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1.
Sci Rep ; 12(1): 14202, 2022 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-35987806

RESUMO

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Incorporation of the sulfur from the fungal volatile into plant metabolism was shown by 34S labeling experiments. Under sulfur deficiency, TMTM down-regulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Glutationa/metabolismo , Homeostase , Metano/metabolismo , Mortierella , Enxofre/metabolismo
2.
J Agric Food Chem ; 70(16): 5186-5196, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35416034

RESUMO

The electron-transfer capabilities of cytochrome b5 reductase (Cyt b5R) and NADPH supply have been shown to be critical factors in microbial fatty acid synthesis. Unfortunately, Cyt b5R substrate specificity is limited to the coenzyme NADH. In this study, we discovered that a novel Cyt b5R from Mortierella alpina (MaCytb5RII) displays affinity for NADPH and NADH. The enzymatic characteristics of high-purity MaCytb5RII were determined with the Km,NADPH and Km,NADH being 0.42 and 0.07 mM, respectively. MaCytb5RII shows high specific activity at 4 °C and pH 9.0. We anchored the residues that interacted with the coenzymes using the homology models of MaCytb5Rs docking NAD(P)H and FAD. The enzyme activity analysis of the purified mutants MaCytb5RII[S230N], MaCytb5RII[Y242F], and MaCytb5RII[S272A] revealed that Ser230 is essential for MaCytb5RII to have dual NAD(P)H dependence, whereas Tyr242 influences MaCytb5RII's NADPH affinity and Ala272 greatly decreases MaCytb5RII's NADH affinity.


Assuntos
Citocromo-B(5) Redutase , NAD , Citocromo-B(5) Redutase/química , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Citocromos b , Cinética , Mortierella , NAD/metabolismo , NADP
3.
Microbiol Spectr ; 10(1): e0130021, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35138146

RESUMO

The present study was designed to explore the possibility of improving lipid production in oleaginous filamentous fungus Mortierella alpina based on an autophagy regulation strategy. According to multiomics information, vacuolate-centered macroautophagy was identified as the main type of autophagy in M. alpina under nitrogen-limited conditions. Mutation of autophagy-related gene MAatg8 led to impaired fatty acid synthesis, while overexpression of both MAatg8 and phosphatidylserine decarboxylases (MApsd2) showed promoting effects on fatty acid synthesis. MAatg8 overexpression strain with external supply of ethanolamine significantly increased arachidonic acid (ARA)-rich triacylglycerol (TAG) and biomass synthesis in M. alpina, and the final fatty acid content increased by approximately 110% compared with that in the wild-type strain. Metabolomics and lipidomics analyses revealed that cell autophagy enhanced the recycling of preformed carbon, nitrogen, and lipid in mycelium, and the released carbon skeleton and energy were contributed to the accumulation of TAG in M. alpina. This study suggests that regulation of autophagy-related MAatg8-phosphatidylethanolamine (MAatg8-PE) conjugation system could be a promising strategy for attaining higher lipid production and biomass growth. The mechanism of autophagy in regulating nitrogen limitation-induced lipid accumulation elucidated in this study provides a reference for development of autophagy-based strategies for improving nutrient use efficiency and high value-added lipid production by oleaginous microorganism. IMPORTANCE Studies have indicated that functional oil accumulation occurs in oleaginous microorganisms under nitrogen limitation. However, until now, large-scale application of nitrogen-deficiency strategies was limited by low biomass. Therefore, the identification of the critical nodes of nitrogen deficiency-induced lipid accumulation is urgently needed to further guide functional oil production. The significance of our research is in uncovering the function of cell autophagy in the ARA-rich TAG accumulation of oleaginous fungus M. alpina and demonstrating the feasibility of improving lipid production based on an autophagy regulation strategy at the molecular and omics levels. Our study proves that regulation of cell autophagy through the MAatg8-PE conjugation system-related gene overexpression or exogenous supply of ethanolamine would be an efficient strategy to increase and maintain biomass productivity when high TAG content is obtained under nitrogen deficiency, which could be useful for the development of new strategies that will achieve more biomass and maximal lipid productivity.


