Cholesterol Synthesis Research Articles

Constructing High-Yielding Serratia marcescens for (-)-α-Bisabolol Production Based on the Exogenous Haloarchaeal MVA Pathway and Endogenous Molecular Chaperones.

(-)-α-Bisabolol exhibits analgesic, anti-inflammatory, and skin-soothing properties and is widely applied in the cosmetic and pharmaceutical industries. The use of plant essential oil distillation or chemical synthesis to produce (-)-α-bisabolol is both inefficient and unsustainable. Currently, the microbial production of (-)-α-bisabolol mainly relies on Escherichia coli and Saccharomyces cerevisiae as chassis strains; however, high concentrations of (-)-α-bisabolol have certain toxicity to the strain. This study uses synthetic biology and metabolic engineering strategies to redesign a solvent-tolerant Serratia marcescens for the efficient production of (-)-α-bisabolol. By introducing the Haloarchaea-type mevalonate (MVA) pathway and the (-)-α-bisabolol biosynthesis pathway, we successfully constructed a strain capable of producing (-)-α-bisabolol. The coexpression of the chaperone protein DnaK/J significantly enhanced the soluble expression of the (-)-α-bisabolol synthase, resulting in a 10% increase in (-)-α-bisabolol titer. Furthermore, knockout of the PhoA gene, which reduced the formation of the byproduct farnesol (FOH), further increased the (-)-α-bisabolol titer to 3.21 g/L. In a 5 L bioreactor, S. marcescens achieved a final (-)-α-bisabolol titer of 30.2 g/L, representing the highest titer reported to date. This research provides guidance for the production of (-)-α-bisabolol in nonmodel microorganisms without the requirement for induction.

Increased Mevalonate Production Using Engineered Citrate Synthase and Phosphofructokinase Variants of Escherichia coli.

Mevalonate is a biochemical precursor to a wide range of isoprenoids. The mevalonate pathway uses three moles of acetyl-CoA, and therefore native pathways which metabolize acetyl-CoA compete with mevalonate synthesis. Moreover, the final step in mevalonate formation, mediated by hydroxymethylglutaryl-CoA reductase, requires NADPH as a co-substrate. This study focuses on chromosomal modification of citrate synthase (GltA) involved in acetyl-CoA utilization and phosphofructokinase (PfkA) involved in NADPH formation to increase the yield and productivity of mevalonate in Escherichia coli overexpressing the three genes of the heterologous mevalonate pathway. Nine GltA variants were compared for mevalonate production with the ΔgltA knockout and the wild-type GltA strain in shake flasks in the absence and presence of casamino acids. In the presence of casamino acids, all variants generated mevalonate at a greater yield than the wild-type control, but less than the GltA knockout. In the absence of casamino acids, the strain expressing wild-type GltA generated the greatest yield of mevalonate, while most variants instead accumulated primarily acetate. Using the wild-type strain and two citrate synthase variants, four phosphofructokinase variants were also compared with the ΔpfkA knockout and the wild-type strain, but PfkA variants generated less mevalonate than the corresponding wild-type PfkA strain. Controlled processes at the 1-liter scale comparing five strains demonstrated the inverse relationship between yield and productivity, with the GltA[K167A] variant showing the best balance for the yield (0.20 g/g) and productivity (0.87 g/L h). A nitrogen-limited process using the GltA[K167A] variant generated 36.9 g/L mevalonate in 31 h at a yield of 0.31 g/g. This study demonstrates that GltA variants offer a means to affect intracellular acetyl-CoA pools for the generation of acetyl-CoA derived products and that the acetyl-CoA pool rather than NADPH availability is the important limiting factor for mevalonate production.

Mechanism of acclimation to chronic intermittent hypoxia in the gills of largemouth bass (Micropterus salmoides).

