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1.
Metab Eng ; 67: 403-416, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34411702

RESUMO

Malonyl-CoA is an important building block for microbial synthesis of numerous pharmaceutically interesting or fatty acid-derived compounds including polyketides, flavonoids, phenylpropanoids and fatty acids. However, the tightly regulated intracellular malonyl-CoA availability often impedes overall product formation. Here, in order to unleash this tightly cellular behavior, we present evolution: dual dynamic regulations-based approaches to write artificial robust and dynamic function into intricate cellular background. Firstly, a conserved core domain based evolutionary principles were incorporated into genome mining to explore the biosynthetic diversities of discrete acetyl-CoA carboxylase (ACC) families, as malonyl-CoA is solely derived from carboxylation of acetyl-CoA by ACC in most organisms. A comprehensive phylogenomic and further experimental analysis, which included genomes of 50 strains throughout representative species, was performed to recapitulate the evolutionary history and reveal that previously unnoticed ACC families from Salmonella enterica exhibited the highest activities among all the candidates. A set of orthogonal and bi-functional quorum-sensing (QS)-based regulation tools were further designed and connected with T7 RNA polymerase as genetic amplifier to achieve dual dynamic control in a high dynamic range, which allowed us to efficiently activate and repress different sets of genes dynamically and independently. These genetic circuits were then combined with ACC of S. enterica and CRISPRi system to reprogram central metabolism that rewired the tightly regulated malonyl-CoA pathway to a robust and autonomous behavior, leading to a 29-fold increase of malony-CoA availability. We applied this dual regulation tool to successfully synthesizing malonyl-CoA-derived compound (2S)-naringenin, and achieved the highest production (1073.8 mg/L) reported to date associate with dramatic decreases of by-product formation. Notably, the whole fermentation presents as an autonomous behavior, totally eliminating human supervision and inducer supplementation. Hence, the constructed evolution: dual dynamic regulations-based approaches pave the way to develop an economically viable and scalable procedure for microbial production of malonyl-CoA derived compounds.


Assuntos
Malonil Coenzima A , Policetídeos , Acetilcoenzima A/genética , Acetil-CoA Carboxilase , Humanos , Engenharia Metabólica
2.
Elife ; 102021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34254587

RESUMO

Volatile anesthetics (VAs) are widely used in medicine, but the mechanisms underlying their effects remain ill-defined. Though routine anesthesia is safe in healthy individuals, instances of sensitivity are well documented, and there has been significant concern regarding the impact of VAs on neonatal brain development. Evidence indicates that VAs have multiple targets, with anesthetic and non-anesthetic effects mediated by neuroreceptors, ion channels, and the mitochondrial electron transport chain. Here, we characterize an unexpected metabolic effect of VAs in neonatal mice. Neonatal blood ß-hydroxybutarate (ß-HB) is rapidly depleted by VAs at concentrations well below those necessary for anesthesia. ß-HB in adults, including animals in dietary ketosis, is unaffected. Depletion of ß-HB is mediated by citrate accumulation, malonyl-CoA production by acetyl-CoA carboxylase, and inhibition of fatty acid oxidation. Adults show similar significant changes to citrate and malonyl-CoA, but are insensitive to malonyl-CoA, displaying reduced metabolic flexibility compared to younger animals.


Assuntos
Anestésicos/metabolismo , Anestésicos/farmacologia , Ácido 3-Hidroxibutírico , Acetil-CoA Carboxilase/metabolismo , Animais , Citratos/metabolismo , Ácido Cítrico/metabolismo , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Hipoglicemia , Isoflurano/metabolismo , Cetose , Masculino , Malonil Coenzima A/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias , Oxirredução
3.
Molecules ; 26(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066831

RESUMO

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.


