributed to a higher accumulation of thymol in Malayer-21. Indeed, 'Zagheh-11' showed higher expression of certain genes (HMGR, CYP71D180, β-amyrin 28-monooxygenase, and sesquiterpene synthases) in the MVA pathway, while some genes in the MEP pathway (including DXR, ispG, and γ-terpinene synthase) were distinctly expressed in Malayer-21. Future efforts in metabolic engineering of MVA/MEP pathways may benefit from these findings to produce increased levels of desired secondary metabolites at commercial scale., (© 2024. The Author(s).)

Tumor suppressor p53 is frequently null or mutated in human cancers. Here in this study, DHX33 protein was found to be induced in p53 null cells in vitro, and in p53 mutant lung tumorigenesis in vivo. Cholesterol metabolism through mevalonate pathway is pivotal for cell proliferation and is frequently altered in human cancers. Mice carrying mutant p53 and Kras G12D alleles showed upregulation of mevalonate pathway gene expression. However upon DHX33 loss, their upregulation was significantly debilitated. Additionally, in many human cancer cells, DHX33 knockdown caused inhibition of mavelonate pathway gene transcription. We propose DHX33 locates downstream of mutant p53 and Ras to regulate mevalonate pathway gene transcription and thereby supports cancer development in vivo., Competing Interests: Declaration of Competing Interest We declare that we have no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Terpenes are valuable industrial chemicals whose demands are increasingly being met by bioengineering microbes such as E. coli. Although the bioengineering efforts commonly involve installing the mevalonate (MVA) pathway in E. coli for terpene production, the less studied methylerythritol phosphate (MEP) pathway is a more attractive target due to its higher energy efficiency and theoretical yield, despite its tight regulation. In this study, we integrated an additional copy of the entire MEP pathway into the E. coli genome for stable, marker-free terpene production. The genomically integrated strain produced more monoterpene geraniol than a plasmid-based system. The pathway genes' transcription was modulated using different promoters to produce geraniol as the reporter of the pathway flux. Pathway genes, including dxs, idi, and ispDF, expressed from a medium-strength promoter, led to the highest geraniol production. Quantifying the MEP pathway intermediates revealed that the highest geraniol producers had high levels of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), but moderate levels of the pathway intermediates upstream of these two building blocks. A principal component analysis demonstrated that 1-deoxy-D-xylulose 5-phosphate (DXP), the product of the first enzyme of the pathway, was critical for determining the geraniol titer, whereas MEP, the product of DXP reductoisomerase (Dxr or IspC), was the least essential. This work shows that an intricate balance of the MEP pathway intermediates determines the terpene yield in engineered E. coli. The genetically stable and intermediate-balanced strains created in this study will serve as a chassis for producing various terpenes. KEY POINTS: • Genome-integrated MEP pathway afforded higher strain stability • Genome-integrated MEP pathway produced more terpene than the plasmid-based system • High monoterpene production requires a fine balance of MEP pathway intermediates., (© 2024. The Author(s).)

Antineoplastic therapies for prostate cancer (PCa) have traditionally centered around the androgen receptor (AR) pathway, which has demonstrated a significant role in oncogenesis. Nevertheless, it is becoming progressively apparent that therapeutic strategies must diversify their focus due to the emergence of resistance mechanisms that the tumor employs when subjected to monomolecular treatments. This review illustrates how the dysregulation of the lipid metabolic pathway constitutes a survival strategy adopted by tumors to evade eradication efforts. Integrating this aspect into oncological management could prove valuable in combating PCa.

Background: Climate plays an essential role in forest health, and climate change may increase forest productivity losses due to abiotic and biotic stress. Increased temperature leads to the increased formation of ozone (O3). Ozone is formed by the interaction of sunlight, molecular oxygen and by the reactions of chemicals commonly found in industrial and automobile emissions such as nitrogen oxides and volatile organic compounds. Although it is well known that productivity of Northern red oak (Quercus rubra) (NRO), an ecologically and economically important species in the forests of eastern North America, is reduced by exposure to O3, limited information is available on its responses to exogenous stimuli at the level of gene expression. Results: RNA sequencing yielded more than 323 million high-quality raw sequence reads. De novo assembly generated 52,662 unigenes, of which more than 42,000 sequences could be annotated through homology-based searches. A total of 4140 differential expressed genes (DEGs) were detected in response to O3 stress, as compared to their respective controls. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the O3-response DEGs revealed perturbation of several biological pathways including energy, lipid, amino acid, carbohydrate and terpenoid metabolism as well as plant-pathogen interaction. Conclusion: This study provides the first reference transcriptome for NRO and initial insights into the genomic responses of NRO to O3. Gene expression profiling reveals altered primary and secondary metabolism of NRO seedlings, including known defense responses such as terpenoid biosynthesis. [ABSTRACT FROM AUTHOR]

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