The acclimation response of fish gills to chronic intermittent hypoxia (CIH) is an important aspect to understand, as anthropogenically induced hypoxia in water bodies has been a stressor for fish for many years and is expected to persist in the future. In order to investigate the acclimation response of fish gills to CIH stress, we conducted a study using largemouth bass (Micropterus salmoides) exposed to intermittent hypoxia (dissolved oxygen level, 2.0mg·L-1) for either 1 or 3h per day, over a period of 8weeks. Our findings indicate that exposure to CIH induced remodeling of the gills and an increase in gill surface area. This remodeling of the gills may be attributed to changes in cell growth and proliferation, which are influenced by the activation of the MAPK signaling pathway. We also observed significant upregulation of genes related to glycolysis (fba, pgam1, pepck, atp-pfk, pfk-2, g6pi, gapd-1, and pk), while genes associated with cholesterol synthesis (3β-hsd, cyp51, dsdr- × 1, dsdr, and dhcr7) were downregulated following CIH exposure. Furthermore, we observed the presence of elongated megamitochondria in mitochondria-rich cells within the gills of fish exposed to hypoxia. Additionally, numerous genes involved in calcium signaling pathways were upregulated in the gills of largemouth bass, suggesting an enhanced sensitivity of gills to environmental cues in hypoxia conditions. However, the expression levels of certain genes related to innate and adaptive immune responses were inhibited following CIH exposure. Moreover, the number of mucous cells decreased after CIH exposure. This may have made the gills more susceptible to infection by pathogens, although it facilitated oxygen uptake. These findings highlight the potential vulnerability of gills to pathogenic organisms in the presence of CIH. Overall, our study contributes to a better understanding of how fish acclimate to CIH.

Exploration of Novel Metabolic Features Reflecting Statin Sensitivity in Lung Cancer Cells.

Statins are cholesterol-lowering drugs often used for the treatment of dyslipidemia. Statins also exert anti-cancer effects by inhibiting hydroxymethylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis. We previously reported that the susceptibility to statin treatment differs among cancer cells and that functional E-cadherin expression on the plasma membrane could be a biomarker of statin sensitivity in cancer cells. However, the detailed qualitative and molecular differences between statin-sensitive and statin-resistant cancer cells remain unclear. Here, we explored novel parameters related to statin sensitivity by comparing gene expression profiles and metabolite contents between statin-sensitive and statin-resistant lung cancer cell lines. We found that the expression of most cholesterol synthesis genes was lower in the statin-sensitive cancer cell line, HOP-92, than in the statin-resistant cancer cell line, NCI-H322M. Moreover, HOP-92 cells originally exhibited lower levels of CoA and HMG-CoA. Additionally, atorvastatin decreased the mRNA expression of PANK2, a rate-limiting enzyme in CoA synthesis. Atorvastatin also reduced the mRNA levels of the cholesterol esterification enzyme SOAT1, which was consistent with a decrease in the ratio of cholesterol ester to total cholesterol in HOP-92 cells. Our data suggest that the cholesterol synthetic flow and CoA content may be limited in statin-sensitive cancer cells. We also suggest that CoA synthesis and cholesterol storage may fluctuate with atorvastatin treatment in statin-sensitive cancer cells.

Human liver organoids are susceptible to Plasmodium vivax infection

BackgroundThe eradication of Plasmodium vivax malaria is complicated due to the presence of hypnozoites, the hidden dormant form of the parasite that is present in the liver. Currently available drug regimens are effective at killing hypnozoites but cause side effects and are difficult to administer. Studies testing drugs for liver-stage malaria remain rare and mainly rely on the use of cancerous or immortalized hepatic cells and primary hepatocytes.MethodsOrganoids were used as platform to model liver-stage vivax malaria. Hepatic endoderm cells, endothelial progenitor cells and mesenchymal cells were generated from human induced pluripotent stem cells and self-assembled into liver organoids on top of Matrigel layer. Liver characteristic and maturity were examined through genes and proteins expression of liver markers, and liver functional tests before infected with Plasmodium vivax sporozoites. The infection was then verified by the detection of parasitophorous vacuole membrane proteins, Upregulated in Infectious Sporozoite 4 (UIS4), and blood-stage infection following co-culture with human reticulocytes.ResultsGenerated liver organoids showed upregulation of liver specific transcripts including hepatic nuclear factor 4A (HNF4A), alpha-fetoprotein (AFP), and albumin (ALB) which also confirmed by the protein expression. Furthermore, those organoids resembled mature hepatocytes in terms of albumin secretion, fat and glycogen storage and cytochrome activity. Following invasion of P. vivax sporozoites, PvUIS4 was detected and the hepatic merozoites could develop into ring-stage and early trophozoites in human reticulocytes. Moreover, differential expression patterns of genes involved in lipid and cholesterol synthesis were also detected.ConclusionsStem cell-derived liver organoids resemble mature liver cells in terms of liver functions and are susceptible to infection with P. vivax sporozoites, paving the way for studies on the mechanism of hypnozoite formation and testing of possible hypnozoitocidal drugs.

