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Background: Lycopene is increasing in demand due to its widespread use in the pharmaceutical and food industries. Metabolic engineering and synthetic biology technologies have been widely used to overexpress the heterologous mevalonate pathway and lycopene pathway in Escherichia coli to produce lycopene. However, due to the tedious metabolic pathways and complicated metabolic background, optimizing the lycopene synthetic pathway using reasonable design approaches becomes difficult., Results: In this study, the heterologous lycopene metabolic pathway was introduced into E. coli and divided into three modules, with mevalonate and DMAPP serving as connecting nodes. The module containing the genes (MVK, PMK, MVD, IDI) of downstream MVA pathway was adjusted by altering the expression strength of the four genes using the ribosome binding sites (RBSs) library with specified strength to improve the inter-module balance. Three RBS libraries containing variably regulated MVK, PMK, MVD, and IDI were constructed based on different plasmid backbones with the variable promoter and replication origin. The RBS library was then transformed into engineered E. coli BL21(DE3) containing pCLES and pTrc-lyc to obtain a lycopene producer library and employed high-throughput screening based on lycopene color to obtain the required metabolic pathway. The shake flask culture of the selected high-yield strain resulted in a lycopene yield of 219.7 mg/g DCW, which was 4.6 times that of the reference strain., Conclusion: A strain capable of producing 219.7 mg/g DCW with high lycopene metabolic flux was obtained by fine-tuning the expression of the four MVA pathway enzymes and visual selection. These results show that the strategy of optimizing the downstream MVA pathway through RBS library design can be effective, which can improve the metabolic flux and provide a reference for the synthesis of other terpenoids., (© 2022. The Author(s).)

Metabolic inflammation, defined as a chronic low-grade inflammation, is implicated in numerous metabolic diseases. In recent years, the role of regulatory T cells (Tregs) as key controllers of metabolic inflammation has emerged, but our comprehension on how different metabolic pathways influence Treg functions needs a deeper understanding. Here we focus on how circulating and intracellular lipid metabolism, in particular cholesterol metabolism, regulates Treg homeostasis, expansion, and functions. Cholesterol is carried through the bloodstream by circulating lipoproteins (chylomicrons, very low-density lipoproteins, low-density lipoproteins). Tregs are equipped with a wide array of metabolic sensors able to perceive and respond to changes in the lipid environment through the activation of different intracellular pathways thus conferring to these cells a crucial metabolic and functional plasticity. Nevertheless, altered cholesterol transport, as observed in genetic dyslipidemias and atherosclerosis, impairs Treg proliferation and function through defective cellular metabolism. The intracellular pathway devoted to the cholesterol synthesis is the mevalonate pathway and several studies have shown that this pathway is essential for Treg stability and suppressive activity. High cholesterol concentrations in the extracellular environment may induce massive accumulation of cholesterol inside the cell thus impairing nutrients sensors and inhibiting the mevalonate pathway. This review summarizes the current knowledge regarding the role of circulating and cellular cholesterol metabolism in the regulation of Treg metabolism and functions. In particular, we will discuss how different pathological conditions affecting cholesterol transport may affect cellular metabolism in Tregs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

HMG-CoA reductase or HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) is a rate-limiting enzyme involved in cholesterol biosynthesis. HMGCR plays an important role in the possible occurrence of hypercholesterolemia leading to atherosclerosis and coronary heart disease. This enzyme is a major target for cholesterol-lowering drugs such as "statin" which blocks the synthesis of mevalonate, a precursor for cholesterol biosynthesis. This study is aimed at characterizing deleterious mutations and classifying functional single nucleotide polymorphisms (SNPs) of the HMGCR gene through analysis of functional and structural evaluation, domain association, solvent accessibility, and energy minimization studies. The functional and characterization tools such as SIFT, PolyPhen, SNPs and GO, Panther, I-Mutant, and Pfam along with programming were employed to explore all the available SNPs in the HMGCR gene in the database. Among 6815 SNP entries from different databases, approximately 388 SNPs were found to be missense. Analysis showed that seven missense SNPs are more likely to have deleterious effects. A tertiary model of the mutant protein was constructed to determine the functional and structural effects of the HMGCR mutation. In addition, the location of the mutations suggests that they may have deleterious effects because most of the mutations are residing in the functional domain of the protein. The findings from the analysis predicted that rs147043821 and rs193026499 missense SNPs could cause significant structural and functional instability in the mutated proteins of the HMGCR gene. The findings of the current study will likely be useful in future efforts to uncover the mechanism and cause of hypercholesterolemia. In addition, the identified SNPs of HMGCR gene could set up a strong foundation for further therapeutic discovery., Competing Interests: The authors declare that they do not have competing interests on this work., (Copyright © 2022 Keshob Chandra Das et al.)

Background: The flux of isoprenoids and the total accumulation of triterpenoid saponins known as centellosides in C. asiatica are controlled by the key genes of the Mevalonate pathway (MVA). These genes were reported to have positive regulation of the pathway in providing isoprenoid moieties. Though, some information is available on the pathway and secondary metabolites. However, most of the pathway steps are not characterized functionally., Methodology and Results: For the study, full-length pathway gene Hydroxymethyl glutaryl-CoA-synthase (CaHMGS; GenBank accession number: MZ997833), was isolated from previously annotated transcriptome data of Centella asiatica leaves. HMGS has been successfully cloned and heterologously expressed in bacteria E. coli strain DH5α. The cloned gene has been sequenced and further characterized through in silico studies by different bioinformatics tools. Also, the gene sequences have been submitted in NCBI. In silico studies of isolated gene sequence revealed the nature, characteristics of genes. The ORF of HMGS is 1449 bp encoding 482 amino acids. Predicted molecular weight (MW) of HMGS was 48.09 kDa and theoretical pI was 5.97. Blast results and Multiple sequence alignments of the gene showing the similarity with HMGS of other plants of their respective families. The Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6 was used to construct a phylogenetic tree. Differential tissue-specific expression of different plant parts was also checked. Tissue expression patterns unveiled that the highest expression level of the CaHMGS had been seen in the roots and lowest in the node of the plant. Functional complementation experiment of the CaHMGS in Saccharomyces cerevisiae wild strain YSC1021 and haploid strain YSC1021 which lack HMGS protein confirmed that the CaHMGS gene encodes functional CaHMGS that catalyzed the biosynthesis of mevalonate in yeast., Conclusions: The gene was reported, cloned and characterized first time in Centella asiatica. Understanding this biosynthetic pathway gene will further help in the improvement of plants for enhanced secondary metabolites production., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the metabolite HMG-CoA into mevalonate. Recent discoveries have shown HMG-CoA is a reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins might increase HMG-CoA levels and protein modifications. Upon statin treatment, we observe a strong increase in HMG-CoA levels and modification of only a single protein. Mass spectrometry identifies this protein as fatty acid synthase (FAS), which is modified on active site residues and, importantly, on non-lysine side-chains. The dynamic modifications occur only on a sub-pool of FAS that is located near HMGCR and alters cellular signaling around the ER and Golgi. These results uncover communication between cholesterol and lipid biosynthesis by the substrate of one pathway inhibiting another in a rapid and reversible manner., (© 2022. The Author(s).)

