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1.
Microb Cell Fact ; 22(1): 14, 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36658566

RESUMO

BACKGROUND: Pseudomonas putida has received increasing interest as a cell factory due to its remarkable features such as fast growth, a versatile and robust metabolism, an extensive genetic toolbox and its high tolerance to oxidative stress and toxic compounds. This interest is driven by the need to improve microbial performance to a level that enables biologically possible processes to become economically feasible, thereby fostering the transition from an oil-based economy to a more sustainable bio-based one. To this end, one of the current strategies is to maximize the product-substrate yield of an aerobic biocatalyst such as P. putida during growth on glycolytic carbon sources, such as glycerol and xylose. We demonstrate that this can be achieved by implementing the phosphoketolase shunt, through which pyruvate decarboxylation is prevented, and thus carbon loss is minimized. RESULTS: In this study, we introduced the phosphoketolase shunt in the metabolism of P. putida KT2440. To maximize the effect of this pathway, we first tested and selected a phosphoketolase (Xfpk) enzyme with high activity in P. putida. Results of the enzymatic assays revealed that the most efficient Xfpk was the one isolated from Bifidobacterium breve. Using this enzyme, we improved the P. putida growth rate on glycerol and xylose by 44 and 167%, respectively, as well as the biomass yield quantified by OD600 by 50 and 30%, respectively. Finally, we demonstrated the impact on product formation and achieved a 38.5% increase in mevalonate and a 25.9% increase in flaviolin yield from glycerol. A similar effect was observed on the mevalonate-xylose and flaviolin-xylose yields, which increased by 48.7 and 49.4%, respectively. CONCLUSIONS: Pseudomonas putida with the implemented Xfpk shunt grew faster, reached a higher final OD600nm and provided better product-substrate yields than the wild type. By reducing the pyruvate decarboxylation flux, we significantly improved the performance of this important workhorse for industrial applications. This work encompasses the first steps towards full implementation of the non-oxidative glycolysis (NOG) or the glycolysis alternative high carbon yield cycle (GATCHYC), in which a substrate is converted into products without CO2 loss These enhanced properties of P. putida will be crucial for its subsequent use in a range of industrial processes.


Assuntos
Pseudomonas putida , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Xilose/metabolismo , Glicerol/metabolismo , Ácido Mevalônico/metabolismo , Piruvatos/metabolismo , Carbono/metabolismo
2.
Anticancer Res ; 43(2): 547-555, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36697063

RESUMO

BACKGROUND/AIM: The prognosis of patients with multiple myeloma (MM) has recently improved due to the emergence of new molecular targeting agents. However, MM remains incurable because MM stem cells are resistant to these agents. Therefore, it is essential to develop strategies to eradicate MM stem cells. We have previously demonstrated that MM cells cultured under prolonged hypoxic conditions (1% O2) (i.e., hypoxia-adapted MM cells; MM-HA cells) exhibited stem-cell-like characteristics. γδ T cells attack tumor cells by recognizing butyrophilin (BTN) 3A1 and BTN2A1, which are activated by the intracellular accumulation of isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway. In the present study, we investigated the cytotoxicity of γδ T cells against MM-HA stem-like cells. MATERIALS AND METHODS: We used a combination of flow cytometry, liquid chromatography-tandem mass spectrometry, and western blotting methods to investigate the cytotoxicity of γδ T cells against MM-HA cells and measured the amounts of IPP in MM-HA cells and their supernatants. RESULTS: The cytotoxicity of γδ T cells against MM-HA cells was significantly lower than that against MM cells cultured under normoxic conditions (20% O2; MM-Normo). Furthermore, the concentration of IPP in MM-HA cells was lower than that in MM-Normo cells. The expression of mevalonate decarboxylase and farnesyl diphosphate synthase proteins were decreased in MM-HA-cells. CONCLUSION: The cytotoxicity of γδ T cells against MM-HA cells was suppressed by the reduced IPP accumulation by modulating the mevalonate pathway in MM-HA cells.