Extracellular vesicles from fifth-stage larval Angiostrongylus cantonensis upregulate cholesterol biosynthesis and suppress NLRP2-associated inflammatory responses in mouse astrocytes.

Angiostrongylus cantonensis is a zoonotic parasite that causes severe symptoms in humans, including eosinophilic meningitis and eosinophilic meningoencephalitis. Extracellular vesicles (EVs) derived from helminthes have been implicated in regulating host survival and immune response. However, the roles of A. cantonensis EVs in modulating parasite pathogenesis and host immune response remain poorly understood. Herein, we characterized EVs derived from A. cantonensis fifth-stage larvae (L5) and adult worms. Ultrastructural features showed that EVs from adult worms are smaller in size compared with those from L5. Proteomic analysis identified stage-specific proteins packaged in L5 and adult worm EVs. To investigate the crosstalk between L5 EVs and host cells, RNA sequencing analysis was conducted to identify the differentially expressed genes (DEGs) and enriched biological pathways in mouse astrocytes treated with L5 EVs. GO and KEGG enrichment analysis demonstrated that the pathways related to "cholesterol biosynthesis" are significantly upregulated in L5 EV-treated astrocytes. Based on the transcriptomic data, we observed a downregulated trend of NOD-like receptors (NLRs) protein 2 (NLRP2), a key regulator of brain inflammation, in mouse astrocytes treated with L5 EVs. To validate this result, we utilized ATP to induce the expression of NLRP2 inflammasome-related genes and proteins, as well as the secretion of downstream cytokines. Notably, ATP-induced overexpression of NLRP2 inflammasome-related molecules was significantly reduced in mouse astrocytes upon L5 EV treatment. Collectively, our data suggest that A. cantonensis L5 EVs enhance cholesterol synthesis and potentially modulate immune response by reducing NLRP2 inflammasome-related signaling in non-permissive host cells.IMPORTANCEAngiostrongylus cantonensis is a significant causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Helminth-derived extracellular vesicles (EVs) are known to play a crucial role in parasite pathogenesis and host immunomodulation. However, the protein compositions of A. cantonensis EVs and their roles in parasite pathogenesis and host immune response remain unclear. Our results demonstrate for the first time the distinct protein compositions in A. cantonensis L5 and adult worm EVs. The highly abundant proteins in L5 EVs that have immunomodulatory or pathogenic potential in the host deserve further investigation. Additionally, the uptake of L5 EVs by mouse astrocytes significantly upregulates cholesterol synthesis and suppresses ATP-induced NLRP2 inflammasome-related signaling. This study highlights the immunomodulatory roles of L5 EVs in non-permissive hosts, suggesting their potential as therapeutic targets and vaccine candidates against A. cantonensis.

Simultaneous visualization of membrane fluidity and morphology defines adhesion signatures of cancer cells

We developed an advanced optical microscope for the simultaneous visualization of membrane fluidity and morphology to define cell adhesion signatures. This microscope combines ratiometric spectral imaging of membrane fluidity and interferometric imaging of membrane morphology. As a preliminary demonstration, we simultaneously visualized the interface between a giant unilamellar vesicle (GUV) and a glass substrate at different temperatures. We identified more fluid regions of the membrane and membrane adhesion sites (conversely, low-fluidic, ordered membrane domains correlate with nonadhered regions). This microscopic system was applied to human breast cancer cell lines with different malignancies; then, we identified adhesion signature of cancer cells: 1) low-fluidic, ordered membrane domains at the cell periphery and 2) large fluidic deviation at the nonadhered region. Inhibition of the cholesterol synthesis pathway suppresses the ordered membrane domains at the cancer cell periphery; thus, high level of cholesterol supports the appearance. Furthermore, an inhibitor of the unsaturated lipid synthesis pathway suppressed the large fluidic deviation at the nonadhered region; variation of unsaturated lipids contributes to heterogeneity of the cancer membrane. Therefore, our advanced optical microscopy enables us to couple membrane physical properties with cell adhesion, leading to definition of adhesion signatures of broad cell types, not just for cancer cells, that regulate life phenomena.