Statins are a class of drugs widely used worldwide to manage hypercholesterolemia and the prevention of secondary heart attacks. Currently, available statins vary in terms of their pharmacokinetic and pharmacodynamic profiles. Although the primary target of statins is the inhibition of HMG-CoA reductase (HMGR), the rate-limiting enzyme in cholesterol biosynthesis, statins exhibit many pleiotropic effects downstream of the mevalonate pathway. These pleiotropic effects include the ability to reduce myocardial fibrosis, pathologic cardiac disease states, hypertension, promote bone differentiation, anti-inflammatory, and antitumor effects through multiple mechanisms. Although these pleiotropic effects of statins may be a cause for enthusiasm, there are many adverse effects that, for the most part, are unappreciated and need to be highlighted. These adverse effects include myopathy, new-onset type 2 diabetes, renal and hepatic dysfunction. Although these adverse effects may be relatively uncommon, considering the number of people worldwide who use statins daily, the actual number of people affected becomes quite large. Also, co-administration of statins with several other medications, herbal agents, and foods, which interact through common enzymatic pathways, can have untoward clinical consequences. In this review, we address these concerns., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this study, we used bioinformatic tools to analyze the 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR) genes from Glycyrrhiza uralensis, Artemisia annua, and Arabidopsis thaliana. The results indicated that GuHMGR and AaHMGR contained two transmembrane regions while AtHMGR had three transmembrane regions. GuHMGR, AaHMGR, and AtHMGR all had the active center for catalysis. Three truncated HMGR genes(tHMGRs) of G. uralensi, A. annua, and A. thaliana were respectively ligated to pYES3 vector to construct the recombinant plasmids pYES3-tGuHMGR,pYES3-tAaHMGR,and pYES3-tAtHMGR. Afterwards, the control plasmid pYES3 and the three plasmids and were respectively introduced into Saccharomyces cerevisiae Cen.pk2-1 D, which yielded strains Y0, Y1, Y2, and Y3, respectively. The content of squalene, lanosterol, and ergosterol in these strains was measured by GC-MS. The relative expression of tGuHMGR, tAaHMGR, and tAtHMGR in strains Y1, Y2, and Y3 was determined by quantitative real-time PCR. The results showed that the strain overexpressing tAaHMGR had the highest yield of squalene and the highest total yield of squalene, ergosterol, and lanosterol. The quantitative real-time PCR showed higher relative expression of tAaHMGR than tGuHMGR, consistent with the strain fermentation result. We selected a superior tHMGR by comparing the effects of different tHMGRs on the mevalonate(MVA) pathway flux in S. cerevisiae. The findings can provide a reference for the construction of S. cerevisiae strains with high yields of squalene and terpenoid precursors.

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1,BcABA2,BcABA3,BcABA4 and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of the mevalonate pathway and ABA biosynthesis encoding genes, and we expressed plant ABA transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g dry cell weight (DCW) ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA; however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms Y. lipolytica as a potential alternative host for the ABA production., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Saccharomyces cerevisiae is increasingly being used for the production of chemicals derived from acetyl coenzyme A (acetyl-CoA). However, the inadequate supply of cytosolic acetyl-CoA often leads to low yields. Here, we developed a novel strategy for balancing acetyl-CoA metabolism and increasing the amount of the downstream product. First, the combination of acetaldehyde dehydrogenase (eutE) and acetoacetyl-CoA thiolase (AtoB) was optimized to redirect the acetyl-CoA flux toward the target pathway, with a 21-fold improvement in mevalonic acid production. Second, pathway engineering and evolutionary engineering were conducted to attenuate the growth deficiency, and a 10-fold improvement of the maximum productivity was achieved. Third, acetyl-CoA carboxylase (ACC 1 ) was dynamically downregulated as the complementary acetyl-CoA pathway, and the yield was improved more than twofold. Fourth, the most efficient and complementary acetyl-CoA pathways were combined, and the final strain produced 68 mg/g CDW lycopene, which was among the highest yields reported in S. cerevisiae . This study demonstrates a new method of producing lycopene products by regulating acetyl-CoA metabolism.

Growing evidence suggests that men prescribed a statin for cholesterol control have a lower risk of advanced prostate cancer (PCa) and improved treatment outcomes; however, the mechanism by which statins elicit their anti-neoplastic effects is not well understood and is likely multifaceted. Statins are potent and specific inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme of the mevalonate (MVA) metabolic pathway. This two-part series is a review of the observational and experimental data on statins as anti-cancer agents in PCa. In this article, we describe the functional role that deregulated MVA metabolism plays in PCa progression and summarize the biological evidence and rationale for targeting the MVA pathway, with statins and other agents, for the treatment of PCa., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism., (© 2022. The Author(s).)

