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1.
Life Sci ; 259: 118375, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891612

RESUMO

OBJECTIVE: Short-chain fatty acids were reported to be the precursors of milk fat and can stimulate the de novo synthesis of fatty acids in bovine mammary epithelial cells (bMECs). However, the mechanism has not been elucidated. The purpose of this study was to investigate the effects of sodium butyrate (NaB) on milk fat synthesis in bMECs and explore its potential mechanism. METHODS: Bovine mammary epithelial cells (bMECs) were isolated for subsequent experimental uses. BODIPY staining and triglyceride kit were used to detect the milk fat synthesis in bMECs. Western blotting and RT-PCR assays were performed to detect the expression of related genes in bMECs. Immunoprecipitation was used to detect the acetylation of SREBP1 in bMECs. RESULTS: The results showed that NaB significantly promoted milk fat synthesis, promoted the activity of mechanistic target of rapamycin (mTOR) and S6 kinase (S6K), inhibited the activity of AMP-activated protein kinase (AMPK), and promoted the gene expression of G protein-coupled receptor 41 (GPR41). Knockdown of GPR41 and sterol regulatory element binding protein 1 (SREBP1) and overexpression of sirtuin1 (SIRT1), mTOR inhibitor (rapamycin), and AMPK activator (AICIR) eliminated these effects. These results indicated that NaB increased the nuclear translocation of SREBP1 via the GPR41/AMPK/mTOR/S6K signalling pathway, promoted the acetylation of mature SREBP1a via GPR41/AMPK/SIRT1, and then promoted milk fat synthesis. CONCLUSION: Taken together, these results demonstrated that NaB increased nuclear translocation and acetylation of SREBP1 to promote milk fat synthesis by activating GPR41 and its downstream signalling pathways.


Assuntos
Ácido Butírico/farmacologia , Glicolipídeos/biossíntese , Glicoproteínas/biossíntese , Glândulas Mamárias Animais/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Carbazóis , Bovinos , Células Cultivadas , Feminino , Imunoprecipitação , Gotículas Lipídicas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Naftalenos , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
2.
PLoS One ; 15(5): e0229700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379829

RESUMO

One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.


Assuntos
Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Glicolipídeos/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Policetídeo Sintases/genética , Transcriptoma , Parede Celular/metabolismo , Simulação por Computador , Redes Reguladoras de Genes , Loci Gênicos , Genoma Bacteriano/genética , Ligases/genética , RNA-Seq , Virulência/genética
3.
Arch Microbiol ; 202(6): 1407-1417, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32173773

RESUMO

Pseudomonas spp. are the main producers of rhamnolipids. These products have applications in pharmaceuticals, cosmetics, food industry and bioremediation. The biosynthesis of rhamnolipids is influenced by nutrient composition, pH and temperature. In this study, the impact of nutrients on the expression levels of rhamnolipid synthesis genes was evaluated in P. aeruginosa ATCC 15442. Glucose and glycerol were used as carbon sources; while, NaNO3, NH4NO3 and yeast extract/peptone were employed as nitrogen sources. The effect of different concentrations of Fe2+ and Fe3+ on rhamnolipid synthesis genes was also evaluated. Highest biosurfactant production was obtained in minimal medium supplemented with glucose, NaNO3 and Fe2+. Two rhamnolipid synthesis genes, rhlA and rhlB, were amplified with PCR. CapLC ESI-Ion trap-MS/MS detected only mono-rhamnolipid Rha-C10-C10 in the extract. Although similar induction levels were recorded in the presence of 0.05 g/L iron ions, the presence of Fe2+ resulted in higher expression levels than Fe3+ at concentrations equivalent to 0.025 and 0.075 g/L.