Tumor-derived miR-9-5p-loaded EVs regulate cholesterol homeostasis to promote breast cancer liver metastasis in mice

Cancer cells secrete extracellular vesicles (EV) encapsulating bioactive cargoes to facilitate inter-organ communication in vivo and are emerging as critical mediators of tumor progression and metastasis, a condition which is often accompanied by a dysregulated cholesterol metabolism. Whether EVs are involved in the control of cholesterol homeostasis during tumor metastasis is still undefined and warrant further investigation. Here, we find that breast cancer-derived exosomal miR-9-5p induces the expression of HMGCR and CH25H, two enzymes involved in cholesterol synthesis and the conversion of 25-hydroxycholesterol from cholesterol by targeting INSIG1, INSIG2 and ATF3 genes in the liver. Notably, in vivo miR-9-5p antagomir treatment and genetic CH25H ablation prevents tumor metastasis in a mouse model of breast cancer. Thus, our findings reveal the regulatory mechanism of tumor-derived miR-9-5p in liver metastasis by linking oxysterol metabolism and Kupffer cell polarization, shedding light on future applications for cancer diagnosis and treatment.

Characterization of primary aroma compounds in Pu‐erh raw tea sourced from various regions using gas chromatography‐mass spectrometry and headspace solid‐phase microextraction

AbstractThe assessment of aroma plays a crucial role in determining the characteristics and quality of Pu‐erh tea. The volatile compounds present in Pu‐erh raw tea sourced from five regions in Yunnan Province have been studied using gas chromatography‐mass spectrometry (GC‐MS) in conjunction with headspace solid phase microextraction (HS‐SPME). A total of 69 aroma‐active compounds were identified, with alcohols, ketones, and aldehydes being the predominant component types. Furthermore, notable variations were observed among four pairs of volatile terpenoid enantiomers. Significant discrimination was achieved using orthogonal partial least squares discrimination analysis (R(x2) = 0.926, R(y2) = 0.843, Q2 = 0.501). Moreover, 33 important differential compounds were identified based on variable importance in the projection values (VIP > 1.0) and one‐way ANOVA analysis (p < 0.05). Subsequently, a KEGG pathway analysis was conducted, revealing enrichment pathways primarily focused on the mevalonate pathway, steroid biosynthesis, and fatty acid biosynthesis. Ultimately, the key volatile compounds and major aroma‐contributing compounds were successfully identified.

Development of Komagataella phaffii as a cell factory for efficient de novo production of β-caryophyllene.

β-Caryophyllene is a natural bicyclic sesquiterpene found in a large number of plants around the world. It has anti-inflammatory, anticancer and analgesic biological activities associated with its important medicinal value, and has also attracted attention in the field of bioenergy with high energy density. Due to the low amount of β-caryophyllene in plants and complex purification process, microbial biosynthesis is considered as a promising alternative for the industrial development of β-caryophyllene. Komagataella phaffii has a robust transcriptional regulatory system and has many advantages in protein expression, high-density culture, making it suitable for large-scale industrial production. However, there are no systematic studies on the efficient biosynthesis of β-caryophyllene in K. phaffii. In this study, firstly, farnesyl diphosphate synthase ERG20 and β-caryophyllene synthase AaCPS were fused and expressed with different linkers. Secondly, we enhanced the mevalonate pathway and inhibited the branch pathway. At last, the copy number of ERG20-(PA)5-AaCPS were adjusted for the biosynthesis of β-caryophyllene, a highly efficient β-caryophyllene production strain AaCPS16 was constructed. AaCPS16 could produce 136.4mg/L β-caryophyllene in shake flask level, which was 37 times higher than the initial strain AaCPS1. To the best of our knowledge, this is the first report of caryophyllene biosynthesis in Komagataella phaffii. This established a good foundation for the synthesis of sesquiterpenes in K. phaffii.

Can Rosuvastatin Reduce the Risk of Thrombosis in Patients with Hypercholesterolemia with its Effect on Coagulation Factors and Homocysteine Levels?