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO + CD4 + T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation., (© 2021 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The bisphosphonates ((HO) 2 P(O)CR 1 R 2 P(O)(OH) 2 , BPs) were first shown to inhibit bone resorption in the 1960s, but it was not until 30 years later that a detailed molecular understanding of the relationship between their varied chemical structures and biological activity was elucidated. In the 1990s and 2000s, several potent bisphosphonates containing nitrogen in their R 2 side chains (N-BPs) were approved for clinical use including alendronate, risedronate, ibandronate, and zoledronate. These are now mostly generic drugs and remain the leading therapies for several major bone-related diseases, including osteoporosis and skeletal-related events associated with bone metastases. The early development of chemistry in this area was largely empirical and only a few common structural features related to strong binding to calcium phosphate were clear. Attempts to further develop structure-activity relationships to explain more dramatic pharmacological differences in vivo at first appeared inconclusive, and evidence for mechanisms underlying cellular effects on osteoclasts and macrophages only emerged after many years of research. The breakthrough came when the intracellular actions on the osteoclast were first shown for the simpler bisphosphonates, via the in vivo formation of P-C-P derivatives of ATP. The synthesis and biological evaluation of a large number of nitrogen-containing bisphosphonates in the 1980s and 1990s led to the key discovery that the antiresorptive effects of these more complex analogs on osteoclasts result mostly from their potency as inhibitors of the enzyme farnesyl diphosphate synthase (FDPS/FPPS). This key branch-point enzyme in the mevalonate pathway of cholesterol biosynthesis is important for the generation of isoprenoid lipids that are utilized for the post-translational modification of small GTP-binding proteins essential for osteoclast function. Since then, it has become even more clear that the overall pharmacological effects of individual bisphosphonates on bone depend upon two key properties: the affinity for bone mineral and inhibitory effects on biochemical targets within bone cells, in particular FDPS. Detailed enzyme-ligand crystal structure analysis began in the early 2000s and advances in our understanding of the structure-activity relationships, based on interactions with this target within the mevalonate pathway and related enzymes in osteoclasts and other cells have continued to be the focus of research efforts to this day. In addition, while many members of the bisphosphonate drug class share common properties, now it is more clear that chemical modifications to create variations in these properties may allow customization of BPs for different uses. Thus, as the appreciation for new potential opportunities with this drug class grows, new chemistry to allow ready access to an ever-widening variety of bisphosphonates continues to be developed. Potential new uses of the calcium phosphate binding mechanism of bisphosphonates for the targeting of other drugs to the skeleton, and effects discovered on other cellular targets, even at non-skeletal sites, continue to intrigue scientists in this research field., (Copyright © 2021. Published by Elsevier Inc.)

Co-culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity. In the present study, we demonstrated the enhancement of mevalonate consumption in Escherichia coli by adaptive laboratory evolution and applied the evolved strain to isoprenol production in an E. coli (upstream: glucose to mevalonate)-E. coli (downstream: mevalonate to isoprenol) co-culture. An engineered mevalonate auxotroph strain was repeatedly sub-cultured in a synthetic medium supplemented with mevalonate, where the mevalonate concentration was decreased stepwise from 100 to 20 µM. In five parallel evolution experiments, all growth rates gradually increased, resulting in five evolved strains. Whole-genome re-sequencing and reverse engineering identified three mutations involved in enhancing mevalonate consumption. After introducing nudF gene for producing isoprenol, the isoprenol-producing parental and evolved strains were respectively co-cultured with a mevalonate-producing strain. At an inoculation ratio of 1:3 (upstream:downstream), isoprenol production using the evolved strain was 3.3 times higher than that using the parental strain., (© 2021 Wiley Periodicals LLC.)

Early T-cell acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematologic malignancy associated with early relapse and poor prognosis that is genetically, immunophenotypically, and transcriptionally distinct from more mature T-cell acute lymphoblastic leukemia (T-ALL) tumors. Here, we leveraged global metabolomic and transcriptomic profiling of primary ETP- and T-ALL leukemia samples to identify specific metabolic circuitries differentially active in this high-risk leukemia group. ETP-ALLs showed increased biosynthesis of phospholipids and sphingolipids and were specifically sensitive to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in the mevalonate pathway. Mechanistically, inhibition of cholesterol synthesis inhibited oncogenic AKT1 signaling and suppressed MYC expression via loss of chromatin accessibility at a leukemia stem cell-specific long-range MYC enhancer. In all, these results identify the mevalonate pathway as a druggable novel vulnerability in high-risk ETP-ALL cells and uncover an unanticipated critical role for cholesterol biosynthesis in signal transduction and epigenetic circuitries driving leukemia cell growth and survival., Significance: Overtly distinct cell metabolic pathways operate in ETP- and T-ALL pointing to specific metabolic vulnerabilities. Inhibition of mevalonate biosynthesis selectively blocks oncogenic AKT-MYC signaling in ETP-ALL and suppresses leukemia cell growth. Ultimately, these results will inform the development of novel tailored and more effective treatments for patients with high-risk ETP-ALL. This article is highlighted in the In This Issue feature, p. 587., (©2021 American Association for Cancer Research.)

Statin use accompanies with increased risk of new onset of type 2 diabetes, however, the underlying mechanisms remain not be fully understood and effective prevention strategies are still lacking. Herein, we find that both pharmacological and genetic inhibition of GGTase II mimic the disruption of simvastatin on hepatic insulin signaling and glucose metabolism in vitro. AAV8-mediated knockdown of liver RABGGTA, the specific subunit of GGTase II, triggers systemic glucose metabolism disorders in vivo. By adopting a small-scale siRNA screening, we identify RAB14 as a regulator of hepatic insulin signaling and glucose metabolism. Geranylgeranylation deficiency of RAB14 inhibits the phosphorylation of AKT (Ser473) and disrupts hepatic insulin signaling and glucose metabolism possibly via impeding mTORC2 complex assembly. Finally, geranylgeranyl pyrophosphate (GGPP) supplementation is sufficient to prevent simvastatin-caused disruption of hepatic insulin signaling and glucose metabolism in vitro. Geranylgeraniol (GGOH), a precursor of GGPP, is able to ameliorate simvastatin-induced systemic glucose metabolism disorders in vivo. In conclusion, our data indicate that statins-targeted mevalonate pathway regulates hepatic insulin signaling and glucose metabolism via geranylgeranylation of RAB14. GGPP/GGOH supplementation might be an effective strategy for the prevention of the diabetic effects of statins., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interests., (Copyright © 2021. Published by Elsevier Inc.)