Assuntos
Carbono/metabolismo , Glicolipídeos/biossíntese , Ferro/metabolismo , Nitrogênio/metabolismo , Pseudomonas aeruginosa/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Íons/metabolismo , Nitratos/metabolismo , Peptonas/metabolismo , Pseudomonas aeruginosa/genética , Tensoativos/química , Tensoativos/metabolismo , Espectrometria de Massas em Tandem
4.
Yeast ; 37(4): 313-320, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061177

RESUMO

The Wickerhamiella and Starmerella genera form a clade (W/S clade) that branches close to Yarrowia lipolytica in the Saccharomycotina species tree. It comprises approximately 90 recognized species and 50 putative new species not formally described yet. The large majority of the members of the W/S clade are ecologically associated with flowers and floricolous insects. Many species exhibit unusual metabolic traits, like fructophily and the production of sophorolipids, which are glycolipids that can be used as environmentally friendly biosurfactants. Genomic data have not only firmly established the W/S clade but have also revealed a tumultuous evolution of metabolism marked by losses and gains of important metabolic pathways, among which alcoholic fermentation. Possibly the most surprising finding brought to light by comparative genomics concerned the large number of genes acquired by some species of the W/S clade from bacteria through horizontal gene transfer, many of which were shown to be functional in their new setting. This was facilitated by the genetic tractability of one species in the clade, Starmerella bombicola, which is used for the industrial production of sophorolipids. We suggest that high-density coverage of genome sequencing in this clade, combined with the possibility to conduct molecular genetics experiments in at least one species, has the potential to set the stage for yet more exciting discoveries concerning the evolution of yeast metabolism.


Assuntos
DNA Fúngico/isolamento & purificação , Saccharomycetales/classificação , Saccharomycetales/genética , Animais , DNA Fúngico/genética , Evolução Molecular , Flores/microbiologia , Transferência Genética Horizontal , Glicolipídeos/biossíntese , Glicolipídeos/genética , Insetos/microbiologia , Redes e Vias Metabólicas , Filogenia , Saccharomycetales/isolamento & purificação
5.
PLoS One ; 15(1): e0227295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923270

RESUMO

Pseudozyma antarctica is a nonpathogenic phyllosphere yeast known as an excellent producer of industrial lipases and mannosylerythritol lipids (MELs), which are multi-functional glycolipids. The fungus produces a much higher amount of MELs from vegetable oil than from glucose, whereas its close relative, Ustilago maydis UM521, produces a lower amount of MELs from vegetable oil. In the present study, we used previous gene expression profiles measured by DNA microarray analyses after culturing on two carbon sources, glucose and soybean oil, to further characterize MEL biosynthesis in P. antarctica T-34. A total of 264 genes were found with induction ratios and expression intensities under oily conditions with similar tendencies to those of MEL cluster genes. Of these, 93 were categorized as metabolic genes using the Eukaryotic Orthologous Groups classification. Within this metabolic category, amino acids, carbohydrates, inorganic ions, and secondary metabolite metabolism, as well as energy production and conversion, but not lipid metabolism, were enriched. Furthermore, genes involved in central metabolic pathways, such as glycolysis and the tricarboxylic acid cycle, were highly induced in P. antarctica T-34 under oily conditions, whereas they were suppressed in U. maydis UM521. These results suggest that the central metabolism of P. antarctica T-34 under oily conditions contributes to its excellent oil utilization and extracellular glycolipid production.


Assuntos
Glicolipídeos/biossíntese , Redes e Vias Metabólicas/genética , Transcriptoma , Ustilago/genética , Ustilago/metabolismo , Ciclo do Ácido Cítrico/genética , Perfilação da Expressão Gênica , Glucose/metabolismo , Glicólise/genética , Família Multigênica , Óleo de Soja/metabolismo
6.
Appl Environ Microbiol ; 86(6)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31924623