Assuntos
Ácido Araquidônico/metabolismo , Autofagia , Mortierella/citologia , Mortierella/metabolismo , Triglicerídeos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metabolismo dos Lipídeos , Mortierella/genética
4.
Sci Rep ; 12(1): 2815, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181683

RESUMO

Soil microorganisms could affect the quality of tobacco leaves, however, little is known about the association of tobacco chemical components and soil fungal communities. In the present study, the relationship between soil fungi and tobacco quality based on chemical components in Bijie was investigated. The results showed that the total harmony scores (THS) of the analyzed tobacco leaves ranged from 46.55 ± 3.5 to 91.55 ± 2.25. Analyses of chemical components revealed that high contents of nicotine (≥ 1.06%) and sugar (total sugar: ≥ 22.96%, reducing sugar: ≥ 19.62%), as well as low potassium level (≤ 2.68%) were the main factors limiting the quality of flue-cured tobacco leaves. Pearson correlation analysis indicated that soil nitrate, available potassium/phosphorous, and organic matter significantly correlated with tobacco nicotine, potassium, and chloride levels (p < 0.05). Besides, the analysis of alpha- and beta-diversity of soil fungal communities implied that fungal structure rather than the richness affected the chemical quality of tobacco. In detail, the relative abundance of Humicola olivacea species in soils was positively correlated with the THS of tobaccos (r = 0.52, p < 0.05). Moreover, the species including Mortierella alpina, Mortierella hyalina, Tausonia pullulan, and Humicola olivacea were negatively correlated with tobacco sugar (r ≤ - 0.45, p < 0.05) while, Codinaea acaciae and Saitozyma podzolica species were negatively correlated with tobacco nicotine (r ≤ - 0.51, p < 0.05). The present study provides a preliminary basis for utilizing fungal species in soils to improve the chemical quality of tobacco in the studied area.


Assuntos
Micobioma/genética , Folhas de Planta/química , Microbiologia do Solo , Tabaco/química , Gênero de Fungos Humicola/química , Fungos/química , Fungos/genética , Mortierella/química , Folhas de Planta/microbiologia , Potássio/metabolismo , Tabaco/microbiologia , Produtos do Tabaco/análise
5.
Biotechnol Adv ; 54: 107794, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34245810

RESUMO

The oleaginous fungus Mortierella alpina has distinct advantages in long-chain PUFAs production, and it is the only source for dietary arachidonic acid (ARA) certificated by FDA and European Commission. This review provides an overall introduction to M. alpina, including its major research methods, key factors governing lipid biosynthesis, metabolic engineering and omics studies. Currently, the research interests in M. alpina focus on improving lipid yield and fatty acid desaturation degree by enhancing fatty acid precursors and the reducing power NADPH, and genetic manipulation on PUFAs synthetic pathways is carried to optimise fatty acid composition. Besides, multi-omics studies have been applied to elucidate the global regulatory mechanism of lipogenesis in M. alpina. However, research challenges towards achieving a lipid cell factory lie in strain breeding and cost control due to the coenocytic mycelium, long fermentation period and insufficient conversion rate from carbon to lipid. We also proposed future research goals based on a multilevel regulating strategy: obtaining ideal chassis by directional evolution and high-throughput screening; rewiring central carbon metabolism and inhibiting competitive pathways by multi-gene manipulation system to enhance carbon to lipid conversion rate; optimisation of protein function based on post-translational modification; application of dynamic fermentation strategies suitable for different fermentation phases. By reviewing the comprehensive research progress of this oleaginous fungus, we aim to further comprehend the fungal lipid metabolism and provide reference information and guidelines for the exploration of microbial oils from the perspectives of fundamental research to industrial application.


Assuntos
Metabolismo dos Lipídeos , Mortierella , Ácido Araquidônico/metabolismo , Lipogênese/genética , Mortierella/genética , Mortierella/metabolismo
6.
Z Naturforsch C J Biosci ; 77(1-2): 11-19, 2022 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-34265877

RESUMO

The fungus, Mortierella polycephala is one of the most productive sources of anticancer bioactive compounds namely those of pigment nature. During our investigation of the produced bioactive metabolites by the terrestrial M. polycephala AM1 isolated from Egyptian poultry feather waste, two main azaphilonoid pigments, monascin (1) and monascinol (2) were obtained as major products; their structures were identified by 1D (1H&13C) and 2D (1H-1H COSY, HMBC) NMR and HRESI-MS spectroscopic data. Biologically, cytotoxic activities of these compounds were broadly studied compared with the fungal extract. To predict the biological target for the presumed antitumor activity, an in silico study was run toward three proteins, topoisomerase IIα, topoisomerase IIß, and VEGFR2 kinase. Monascinol (2) was expected to be moderately active against VEGFR2 kinase without any anticipated inhibition toward topo II isoforms. The in vitro study confirmed the docked investigation consistently and introduced monascinol (2) rather than its counterpart (1) as a potent inhibitor to the tested VEGFR2 kinase. Taxonomically, the fungus was identified using morphological and genetic assessments.