Mitochondrial dysfunction induces a strong adaptive retrograde signaling response; however, many of the downstream effectors of this response remain to be discovered. Here, we studied the shared transcriptional responses to three different mitochondrial respiratory chain inhibitors in human primary skin fibroblasts using QuantSeq 3'-RNA-sequencing. We found that genes involved in the mevalonate pathway were concurrently downregulated, irrespective of the respiratory chain complex affected. Targeted metabolomics demonstrated that impaired mitochondrial respiration at any of the three affected complexes also had functional consequences on the mevalonate pathway, reducing levels of cholesterol precursor metabolites. A deeper study of complex I inhibition showed a reduced activity of endoplasmic reticulum-bound sterol-sensing enzymes through impaired processing of the transcription factor Sterol Regulatory Element-Binding Protein 2 and accelerated degradation of the endoplasmic reticulum cholesterol-sensors squalene epoxidase and HMG-CoA reductase. These adaptations of mevalonate pathway activity affected neither total intracellular cholesterol levels nor the cellular free (nonesterified) cholesterol pool. Finally, measurement of intracellular cholesterol using the fluorescent cholesterol binding dye filipin revealed that complex I inhibition elevated cholesterol on intracellular compartments. Taken together, our study shows that mitochondrial respiratory chain dysfunction elevates intracellular free cholesterol levels and therefore attenuates the expression of mevalonate pathway enzymes, which lowers endogenous cholesterol biosynthesis, disrupting the metabolic output of the mevalonate pathway. We conclude that intracellular disturbances in cholesterol homeostasis may alter systemic cholesterol management in diseases associated with declining mitochondrial function., Competing Interests: Conflict of interest All authors were employed by Société des Produits Nestlé S.A. during the generation of this manuscript., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are a primary treatment for hyperlipidemic cardiovascular diseases which are a leading global cause of death. Statin therapy is life saving and discontinuation due to adverse events such as myotoxicity may lead to unfavourable outcomes. There is no known mechanism for statin-induced myotoxicity although it is theorized that it is due to inhibition of downstream products of the HMG-CoA pathway. It is known that drug-drug interactions with conventional medicines exacerbate the risk of statin-induced myotoxicity, though little attention has been paid to herb-drug interactions with complementary medicines. Flavonoids are a class of phytochemicals which can be purchased as high dose supplements. There is evidence that flavonoids can raise statin plasma levels, increasing the risk of statin-induced myopathy. This could be due to pharmacokinetic interactions involving hepatic cytochrome 450 (CYP450) metabolism and organic anion transporter (OATP) absorption. There is also the potential for flavonoids to directly and indirectly inhibit HMG-CoA reductase which could contraindicate statin-therapy. This review aims to discuss what is currently known about the potential for high dose flavonoids to interact with the hepatic CYP450 metabolism, OATP uptake of statins or their ability to interact with HMG-CoA reductase. Flavonoids of particular interest will be covered and the difficulties of examining herbal products will be discussed throughout., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The mevalonate pathway is responsible for the synthesis of isoprenoids, including sterols and other metabolites that are essential for diverse biological functions. Cholesterol, the main sterol in mammals, and non-sterol isoprenoids are in high demand by rapidly dividing cells. As evidence of its importance, many cell signaling pathways converge on the mevalonate pathway and these include those involved in proliferation, tumor-promotion, and tumor-suppression. As well as being a fundamental building block of cell membranes, cholesterol plays a key role in maintaining their lipid organization and biophysical properties, and it is crucial for the function of proteins located in the plasma membrane. Importantly, cholesterol and other mevalonate derivatives are essential for cell cycle progression, and their deficiency blocks different steps in the cycle. Furthermore, the accumulation of non-isoprenoid mevalonate derivatives can cause DNA replication stress. Identification of the mechanisms underlying the effects of cholesterol and other mevalonate derivatives on cell cycle progression may be useful in the search for new inhibitors, or the repurposing of preexisting cholesterol biosynthesis inhibitors to target cancer cell division. In this review, we discuss the dependence of cell division on an active mevalonate pathway and the role of different mevalonate derivatives in cell cycle progression., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca 2+ -imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism., (© 2022. The Author(s).)

Objective: We were aimed at evaluating the long-term impact of perinatal an omega-3 fatty acid-enriched diet on the mevalonate/cholesterol pathway in the brain of male offspring. Methods: Female rats were fed with standard or omega-3 fatty acid-enriched diet during pregnancy and lactation. Liver, brain and plasma were collected from infant, adolescent and adult male offspring for subsequent biochemical and morphological analyses. Results: The omega-3 enriched diet induced region-dependent changes of the 3-hydroxy 3-methylglutaryl Coenzyme A reductase in the brain and affected notably RhoA/CREB signaling and the nerve growth factor content in the hippocampus. Our data reveal a long-lasting impact of perinatal omega-3 fatty acid supplementation on hippocampal nerve growth factor levels mediated by reduced 3-hydroxy 3-methylglutaryl Coenzyme A reductase activation state and enhanced CREB signaling. Discussion: These data underline the importance of the perinatal omega-3 enriched diet for adult brain function and reveal a new pathway important for nerve growth factor regulation.

Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Although brain tumors occur less frequently than other forms of cancer, they have one of the bleakest prognoses with low survival rates. The conventional treatment for brain tumors includes surgery, radiotherapy, and chemotherapy. However, resistance to treatment remains a problem with recurrence shortly following. The resistance to treatment may be caused by cancer stem cells (CSCs), a subset of brain tumor cells with the affinity for self-renewal and differentiation into multiple cell lineages. An emerging approach to targeting CSCs in brain tumors is through repurposing the lipid-lowering medication, lovastatin. Lovastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that impacts the mevalonate pathway. The inhibition of intermediates in the mevalonate pathway affects signaling cascades and oncogenes associated with brain tumor stem cells (BTSC). In this review, we show the possible mechanisms where lovastatin can target BTSC for different varieties of malignant brain tumors.