RESUMO

While rhamnolipids of the Pseudomonas aeruginosa type are commercially available, the natural diversity of rhamnolipids and their origin have barely been investigated. Here, we collected known and identified new rhlA genes encoding the acyltransferase responsible for the synthesis of the lipophilic rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA). Generally, all homologs were found in Betaproteobacteria and Gammaproteobacteria A likely horizontal gene transfer event into Actinobacteria is the only identified exception. The phylogeny of the RhlA homologs from Pseudomonas and Burkholderia species is consistent with the organism phylogeny, and genes involved in rhamnolipid synthesis are located in operons. In contrast, RhlA homologs from the Enterobacterales do not follow the organisms' phylogeny but form their own branch. Furthermore, in many Enterobacterales and Halomonas from the Oceanospirillales, an isolated rhlA homolog can be found in the genome. The RhlAs from Pseudomonas aeruginosa PA01, Pseudomonas fluorescens LMG 05825, Pantoea ananatis LMG 20103, Burkholderia plantarii PG1, Burkholderia ambifaria LMG 19182, Halomonas sp. strain R57-5, Dickeya dadantii Ech586, and Serratia plymuthica PRI-2C were expressed in Escherichia coli and tested for HAA production. Indeed, except for the Serratia RhlA, HAAs were produced with the engineered strains. A detailed analysis of the produced HAA congeners by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) highlights the congener specificity of the RhlA proteins. The congener length varies from 4 to 18 carbon atoms, with the main congeners consisting of different combinations of saturated or monounsaturated C10, C12, and C14 fatty acids. The results are discussed in the context of the phylogeny of this unusual enzymatic activity.IMPORTANCE The RhlA specificity explains the observed differences in 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) congeners. Whole-cell catalysts can now be designed for the synthesis of different congener mixtures of HAAs and rhamnolipids, thereby contributing to the envisaged synthesis of designer HAAs.


Assuntos
Aciltransferases/genética , Bactérias/genética , Proteínas de Bactérias/genética , Ácidos Carboxílicos/metabolismo , Glicolipídeos/metabolismo , Aciltransferases/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Glicolipídeos/biossíntese
7.
Bioprocess Biosyst Eng ; 43(3): 385-392, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31724063

RESUMO

To be competitive with common synthetic surfactants, the cost of production of rhamnolipid must be minimized by the fermentation process of non-foaming and low impurities. Herein, a novel solid-state fermentation process was developed for production of rhamnolipid by Pseudomonas aeruginosa SKY. The results were shown that high-density polyurethane foam is a satisfactory alternative to agro-industrial by-products for SSF of rhamnolipid. Palm oil and NaNO3 were promising carbon source and nitrogen source, respectively. Response surface methodology was employed to enhance the production of rhamnolipid. Palm oil, NaNO3 and liquid-to-solid ratios were significant factors. The optimal medium was developed as: 73.6 g/l palm oil; 3.0 g/l g NaNO3; 1.1 g NaCl; 1.1 g KCl; 3.4 g KH2PO4; 4.4 g K2HPO4; 0.5 g MgSO4·7H2O and 37.2 liquid-to-solid ratios. An overall 1.39-fold increase in rhamnolipid production was achieved in the optimized medium as compared with the unoptimized basal medium. Air pressure pulsation solid-state fermentation (APP-SSF) was applied to the experiment of scale-up for improving transfer efficiency of heat and mass. The yield of rhamnolipid reached 39.8 g/l in a 30 l APP-SSF fermenter. The crude extract of rhamnolipid lowered the surface tension of water to 28 mN/m and kept the critical micelle concentration at 50 mg/l. The work revealed the SSF with HPUF as an inert support was a promising fermentation system that could effectively produce rhamnolipid with low impurities, high productivity and low cost of production at a large scale.


Assuntos
Fermentação , Glicolipídeos/biossíntese , Poliuretanos/química , Pseudomonas aeruginosa/metabolismo , Cromatografia Líquida de Alta Pressão , Nitratos/química , Óleo de Palmeira/química
8.
J Biol Chem ; 295(5): 1225-1239, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31819007

RESUMO

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo, signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE -/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome.


Assuntos
Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Açúcares/metabolismo , UDPglucose 4-Epimerase/química , UDPglucose 4-Epimerase/metabolismo , Receptor fas/metabolismo , Apoptose/genética , Cromatografia Líquida , Desoxiaçúcares/metabolismo , Técnicas de Inativação de Genes , Glicolipídeos/biossíntese , Glicolipídeos/química , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicosilação , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Receptores de Superfície Celular/metabolismo , UDPglucose 4-Epimerase/genética , Receptor fas/química
9.
Microb Cell Fact ; 18(1): 164, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31597569