Assuntos
Benzoatos/metabolismo , Benzofenonas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Xantonas/metabolismo , Biotransformação , Carbono-Carbono Ligases/metabolismo , Cromatografia Líquida , Coenzima A Ligases/metabolismo , Simulação por Computador , Meios de Cultura , Garcinia mangostana/enzimologia , Garcinia mangostana/genética , Malonil Coenzima A/metabolismo , Plasmídeos/genética , Rodopseudomonas/enzimologia , Rodopseudomonas/genética , Espectrometria de Massas em Tandem
4.
Metab Eng ; 67: 41-52, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34052445

RESUMO

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.


Assuntos
Flavanonas , Malonil Coenzima A , Escherichia coli/genética , Engenharia Metabólica
5.
Nat Commun ; 12(1): 2193, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850151

RESUMO

Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Aciltransferases/genética , Domínio Catalítico , Malonil Coenzima A , Simulação de Dinâmica Molecular , Mutagênese , Policetídeo Sintases/genética , Policetídeos , Engenharia de Proteínas , Metabolismo Secundário , Especificidade por Substrato
6.
Cardiol Young ; 31(9): 1535-1537, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33745485

RESUMO

Malonyl-CoA, a product of acetyl-CoA carboxylase is a metabolic intermediate in lipogenic tissues that include liver and adipose tissue, where it is involved in the de novo fatty acid synthesis and elongation. Malonyl-CoA decarboxylase (MLYCD, E.C.4.1.1.9), a 55-kDa enzyme catalyses the conversion of malonyl-CoA to acetyl-CoA and carbon dioxide, thus providing a route for disposal of malonyl-CoA from mitochondria and peroxisomes, whereas in the cytosol, the malonyl-CoA pool is regulated by the balance of MLYCD and acetyl-CoA carboxylase activities. So far, 34 cases with different MLYCD gene defects comprising point mutations, stop codons, and frameshift mutations have been reported in the literature. Here, we describe the follow-up of a patient affected by malonic aciduria upon neonatal onset. Molecular analysis showed novel homozygous mutations in the MLYCD gene. Our findings expand the number of reported cases and add a novel variant to the repertoire of MLYCD mutations.


Assuntos
Carboxiliases , Erros Inatos do Metabolismo , Carboxiliases/deficiência , Carboxiliases/genética , Humanos , Recém-Nascido , Malonil Coenzima A , Ácido Metilmalônico , Mutação
7.
Cell ; 184(7): 1693-1705.e17, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33770502

RESUMO

Plants protect themselves with a vast array of toxic secondary metabolites, yet most plants serve as food for insects. The evolutionary processes that allow herbivorous insects to resist plant defenses remain largely unknown. The whitefly Bemisia tabaci is a cosmopolitan, highly polyphagous agricultural pest that vectors several serious plant pathogenic viruses and is an excellent model to probe the molecular mechanisms involved in overcoming plant defenses. Here, we show that, through an exceptional horizontal gene transfer event, the whitefly has acquired the plant-derived phenolic glucoside malonyltransferase gene BtPMaT1. This gene enables whiteflies to neutralize phenolic glucosides. This was confirmed by genetically transforming tomato plants to produce small interfering RNAs that silence BtPMaT1, thus impairing the whiteflies' detoxification ability. These findings reveal an evolutionary scenario whereby herbivores harness the genetic toolkit of their host plants to develop resistance to plant defenses and how this can be exploited for crop protection.


Assuntos
Hemípteros/genética , Proteínas de Insetos/metabolismo , Lycopersicon esculentum/genética , Toxinas Biológicas/metabolismo , Animais , Transferência Genética Horizontal , Genes de Plantas , Glucosídeos/química , Glucosídeos/metabolismo , Hemípteros/fisiologia , Herbivoria , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Mucosa Intestinal/metabolismo , Lycopersicon esculentum/metabolismo , Malonil Coenzima A/metabolismo , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Toxinas Biológicas/química
8.
mBio ; 12(1)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531402