Hypercholesterolemia is one of the main risk factors for vascular thrombosis in individuals. Therefore, the use of statins is very effective in reducing cholesterol and can reduce the risk of thrombosis in these patients. Rosuvastatin, a member of the statin family which, inhibits cholesterol synthesis. Very few studies have been done in relation to how rosuvastatin can affect thrombosis. So, this research has been tried whether rosuvastatin can have an effect on coagulation factors and homocysteine as risk factors for thrombosis in hypercholesterolemia? In this experimental study, 60 patients (30 men and 30 women with a mean age of 40- 70 years) diagnosed with hypercholesterolemia (cholesterol > 250 mg/dl) participated in this research. 30 patients were prescribed rosuvastatin (20 mg/day), and 30 patients were simultaneously taken placebo for three months. All parameters, including FVIII, FV, Fibrinogen, DDimer, plasma homocysteine level and lipid profile, were measured before and after treatment. All the results were statistically compared between the two groups. In patients who took rosuvastatin, the drug was able to significantly reduce the concentrations of total cholesterol, triglycerides, and low-density lipoprotein (LDL) (P < 0.001). Also, rosuvastatin was able to reduce the concentrations of homocysteine significantly, D-Dimer (P < 0.001), coagulation factor VIII and factor V (P < 0.05). In patients with hypercholesterolemia who took the placebo, did not affect the mentioned variables (P > 0.05). According to the results, it seems that rosuvastatin may be able to reduce the risk of thrombosis in patients by affecting coagulation factors and homocysteine levels.

Metabolomic and transcriptomic analysis of Panax Notoginseng root: Mechanistic insights into the effects of flower bud removal on yield and phytochemical composition

The roots of Panax notoginseng (Burk.) F. H. Chen are used to treat organ damage and cardiovascular diseases in several East Asian countries. The removal of floral buds before inflorescence formation is widely employed to increase rhizome yield in the cultivation of Panax notoginseng. However, the molecular mechanisms regulating the growth and the accumulation of secondary metabolites remain unclear. We find that after the removal of flower buds, the root biomass accumulated in three-year-old Panax notoginseng after growing for four months increased by 27 %. Comparative transcriptomic and metabolomic analyses revealed that disbudding promoted the conversion of small molecule sugars to polysaccharides by regulating the expression of genes in the glycogen metabolism (GLG) family. It facilitated the accumulation of metabolites in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by upregulating the expression of isoprenoid synthase (ISP) family genes and inhibited the conversion from the tricarboxylic acid (TCA) cycle to the mevalonate (MVA) pathway by downregulating the expression of acetyl-CoA acetyltransferase (ACAT) family genes. At the same time, the total content of triterpene saponins, main active ingredients, basically remained unchanged. However, the content of intermediate saponins increased after the upregulation of cytochrome P450 monooxygenase (CYP) transcripts, while that of downstream saponins decreased after the downregulation of UDP-glucuronosyltransferase (UGT) transcripts. Additionally, scanning electron microscopy (SEM) observations of root cross-sections indicated that the disbudding potentially promoted the development of the primary root cortex and epidermis by regulating auxin-cytokinin (AUX-CTK) signaling, thus alleviating xylem cavitation. This study confirms the removal of flower buds as an effective way to enhance yield of Panax notoginseng, offering new insights into the basic mechanisms of growth regulation during plant reproductive development and providing valuable reference for studying metabolite accumulation patterns in Panax notoginseng and other medicinal plants in the Panax genus.

Effects of Wedelia Ethanol Extract on Sperm Quality and Blood Cholesterol Levels in Obese Rats