RESUMO

BACKGROUND: In comparison to synthetically derived surfactants, biosurfactants produced from microbial culture are generally regarded by industry as being more sustainable and possess lower toxicity. One major class of biosurfactants are rhamnolipids primarily produced by Pseudomonas aeruginosa. Due to its pathogenicity rhamnolipid synthesis by this species is viewed as being commercially nonviable, as such there is a significant focus to identify alternative producers of rhamnolipids. RESULTS: To achieve this, we phenotypically screened marine bacteria for biosurfactant production resulting in the identification of rhamnolipid biosynthesis in a species belonging to the Marinobacter genus. Preliminary screening showed the strain to reduce surface tension of cell-free supernatant to 31.0 mN m-1. A full-factorial design was carried out to assess the effects of pH and sea salt concentration for optimising biosurfactant production. When cultured in optimised media Marinobacter sp. MCTG107b produced 740 ± 28.3 mg L-1 of biosurfactant after 96 h of growth. Characterisation of this biosurfactant using both HPLC-MS and tandem MS showed it to be a mixture of different rhamnolipids, with di-rhamnolipid, Rha-Rha-C10-C10 being the most predominant congener. The strain exhibited no pathogenicity when tested using the Galleria mellonella infection model. CONCLUSIONS: This study expands the paradigm of rhamnolipid biosynthesis to a new genus of bacterium from the marine environment. Rhamnolipids produced from Marinobacter have prospects for industrial application due to their potential to be synthesised from cheap, renewable feed stocks and significantly reduced pathogenicity compared to P. aeruginosa strains.


Assuntos
Glicolipídeos/biossíntese , Marinobacter/metabolismo , Tensoativos/química , Fermentação
10.
Colloids Surf B Biointerfaces ; 181: 943-952, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382344

RESUMO

Rhamnolipids produced by P. aeruginosa MR01 were fractionated into mono- and di-rhamnolipids, and their dominant congeners, Rha-C10-C10 and Rha-Rha-C10-C10, were shown by mass spectrometry. Minimum surface tensions and critical micelle concentrations (CMC) were determined as "≃34 mN/m; ≃26.17 mg/l;" and "≃29 mN/m; ≃29.63 mg/l" for mono- and di-rhamnolipids, respectively. Spectrophotometry measurements provided a close approximation of CMC. Contact angle and diameter of wet area were determined for rhamnolipid-containing drops on hydrophobic paper to display their capability for alteration of surface wettability. Wet area measurement is a simple, reliable method not requiring a Drop Shape Analyzer. Cell viabilities determined by MTT assay showed a decline in a dose-dependent manner and estimated IC50 values were 25.87 µg/ml and 31.00 µg/ml for mono- and di-rhamnolipids treating MCF-7 cells for 48 h. Morphological observations using the inverted phase-contrast microscopy and fluorescence microscopy via Hoechst staining revealed the apoptotic characteristics in treated MCF-7 cells. The semi-quantitative RT-PCR method demonstrated that expression of the p53 gene in mRNA levels significantly (P < 0.05) increased when treated with 30 µg/ml of each rhamnolipid compound for 12 h. It can be concluded that rhamnolipids derived from MR01 show significant anticancer potential against MCF-7 cell line and should be further investigated as natural, therapeutic anti-tumor agents.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Glicolipídeos/farmacologia , Pseudomonas aeruginosa/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Glicolipídeos/biossíntese , Glicolipídeos/química , Humanos , Células MCF-7 , Microscopia de Fluorescência , Tamanho da Partícula , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Relação Estrutura-Atividade , Propriedades de Superfície , Proteína Supressora de Tumor p53/genética
11.
Colloids Surf B Biointerfaces ; 182: 110358, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325779