Assuntos
Ácido Mevalônico , Mieloma Múltiplo , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Hipóxia , Células-Tronco , Ativação Linfocitária
3.
Talanta ; 254: 124182, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36527912

RESUMO

Isoprenoids give rise to many functional products used today such as flavours, fragrances and even pharmaceutical compounds. Mevalonate pathway metabolites are the key intermediates that affect the production yield of isoprenoids. With increasing demand and benefit of isoprenoids, the present study adopts Analytical Quality-by-Design (AQbD) approach to establish an efficacious extraction protocol prior to the determination of mevalonate pathway metabolites in an engineered Escherichia coli model. The statistical experimental design approach, described in this work, has successfully validated an optimised sample preparation method i.e., using acetonitrile: 50 mM ammonium formate (pH 9.5) (7:3) (ACN73) at -20 °C for 10 min without solvent evaporation to retain the targeted mevalonate metabolites in engineered E. coli strain. The study also demonstrates the use of liquid chromatography paired with a Time-of-Flight Mass Spectrometer (LC-ToF-MS) for the quantitative analysis of the mevalonate pathway metabolites in E. coli. The analytical method was validated in accordance with guidelines in Metabolomics Standards Initiative and ICH Q2 (R1) with analyte spike recoveries at 80% and above. In short, the present study overcomes the one-variable-at-a-time (OVAT) limitations in analytical development, minimises metabolite losses and gives better cost and time efficiencies by eliminating the solvent evaporation and swapping process. This work highlights the importance of analytical methods development in microbial metabolomics studies.


Assuntos
Escherichia coli , Ácido Mevalônico , Escherichia coli/metabolismo , Ácido Mevalônico/metabolismo , Projetos de Pesquisa , Cromatografia Líquida/métodos , Terpenos , Solventes
4.
Metab Eng ; 74: 139-149, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36341776

RESUMO

The production of terpenoids from engineered microbes contributes markedly to the bioeconomy by providing essential medicines, sustainable materials, and renewable fuels. The mevalonate pathway leading to the synthesis of terpenoid precursors has been extensively targeted for engineering. Nevertheless, the importance of individual pathway enzymes to the overall pathway flux and final terpenoid yield is less known, especially enzymes that are thought to be non-rate-limiting. To investigate the individual contribution of the five non-rate-limiting enzymes in the mevalonate pathway, we created a combinatorial library of 243 Saccharomyces cerevisiae strains, each having an extra copy of the mevalonate pathway integrated into the genome and expressing the non-rate-limiting enzymes from a unique combination of promoters. High-throughput screening combined with machine learning algorithms revealed that the mevalonate kinase, Erg12p, stands out as the critical enzyme that influences product titer. ERG12 is ideally expressed from a medium-strength promoter which is the 'sweet spot' resulting in high product yield. Additionally, a platform strain was created by targeting the mevalonate pathway to both the cytosol and peroxisomes. The dual localization synergistically increased terpenoid production and implied that some mevalonate pathway intermediates, such as mevalonate, isopentyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP), are diffusible across peroxisome membranes. The platform strain resulted in 94-fold, 60-fold, and 35-fold improved titer of monoterpene geraniol, sesquiterpene α-humulene, and triterpene squalene, respectively. The terpenoid platform strain will serve as a chassis for producing any terpenoids and terpene derivatives.


Assuntos
Ácido Mevalônico , Saccharomyces cerevisiae , Ácido Mevalônico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Difosfatos/metabolismo , Engenharia Metabólica/métodos , Aprendizado de Máquina
5.
Sci Rep ; 12(1): 18443, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36323841