Leucine, isoleucine and valine, known as branched chain amino acids (BCAAs), have been reported to be degraded by different cancer cells, and their biodegradation pathways have been suggested as anticancer targets. However, the mechanisms by which the degradation of BCAAs could support the growth of cancer cells remains unclear. In this work, 13 C experiments have been carried out in order to elucidate the metabolic role of BCAA degradation in two breast cancer cell lines (MCF-7 and BCC). The results revealed that up to 36% of the energy production via respiration by MCF-7 cells was supported by the degradation of BCAAs. Also, 67% of the mevalonate (the precursor of cholesterol) synthesized by the cells was coming from the degradation of leucine. The results were lower for BCC cells (14 and 30%, respectively). The non-tumorigenic epythelial cell line MCF-10A was used as a control, showing that 10% of the mitochondrial acetyl-CoA comes from the degradation of BCAAs and no mevalonate production. Metabolic flux analysis around the mevalonate node, also revealed that significant amounts of acetoacetate are being produced from BCAA derived carbon, which could be at the source of lipid synthesis. From these results we can conclude that the degradation of BCAAs is an important energy and carbon source for the proliferation of some cancer cells and its therapeutic targeting could be an interesting option., Competing Interests: Declaration of competing interest The authors declare no financial and non-financial competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein glycosylation requires dolichyl phosphate as a carbohydrate carrier. Dolichols are α-saturated polyprenols, and their saturation in S. cerevisiae is catalyzed by polyprenyl reductase Dfg10 together with some other unknown enzymes. The aim of this study was to identify such enzymes in Candida . The Dfg10 polyprenyl reductase from S. cerevisiae comprises a C-terminal 3-oxo-5-alpha-steroid 4-dehydrogenase domain. Alignment analysis revealed such a domain in two ORFs (orf19.209 and orf19.3293) from C. albicans , which were similar, respectively, to Dfg10 polyprenyl reductase and Tsc13 enoyl-transferase from S. cerevisiae . Deletion of orf19.209 in Candida impaired saturation of polyprenols. The Tsc13 homologue turned out not to be capable of saturating polyprenols, but limiting its expression reduce the cellular level of dolichols and polyprenols. This reduction was not due to a decreased expression of genes encoding cis -prenyltransferases from the dolichol branch but to a lower expression of genes encoding enzymes of the early stages of the mevalonate pathway. Despite the resulting lower consumption of acetyl-CoA, the sole precursor of the mevalonate pathway, it was not redirected towards fatty acid synthesis or elongation. Lowering the expression of TSC13 decreased the expression of the ACC1 gene encoding acetyl-CoA carboxylase, the key regulatory enzyme of fatty acid synthesis and elongation.

Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently labelled bisphosphonate is internalised by alveolar macrophages and large peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia., Competing Interests: MM, SS, FE, JC FHE is an employee of BioVinc (Pasadena, CA, USA). SS also co-affiliates with BioVinc. JRC participates on advisory boards of Amgen and Bayer and receives payment from Amgen for educational activities, EF, OS, JJ, EK, MH, DC, PH, MR No competing interests declared, (© 2021, Munoz et al.)

Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

7-Ketocholesterol (7KCh) is a major oxidized cholesterol product abundant in lipoprotein deposits and atherosclerotic plaques. Our previous study has shown that 7KCh accumulates in erythrocytes of heart failure patients, and further investigation centered on how 7KCh may affect metabolism in cardiomyocytes. We applied metabolomics to study the metabolic changes in cardiac cell line HL-1 after treatment with 7KCh. Mevalonic acid (MVA) pathway-derived metabolites, such as farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate, phospholipids, and triacylglycerols levels significantly declined, while the levels of lysophospholipids, such as lysophosphatidylcholines (lysoPCs) and lysophosphatidylethanolamines (lysoPEs), considerably increased in 7KCh-treated cells. Furthermore, the cholesterol content showed no significant change, but the production of cholesteryl esters was enhanced in the treated cells. To explore the possible mechanisms, we applied mRNA-sequencing (mRNA-seq) to study genes differentially expressed in 7KCh-treated cells. The transcriptomic analysis revealed that genes involved in lipid metabolic processes, including MVA biosynthesis and cholesterol transport and esterification, were differentially expressed in treated cells. Integrated analysis of both metabolomic and transcriptomic data suggests that 7KCh induces cholesteryl ester accumulation and reprogramming of lipid metabolism through altered transcription of such genes as sterol O-acyltransferase- and phospholipase A2-encoding genes. The 7KCh-induced reprogramming of lipid metabolism in cardiac cells may be implicated in the pathogenesis of cardiovascular diseases.

Terpenoids constitute the largest class of natural compounds and are extremely valuable from an economic point of view due to their extended physicochemical properties and biological activities. Due to recent environmental concerns, terpene extraction from natural sources is no longer considered as a viable option, and neither is the chemical synthesis to access such chemicals due to their sophisticated structural characteristics. An alternative to produce terpenoids is the use of biotechnological tools involving, for example, the construction of enzymatic cascades (cell-free synthesis) or a microbial bio-production thanks to metabolic engineering techniques. Despite outstanding successes, these approaches have been hampered by the length of the two natural biosynthetic routes (the mevalonate and the methyl erythritol phosphate pathways), leading to dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), the two common universal precursors of all terpenoids. Recently, we, and others, developed what we called the terpene mini-path, a robust two enzyme access to DMAPP and IPP starting from their corresponding two alcohols, dimethylallyl alcohol and isopentenol. The aim here is to present the potential of this artificial bio-access to terpenoids, either in vitro or in vivo, through a review of the publications appearing since 2016 on this very new and fascinating field of investigation.