Assuntos
Antineoplásicos , Mortierella , Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis
7.
Lett Appl Microbiol ; 74(2): 194-203, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34755357

RESUMO

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.


Assuntos
Diacilglicerol O-Aciltransferase , Mortierella , Diacilglicerol O-Aciltransferase/genética , Ácidos Graxos , Mortierella/genética , Triglicerídeos
8.
Ultrason Sonochem ; 78: 105720, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34469850

RESUMO

Ultrasound assisted enzymatic method was applied to the degumming of arachidonic acid (ARA) oil produced by Mortierella alpina. The conditions of degumming process were optimized by response surface methodology with Box- Behnken design. A dephosphorization rate of 98.82% was achieved under optimum conditions of a 500 U/kg of Phospholipase A1 (PLA1) dosage, 2.8 mL/100 g of water volume, 120 min of ultrasonic time, and 135 W of ultrasonic power. The phosphorus content of ultrasonic assisted enzymatic degumming oil (UAEDO) was 4.79 mg/kg, which was significantly lower than that of enzymatic degumming oil (EDO, 17.98 mg/kg). Crude Oil (CO), EDO and UAEDO revealed the similar fatty acid compositions, and ARA was dominated (50.97 ~ 52.40%). The oxidation stability of UAEDO was equivalent to EDO and weaker than CO, while UAEDO presented the strongest thermal stability, followed by EDO and CO. Furthermore, aldehydes, acids and alcohols were identified the main volatile flavor components for the three oils. The proportions of major contributing components such as hexanal, nonanal, (E)-2-nonanal, (E, E)-2,4-decadienal, (E)-2-nonenal and aldehydes in UAEDO and EDO were all lower than CO. Overall, Ultrasound assisted enzymatic degumming proved to be an efficient and superior method for degumming of ARA oil.


Assuntos
Ácido Araquidônico , Ácidos Graxos , Óleos Vegetais , Aldeídos/química , Ácido Araquidônico/química , Ácidos Graxos/química , Mortierella/química , Óleos Vegetais/química , Ondas Ultrassônicas , Água/química
9.
F1000Res ; 10: 895, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745563

RESUMO

Background: A large number of undiscovered fungal species still exist on earth, which can be useful for bioprospecting, particularly for single cell oil (SCO) production. Mortierella is one of the significant genera in this field and contains about hundred species. Moreover, M. alpina is the main single cell oil producer at commercial scale under this genus. Methods: Soil samples from four unique locations of North-East Libya were collected for the isolation of oleaginous Mortierellaalpina strains by a serial dilution method. Morphological identification was carried out using light microscopy (Olympus, Japan) and genetic diversity of the isolated Mortierella alpina strains was assessed using conserved internal transcribed spacer (ITS) gene sequences available on the NCBI GenBank database for the confirmation of novelty. The nucleotide sequences reported in this study have been deposited at GenBank (accession no. MZ298831:MZ298835). The MultAlin program was used to align the sequences of closely related strains. The DNA sequences were analyzed for phylogenetic relationships by molecular evolutionary genetic analysis using MEGA X software consisting of Clustal_X v.2.1 for multiple sequence alignment. The neighbour-joining tree was constructed using the Kimura 2-parameter substitution model. Results: The present research study confirms four oleaginous fungal isolates from Libyan soil. These isolates (barcoded as MSU-101, MSU-201, MSU-401 and MSU-501) were discovered and reported for the first time from diverse soil samples of district Aljabal Al-Akhdar in North-East Libya and fall in the class: Zygomycetes; order: Mortierellales. Conclusions: Four oleaginous fungal isolates barcoded as MSU-101, MSU-201, MSU-401 and MSU-501 were identified and confirmed by morphological and molecular analysis. These fungal isolates showed highest similarity with Mortierella alpina species and can be potentialistic single cell oil producers. Thus, the present research study provides insight to the unseen fungal diversity and contributes to more comprehensive Mortierella alpina reference collections worldwide.