RESUMO

The microRNA (miR) miR-874, a potential tumour suppressor, causes cell death via target gene suppression in various cancer types. Mevalonate pathway inhibition also causes cell death in breast cancer. However, the relationship between the mevalonate pathway and miR-874-induced apoptosis or its association with the tumour suppressor p53 has not been elucidated. We identified phosphomevalonate kinase (PMVK), a key mevalonate pathway enzyme, and sterol regulatory element-binding factor 2 (SREBF2), the master cholesterol biosynthesis regulator, as direct miR­874 targets. Next-generation sequencing analysis revealed a significant miR-874-mediated downregulation of PMVK and SREBF2 gene expression and p53 pathway enrichment. Luciferase reporter assays showed that miR-874 directly regulated PMVK and SREBF2. miR-874-induced apoptosis was p53 dependent, and single-cell RNA sequencing analysis demonstrated that miR-874 transfection resulted in apoptosis and p53 pathway activation. Downregulation of PMVK expression also caused cell cycle arrest and p53 pathway activation, which was rescued by geranylgeranyl pyrophosphate (GGPP) supplementation. Analysis of The Cancer Genome Atlas (TCGA) database indicated a negative correlation between miR-874 and PMVK expression and between miR-874 and SREBF2 expression. These findings suggest that miR-874 suppresses the mevalonate pathway by targeting SREBF2 and PMVK, resulting in GGPP depletion, which activates the p53 pathway and promotes cycle arrest or apoptosis.


Assuntos
MicroRNAs , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ácido Mevalônico/metabolismo , Linhagem Celular Tumoral , MicroRNAs/metabolismo , Apoptose/genética , Transformação Celular Neoplásica/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica
6.
Biomed Res Int ; 2022: 7441296, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36246988

RESUMO

The saponins of Polygonatum sibiricum had many pharmacological activities such as antitumor, antioxidation, and blood sugar lowering, which were synthesized by two pathways: mevalonate (MVA) and methylerythritol phosphate (MEP). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) was the key enzyme in the MVA synthesis pathway, and its expression level may affect the accumulation of saponins which were the main active ingredients of P. sibiricum. In this study, we successfully cloned HMGS1 and HMGS2 from P. sibiricum and their sequence similarity was 93.71% with 89 different sites. The multiple sequence alignment results indicated that the N-terminal sequences of HMGS were conserved. Phylogenetic analysis showed that P. sibiricum, A. officinalis, N. tazetta, D. nobile, and other relatives had a common evolutionary ancestor. The expression levels of both HMGSs and the total saponin content in different tissues revealed that HMGS expression in rhizomes was positively correlated with total saponin content. Further study of the abiotic stress effect of Methyl Jasmonate (MeJA) demonstrated that the expression of HMGS1 and HMGS2 genes was induced by MeJA, peaked at 24 h, and fell by 48 h. Our present findings would provide a blueprint for future studies of HMGS and its role in triterpenoid biosynthesis in P. sibiricum.


Assuntos
Polygonatum , Saponinas , Triterpenos , Acetatos , Glicemia , Clonagem Molecular , Coenzima A/metabolismo , Ciclopentanos , Regulação da Expressão Gênica de Plantas , Ácido Mevalônico/metabolismo , Oxilipinas , Fosfatos/metabolismo , Filogenia , Polygonatum/genética
7.
J Microbiol ; 60(12): 1191-1200, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36279103

RESUMO

Terpenes have many applications and are widely found in nature, but recent progress in synthetic biology has enabled the use of microorganisms as chassis cells for the synthesis of these compounds. Candida glycerinogenes (C. glycerinogenes) is an industrial strain that may be developed as a chassis for the synthesis of terpenes since it has a tolerance to hyperosmolality and high sugar, and has a complete mevalonate (MVA) pathway. However, monoterpenes such as pinene are highly toxic, and the tolerance of C. glycerinogenes to pinene was investigated. We also measured the content of mevalonate and squalene to evaluate the strength of the MVA pathway. To determine terpene synthesis capacity, a pathway for the synthesis of pinene was constructed in C. glycerinogenes. Pinene production was improved by overexpression, gene knockdown and antisense RNA inhibition. Pinene production was mainly enhanced by strengthening the upstream MVA pathway and inhibiting the production of by-products from the downstream pathway. With these strategies, yield could be increased by almost 16 times, to 6.0 mg/L. Overall, we successfully constructed a pinene synthesis pathway in C. glycerinogenes and enhanced pinene production through metabolic modification.