New therapeutic targets are revolutionizing colorectal cancer clinical management, opening new horizons in metastatic patients' outcome. Polo Like Kinase1 (PLK1) inhibitors have high potential as antitumoral agents, however, the emergence of drug resistance is a major challenge for their use in clinical practice. Overcoming this challenge represents a hot topic in current drug discovery research. BI2536-resistant colorectal cancer cell lines HT29 R , RKO R , SW837 R and HCT116 R , were generated in vitro and validated by IG 50 assays and xenografts models by the T/C ratio. Exons 1 and 2 of PLK1 gene were sequenced by Sanger method. AXL pathway, Epithelial-to-Mesenchymal transition (EMT) and Multidrug Resistance (MDR1) were studied by qPCR and western blot in resistant cells. Simvastatin as a re-sensitizer drug was tested in vitro and the drug combination strategies were validated in vitro and in vivo. PLK1 gene mutation R136G was found for RKO R . AXL pathway trough TWIST1 transcription factor was identified as one of the mechanisms involved in HT29 R , SW837 R and HCT116 R lines, inducing EMT and upregulation of MDR1. Simvastatin was able to impair the mechanisms activated by adaptive resistance and its combination with BI2536 re-sensitized resistant cells in vitro and in vivo. Targeting the mevalonate pathway contributes to re-sensitizing BI2536-resistant cells in vitro and in vivo, raising as a new strategy for the clinical management of PLK1 inhibitors., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

N6-Isopentenyladenosine ( i6A ) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives' (compounds 2a-m ) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2 , 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

The enzymes immobilized through yeast surface display (YSD) can be used in in vitro metabolic pathway reconstruction as alternatives to the enzymes isolated or purified through conventional biochemistry methods. They can be easily prepared by growing and collecting yeast cells harboring display constructs. This may provide an economical method for enriching certain enzymes for biochemistry characterization and application. Herein, we took the advantage of one-pot cascade reactions catalyzed by YSD-immobilized enzymes in the mevalonate pathway to produce geraniol in vitro . YSD-immobilized enzymes of 10 cascade reactions for geraniol production, together with optimization of catalytic components, cofactor regeneration, and byproduct removal, achieved a final yield of 7.55 mg L -1 after seven cycles. This study demonstrated that it is feasible to reconstitute a complex multi-enzymatic system for the chemical biosynthesis in vitro by exploiting YSD-immobilized cascade enzymes.

Yarrowia lipolytica has recently emerged as a prominent microbial host for production of terpenoids. Its robust metabolism and growth in wide range of substrates offer several advantages at industrial scale. In the present study, we investigate the metabolic potential of Y. lipolytica to produce isoprene. Sustainable production of isoprene has been attempted through engineering several microbial hosts; however, the engineering studies performed so far are challenged with low titers. Engineering of Y. lipolytica, which have inherent high acetyl-CoA flux could fuel precursors into the biosynthesis of isoprene and thus is an approach that would offer sustainable production opportunities. The present work, therefore, explores this opportunity wherein a codon-optimized IspS gene (single copy) of Pueraria montana was integrated into the Y. lipolytica genome. With no detectable isoprene level during the growth or stationary phase of modified strain, attempts were made to overexpress enzymes from MVA pathway. GC-FID analyses of gas collected during stationary phase revealed that engineered strains were able to produce detectable isoprene only after overexpressing HMGR (or tHMGR). The significant role of HMGR (tHMGR) in diverting the pathway flux toward DMAPP is thus highlighted in our study. Nevertheless, the final recombinant strains overexpressing HMGR (tHMGR) along with Erg13 and IDI showed isoprene titers of ~500 μg/L and yields of ~80 μg/g. Further characterization of the recombinant strains revealed high lipid and squalene content compared to the unmodified strain. Overall, the preliminary results of our laboratory-scale studies represent Y. lipolytica as a promising host for fermentative production of isoprene., (© 2021 American Institute of Chemical Engineers.)

Soyasapogenol B is an oleanane-type pentacyclic triterpene that has various applications in food and healthcare and has a higher biological activity than soyasaponin. Saccharomyces cerevisiae is a potential platform for terpenoid production with mature genetic tools for metabolic pathway manipulation. In this study, we developed a biosynthesis method to produce soyasapogenol B. First, we expressed β-amyrin synthase derived from Glycyrrhiza glabra in S. cerevisiae to generate β-amyrin, as the precursor of soyasapogenol B. Several different types of promoters were then used to regulate the expression of key genes in the mevalonate pathway (MVA), and this subsequently increased the yield of β-amyrin to 17.6 mg/L, 25-fold more than that produced in the original strain L01 (0.68 mg/L). Then, using the β-amyrin-producing strain, we expressed soyasapogenol B synthases from Medicago truncatula (CYP93E2 and CYP72A61V2) and from G. glabra (CYP93E3 and CYP72A566). Soyasapogenol B yields were then optimized by using soyasapogenol B synthases and cytochrome P450 reductase from G. glabra. The most effective soyasapogenol B production strain was used for fermentation, and the yield of soyasapogenol B reached 2.9 mg/L in flask and 8.36 mg/L in a 5-L bioreactor with fed glucose and ethanol. This study demonstrated the heterologous synthesis of soyasapogenol B in S. cerevisiae using the combined expression of CYP93E3 and CYP72A566 in the synthesis pathway, which significantly increased the production of soyasapogenol B and provides a reference method for the biosynthesis of other triterpenes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mevalonate Kinase Deficiency (MKD) is a rare inborn disease belonging to the family of periodic fever syndromes. The MKD phenotype is characterized by systemic inflammation involving multiple organs, including the nervous system. Current anti-inflammatory approaches to MKD are only partially effective and do not act specifically on neural inflammation. According to the new emerging pharmacology trends, the repositioning of drugs from the indication for which they were originally intended to another one can make mechanistic-based medications easily available to treat rare diseases. According to this perspective, the squalene synthase inhibitor Lapaquistat (TAK-475), originally developed as a cholesterol-lowering drug, might find a new indication in MKD, by modulating the mevalonate cholesterol pathway, increasing the availability of anti-inflammatory isoprenoid intermediates. Using an in vitro model for MKD, we mimicked the blockade of the cholesterol pathway and evaluated the potential anti-inflammatory effect of Lapaquistat. The results obtained showed anti-inflammatory effects of Lapaquistat in association with a low blockade of the metabolic pathway, while this effect did not remain with a tighter blockade. On these bases, Lapaquistat could be configured as an effective treatment for MKD's mild forms, in which the residual enzymatic activity is only reduced and not almost completely absent as in the severe forms.