Assuntos
Mortierella , Variação Genética , Líbia , Mortierella/genética , Filogenia
10.
J Agric Food Chem ; 69(46): 13849-13858, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34779198

RESUMO

A sucrose nonfermenting protein kinase 1 (SNF1) complex is an important metabolic regulator in fungi that is critical to cell metabolism and stress response. In this study, the role of an SNF1 ß-subunit in the oleaginous fungus Mortierella alpina (MaSip2) was investigated. The MaSip2 contained a glycogen-binding domain and a conserved SNF1-complex interaction region; its transcriptional level during lipogenesis shared high consistency with a previously reported SNF1 γ-subunit (MaSnf4). Overexpression of MaSip2 in M. alpina significantly promoted glucose uptake and resulted in 34.1% increased total biomass, leading to 44.8% increased arachidonic acid yield after 7 day fermentation. MaSip2 also regulated the balance between polyunsaturated fatty acids and carbohydrates in M. alpina. Intracellular metabolite analysis revealed increased carbohydrate-related metabolite accumulation in MaSip2 overexpression strains. On the contrary, knockdown of MaSip2 increased the total fatty acid unsaturation degree, especially under low-temperature conditions. This research improved our knowledge of SNF1 complex in M. alpina and provided a target gene for enhancing glucose utilization and modulating fatty acid composition for better application of oleaginous fungi.


Assuntos
Mortierella , Ácidos Graxos , Ácidos Graxos Insaturados , Glucose , Mortierella/genética
11.
Chem Biodivers ; 18(12): e2100741, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34786854

RESUMO

Three new hydroxyphenylacetic acid derivatives, stachylines E-G (1-3), and a new alkaloid, mortieridinone (4), along with six known compounds (5-10), were isolated from endophytic fungus Mortierella sp. in Epimedium acuminatum Franch. Their structures were determined by their spectroscopic analyses and by comparison with the literature data. Compounds 7 and 10 showed selective antibacterial activity against tested multidrug-resistant bacteria with minimum inhibitory concentration (MIC) values ranging from 25 to 3.13 µg/mL.


Assuntos
Alcaloides/farmacologia , Antibacterianos/farmacologia , Epimedium/microbiologia , Mortierella/química , Fenilacetatos/farmacologia , Alcaloides/química , Alcaloides/isolamento & purificação , Antibacterianos/química , Antibacterianos/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Fenilacetatos/química , Fenilacetatos/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos
12.
Appl Microbiol Biotechnol ; 105(16-17): 6275-6289, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34424385

RESUMO

Mortierella alpina is an oleaginous filamentous fungus with considerable lipid productivity, and it has been widely used for industrial production of arachidonic acid. The fermentation process of M. alpina is complicated and can be affected by various factors; therefore, a comprehensive knowledge of its metabolic characteristics and key factors governing lipid biosynthesis is required to further improve its industrial performance. In this review, we discuss the metabolic features and extracellular factors that affect lipid biosynthesis in M. alpina. The current progress in fermentation optimisation and metabolic engineering to improve lipid yield are also summarised. Moreover, we review the applications of M. alpina in the food industry and propose fermentation strategies for better utilisation of this genus in the future. In our opinion, the economic performance of M. alpina should be enhanced from multiple levels, including strains with ideal traits, efficient fermentation strategies, controllable fermentation costs, and competitive products of both high value and productivity. By reviewing the peculiarities of M. alpina and current progress to improve its suitability for biotechnological production, we wish to provide more efficient strategies for future development of M. alpina as a high-value lipid cell factory. KEY POINTS: • Understanding M. alpina metabolism is helpful for rational design of its fermentation processes. • Nitrogen source is a key point that affects PUFA's component and fermentation cost in M. alpina. • Dynamic fermentation strategy combined with breeding is needed to increase lipid yield in M. alpina.


Assuntos
Mortierella , Ácido Araquidônico , Ácidos Graxos Insaturados , Fermentação , Mortierella/genética
13.
Microbiology (Reading) ; 167(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34402775

RESUMO

Phenylalanine hydroxylase (PAH) catalyses the irreversible hydroxylation of phenylalanine to tyrosine, which is the rate-limiting reaction in phenylalanine metabolism in animals. A variety of polyunsaturated fatty acids can be synthesized by the lipid-producing fungus Mortierella alpina, which has a wide range of industrial applications in the production of arachidonic acid. In this study, RNA interference (RNAi) with the gene PAH was used to explore the role of phenylalanine hydroxylation in lipid biosynthesis in M. alpina. Our results indicated that PAH knockdown decreased the PAH transcript level by approximately 55% and attenuated cellular fatty acid biosynthesis. Furthermore, the level of NADPH, which is a critical reducing agent and the limiting factor in lipogenesis, was decreased in response to PAH RNAi, in addition to the downregulated transcription of other genes involved in NADPH production. Our study indicates that PAH is part of an overall enzymatic and regulatory mechanism supplying NADPH required for lipogenesis in M. alpina.