Assuntos
Vias Biossintéticas , Ácido Mevalônico , Ácido Mevalônico/metabolismo , Fermentação , Terpenos
8.
Nat Commun ; 13(1): 6323, 2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36280687

RESUMO

Statins, a family of FDA-approved cholesterol-lowering drugs that inhibit the rate-limiting enzyme of the mevalonate metabolic pathway, have demonstrated anticancer activity. Evidence shows that dipyridamole potentiates statin-induced cancer cell death by blocking a restorative feedback loop triggered by statin treatment. Leveraging this knowledge, we develop an integrative pharmacogenomics pipeline to identify compounds similar to dipyridamole at the level of drug structure, cell sensitivity and molecular perturbation. To overcome the complex polypharmacology of dipyridamole, we focus our pharmacogenomics pipeline on mevalonate pathway genes, which we name mevalonate drug-network fusion (MVA-DNF). We validate top-ranked compounds, nelfinavir and honokiol, and identify that low expression of the canonical epithelial cell marker, E-cadherin, is associated with statin-compound synergy. Analysis of remaining prioritized hits led to the validation of additional compounds, clotrimazole and vemurafenib. Thus, our computational pharmacogenomic approach identifies actionable compounds with pathway-specific activities.


Assuntos
Neoplasias da Mama , Inibidores de Hidroximetilglutaril-CoA Redutases , Humanos , Feminino , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Ácido Mevalônico/metabolismo , Farmacogenética , Vemurafenib/uso terapêutico , Nelfinavir/uso terapêutico , Clotrimazol/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Caderinas , Colesterol , Dipiridamol
9.
Atherosclerosis ; 362: 38-46, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36253169

RESUMO

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) regulates lipid and glucose metabolism. Loss-of-function mutations in its gene, leading to ANGPTL3 deficiency, cause in humans the familial combined hypolipidemia type 2 (FHBL2) phenotype, characterized by very low concentrations of circulating lipoproteins and reduced risk of atherosclerotic cardiovascular disease. Whether this condition is accompanied by immune dysfunctions is unknown. Regulatory T cells (Tregs) are CD4 T lymphocytes endowed with immune suppressive and atheroprotective functions and sensitive to metabolic signals. By investigating FHBL2, we explored the hypothesis that Tregs expand in response to extreme hypolipidemia, through a modulation of the Treg-intrinsic lipid metabolism. METHODS: Treg frequency, phenotype, and intracellular lipid content were assessed ex vivo from FHBL2 subjects and age- and sex-matched controls, through multiparameter flow cytometry. The response of CD4 T cells from healthy controls to marked hypolipidemia was tested in vitro in low-lipid culture conditions. RESULTS: The ex vivo analysis revealed that FHBL2 subjects showed higher percentages of Tregs with a phenotype undistinguishable from controls and with a lower lipid content, which directly correlated with the concentrations of circulating lipoproteins. In vitro, lipid restriction induced the upregulation of genes of the mevalonate pathway, including those involved in isoprenoid biosynthesis, and concurrently increased the expression of the Treg markers FOXP3 and Helios. The latter event was found to be prenylation-dependent, and likely related to increased IL-2 production and signaling. CONCLUSIONS: Our study demonstrates that FHBL2 is characterized by high Treg frequencies, a feature which may concur to the reduced atherosclerotic risk in this condition. Mechanistically, hypolipidemia may directly favor Treg expansion, through the induction of the mevalonate pathway and the prenylation of key signaling proteins.


