Glucose-oxygen deprivation constrains HMGCR function and Rac1 prenylation and activates the NLRP3 inflammasome in human monocytes.

Hypoxia and low glucose abundance often occur simultaneously at sites of inflammation. In monocytes and macrophages, glucose-oxygen deprivation stimulates the assembly of the NLRP3 inflammasome to generate the proinflammatory cytokine IL-1ß. We found that concomitant glucose deprivation and hypoxia activated the NLRP3 inflammasome by constraining the function of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate kinase pathway. HMGCR is involved in the synthesis of geranylgeranyl pyrophosphate (GGPP), which is required for the prenylation and lipid membrane integration of proteins. Under glucose-oxygen deprivation, GGPP synthesis was decreased, leading to reduced prenylation of the small GTPase Rac1, increased binding of nonprenylated Rac1 to the scaffolding protein IQGAP1, and enhanced activation of the NLRP3 inflammasome. In response to restricted oxygen and glucose supply, patient monocytes with a compromised mevalonate pathway due to mevalonate kinase deficiency or Muckle-Wells syndrome released more IL-1ß than did control monocytes. Thus, reduced GGPP synthesis due to inhibition of HMGCR under glucose-oxygen deprivation results in proinflammatory innate responses, which are normally kept in check by the prenylation of Rac1. We suggest that this mechanism is also active in inflammatory autoimmune conditions.

Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

Induction of resistance to neurotrophic tropomyosin-receptor kinase inhibitors by HMGCS2 via a mevalonate pathway.

INTRODUCTION: A neurotrophic tropomyosin receptor kinase (NTRK)-tyrosine kinase inhibitor (TKI) has shown dramatic efficacy against malignant tumors harboring an NTRK fusion gene. However, almost all tumors eventually acquire resistance to NTRK-TKIs. METHOD: To investigate the mechanism of resistance to NTRK-TKIs, we established cells resistant to three types of NTRK-TKIs (larotrectinib, entrectinib, and selitrectinib) using KM12 colon cancer cells with a TPM3-NTRK1 rearrangement. RESULT: Overexpression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) was observed in three resistant cells (KM12-LR, KM12-ER, and KM12-SR) by microarray analysis. Lower expression of sterol regulatory element-binding protein 2 (SREBP2) and peroxisome proliferator activated receptor α (PPARα) was found in two cells (KM12-ER and KM12-SR) in which HMGCS2 was overexpressed compared to the parental KM12 and KM12-LR cells. In resistant cells, knockdown of HMGCS2 using small interfering RNA improved the sensitivity to NTRK-TKI. Further treatment with mevalonolactone after HMGCS2 knockdown reintroduced the NTRK-TKI resistance. In addition, simvastatin and silibinin had a synergistic effect with NTRK-TKIs in resistant cells, and delayed tolerance was observed after sustained exposure to clinical concentrations of NTRK-TKI and simvastatin in KM12 cells. In xenograft mouse models, combination treatment with entrectinib and simvastatin reduced resistant tumor growth compared with entrectinib alone. CONCLUSION: These results suggest that HMGCS2 overexpression induces resistance to NTRK-TKIs via the mevalonate pathway in colon cancer cells. Statin inhibition of the mevalonate pathway may be useful for overcoming this mechanistic resistance.

The picky mTORC1 in metabolic enzyme degradation.

In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.

Activation of the Mevalonate Pathway in Response to Anti-cancer Treatments Drives Glioblastoma Recurrences Through Activation of Rac-1.

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

HY5 and PIF antagonistically regulate HMGR expression and sterol biosynthesis in Arabidopsis thaliana.

Secondary metabolites play multiple crucial roles in plants by modulating various regulatory networks. The biosynthesis of these compounds is unique to each species and is intricately controlled by a range of developmental and environmental factors. While light's role in certain secondary metabolites is evident, its impact on sterol biosynthesis remains unclear. Previous studies indicate that ELONGATED HYPOCOTYL5 (HY5), a bZIP transcription factor, is pivotal in skotomorphogenesis to photomorphogenesis transition. Additionally, PHYTOCHROME INTERACTING FACTORs (PIFs), bHLH transcription factors, act as negative regulators. To unveil the light-dependent regulation of the mevalonic acid (MVA) pathway, a precursor for sterol biosynthesis, mutants of light signaling components, specifically hy5-215 and the pifq quadruple mutant (pif 1,3,4, and 5), were analyzed in Arabidopsis thaliana. Gene expression analysis in wild-type and mutants implicates HY5 and PIFs in regulating sterol biosynthesis genes. DNA-protein interaction analysis confirms their interaction with key genes like AtHMGR2 in the rate-limiting pathway. Results strongly suggest HY5 and PIFs' pivotal role in light-dependent MVA pathway regulation, including the sterol biosynthetic branch, in Arabidopsis, highlighting a diverse array of light signaling components finely tuning crucial growth pathways.

mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.

Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.

De novo biosynthesis of 3-hydroxy-3-methylbutyrate as anti-catabolic supplement by metabolically engineered Escherichia coli.

3-Hydroxy-3-methylbutyrate (HMB) is a five-carbon branch-chain hydroxy acid currently used as a dietary supplement to treat sarcopenia and exercise training. However, its current production relies on conventional chemical processes which require toxic substances and are generally non-sustainable. While bio-based syntheses of HMB have been developed, they are dependent on biotransformation of its direct precursors which are generally costly. Therefore, in this work, we developed a synthetic de novo HMB biosynthetic pathway that enables HMB production from renewable resources. This novel HMB biosynthesis employs heterologous enzymes from mevalonate pathway and myxobacterial iso-fatty acid pathway for converting acetyl-CoA to HMB-CoA. Subsequently, HMB-CoA is hydrolyzed by a thioesterase to yield HMB. Upon expression of this pathway, our initial Escherichia coli strain produced 660 mg/L of HMB from glucose in 48 hours. Through optimization of coenzyme A removal from HMB-CoA and genetic operon structure, our final strain achieved HMB production titer of 17.7 g/L in glucose minimal media using a bench-top bioreactor. This engineered strain was further demonstrated to produce HMB from other renewable carbon sources such as xylose, glycerol, and acetate. The results from this work provided a flexible and environmentally benign method for producing HMB.

Statin prevents cancer development in chronic inflammation by blocking interleukin 33 expression.

Chronic inflammation is a major cause of cancer worldwide. Interleukin 33 (IL-33) is a critical initiator of cancer-prone chronic inflammation; however, its induction mechanism by environmental causes of chronic inflammation is unknown. Herein, we demonstrate that Toll-like receptor (TLR)3/4-TBK1-IRF3 pathway activation links environmental insults to IL-33 induction in the skin and pancreas inflammation. An FDA-approved drug library screen identifies pitavastatin to effectively suppress IL-33 expression by blocking TBK1 membrane recruitment/activation through the mevalonate pathway inhibition. Accordingly, pitavastatin prevents chronic pancreatitis and its cancer sequela in an IL-33-dependent manner. The IRF3-IL-33 axis is highly active in chronic pancreatitis and its associated pancreatic cancer in humans. Interestingly, pitavastatin use correlates with a significantly reduced risk of chronic pancreatitis and pancreatic cancer in patients. Our findings demonstrate that blocking the TBK1-IRF3-IL-33 signaling axis suppresses cancer-prone chronic inflammation. Statins present a safe and effective prophylactic strategy to prevent chronic inflammation and its cancer sequela.

Unravelling the origin of isoprene in the human body-a forty year Odyssey.

In the breath research community's search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e. for 40 years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is to review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

Squalene Epoxidase: Its Regulations and Links with Cancers.

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

Remodeling the Homologous Recombination Mechanism of Yarrowia lipolytica for High-Level Biosynthesis of Squalene.

Squalene is a high-value antioxidant with many commercial applications. The use of microbial cell factories to produce squalene as an alternative to plant and animal extracts could meet increasing market demand. Yarrowia lipolytica is an excellent host for squalene production due to its high levels of acetyl-CoA and a hydrophobic environment. However, the need for precise and complicated gene editing has hindered the industrialization of this strain. Herein, the rapid construction of a strain with high squalene production was achieved by enhancing the homologous recombination efficiency in Y. lipolytica. First, remodeling of the homologous recombination efficiency resulted in a 10-fold increase in the homologous recombination rate. Next, the whole mevalonate pathway was integrated into the chromosome to enhance squalene production. Then, a higher level of squalene accumulation was achieved by increasing the level of acetyl coenzyme A and regulating the downstream steroid synthesis pathway. Finally, the squalene production reached 35 g/L after optimizing the fermentation conditions and performing a fed-batch culture in a 5 L jar fermenter. This is the highest squalene production ever reported to date by de novo biosynthesis without adding any inhibitors, paving a new path toward the industrial production of squalene and its downstream products.

Multi-modular metabolic engineering and efflux engineering for enhanced lycopene production in recombinant Saccharomyces cerevisiae.