Assuntos
Mortierella , Fenilalanina Hidroxilase , Ácido Araquidônico , Lipogênese/genética , Mortierella/genética , Fenilalanina Hidroxilase/genética
14.
Fungal Genet Biol ; 152: 103572, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34015432

RESUMO

Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.


Assuntos
3-Isopropilmalato Desidrogenase/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipogênese/fisiologia , Mortierella/enzimologia , Mortierella/metabolismo , 3-Isopropilmalato Desidrogenase/genética , Acetilcoenzima A , Sequência de Aminoácidos , Aminoácidos/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Cetoácidos/metabolismo , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Metabolômica , Mortierella/genética , Mucor/metabolismo , Alinhamento de Sequência
15.
Biotechnol Lett ; 43(7): 1289-1301, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33864523

RESUMO

OBJECTIVES: To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. RESULTS: The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. CONCLUSIONS: This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.


Assuntos
Carboidratos/análise , Lipídeos/análise , Mortierella/química , Carboidratos/química , Cromatografia Líquida de Alta Pressão/instrumentação , Colorimetria , Ácidos Graxos/isolamento & purificação , Fermentação , Glucose/isolamento & purificação , Lipídeos/química , Fotometria , Trealose/isolamento & purificação
16.
Biotechnol Lett ; 43(7): 1455-1466, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33907945

RESUMO

OBJECTIVES: The transport of citrate from the mitochondria to the cytoplasm is essential during lipid accumulation. This study aimed to explore the role of mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina. RESULTS: Homologous MaYHM (the gene encoding the mitochondrial citrate-oxoglutarate carrier) was overexpressed in M. alpina. The fatty acid content of MaYHM-overexpressing recombinant strains was increased by up to 30% compared with the control. Moreover, the intracellular α-ketoglutarate level in recombinant strains was increased by 2.2 fold, together with a 23-35% decrease in NAD+-isocitrate dehydrogenase activity compared with the control. The overexpression of MaYHM altered the metabolic flux in the glutamate dehydrogenase shunt and 4-aminobutyric acid shunt during metabolic reprogramming, supplying more carbon to synthesize fatty acids. CONCLUSIONS: Overexpression of MaYHM resulted in more efflux of citrate from mitochondria to the cytoplasm and enhanced lipid accumulation. These findings provide new perspectives for the improvement of industrial lipid production in M. alpina.


Assuntos
Ácido Cítrico/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mortierella/crescimento & desenvolvimento , Clonagem Molecular , Citoplasma/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Mortierella/genética , Proteínas Recombinantes/metabolismo , Ácido gama-Aminobutírico/metabolismo
17.
In Vitro Cell Dev Biol Anim ; 57(4): 395-403, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33904018

RESUMO

This study presents an in vitro evaluation of the antitumor potential of a chitin-like exopolysaccharide (EPS, produced by Mortierella alpina) on Adrenocortical carcinoma cells (ACC) compared to mitotane, a commercial drug commonly used in ACC treatment, and known for its side effects. Techniques of cellular viability determination such as MTT and fluorescence were used to measure the cytotoxic effects of the EPS and mitotane in tumoral cells (H295R) and non-tumoral cells (VERO), observing high cytotoxicity of mitotane and a 10% superior pro-apoptotic effect of the EPS compared to mitotane (p < 0.05). The cytotoxic effect of the EPS was similar to the effect of 50 µM mitotane on tumoral cells (p < 0.05). A decrement of the lysosomal volume was also noted in tumoral cells treated with the EPS. To enhance the antitumor effect, a combination of mitotane at a lower dosage and the EPS (as adjuvant) was also tested, showing a slight improvement of the cytotoxicity effect on tumoral cells. Therefore, the results indicate a cytotoxic effect of the EPS produced by Mortierella alpina on adrenocortical carcinoma, and a possible application in biomedical formulations or additional treatments.


Assuntos
Carcinoma Adrenocortical/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Quitina/farmacologia , Mortierella/química , Carcinoma Adrenocortical/patologia , Animais , Linhagem Celular Tumoral , Quitina/química , Chlorocebus aethiops , Humanos , Mitotano/farmacologia , Polissacarídeos , Células Vero
18.
Biosci Biotechnol Biochem ; 85(5): 1252-1265, 2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33728459

RESUMO

ω3 Polyunsaturated fatty acids are currently obtained mainly from fisheries; thus, sustainable alternative sources such as oleaginous microorganisms are required. Here, we describe the isolation, characterization, and application of 3 novel ω3 desaturases with ω3 polyunsaturated fatty acid-producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17, respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid-producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources.