RESUMO

In this study, rhamnolipid (RL) production by Pseudomonas aeruginosa SS14 utilizing rice based Distillers Dried Grains with Solubles (rDDGS) as the sole carbon source was evaluated and the production parameters were optimized using response surface methodology. Highest RL (RL-rDDGS) yield was 14.87 g/L in a culture medium containing 12% (w/v) rDDGS and 11% (v/v) inoculum concentration after 48 h of fermentation at 35 °C. RL-rDDGS was produced as a mixture of mono and di-RL congeners with four novel homologues Rha-C18:2, Rha-C19, Rha-C9, and Rha-Rha-C19. The RL reduced the surface tension of water to 34.8 mN/m at a critical micelle concentration (CMC) value of 100 mg/L, exhibited high stability at a wide range of pH (6-12), heating time (0-120 min), and salinity (2-12% NaCl). Furthermore, RL-rDDGS demonstrated appreciable biofilm disruptive property against Candida tropicalis. This is the first report on the usage of rDDGS for sustainable and low cost production of RL.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida tropicalis/efeitos dos fármacos , Glicolipídeos/farmacologia , Oryza/química , Tensoativos/farmacologia , Antifúngicos/economia , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Biofilmes/crescimento & desenvolvimento , Candida tropicalis/crescimento & desenvolvimento , Estabilidade de Medicamentos , Análise Fatorial , Fermentação , Glicolipídeos/biossíntese , Glicolipídeos/economia , Glicolipídeos/isolamento & purificação , Temperatura Alta , Concentração de Íons de Hidrogênio , Micelas , Testes de Sensibilidade Microbiana , Oryza/metabolismo , Pseudomonas aeruginosa , Salinidade , Sementes/química , Sementes/metabolismo , Tensão Superficial , Tensoativos/economia , Tensoativos/isolamento & purificação , Tensoativos/metabolismo , Água/química
12.
Nat Chem Biol ; 15(6): 632-640, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31036922

RESUMO

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2→4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.


Assuntos
Cápsulas Bacterianas/metabolismo , Glicolipídeos/biossíntese , Bactérias Gram-Negativas/metabolismo , Transferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Transferases/química
13.
Fungal Genet Biol ; 130: 91-97, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31103599

RESUMO

Mannosylerythritol lipids (MELs) are surface active molecules produced by many basidiomycetous fungi. MELs consist of a mannosylerythritol disaccharide, which is acylated with short and medium chain fatty acids at the mannosyl moiety. A gene cluster composed of five genes is required for MEL biosynthesis. Here we show that the plant pathogenic fungus Ustilago hordei secretes these glycolipids under nitrogen starvation conditions. In contrast to MELs produced by the closely related fungus Ustilago maydis those secreted by U. hordei are mostly mono-acetylated and contain a different mixture of acyl groups. Cross-species complementation between these fungi revealed that these differences result from different catalytic activities of the acetyltransferase Mat1 and the acyltransferases Mac1 and Mac2. U. maydis mat1 mutants expressing the homologous mat1 gene from U. hordei produced mostly mono-acetylated variants and lack di-acetylated MELs normally produced by U. maydis. Furthermore, we determined that the acyltransferase Mac1 acylates the mannosylerythritol moiety at position C2 while Mac2 acylates C3. The identification of decorating enzymes with different substrate specificities will allow the tailor-made production of novel subsets of MELs.


Assuntos
Glicolipídeos/biossíntese , Ustilaginales/enzimologia , Ustilaginales/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Família Multigênica , Nitrogênio/metabolismo , Especificidade por Substrato , Transcriptoma , Ustilaginales/genética
14.
Sci Rep ; 9(1): 7085, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068620

RESUMO

Because of the lack of cell wall, Micoplasma species require a fine control of membrane fluidity and integrity. mg517 is an essential gene of Mycoplasma genitalium responsible for the biosynthesis of membrane glycoglycerolipids. It encodes for a unique glycosyltransferase (MG517) with processive activity, transferring activated glycosyl donors to either nude diacylglycerol or already glycosylated diacylglycerol. This dual activity, asserted to different enzymes in other species, is sensitive to and regulated by the presence of anionic lipid vesicles in vitro. We present here a computational model of the C-terminus domain of MG517 that complements a previous structural model of the N-terminus domain. By means of sequence analysis, molecular dynamics and metadynamics simulations, we have identified a short α-helix at the apical C-terminus of MG517 with clear amphipathic character. Binding to a membrane model is thermodynamically favored which suggests that this structural element guides the adhesion of MG517 to the cell membrane. We have experimentally verified that truncation of part of this helix causes a substantial reduction of glycoglycerolipids synthesis. The model proposes that MG517 recognizes and binds the diacylglycerol substrate embedded in the membrane by means of this α-helix at the C-terminus together with a previously identified binding pocket at the N-terminus.