Assuntos
Doenças Metabólicas , Linfócitos T Reguladores , Humanos , Linfócitos T Reguladores/metabolismo , Proteínas Semelhantes a Angiopoietina/genética , Angiopoietinas/genética , Angiopoietinas/metabolismo , Ácido Mevalônico , Proteína 3 Semelhante a Angiopoietina , Lipoproteínas , Fatores de Transcrição Forkhead/genética
10.
Mar Drugs ; 20(10)2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36286435

RESUMO

This review presents literature data: the history of the discovery of quinoid compounds, their biosynthesis and biological activity. Special attention is paid to the description of the quinoid pigments of the sea urchins Scaphechinus mirabilis (from the family Scutellidae) and Strongylocentrotus intermedius (from the family Strongylocentrotidae). The marine environment is considered one of the most important sources of natural bioactive compounds with extremely rich biodiversity. Primary- and some secondary-mouthed animals contain very high concentrations of new biologically active substances, many of which are of significant potential interest for medical purposes. The quinone pigments are products of the secondary metabolism of marine animals, can have complex structures and become the basis for the development of new natural products in echinoids that are modulators of chemical interactions and possible active ingredients in medicinal preparations. More than 5000 chemical compounds with high pharmacological potential have been isolated and described from marine organisms. There are three well known ways of naphthoquinone biosynthesis-polyketide, shikimate and mevalonate. The polyketide pathway is the biosynthesis pathway of various quinones. The shikimate pathway is the main pathway in the biosynthesis of naphthoquinones. It should be noted that all quinoid compounds in plants and animals can be synthesized by various ways of biosynthesis.


Assuntos
Produtos Biológicos , Mirabilis , Naftoquinonas , Policetídeos , Strongylocentrotus , Animais , Strongylocentrotus/metabolismo , Mirabilis/metabolismo , Ácido Mevalônico/metabolismo , Ouriços-do-Mar/química , Naftoquinonas/química , Policetídeos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/metabolismo , Pigmentos Biológicos/farmacologia , Pigmentos Biológicos/metabolismo
11.
Sci Rep ; 12(1): 17093, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224205

RESUMO

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.


Assuntos
Lithospermum , Naftoquinonas , Acetatos , Ciclopentanos , Lithospermum/genética , Ácido Mevalônico/metabolismo , Naftoquinonas/metabolismo , Oxilipinas , Preparações Farmacêuticas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia
12.
J Biotechnol ; 359: 161-175, 2022 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-36216209

RESUMO

Previously, we reported, based on an untargeted metabolomics, carnitine derivatives are part of a mechanism to overcome impaired mitochondrial functioning triggered by an acyl-group overflow in CHO cells. In this study, we analyzed the cell-specific rates of 24 selected metabolites using two metrics: correlation coefficients and root-mean-square deviations (RMSDs) between glucose-fed versus glucose/lactic acid-fed cultures. The time-course profiles of acetylcarnitine, adipoylcarnitine, glutarylcarnitine, glutamate, and succinate exhibited significant negative correlations between the two culture conditions. Based on RMSDs, seven carnitine derivatives, 3-hydroxy-methyl-glutarate, mevalonate, pyridoxamine-5-phosphate, succinate, and glycine were substantially different. The analyses from the two metrics reveal a distinctive rearrangement of rates from the following metabolic pathways: (i) high secretion rates of carnitines as part of the acyl-group removal, (ii) low secretion rates of succinate, related to the tricarboxylic acid cycle and the electron-transport chain, (iii) low secretion rates of pyridoxamine-5-phosphate - a co-factor for amino acid catabolism, transaminations, and transsulfuration, and (iv) increases in the consumption rates of glutamate and glycine, both used to produce glutathione. The rewiring in rates observed upon feeding lactic acid is best explained by the activation of pathways supporting homeostasis of acyl-groups and antioxidant synthesis, which are required for continuous proper functioning of oxidative phosphorylation.