RESUMO

Fatty acid biosynthesis (FASII) enzymes are considered valid targets for antimicrobial drug development against the human pathogen Staphylococcus aureus However, incorporation of host fatty acids confers FASII antibiotic adaptation that compromises prospective treatments. S. aureus adapts to FASII inhibitors by first entering a nonreplicative latency period, followed by outgrowth. Here, we used transcriptional fusions and direct metabolite measurements to investigate the factors that dictate the duration of latency prior to outgrowth. We show that stringent response induction leads to repression of FASII and phospholipid synthesis genes. (p)ppGpp induction inhibits synthesis of malonyl-CoA, a molecule that derepresses FapR, a key regulator of FASII and phospholipid synthesis. Anti-FASII treatment also triggers transient expression of (p)ppGpp-regulated genes during the anti-FASII latency phase, with concomitant repression of FapR regulon expression. These effects are reversed upon outgrowth. GTP depletion, a known consequence of the stringent response, also occurs during FASII latency, and is proposed as the common signal linking these responses. We next showed that anti-FASII treatment shifts malonyl-CoA distribution between its interactants FapR and FabD, toward FapR, increasing expression of the phospholipid synthesis genes plsX and plsC during outgrowth. We conclude that components of the stringent response dictate malonyl-CoA availability in S. aureus FASII regulation, and contribute to latency prior to anti-FASII-adapted outgrowth. A combinatory approach, coupling a (p)ppGpp inducer and an anti-FASII, blocks S. aureus outgrowth, opening perspectives for bi-therapy treatment.IMPORTANCE Staphylococcus aureus is a major human bacterial pathogen for which new inhibitors are urgently needed. Antibiotic development has centered on the fatty acid synthesis (FASII) pathway, which provides the building blocks for bacterial membrane phospholipids. However, S. aureus overcomes FASII inhibition and adapts to anti-FASII by using exogenous fatty acids that are abundant in host environments. This adaptation mechanism comprises a transient latency period followed by bacterial outgrowth. Here, we use metabolite sensors and promoter reporters to show that responses to stringent conditions and to FASII inhibition intersect, in that both involve GTP and malonyl-CoA. These two signaling molecules contribute to modulating the duration of latency prior to S. aureus adaptation outgrowth. We exploit these novel findings to propose a bi-therapy treatment against staphylococcal infections.


Assuntos
Antibacterianos/farmacologia , Ácidos Graxos/antagonistas & inibidores , Guanosina Pentafosfato/fisiologia , Guanosina Trifosfato/fisiologia , Malonil Coenzima A/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Ácidos Graxos/biossíntese , Humanos , Malonil Coenzima A/análise , Mupirocina/farmacologia , Fosfolipídeos/biossíntese , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/fisiologia
9.
Biochemistry ; 60(5): 365-372, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33482062

RESUMO

LnmK stereospecifically accepts (2R)-methylmalonyl-CoA, generating propionyl-S-acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2R)-methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl-O-LnmK acyl-enzyme intermediate. Due to the inherent reactivity of LnmK and methylmalonyl-CoA, a substrate-bound structure could not be obtained. To gain insight into substrate specificity, stereospecificity, and catalytic mechanism, we determined the structures of LnmK with bound substrate analogues that bear malonyl-thioester isosteres where the carboxylate is represented by a nitro or sulfonate group. The nitro-bearing malonyl-thioester isosteres bind in the nitronate form, with specific hydrogen bonds that allow modeling of the (2R)-methylmalonyl-CoA substrate and rationalization of stereospecificity. The sulfonate isosteres bind in multiple conformations, suggesting the large active site of LnmK allows multiple binding modes. Considering the smaller malonyl group has more conformational freedom than the methylmalonyl group, we hypothesized the active site can entropically screen against catalysis with the smaller malonyl-CoA substrate. Indeed, our kinetic analysis reveals malonyl-CoA is accepted at 1% of the rate of methylmalonyl-CoA. This study represents another example of how our nitro- and sulfonate-bearing methylmalonyl-thioester isosteres are of use for elucidating enzyme-substrate binding interactions and revealing insights into catalytic mechanism. Synthesis of a larger panel of analogues presents an opportunity to study enzymes with complicated structure-function relationships such as acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases, and ß-ketoacylsynthases.