Obesity is a complex, multifactorial condition contributing to male infertility by increasing oxidative stress, which negatively affects reproductive hormones, sperm quality, and blood cholesterol levels. The Wedelia plant (Sphagneticola trilobata (L.) Pruski) is a natural herbaceous plant containing flavonoids and tannins, known for enhancing sperm quality and inhibiting cholesterol synthesis in the blood. This study aims to determine the potential of Wedelia in improving sperm quality and regulating blood cholesterol levels. The study used 24 male Wistar rats divided into four groups, consisting of C (no HFF, distilled water only), NC (HFF, distilled water), T1 (HFF and 100 mg/Kg BW Wedelia extract), and T2 (HFF and 200 mg/Kg BW Wedelia extract). HFF was administered from weeks 1 to 3, followed with extract treatments from weeks 3 to 5. On day 36, the rats were euthanized for blood collection using hematocrit into EDTA tubes. The rats were dissected for the epididymis, which was then incised in a 10% PBS to release sperm. Sperm motility and count were observed on glass slides, while sperm viability and morphology were analyzed with hematoxylin-eosin using an Optilab microscope (400x magnification). The observation parameters included sperm morphology, count, motility, viability, total cholesterol, LDL, and HDL levels. Data were analyzed using ANOVA with DMRT post hoc testing (p ≤ 0.05). The results showed that T2 exhibited an increase in sperm count, motility, viability, and morphology in HFF-induced obese rats. T1 and T2 also showed enhanced HDL cholesterol and reduced total and LDL cholesterol levels. In conclucions, a Wedelia leaf extract dose of 200 mg/kg BW optimally improves sperm quality and reduces total and LDL cholesterol levels.

HDL Cholesterol-Associated Shifts in the Expression of Preselected Genes Reveal both Pro-Atherogenic and Atheroprotective Effects of HDL in Coronary Artery Disease.

The associations of high-density lipoprotein (HDL) level and functionality with lipid metabolism, inflammation, and innate immunity in coronary artery disease (CAD) remain controversial. The differential expression of a set of genes related to HDL metabolism (24 genes) and atherogenesis (41 genes) in peripheral blood mononuclear cells (PBMC) from CAD and control patients with varied HDL cholesterol (HDL-C) levels was compared. 76 male patients 40-60 years old with CAD diagnosed by angiography and 63 control patients were divided into three groups with low, normal (1.0-1.4 mM), and increased HDL-C levels. Transcript levels were measured by real-time PCR. The differentially expressed genes (DEGs) and associated metabolic pathways were analyzed for three groups, with prevalent CAD as an outcome. The common feature was the increased odds ratio values for liver X receptor (LXR) gene expression for three patient groups. CAD patients with low HDL-C possessed 24 DEGs with lower expression of genes involved in cholesterol efflux, and down-regulated SREBF1 and ABCG1 are suggested as gene signatures. CAD patients with normal HDL-C possessed nine DEGs with down-regulated ITGAM and ALB as gene signatures. CAD patients with increased HDL-C possessed 19 DEGs with down-regulated APOA1 and HMGCR as gene signatures. With gene expression signatures, one standard deviation higher average gene expressions were associated with 5.1-, 48.8-, and 38.9-fold fewer CAD cases for three patient groups. As HDL-C increased in CAD patients, the expression of ABCG1, CUBN, and HDLBP genes increased, while the expression of HMGCR and NPC2 genes, involved in cholesterol synthesis and trafficking, decreased. The expression of CD14, CD36, S100A8, S100A9, S100A12, TLR5, TLR8, and VEGFA genes, involved in angiogenesis and inflammation mainly via nuclear factor-κB (NF-κB), decreased. The increased accumulation of cholesteryl ester in PBMC from patients with low HDL-C was suggested. This assumption contrasts with the suggested accumulation of free cholesterol in PBMC from patients with increased HDL-C, concomitant with suppression of cholesterol synthesis and traffic to the plasma membrane, and with an inflammatory state controlled by depressed CD36-mediated and upregulated apoE-mediated immunometabolic signaling. Gene signatures may be used for the diagnosis, prognosis, and treatment of CAD in dependence on HDL-C levels.

Targeting the mevalonate pathway enhances the efficacy of 5-fluorouracil by regulating pyroptosis

The 5-fluorouracil (5-FU)-based chemotherapy regimen is a primary strategy for treating pancreatic cancer (PC). However, challenges related to 5-FU resistance persist. Investigating the mechanisms of 5-FU resistance and identifying a clinically viable therapeutic strategy are crucial for improving the prognosis of PC. Here, through clinical samples analysis, we found that the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-limiting enzyme in mevalonate metabolism, is negatively correlated with the efficacy of 5-FU treatment. There is a significant correlation between HMGCR and the pyroptosis marker gasdermin D (GSDMD), and the HMGCR inhibitor simvastatin can significantly inhibit the activation of pyroptosis signaling. The exogenous addition of geranylgeranyl pyrophosphate (GGPP), a key metabolite of the mevalonate pathway, can significantly reduce sensitivity to 5-FU, and simvastatin combined with 5-FU demonstrates a strong synergistic effect. Furthermore, in organoid models and genetically engineered mice with spontaneous PC, the combination of simvastatin and 5-FU significantly inhibits tumor growth. In conclusion, our study reveals the critical role of the mevalonate pathway in 5-FU resistance and proposes a clinically feasible combination therapy strategy.