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group., (© 2021. The Author(s).)

Mevalonic aciduria (MVA) and hyperimmunoglobulinemia D syndrome (MKD/HIDS) are disorders of cholesterol biosynthesis caused by variants in the MVK gene and characterized by increased urinary excretion of mevalonic acid. So far, 30 MVA patients have been reported, suffering from recurrent febrile crises and neurologic impairment. Here, we present an in-depth analysis of the phenotypic spectrum of MVA and provide an in-silico pathogenicity model analysis of MVK missense variants. The phenotypic spectrum of 11 MVA patients (age range 0-51 years) registered in the Unified European Registry for Inherited Metabolic Disorders database was systematically analyzed using terms of the Human Phenotype Ontology. Biochemical, radiological as well as genetic characteristics were investigated. Six of eleven patients have reached adulthood and four have reached adolescence. One of the adolescent patients died at the age of 16 years and one patient died shortly after birth. Symptoms started within the first year of life, including episodic fever, developmental delay, ataxia, and ocular involvement. We also describe a case with absence of symptoms despite massive excretion of mevalonic acid. Pathogenic variants causing MVA cluster within highly conserved regions, which are involved in mevalonate and ATP binding. The phenotype of adult and adolescent MVA patients is more heterogeneous than previously assumed. Outcome varies from an asymptomatic course to early death. MVK variants cluster in functionally important and highly conserved protein domains and show high concordance regarding their expected pathogenicity., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The Chinese white pine beetle (Dendroctonus armandi Tsai and Li) is a significant pest of pine forests in the Qinling and Bashan Mountains of China. Adult males commonly produce frontalin using precursors synthesized through the mevalonate pathway, which is regulated by juvenile hormone III (JHIII). In this study, the expression levels of mevalonate pathway genes were quantified after phloem feeding and topical application of the JHIII solution. The frontalin was quantified by gas chromatography-mass spectrometry. Both the phloem feeding and JHIII treatments produced an evident upregulation in the male gut, mainly in 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). Moreover, HMGS, HMGR, isopentenyl diphosphate isomerase, and geranyl diphosphate synthase/farnesyl diphosphate synthase were upregulated in fed and JHIII-stimulated males of D. armandi under both conditions (solitary and paired). The expression levels were higher in paired compared to solitary males. Males had higher expression levels compared with females. Correspondingly, the phloem-feeding males produced more frontalin than JHIII-treated males, and the production of frontalin was higher in paired males than in solitary males. The knockdown of mevalonate pathway genes using RNAi in vivo effectively reduced the messenger RNA level of these genes and inhibited the production of frontalin. Among them, the silencing of HMGR or HMGS genes reduced the synthesis of frontalin most significantly., (© 2021 Wiley Periodicals LLC.)

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus ), dengue virus ( Flavivirus ), African swine fever virus ( Asfarviridae ) and poliovirus ( Picornaviridae ). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR (-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Isoprenoids are among the largest and most chemically diverse classes of organic compounds in nature and are involved in the processes of photosynthesis, respiration, growth, development, and plant responses to stress. The basic building block units for isoprenoid synthesis-isopentenyl diphosphate and its isomer dimethylallyl diphosphate-are generated by the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. Here, we summarize recent advances on the roles of the MEP and MVA pathways in plant growth, development and stress responses, and attempt to define the underlying gene networks that orchestrate the MEP and MVA pathways in response to developmental or environmental cues. Through phylogenomic analysis, we also provide a new perspective on the evolution of the plant isoprenoid pathway. We conclude that the presence of the MVA pathway in plants may be associated with the transition from aquatic to subaerial and terrestrial environments, as lineages for its core components are absent in green algae. The emergence of the MVA pathway has acted as a key evolutionary event in plants that facilitated land colonization and subsequent embryo development, as well as adaptation to new and varied environments., (© 2021 Institute of Botany, Chinese Academy of Sciences.)

Cholesterol metabolism is tightly associated with colorectal cancer (CRC). Nevertheless, the clinical benefit of statins, the inhibitor of cholesterol biogenesis mevalonate (MVA) pathway, is inconclusive, possibly because of a lack of patient stratification criteria. Here, we describe that YAP-mediated zinc finger MYND-type containing 8 (ZMYND8) expression sensitizes intestinal tumors to the inhibition of the MVA pathway. We show that the oncogenic activity of YAP relies largely on ZMYND8 to enhance intracellular de novo cholesterol biogenesis. Disruption of the ZMYND8-dependent MVA pathway greatly restricts the self-renewal capacity of Lgr5 + intestinal stem cells (ISCs) and intestinal tumorigenesis. Mechanistically, ZMYND8 and SREBP2 drive the enhancer-promoter interaction to facilitate the recruitment of Mediator complex, thus upregulating MVA pathway genes. Together, our results establish that the epigenetic reader ZMYND8 endows YAP-high intestinal cancer with metabolic vulnerability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Understanding carbon flux controlling mechanisms in a tangled metabolic network is an essential question of cell metabolism. Secondary metabolism, such as terpene biosynthesis, has evolved with low carbon flux due to inherent pathway constraints. Thraustochytrids are a group of heterotrophic marine unicellular protists and can accumulate terpenoids under the high-salt conditions in their natural environment. However, the mechanism behind terpene accumulation is not well understood. Here, we show that terpene biosynthesis in Thraustochytrium sp. ATCC 26185 is constrained by local thermodynamics in the mevalonate pathway. Thermodynamic analysis reveals metabolite limitation in the nondecarboxylative Claisen condensation of acetyl-coenzyme A (CoA) to the acetoacetyl-CoA step, catalyzed by the acetyl-CoA acetyltransferase (ACAT). Through a sodium-elicited mechanism, higher respiration leads to increased ATP investment into the mevalonate pathway, providing a strong thermodynamic driving force for enhanced terpene biosynthesis. Proteomic and metabolomic analyses further show that the increased ATP demands are fulfilled by shifting energy generation from carbohydrate to lipid oxidation. This study demonstrates a unique strategy in nature that uses ATP to drive a low-flux metabolic pathway, providing an alternative solution for efficient terpene metabolic engineering. IMPORTANCE Terpenoids are a large class of lipid molecules with important biological functions and diverse industrial and medicinal applications. Metabolic engineering for terpene production has been hindered by the low-flux distribution to its biosynthesis pathway. In practice, a high substrate load is generally required to reach high product titers. Here, we show that mevalonate-derived terpene biosynthesis is constrained by local pathway thermodynamics, which can only be partially relieved by increasing substrate levels. Through comparative omics and biochemical analyses, we discovered a unique mechanism for high terpene accumulation in marine protist thraustochytrids. Through a sodium-induced mechanism, thraustochytrids shift their energy metabolism from carbohydrate to lipid oxidation for enhanced ATP production, providing a strong thermodynamic driving force for efficient terpene biosynthesis. This study reveals an important mechanism in eukaryotes to overcome the thermodynamic constraint in low-flux pathways by increased ATP consumption. Engineering energy metabolism thus provides an important alternative to relieve flux constraints in low-flux and energy-consuming pathways.