Assuntos
Aciltransferases/química , Carboxiliases/química , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/química , Carbono-Carbono Ligases/química , Catálise , Domínio Catalítico , Malonil Coenzima A/metabolismo , Streptomyces/metabolismo , Streptomyces coelicolor/metabolismo , Especificidade por Substrato
10.
Br J Pharmacol ; 178(7): 1507-1523, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33444462

RESUMO

BACKGROUND AND PURPOSE: The enzyme α/ß-hydrolase domain containing 6 (ABHD6), a new member of the endocannabinoid system, is a promising therapeutic target against neuronal-related diseases. However, how ABHD6 activity is regulated is not known. ABHD6 coexists in protein complexes with the brain-specific carnitine palmitoyltransferase 1C (CPT1C). CPT1C is involved in neuro-metabolic functions, depending on brain malonyl-CoA levels. Our aim was to study CPT1C-ABHD6 interaction and determine whether CPT1C is a key regulator of ABHD6 activity depending on nutritional status. EXPERIMENTAL APPROACH: Co-immunoprecipitation and FRET assays were used to explore ABHD6 interaction with CPT1C or modified malonyl-CoA-insensitive or C-terminal truncated CPT1C forms. Cannabinoid CB1 receptor-mediated signalling was investigated by determining cAMP levels. A novel highly sensitive fluorescent method was optimized to measure ABHD6 activity in non-neuronal and neuronal cells and in brain tissues from wild-type (WT) and CPT1C-KO mice. KEY RESULTS: CPT1C interacted with ABHD6 and negatively regulated its hydrolase activity, thereby regulating 2-AG downstream signalling. Accordingly, brain tissues of CPT1C-KO mice showed increased ABHD6 activity. CPT1C malonyl-CoA sensing was key to the regulatory role on ABHD6 activity and CB1 receptor signalling. Fasting, which attenuates brain malonyl-CoA, significantly increased ABHD6 activity in hypothalamus from WT, but not CPT1C-KO, mice. CONCLUSIONS AND IMPLICATIONS: Our finding that negative regulation of ABHD6 activity, particularly in the hypothalamus, is sensitive to nutritional status throws new light on the characterization and the importance of the proteins involved as potential targets against diseases affecting the CNS.


Assuntos
Carnitina O-Palmitoiltransferase , Monoacilglicerol Lipases/metabolismo , Estado Nutricional , Animais , Carnitina O-Palmitoiltransferase/genética , Hidrolases , Malonil Coenzima A , Camundongos
11.
J Exp Bot ; 72(4): 1349-1369, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33130852

RESUMO

Malonyl-CoA:flavonoid acyltransferases (MaTs) modify isoflavones, but only a few have been characterized for activity and assigned to specific physiological processes. Legume roots exude isoflavone malonates into the rhizosphere, where they are hydrolyzed into isoflavone aglycones. Soybean GmMaT2 was highly expressed in seeds, root hairs, and nodules. GmMaT2 and GmMaT4 recombinant enzymes used isoflavone 7-O-glucosides as acceptors and malonyl-CoA as an acyl donor to generate isoflavone glucoside malonates. GmMaT2 had higher activity towards isoflavone glucosides than GmMaT4. Overexpression in hairy roots of GmMaT2 and GmMaT4 produced more malonyldaidzin, malonylgenistin, and malonylglycitin, and resulted in more nodules than control. However, only GmMaT2 knockdown (KD) hairy roots showed reduced levels of malonyldaidzin, malonylgenistin, and malonylglycitin, and, likewise, reduced nodule numbers. These were consistent with the up-regulation of only GmMaT2 by rhizobial infection, and higher expression levels of early nodulation genes in GmMaT2- and GmMaT4-overexpressing roots, but lower only in GmMaT2-KD roots compared with control roots. Higher malonyl isoflavonoid levels in transgenic hairy roots were associated with higher levels of isoflavones in root exudates and more nodules, and vice versa. We suggest that GmMaT2 participates in soybean nodulation by catalyzing isoflavone malonylation and affecting malonyl isoflavone secretion for activation of Nod factor and nodulation.