Efficient Production of the Colorless Carotenoid Phytoene in Yarrowia lipolytica through Metabolic Engineering.

Phytoene, a colorless carotenoid with unique ultraviolet (UV)-B absorption properties, offers potential for applications in functional food, cosmetics, and therapeutics. However, their low natural yield poses a challenge for large-scale production. This study aims to enhance phytoene production in the oleaginous yeast Yarrowia lipolytica by introducing a heterologous phytoene synthase gene combined with metabolic engineering approaches. We enhanced phytoene synthesis by overexpressing key genes in the mevalonate pathway and compartmentalizing the biosynthetic pathway within peroxisomes. Moreover, we inhibited the glyoxylate cycle to increase the accumulation of peroxisomal acetyl-CoA available for phytoene production. Our engineered strains demonstrated a significant increase in phytoene production, reaching up to 1.34 g/L titer and 58.74 mg/gDCW yield in the flask-scale fed-batch culture, which are the highest levels reported to date. These results underscore the potential of Y. lipolytica as a robust platform for producing phytoenes and other terpenoids on an industrial scale, offering valuable insights for future efforts in metabolic engineering.

Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization

Microbial biosynthesis has become the leading commercial approach for large-scale production of terpenoids, a valuable class of natural products. Enhancing terpenoid production, however, requires complex modifications on the host organism. Recently, a two-step isopentenol utilization (IU) pathway relying solely on ATP as the cofactor has been proposed as an alternative to the mevalonate (MVA) pathway, streamlining the synthesis of the common terpenoid precursors. Herein, we find that isopentenol inhibits energy metabolism, leading to reduced efficiency of the IU pathway in Saccharomyces cerevisiae. To overcome this, we engineer an IU pathway-dependent (IUPD) strain, designed for growth-coupled production. The IUPD strain is compelled to enhance the ATP supply, essential for the IU pathway, and incorporates a high-throughput screening method for enzyme evolution. The refined IU pathway surpasses the MVA pathway in synthesizing complex terpenoids. Our work offers valuable insights into developing growth-coupled strains adapted to efficient natural product synthesis.

Lipophilic bisphosphonates reduced cyst burden and ameliorated hyperactivity of mice chronically infected with Toxoplasma gondii.

The current treatments for toxoplasmosis are only active against fast-growing tachyzoites, present in acute infections, with little effect on slow-growing bradyzoites within tissue cysts, present in latent chronic infections. The mitochondrion of Toxoplasma gondii is essential for its survival, and one of the major anti-parasitic drugs, atovaquone, inhibits the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase site. Coenzyme Q (also known as ubiquinone [UQ]) consists of a quinone head and a lipophilic, isoprenoid tail that anchors UQ to membranes. The synthesis of the isoprenoid unit is essential for cell growth and is inhibited by lipophilic bisphosphonates, which inhibit the parasite growth. In this work, we investigated the effect of lipophilic bisphosphonates on the chronic stages of T. gondii. We discovered that three lipophilic bisphosphonates (BPH-1218, BPH-1236, and BPH-1238), effective for the acute infection, were also effective in controlling the development of chronic stages. We showed effectiveness by testing them against in vitro cysts and in vivo derived tissue cysts and, most importantly, these compounds reduced the cyst burden in the brains of chronically infected mice. We monitored the activity of infected mice non-invasively and continuously with a novel device termed the CageDot. A decrease in activity accompanied the acute phase, but mice recovered to normal activity and showed signs of hyperactivity when the chronic infection was established. Moreover, treatment with atovaquone or BPH-1218 ameliorated the hyperactivity observed during the chronic infection.IMPORTANCETreatment for toxoplasmosis is challenged by a lack of effective drugs to eradicate the chronic stages. Most of the drugs currently used are poorly distributed to the central nervous system, and they trigger allergic reactions in a large number of patients. There is a compelling need for safe and effective treatments for toxoplasmosis. Bisphosphonates (BPs) are analogs of inorganic pyrophosphate and are used for the treatment of bone disorders. BPs target the isoprenoid pathway and are effective against several experimental parasitic infections. Some lipophilic BPs can specifically inhibit the mitochondrial activity of Toxoplasma gondii by interfering with the mechanism by which ubiquinone is inserted into the inner mitochondrial membrane. In this work, we present the effect of three lipophilic BPs against T. gondii chronic stages. We also present a new strategy for the monitoring of animal activity during disease and treatment that is non-invasive and continuous.