Assuntos
Membrana Celular/metabolismo , Glicolipídeos/biossíntese , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Mycoplasma genitalium/enzimologia , Sequência de Aminoácidos , Adesão Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosiltransferases/genética , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Mycoplasma genitalium/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos
15.
mBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940701

RESUMO

Caulobacter crescentus adapts to phosphate starvation by elongating its cell body and a polar stalk structure. The stalk is an extension of the Gram-negative envelope containing inner and outer membranes as well as a peptidoglycan cell wall. Cellular elongation requires a 6- to 7-fold increase in membrane synthesis, yet phosphate limitation would preclude the incorporation of additional phospholipids. In the place of phospholipids, C. crescentus can synthesize several glycolipid species, including a novel glycosphingolipid (GSL-2). While glycosphingolipids are ubiquitous in eukaryotes, the presence of GSL-2 in C. crescentus is surprising since GSLs had previously been found only in Sphingomonas species, in which they play a role in outer membrane integrity. In this paper, we identify three proteins required for GSL-2 synthesis: CcbF catalyzes the first step in ceramide synthesis, while Sgt1 and Sgt2 sequentially glycosylate ceramides to produce GSL-2. Unlike in Sphingomonas, GSLs are nonessential in C. crescentus; however, the presence of ceramides does contribute to phage resistance and susceptibility to the cationic antimicrobial peptide polymyxin B. The identification of a novel lipid species specifically produced upon phosphate starvation suggests that bacteria may be able to synthesize a wider variety of lipids in response to stresses than previously observed. Uncovering these lipids and their functional relevance will provide greater insight into microbial physiology and environmental adaptation.IMPORTANCE Bacteria adapt to environmental changes in a variety of ways, including altering their cell shape. Caulobacter crescentus adapts to phosphate starvation by elongating its cell body and a polar stalk structure containing both inner and outer membranes. While we generally think of cellular membranes being composed largely of phospholipids, cellular elongation occurs when environmental phosphate, and therefore phospholipid synthesis, is limited. In order to adapt to these environmental constraints, C. crescentus synthesizes several glycolipid species, including a novel glycosphingolipid. This finding is significant because glycosphingolipids, while ubiquitous in eukaryotes, are extremely rare in bacteria. In this paper, we identify three proteins required for GSL-2 synthesis and demonstrate that they contribute to phage resistance. These findings suggest that bacteria may synthesize a wider variety of lipids in response to stresses than previously observed.


Assuntos
Adaptação Fisiológica , Caulobacter crescentus/fisiologia , Glicolipídeos/biossíntese , Glicoesfingolipídeos/biossíntese , Fosfatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Redes e Vias Metabólicas/genética
16.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979013

RESUMO

Rhamnolipids are a mixture of the homologs species due to variations in the rhamnose units and ß-hydroxy fatty acid moieties, mainly including Rha-C10-C10, Rha-Rha-C10-C10, and Rha-C10. In this study, strain P. aeruginosa YM4 was selected for its capacity to efficiently produce di-rhamnolipid (Rha-Rha-C10-C10) as the predominant component with soybean oil and glycerol as carbon source, accounting for 64.8% and 85.7% of total products, respectively. The critical micelle concentration (CMC) of rhamnolipid products varies with the content of di-rhamnolipid, whereby lower CMC values corresponding to higher di-rhamnolipid contents. The rhamnolipids containing 85.7% di-rhamnolipid had the lowest CMC value of 50 mg/L. Accordingly the viscosity-reducing efficiency and oil-washing efficiency of rhamnolipids increased with higher di-rhamnolipid component. At a concentration of 500 mg/L, the rhamnolipids containing 85.7% di-rhamnolipid worked best and showed 82.5% oil-washing efficiency, which offered great promise for applications in enhanced oil recovery. The results showed the variation of structure and composition of rhamnolipids had a significant effect on their application.