Assuntos
Glucose , Ácido Láctico , Cricetinae , Animais , Glucose/metabolismo , Ácido Láctico/metabolismo , Cricetulus , Ácido Mevalônico , Acetilcarnitina , Antioxidantes , Piridoxamina , Células CHO , Carnitina/metabolismo , Ácido Succínico , Aminoácidos , Glutamatos , Glicina , Glutaratos , Glutationa , Fosfatos
13.
BMJ Open ; 12(10): e066255, 2022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36207048

RESUMO

INTRODUCTION: Whether bisphosphonates and RANKL inhibitors play a novel role in delaying cardiovascular calcification is unknown. Their action on regulatory enzymes in the mevalonic acid pathway, which is implicated in both bone and lipid metabolism, may be a novel therapeutic target to manage coronary artery disease (CAD). Such therapies may particularly be relevant in those for whom traditional cardiovascular therapies are no longer sufficient to control disease progression. METHODS AND ANALYSIS: We will perform a systematic review which aims to synthesise evidence regarding whether use of bisphosphonates or use of the RANKL inhibitor denosumab delays coronary artery calcium (CAC) progression. Eligible studies will include longitudinal studies investigating CAC progression in patients aged >18 years taking either a bisphosphonate or denosumab compared with those who do not. Embase, MEDLINE and Cochrane will be searched using prespecified search terms. Studies will be screened by title and abstract independently and then in full to determine suitability for inclusion in the review. Extracted data will include that relating to study and participant characteristics. The primary outcome will be the CAC score. Secondary outcomes will include aortic and carotid artery calcification. Meta-analysis will be performed if sufficient data are available. ETHICS AND DISSEMINATION: This study does not require ethics as it is a systematic review of the literature. The results of the review described within this protocol will be distributed via presentations at relevant conferences and publication within a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: The systematic review pertaining to this protocol is registered with PROSPERO (Registration ID: CRD42022312377).


Assuntos
Doença da Artéria Coronariana , Cálcio , Doença da Artéria Coronariana/tratamento farmacológico , Denosumab/farmacologia , Denosumab/uso terapêutico , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Humanos , Metanálise como Assunto , Ácido Mevalônico , Revisões Sistemáticas como Assunto
14.
ACS Synth Biol ; 11(10): 3305-3317, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36198145

RESUMO

Co-localizing biochemical processes is a great strategy when expressing the heterologous metabolic pathway for product biosynthesis. The RNA scaffold is a flexible and efficient synthetic compartmentalization method to co-localize the enzymes involved in the metabolic pathway by binding to the specific RNA, binding domains fused with the engineered enzymes. Herein, we designed two artificial RNA scaffold structures─0D RNA scaffolds and 2D RNA scaffolds─using the reported aptamers PP7 and BIV-Tat and the corresponding RNA-binding domains (RBDs). We verified the interaction of the RBD and RNA aptamer in vitro and in vivo. Then, we determined the efficiencies of these RNA scaffolds by co-localizing fluorescent proteins. We employed the RNA scaffolds combined with the enzyme fusion strategies to increase the metabolic flux involved in the enzymes of the mevalonate pathway for mevalonate and isoprene production. Compared with the no RNA scaffold strain, the mevalonate levels of the 0D RNA scaffolds and 2D RNA scaffolds increased by 84.1% (3.13 ± 0.03 g/L) and 76.5% (3.00 ± 0.09 g/L), respectively. We applied the 0D RNA scaffolds for increasing the isoprene production by localizing the enzymes involved in a heterologous multi-enzyme pathway. When applying the RNA scaffolds for co-localizing the enzymes mvaE and mvaS, the isoprene production reached to 609.3 ± 57.9 mg/L, increasing by 142% compared with the no RNA scaffold strain. Our results indicate that the RNA scaffold is a powerful tool for improving the efficiencies of the reaction process in the metabolic pathway.


Assuntos
Aptâmeros de Nucleotídeos , Engenharia Metabólica , Engenharia Metabólica/métodos , Ácido Mevalônico/metabolismo , Escherichia coli/metabolismo , RNA/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Redes e Vias Metabólicas/genética
15.
Microb Cell Fact ; 21(1): 212, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36243714

RESUMO

BACKGROUND: Linalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites. RESULTS: In this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20F96W-N127W (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20F96W-N127W genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg2+ addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation. CONCLUSIONS: The results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.