Lycopene has been widely used in the food industry and medical field due to its antioxidant, anti-cancer, and anti-inflammatory properties. However, achieving efficient manufacture of lycopene using chassis cells on an industrial scale remains a major challenge. Herein, we attempted to integrate multiple metabolic engineering strategies to establish an efficient and balanced lycopene biosynthetic system in Saccharomyces cerevisiae. First, the lycopene synthesis pathway was modularized to sequentially enhance the metabolic flux of the mevalonate pathway, the acetyl-CoA supply module, and lycopene exogenous enzymatic module. The modular operation enabled the efficient conversion of acetyl-CoA to downstream pathway of lycopene synthesis, resulting in a 3.1-fold increase of lycopene yield. Second, we introduced acetate as an exogenous carbon source and utilized an acetate-repressible promoter to replace the natural ERG9 promoter. This approach not only enhanced the supply of acetyl-CoA but also concurrently diminished the flux toward the competitive ergosterol pathway. As a result, a further 42.3% increase in lycopene production was observed. Third, we optimized NADPH supply and mitigated cytotoxicity by overexpressing ABC transporters to promote lycopene efflux. The obtained strain YLY-PDR11 showed a 12.7-fold increase in extracellular lycopene level compared to the control strain. Finally, the total lycopene yield reached 343.7 mg/L, which was 4.3 times higher than that of the initial strain YLY-04. Our results demonstrate that combining multi-modular metabolic engineering with efflux engineering is an effective approach to improve the production of lycopene. This strategy can also be applied to the overproduction of other desirable isoprenoid compounds with similar synthesis and storage patterns in S. cerevisiae. ONE-SENTENCE SUMMARY: In this research, lycopene production in yeast was markedly enhanced by integrating a multi-modular approach, acetate signaling-based down-regulation of competitive pathways, and an efflux optimization strategy.

Effects of hindlimb unloading on the mevalonate and mechanistic target of rapamycin complex 1 signaling pathways in a fast-twitch muscle in rats.

Fast-twitch muscles are less susceptible to disuse atrophy, activate the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, and increase protein synthesis under prolonged muscle disuse conditions. However, the mechanism underlying prolonged muscle disuse-induced mTORC1 signaling activation remains unclear. The mevalonate pathway activates the mTORC1 signaling pathway via the prenylation and activation of Ras homolog enriched in brain (Rheb). Therefore, we investigated the effects of hindlimb unloading (HU) for 14 days on the mevalonate and mTORC1 signaling pathways in the plantaris muscle, a fast-twitch muscle, in adult male rats. Rats were divided into HU and control groups. The plantaris muscles of both groups were harvested after the treatment period, and the expression and phosphorylation levels of metabolic and intracellular signaling proteins were analyzed using Western blotting. We found that HU increased the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, and activated the mTORC1 signaling pathway without activating AKT, an upstream activator of mTORC1. Furthermore, HU increased prenylated Rheb. Collectively, these findings suggest that the activated mevalonate pathway may be involved in the activation of the Rheb/mTORC1 signaling pathway without AKT activation in fast-twitch muscles under prolonged disuse conditions.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclusion: Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Structural Characterization and Functional Analysis of Mevalonate Kinase from Tribolium castaneum (Red Flour Beetle).

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/ß conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.

RNA-seq analysis reveals narrow differential gene expression in MEP and MVA pathways responsible for phytochemical divergence in extreme genotypes of Thymus daenensis Celak.

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

The non-mevalonate pathway requires a delicate balance of intermediates to maximize terpene production.

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

Targeting mevalonate pathway by zoledronate ameliorated pulmonary fibrosis in a rat model: Promising therapy against post-COVID-19 pulmonary fibrosis.

BACKGROUND: Rho kinase (ROCK) pathway plays a critical role in post-COVID-19 pulmonary fibrosis (PCPF) and its intervention with angiotensin-converting enzyme 2 (ACE2) and vascular endothelial growth factor (VEGF) will be a potential therapeutic target. OBJECTIVES: The present study was conducted to investigate the efficacy of zoledronate (ZA) on carbon tetrachloride (CCl4) induced pulmonary fibrosis (PF) in rats through targeting ACE2, ROCK, and VEGF signaling pathways. METHODS: Fifty male Wistar rats were divided into five groups: control, vehicle-treated, PF, PF-ZA 50, and PF-ZA 100 groups. ZA was given in two different doses 100 and 50 µg/kg/week intraperitoneally. After anesthesia, mean arterial blood pressure (MBP) was measured. After scarification, lung coefficient was calculated. Lung levels of ACE 2, interleukin-1ß (IL-1ß), transforming growth factor-ß (TGF-ß), VEGF, glutathione (GSH), and superoxide dismutase (SOD) were measured. Expression of ROCK, phosphorylated myosin phosphatase target subunit 1 (P-MYPT1), and matrix metalloproteinase (MMP-1), along with histopathological changes and immune-histochemical staining for lung α-smooth muscle actin (α-SMA), tumor necrosis factor-alpha (TNFα), and caspase-3, were evaluated. RESULTS: ZA significantly prevented the decrease in MBP. ZA significantly increased ACE2, GSH, and SOD and significantly decreased IL-1ß, TGF-ß, and VEGF in lung in comparison to PF group. ZA prevented the histopathological changes induced by CCl4. ZA inhibited lung expression of ROCK, P-MYPT1, MMP-1, α-SMA, TNFα, and caspase-3 with significant differences favoring the high dose intervention. CONCLUSION: ZA in a dose-dependent manner prevented the pathological effect of CCl4 in the lung by targeting mevalonate pathway. It could be promising therapy against PCPF.