Assuntos
Glicolipídeos/biossíntese , Poluição por Petróleo/prevenção & controle , Pseudomonas aeruginosa/metabolismo , Ramnose/biossíntese , Carbono/química , Ácidos Graxos/química , Glicerol/química , Glicolipídeos/química , Humanos , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Ramnose/química , Óleo de Soja/química , Tensoativos/química
17.
Sci Rep ; 9(1): 5992, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30979908

RESUMO

In this paper, we describe the isolation of an unusual type of high molecular weight monorhamnolipid attached to esters of palmitic, stearic, hexa and octadecanoic acids as against the routinely reported di-rhamnolipids linked to hydroxydecanoic acids. The bioemulsifier was column-purified and the chemical nature of the compound was elucidated using FT-IR, GC-MS and 1D [1H and13C] and 2D NMR. This monorhamnolipid is extracted from a bacterium, Pseudomonas guganensis and is not reported to have biological activities, let alone emulsification abilities. The bacterium continually produced rhamnolipids when nourished with n-hexadecane as its lone carbon source. The extracellularly secreted monorhamnolipids are capable of degrading hydrocarbons, with most preference to n-hexadecane [EI24 of 56 ± 1.42% by 2 mL of the spent medium]. Whilst the crude ethyl acetate partitioned extract had an EI24 of 65 ± 1.43%; the purified rhamnolipid product showed 78 ± 1.75% both at 12.5 mg/mL concentration. The used-up n-hexadecane is biotransformed to prepare its own rhamnolipids which in return is utilized to degrade n-alkanes thus creating a circular pathway which is proposed herein. This bacterium can be seen as a new source of bioemulsifier to reduce hydrocarbon in polluted waters.


Assuntos
Glicolipídeos/biossíntese , Glicolipídeos/química , Pseudomonas/metabolismo , Emulsões , Índia , Pseudomonas/isolamento & purificação
18.
Bioprocess Biosyst Eng ; 42(8): 1301-1315, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31028463

RESUMO

With the ever growing increase in the demands of biosurfactants, the present study was focused in developing a set of parameters influencing biosurfactant production using one-factor-at-a-time (OFAT) approach in chemically defined medium from an indigenous isolate of Achromobacter sp. (PS1). Subsequently, the feasibility of biosurfactant production was examined using influential OFAT parameters in same medium, replacing only carbon source with lignocellulosic hydrolyzed sugars. These sugars were obtained from ammonia (15% v/v) soaking pretreatment of lignocellulosic residues (7.5% solid loading at 70 °C for 72 h) with subsequent saccharification using lignocellulolytic enzymes. OFAT influential parameters observed were dextrose (3-4% w/v); C/N ratio 8.3 using sodium nitrate and beef extract; 2 × 10-5 grams equivalents Fe2+; 1500 mM PO43- in minimal salt medium (MSM) at pH 7.0, 120 rpm, 30 °C resulting in 4.13 ± 0.12 g/L rhamnolipid in 192 h with 30.42 mN/m surface tension and 136 mg/L critical micelle concentration (CMC). Biosurfactant was characterized using tandem-MS and NMR as rhamnolipid with six-congeners, Rha-C10-C10 and Rha-Rha-C10-C10 being the most abundant. Rhamnolipid showed broad range stability at temperatures (30-121 °C), pH (6-12), and salinity (0.5-5% w/v) of NaCl. In Rice-straw (RS) hydrolysate, maximum glucan (73.10%) and xylan (91.13%) were obtained and the RS-hydrolysate medium with a total of 4.55% (w/v) sugars under optimum OFAT parameters (other than dextrose) showed at par production of 3.55 ± 0.06 g/L of rhamnolipid in 192 h with YBS/S (biosurfactant yield per gram of sugar consumed) of 0.08 g/g and YBS/CDW (biosurfactant yield per gram of cell biomass) of 0.68 g/g.


Assuntos
Achromobacter/crescimento & desenvolvimento , Amônia/química , Glicolipídeos/biossíntese , Lignina/química , Tensoativos/metabolismo
19.
Appl Microbiol Biotechnol ; 103(9): 3753-3760, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30919102

RESUMO

Pseudomonas aeruginosa are ubiquitous γ-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the ß-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(R)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and ß-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (R)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through ß-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (S)-3-hydroxyacyl-CoA).


Assuntos
Ácidos Graxos/metabolismo , Glicolipídeos/biossíntese , Poli-Hidroxialcanoatos/biossíntese , Pseudomonas aeruginosa/metabolismo , Aciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Oxirredução , Pseudomonas aeruginosa/enzimologia
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