Assuntos
Aciltransferases/fisiologia , Isoflavonas , Malonil Coenzima A/fisiologia , Nodulação , Soja , Aciltransferases/genética , Malonil Coenzima A/genética , Soja/enzimologia , Soja/genética
12.
J Biotechnol ; 325: 83-90, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-33278463

RESUMO

To reduce dependence on petroleum, the biosynthesis of important chemicals from simple substrates using industrial microorganisms has attracted increased attention. Metabolic engineering of Saccharomyces cerevisiae offers a sustainable and flexible alternative for the production of various chemicals. As a key metabolic intermediate, malonyl-CoA is a precursor for many useful compounds. However, the productivity of malonyl-CoA derivatives is restricted by the low cellular level of malonyl-CoA and enzymatic performance. In this review, we focused on how to increase the intracellular malonyl-CoA level and summarize the recent advances in different metabolic engineering strategies for directing intracellular malonyl-CoA to the desired malonyl-CoA derivatives, including strengthening the malonyl-CoA supply, reducing malonyl-CoA consumption, and precisely controlling the intracellular malonyl-CoA level. These strategies provided new insights for further improving the synthesis of malonyl-CoA derivatives in microorganisms.


Assuntos
Malonil Coenzima A , Engenharia Metabólica , Saccharomyces cerevisiae/genética
13.
Org Lett ; 22(20): 7837-7841, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33006285

RESUMO

A highly oxygenated phenethyl derivative ustethylin A was isolated from Aspergillus ustus. Gene deletion, isotope labeling, and heterologous expression proved that the phenethyl core structure is assembled from malonyl-CoA by a polyketide synthase harboring a methyltransferase domain. Propionate was converted via acetyl-CoA to malonyl-CoA and incorporated into the molecule. Modifications on the core structure by three different oxidoreductases and one O-methyltransferase lead to the final product, ustethylin A.


Assuntos
Acetilcoenzima A/química , Aspergillus/química , Malonil Coenzima A/química , Metiltransferases/metabolismo , Oxirredutases/metabolismo , Policetídeo Sintases/metabolismo , Acetilcoenzima A/metabolismo , Malonil Coenzima A/isolamento & purificação , Malonil Coenzima A/metabolismo , Metiltransferases/química , Estrutura Molecular , Oxirredutases/química , Policetídeo Sintases/química
14.
J Cell Biol ; 219(10)2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-32931550

RESUMO

Carnitine palmitoyltransferase 1C (CPT1C) is a sensor of malonyl-CoA and is located in the ER of neurons. AMPA receptors (AMPARs) mediate fast excitatory neurotransmission in the brain and play a key role in synaptic plasticity. In the present study, we demonstrate across different metabolic stress conditions that modulate malonyl-CoA levels in cortical neurons that CPT1C regulates the trafficking of the major AMPAR subunit, GluA1, through the phosphatidyl-inositol-4-phosphate (PI(4)P) phosphatase SAC1. In normal conditions, CPT1C down-regulates SAC1 catalytic activity, allowing efficient GluA1 trafficking to the plasma membrane. However, under low malonyl-CoA levels, such as during glucose depletion, CPT1C-dependent inhibition of SAC1 is released, facilitating SAC1's translocation to ER-TGN contact sites to decrease TGN PI(4)P pools and trigger GluA1 retention at the TGN. Results reveal that GluA1 trafficking is regulated by CPT1C sensing of malonyl-CoA and provide the first report of a SAC1 inhibitor. Moreover, they shed light on how nutrients can affect synaptic function and cognition.