Ferroptosis is a novel form of nonapoptotic regulated cell death (RCD). It features iron-dependent lipid peroxide accumulation accompanied by inadequate redox enzymes, especially glutathione peroxidase 4 (GPX4). RAS-selective lethal 3 (RSL3), erastin, and ferroptosis inducing 56 (FIN56) induce ferroptosis via different manners targeting GPX4 function. Acyl-CoA synthetase long-chain family 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and lipoxygenases (LOXs) participate in the production of lipid peroxides. Heat shock protein family B member 1 (HSPB1) and nuclear receptor coactivator 4 (NCOA4) regulate iron homeostasis preventing ferroptosis caused by the high concentration of intracellular iron. Ferroptosis is ubiquitous in our body as it exists in both physiologic and pathogenic processes. It is involved in glucose-stimulated insulin secretion (GSIS) impairment and arsenic-induced pancreatic damage in the pathogenesis of diabetes. Moreover, iron and the iron-sulfur (Fe-S) cluster influence each other, causing mitochondrial iron accumulation, more reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, failure in biosynthesis of insulin, and ferroptosis in β -cells. In addition, ferroptosis also engages in the pathogenesis of diabetic complications such as myocardial ischemia and diabetic cardiomyopathy (DCM). In this review, we summarize the mechanism of ferroptosis and especially its association with type 2 diabetes mellitus (T2DM)., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Wenxin Sha et al.)

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus . The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

Statins have been widely used for the treatment of hypercholesterolemia due to their ability to inhibit HMG-CoA reductase, the rate-limiting enzyme of de novo cholesterol synthesis, via the so-called mevalonate pathway. However, their inhibitory action also causes depletion of downstream intermediates of the pathway, resulting in the pleiotropic effects of statins, including the beneficial impact in the treatment of cancer. In our study, we compared the effect of all eight existing statins on the expression of genes, the products of which are implicated in cancer inhibition and suggested the molecular mechanisms of their action in epigenetic and posttranslational regulation, and in cell-cycle arrest, death, migration, or invasion of the cancer cells.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: To asseess whether the mevalonate pathway can be targeted in managing patients with esophageal cancer., Methods: This a narrative review of peer-revieweed publications indexed in MedLine PubMed. The search was conducted by using "Esophageal cancer", "Mevalonate pahtway", "Statins", and "Translational research.", Results: The mevalonate pathway is an important metabolic pathway that is involved in various cellular functions. Its downstream products are essential for cell-signaling, cell membrane integrity, protein synthesis, and cellular respiration. Statins, a class of medications that are best known as lipid-lowering drugs, inhibit the rate-limiting enzyme of this pathway. Many studies have shown that a variety of cancerous cells have a dysregulated mevalonate pathway. Esophageal cancer is a malignancy that has a poor prognosis, which is mainly due to patients presenting once the cancer is in the advances stages. Chemotherapy has been the mainstay for treating esophageal cancer. However, these agents are not consistently effective and fail to differentiate between the different subtypes of esophageal cancers. Identifying other classes of drugs which could possess anti esophageal cancer properties is appealing., Conclusion: There is a growing body of literature that has shown the anti-cancer properties of statins in the setting of various malignancies. Herein, we summarize the current literature as it pertains to how the mevalonate pathway can be targeted by statins for potentially treating esophageal cancer.

Blocking the mevalonate pathway for cholesterol reduction by using statin may have adverse effects including statin-induced colitis. Moreover, one of the predisposing factors for colitis is an imbalanced CD4 + T cell, which can be observed on the complete deletion of HMG-CoA reductase (HMGCR), a target of statins. In this study, we inquired geranylgeranyl pyrophosphate (GGPP) is responsible for maintaining the T-cell homeostasis. Following dextran sulfate sodium (DSS)-induced colitis, simvastatin increased the severity of disease, while cotreatment with GGPP, but not with cholesterol, reversed the disease magnitude. GGPP ameliorated DSS-induced colitis by increasing T reg cells. GGPP amplified T reg differentiation through increased IL-2/STAT 5 signaling. GGPP prenylated Ras protein, a prerequisite for extracellular signal-regulated kinase (ERK) pathway activation, leading to increased IL-2 production. Higher simvastatin dose increased the severity of colitis. GGPP ameliorated simvastatin-increased colitis by increasing T reg cells. T reg cells, which have the capacity to suppress inflammatory T cells and were generated through IL-2/STAT5 signaling, increased IL-2 production through prenylation and activation of the Ras/ERK pathway., (© 2021 Wiley-VCH GmbH.)