Assuntos
Ácido Mevalônico , Saccharomyces cerevisiae , Monoterpenos Acíclicos , Carbono/metabolismo , Variações do Número de Cópias de DNA , Difosfatos/metabolismo , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Hemiterpenos , Engenharia Metabólica/métodos , Ácido Mevalônico/metabolismo , Monoterpenos/metabolismo , Compostos Organofosforados , Pentanóis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
16.
J Clin Invest ; 132(19)2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36189795

RESUMO

Mevalonate kinase deficiency (MKD) is characterized by recurrent fevers and flares of systemic inflammation, caused by biallelic loss-of-function mutations in MVK. The underlying disease mechanisms and triggers of inflammatory flares are poorly understood because of the lack of in vivo models. We describe genetically modified mice bearing the hypomorphic mutation p.Val377Ile (the commonest variant in patients with MKD) and amorphic, frameshift mutations in Mvk. Compound heterozygous mice recapitulated the characteristic biochemical phenotype of MKD, with increased plasma mevalonic acid and clear buildup of unprenylated GTPases in PBMCs, splenocytes, and bone marrow. The inflammatory response to LPS was enhanced in compound heterozygous mice and treatment with the NLRP3 inflammasome inhibitor MCC950 prevented the elevation of circulating IL-1ß, thus identifying a potential inflammasome target for future therapeutic approaches. Furthermore, lines of mice with a range of deficiencies in mevalonate kinase and abnormal prenylation mirrored the genotype-phenotype relationship in human MKD. Importantly, these mice allowed the determination of a threshold level of residual enzyme activity, below which protein prenylation is impaired. Elevated temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and the defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares.


Assuntos
Deficiência de Mevalonato Quinase , Animais , Temperatura Corporal , Febre , GTP Fosfo-Hidrolases/genética , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Lipopolissacarídeos/metabolismo , Deficiência de Mevalonato Quinase/tratamento farmacológico , Deficiência de Mevalonato Quinase/genética , Deficiência de Mevalonato Quinase/metabolismo , Ácido Mevalônico/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Prenilação de Proteína
17.
Skin Res Technol ; 28(6): 804-814, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36148627

RESUMO

BACKGROUND: Fatty acids increase ATP-binding cassette ABC transporter A12 (ABCA12) levels via an increase in peroxisome proliferator-activated receptor ß/δ (PPAR ß/δ). Promoting lipid transport to lamellar granules has been suggested to improve epidermal barrier function in patients with dry skin. OBJECTIVE: We investigated whether mevalonolactone (MVL) produced by Saccharomycopsis fibuligera improves dry skin by promoting ABCA12 expression and the amount of free fatty acids in epidermal keratinocytes. METHODS: We examined whether MVL increases ABCA12 mRNA and protein levels and the amount of Nile red-positive lipids in cultured epidermal keratinocytes and in a three-dimensional epidermal model by cell staining. Promotion of fatty acid production by MVL was analyzed by liquid chromatography-mass spectrometry. We also evaluated whether MVL addition increases PPAR ß/δ mRNA expression in cultured keratinocytes. Based on the results, a randomized controlled trial was conducted in which milky lotions containing MVL and placebo were applied to dry facial skin of healthy female volunteers in winter. RESULTS: MVL increased ABCA12 mRNA and protein levels and lamellar granule number and size. Fatty acid analysis revealed that MVL elevated myristic acid, palmitic acid, and palmitoleic acid levels as well as PPAR ß/δ mRNA expression. In human tests, milky lotions containing MVL were shown to significantly improve transepidermal water loss (TEWL) in the stratum corneum compared to placebo. CONCLUSION: The results suggest that MVL increases fatty acid uptake and ABCA12, promotes fatty acid transport to lamellar granules, and improves epidermal barrier function in dry skin through increased expression of PPAR ß/δ.