Assuntos
Carnitina O-Palmitoiltransferase/genética , Proteínas de Membrana/genética , Neurônios/metabolismo , Receptores de AMPA/genética , Animais , Encéfalo/metabolismo , Glucose/metabolismo , Humanos , Malonil Coenzima A/genética , Camundongos , Nutrientes/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte Proteico/genética , Transmissão Sináptica/genética
15.
Stem Cell Reports ; 15(3): 566-576, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32857979

RESUMO

Fatty acid ß-oxidation (FAO), the breakdown of lipids, is a metabolic pathway used by various stem cells. FAO levels are generally high during quiescence and downregulated with proliferation. The endogenous metabolite malonyl-CoA modulates lipid metabolism as a reversible FAO inhibitor and as a substrate for de novo lipogenesis. Here we assessed whether malonyl-CoA can be exploited to steer the behavior of hematopoietic stem/progenitor cells (HSPCs), quiescent stem cells of clinical relevance. Treatment of mouse HSPCs in vitro with malonyl-CoA increases HSPC numbers compared with nontreated controls and ameliorates blood reconstitution capacity when transplanted in vivo, mainly through enhanced lymphoid reconstitution. Similarly, human HSPC numbers also increase upon malonyl-CoA treatment in vitro. These data corroborate that lipid metabolism can be targeted to direct cell fate and stem cell proliferation. Physiological modulation of metabolic pathways, rather than genetic or pharmacological inhibition, provides unique perspectives for stem cell manipulations in health and disease.


Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Metabolismo dos Lipídeos , Linfócitos/citologia , Metaboloma , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células/genética , Células Cultivadas , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Linfócitos/metabolismo , Malonil Coenzima A/metabolismo , Metaboloma/genética , Camundongos Endogâmicos C57BL , Oxirredução
16.
Life Sci ; 258: 118240, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781072

RESUMO

As a dicarboxylic acid with the structural formula HOOCCH (OH) COOH, tartronic acid is considered as an inhibitor of the transformation of carbohydrates into fat under fat-deficient diet conditions. However, the effect of tartronic acid on lipogenesis under high-fat diet conditions has yet to be established. In this work, we investigated the regulatory role of tartronic acid in lipogenesis in 3T3-L1 adipocytes and C57BL/6J mice. The results confirmed that tartronic acid promoted weight gain (without affecting food intake) and induced adipocyte hypertrophy in epididymal white adipose tissue and lipid accumulation in the livers of high-fat diet-induced obese mice. In vitro, tartronic acid promoted 3T3-L1 adipocyte differentiation by increasing the protein expression of FABP-4, PPARγ and SREBP-1. Moreover, the contents of both acetyl-CoA and malonyl-CoA were significantly upregulated by treatment with tartronic acid, while the protein expression of CPT-1ß were inhibited. In summary, we proved that tartronic acid promotes lipogenesis by serving as substrates for fatty acid synthesis and inhibiting CPT-1ß, providing a new perspective for the study of tartronic acid.


Assuntos
Acetilcoenzima A/biossíntese , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Lipogênese/efeitos dos fármacos , Malonil Coenzima A/biossíntese , Tartronatos/farmacologia , Regulação para Cima/efeitos dos fármacos , Células 3T3-L1 , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Dieta Hiperlipídica/efeitos adversos , Lipogênese/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima/fisiologia
17.
Elife ; 92020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32735213