Assuntos
Ácido Eicosapentaenoico/biossíntese , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Insaturados/biossíntese , Mortierella/genética , Oomicetos/genética , Pythium/genética , Ácido Araquidônico/biossíntese , Clonagem Molecular , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/classificação , Expressão Gênica , Biblioteca Gênica , Engenharia Metabólica/métodos , Mortierella/enzimologia , Oomicetos/classificação , Oomicetos/enzimologia , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Pythium/classificação , Pythium/enzimologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transformação Genética , Transgenes
19.
World J Microbiol Biotechnol ; 37(1): 4, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33392832

RESUMO

Arachidonic acid (ARA, 5, 8, 11, 14-cis-eicosatetraenoic acid) is a relevant ω-6 polyunsaturated fatty acid, which plays essential roles in human immune, cardiovascular, and nervous systems. It is widely used in medicine, cosmetics, nutrition, and other fields. Traditionally, ARA is obtained from animal tissues. However, due to the limitation and unsustainability of existing resources, microorganisms are a potential alternative resource for ARA production. In this regard, major efforts have been made on algae and filamentous fungi, among which Mortierella alpina is the most effective strain for industrial ARA production. In this review, we summarized the recent progress in enhancing M. alpina production by optimization of culture medium and fermentation process and genetic modification. In addition, we provided perspectives in synthetic biology methods and technologies to further increase ARA production.


Assuntos
Ácido Araquidônico/biossíntese , Ácido Araquidônico/genética , Fermentação , Edição de Genes , Mortierella/genética , Mortierella/metabolismo , Reatores Biológicos , Vias Biossintéticas/genética , Meios de Cultura , Humanos , Engenharia Metabólica/métodos , Oxigênio/metabolismo
20.
Int J Phytoremediation ; 23(8): 846-856, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33397125

RESUMO

Improved knowledge of the ecology of contaminant-degrading organisms is paramount for effective assessment and remediation of aromatic hydrocarbon-impacted sites. DNA stable isotope probing was used herein to identify autochthonous degraders in rhizosphere soil from a hybrid poplar phytoremediation system incubated under semi-field-simulated conditions. High-throughput sequencing of bacterial 16S rRNA and fungal internal transcribed spacer (ITS) rRNA genes in metagenomic samples separated according to nucleic acid buoyant density was used to identify putative toluene degraders. Degrader bacteria were found mainly within the Actinobacteria and Proteobacteria phyla and classified predominantly as Cupriavidus, Rhodococcus, Luteimonas, Burkholderiaceae, Azoarcus, Cellulomonadaceae, and Pseudomonas organisms. Purpureocillium lilacinum and Mortierella alpina fungi were also found to assimilate toluene, while several strains of the fungal poplar endophyte Mortierella elongatus were indirectly implicated as potential degraders. Finally, PICRUSt2 predictive taxonomic functional modeling of 16S rRNA genes was performed to validate successful isolation of stable isotope-labeled DNA in density-resolved samples. Four unique sequences, classified within the Bdellovibrionaceae, Intrasporangiaceae, or Chitinophagaceae families, or within the Sphingobacteriales order were absent from PICRUSt2-generated models and represent potentially novel putative toluene-degrading species. This study illustrates the power of combining stable isotope amendment with advanced metagenomic and bioinformatic techniques to link biodegradation activity with unisolated microorganisms. Novelty statement: This study used emerging molecular biological techniques to identify known and new organisms implicated in aromatic hydrocarbon biodegradation from a field-scale phytoremediation system, including organisms with phyto-specific relevance and having potential for downstream applications (amendment or monitoring) in future and existing systems. Additional novelty in this study comes from the use of taxonomic functional modeling approaches for validation of stable isotope probing techniques. This study provides a basis for expanding existing reference databases of known aromatic hydrocarbon degraders from field-applicable sources and offers technological improvements for future site assessment and management purposes.


Assuntos
Rizosfera , Solo , Biodegradação Ambiental , Fungos/genética , Hypocreales , Isótopos , Mortierella , RNA Ribossômico 16S/genética , Microbiologia do Solo , Tolueno
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