Assuntos
Epiderme , Ácidos Graxos , Corpos Lamelares , Ácido Mevalônico , PPAR beta , Feminino , Humanos , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Ácidos Graxos/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Corpos Lamelares/efeitos dos fármacos , Corpos Lamelares/metabolismo , Ácido Mevalônico/farmacologia , PPAR beta/metabolismo , RNA Mensageiro/metabolismo , Transporte Biológico/efeitos dos fármacos , Adulto , Pessoa de Meia-Idade
18.
Medicina (Kaunas) ; 58(9)2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36143915

RESUMO

Over the last years, repurposed agents have provided growing evidence of fast implementation in oncology treatment such as certain antimalarial, anthelmintic, antibiotics, anti-inflammatory, antihypertensive, antihyperlipidemic, antidiabetic agents. In this study, the four agents of choice were present in our patients' daily treatment for nonmalignant-associated pathology and have known, light toxicity profiles. It is quite common for a given patient's daily administration schedule to include two or three of these drugs for the duration of their treatment. We chose to review the latest literature concerning metformin, employed as a first-line treatment for type 2 diabetes; mebendazole, as an anthelmintic; atorvastatin, as a cholesterol-lowering drug; propranolol, used in cardiovascular diseases as a nonspecific inhibitor of beta-1 and beta-2 adrenergic receptors. At the same time, certain key action mechanisms make them feasible antitumor agents such as for mitochondrial ETC inhibition, activation of the enzyme adenosine monophosphate-activated protein kinase, amelioration of endogenous hyperinsulinemia, inhibition of selective tyrosine kinases (i.e., VEGFR2, TNIK, and BRAF), and mevalonate pathway inhibition. Despite the abundance of results from in vitro and in vivo studies, the only solid data from randomized clinical trials confirm metformin-related oncological benefits for only a small subset of nondiabetic patients with HER2-positive breast cancer and early-stage colorectal cancer. At the same time, clinical studies confirm metformin-related detrimental/lack of an effect for lung, breast, prostate cancer, and glioblastoma. For atorvastatin we see a clinical oncological benefit in patients and head and neck cancer, with a trend towards radioprotection of critical structures, thus supporting the role of atorvastatin as a promising agent for concomitant association with radiotherapy. Propranolol-related increased outcomes were seen in clinical studies in patients with melanoma, breast cancer, and sarcoma.


Assuntos
Anti-Helmínticos , Antimaláricos , Antineoplásicos , Neoplasias da Mama , Diabetes Mellitus Tipo 2 , Inibidores de Hidroximetilglutaril-CoA Redutases , Metformina , Monofosfato de Adenosina/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Anti-Helmínticos/uso terapêutico , Antibacterianos/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Antimaláricos/uso terapêutico , Antineoplásicos/uso terapêutico , Atorvastatina/uso terapêutico , Neoplasias da Mama/patologia , Colesterol , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipoglicemiantes/uso terapêutico , Masculino , Mebendazol/uso terapêutico , Metformina/uso terapêutico , Ácido Mevalônico/uso terapêutico , Propranolol/uso terapêutico , Proteínas Quinases/metabolismo , Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf , Receptores Adrenérgicos beta 2/uso terapêutico , Tirosina
19.
Nat Commun ; 13(1): 5563, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36137996

RESUMO

Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a mode of action target for the development of herbicides. Here, we present the crystal structure of a HMGR from Arabidopsis thaliana (AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enables the rational design of specific HMGR inhibitors and we develop a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.


Assuntos
Arabidopsis , Herbicidas , Inibidores de Hidroximetilglutaril-CoA Redutases , Acil Coenzima A , Arabidopsis/metabolismo , Herbicidas/farmacologia , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Ácido Mevalônico
20.
Cells ; 11(18)2022 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-36139434

RESUMO

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.


Assuntos
Proteínas Proto-Oncogênicas c-akt , Rabdomiossarcoma Embrionário , Animais , Criança , Reparo do DNA , Proteína Quinase Ativada por DNA/genética , Desoxiglucose , Doxorrubicina/farmacologia , Glucose , Glicólise , Hexoquinase/metabolismo , Histonas/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Lovastatina , Inibidores MTOR , Ácido Mevalônico , Oxirredutases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rabdomiossarcoma Embrionário/tratamento farmacológico , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Peixe-Zebra/genética
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