RESUMO

Gap junctions are ubiquitous in metazoans and play critical roles in important biological processes, including electrical conduction and development. Yet, only a few defined molecules passing through gap junction channels have been linked to specific functions. We isolated gap junction channel mutants that reduce coupling between the soma and germ cells in the Caenorhabditis elegans gonad. We provide evidence that malonyl-CoA, the rate-limiting substrate for fatty acid synthesis (FAS), is produced in the soma and delivered through gap junctions to the germline; there it is used in fatty acid synthesis to critically support embryonic development. Separation of malonyl-CoA production from its site of utilization facilitates somatic control of germline development. Additionally, we demonstrate that loss of malonyl-CoA production in the intestine negatively impacts germline development independently of FAS. Our results suggest that metabolic outsourcing of malonyl-CoA may be a strategy by which the soma communicates nutritional status to the germline.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Desenvolvimento Embrionário/genética , Células Germinativas/crescimento & desenvolvimento , Malonil Coenzima A/genética , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Junções Comunicantes/fisiologia , Gônadas/crescimento & desenvolvimento , Malonil Coenzima A/metabolismo
18.
Nat Chem Biol ; 16(12): 1427-1433, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32839605

RESUMO

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.


Assuntos
Canabinoides/biossíntese , Sistema Livre de Células/metabolismo , Malonil Coenzima A/metabolismo , Engenharia Metabólica/métodos , Policetídeos/metabolismo , Terpenos/metabolismo , Trifosfato de Adenosina/biossíntese , Benzoatos/isolamento & purificação , Benzoatos/metabolismo , Canabinoides/isolamento & purificação , Sistema Livre de Células/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Engenharia Metabólica/economia , Organofosfatos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Policetídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Terpenos/química , Termodinâmica
19.
J Ind Microbiol Biotechnol ; 47(9-10): 845-862, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32623653

RESUMO

Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature's biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.


Assuntos
Engenharia Metabólica , Biologia Sintética , Biologia de Sistemas , Yarrowia , Acetilcoenzima A/metabolismo , Acil Coenzima A , Ácidos Graxos/metabolismo , Lipídeos , Malonil Coenzima A/metabolismo , Policetídeos/metabolismo , Terpenos/metabolismo , Yarrowia/metabolismo
20.
Mol Genet Genomic Med ; 8(9): e1379, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32602666

RESUMO

BACKGROUND: Malonic aciduria (MA, OMIM#248360) is an extremely rare inherited metabolic disorder caused by the deficiency of malonyl-CoA decarboxylase. The phenotype exhibited by patients with MA is variable, but may include symptoms, such as developmental delay in early childhood, seizures, vomiting, metabolic acidosis, hypoglycemia, ketosis, and cardiomyopathy. We describe the first case of a Korean child with MA who presented with dilated cardiomyopathy (DCMP) at the age of 3 months. METHODS AND RESULTS: A 3-month-old Korean boy visited our hospital for diagnosis and management of cardiomegaly. Newborn screening for inherited metabolic diseases showed a normal result; therefore, DCMP management was initiated. Biochemical and the MLYCD gene analyses subsequently confirmed diagnosis of MA. Elevated plasma C3DC level and excessive excretion of urinary malonate were observed, and two pathogenic variants, including a novel start codon mutation (c.1A>G), were identified in MLYCD. A low long-chain fat diet with middle-chain triglyceride formula and L-carnitine supplementation was initiated. The patient is now 5 years old and exhibits considerably improved cardiac function. CONCLUSIONS: MA can be diagnosed using newborn screening; however, negative results do not exclude the possibility of disease. Metabolic screening for differential diagnosis of infantile DCMP is recommended to rule out rare, but manageable, metabolic cardiomyopathies.


Assuntos
Carboxiliases/deficiência , Cardiomiopatia Dilatada/genética , Erros Inatos do Metabolismo/genética , Mutação , Carboxiliases/genética , Cardiomiopatia Dilatada/patologia , Códon de Iniciação , Humanos , Lactente , Masculino , Malonatos/metabolismo , Malonil Coenzima A/genética , Erros Inatos do Metabolismo/patologia , Ácido Metilmalônico , Fenótipo
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