Synthetic indole derivatives as an antibacterial agent inhibiting respiratory metabolism of multidrug-resistant gram-positive bacteria

The survival of modern medicine depends heavily on the effective prevention and treatment of bacterial infections, are threatened by antibacterial resistance. The increasing use of antibiotics and lack of stewardship have led to an increase in antibiotic-resistant pathogens, so the growing issue of resistance can be resolved by emphasizing chemically synthesized antibiotics. This study discovered SMJ-2, a synthetic indole derivative, is effective against all multidrug-resistant gram-positive bacteria. SMJ-2 has multiple targets of action, but the primary mechanism inhibits respiratory metabolism and membrane potential disruption. SMJ-2 was discovered to interfere with the mevalonate pathway, ultimately preventing the synthesis of farnesyl diphosphate, a precursor to the antioxidant staphyloxanthin, eventually releasing reactive oxygen species, and leading phagocytic cells to destroy pathogens. Additionally, no discernible biochemical and histopathological alterations were found in the mouse acute toxicity model. This study emphasizes mechanistic insights into SMJ-2 as a potential antibacterial with an unusual method of action.

HMGCS1 variants cause rigid spine syndrome amenable to mevalonic acid treatment in an animal model.

Rigid spine syndrome is a rare childhood-onset myopathy characterised by slowly progressive or non-progressive scoliosis, neck and spine contractures, hypotonia, and respiratory insufficiency. Biallelic variants in SELENON account for most cases of rigid spine syndrome, however, the underlying genetic cause in some patients remains unexplained. We used exome and genome sequencing to investigate the genetic basis of rigid spine syndrome in patients without a genetic diagnosis. In five patients from four unrelated families, we identified biallelic variants in HMGCS1 (3-hydroxy-3-methylglutaryl-coenzyme A synthase). These included six missense variants and one frameshift variant distributed throughout HMGCS1. All patients presented with spinal rigidity primarily affecting the cervical and dorsolumbar regions, scoliosis, and respiratory insufficiency. Creatine kinase levels were variably elevated. The clinical course worsened with intercurrent disease or certain drugs in some patients; one patient died from respiratory failure following infection. Muscle biopsies revealed irregularities in oxidative enzyme staining with occasional internal nuclei and rimmed vacuoles. HMGCS1 encodes a critical enzyme of the mevalonate pathway and has not yet been associated with disease. Notably, biallelic hypomorphic variants in downstream enzymes including HMGCR and GGPS1 are associated with muscular dystrophy resembling our cohort's presentation. Analyses of recombinant human HMGCS1 protein and four variants (p.S447P, p.Q29L, p.M70T, p.C268S) showed that all mutants maintained their dimerization state. Three of the four mutants exhibited reduced thermal stability, and two mutants showed subtle changes in enzymatic activity compared to the wildtype. Hmgcs1 mutant zebrafish displayed severe early defects, including immobility at 2 days and death by day 3 post-fertilisation and were rescued by HMGCS1 mRNA. We demonstrate that the four variants tested (S447P, Q29L M70T, and C268S) have reduced function compared to wildtype HMGCS1 in zebrafish rescue assays. Additionally, we demonstrate the potential for mevalonic acid supplementation to reduce phenotypic severity in mutant zebrafish. Overall, our analyses suggest that these missense variants in HMGCS1 act through a hypomorphic mechanism. Here, we report an additional component of the mevalonate pathway associated with disease and suggest biallelic variants in HMGCS1 should be considered in patients presenting with an unresolved rigid spine myopathy phenotype. Additionally, we highlight mevalonoic acid supplementation as a potential treatment for patients with HMGCS1-related disease.