Characterization of NAD+/NADP+-Specific Isocitrate Dehydrogenases From Oleaginous Fungus Mortierella alpina Involved in Lipid Accumulation
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SUBMITTER: Tang X
PROVIDER: S-EPMC8566678 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50% of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50% of its 12,796 gene models, and 60% of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina's fatty acid synthase is a single polypeptide containing all of the catalytic domains required for fatty acid synthesis from acetyl-CoA and malonyl-CoA, whereas in many fungi this enzyme is comprised of two polypeptides. Major lipids were profiled to confirm the products predicted in the lipogenesis pathway. M. alpina produces a complex mixture of glycerolipids, glycerophospholipids and sphingolipids. In contrast, only two major sterol lipids, desmosterol and 24(28)-methylene-cholesterol, were detected. Phylogenetic analysis based on genes involved in lipid metabolism suggests that oleaginous fungi may have acquired their lipogenic capacity during evolution after the divergence of Ascomycota, Basidiomycota, Chytridiomycota and Mucoromycota. Our study provides the first draft genome and comprehensive lipid profile for M. alpina, and lays the foundation for possible genetic engineering of M. alpina to produce higher levels and diverse contents of dietary lipids.
| S-EPMC3234268 | biostudies-literature
Project description:Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme within the glycolytic pathway. GAPDH catalyzes the transformation of glyceraldehyde 3-phosphate to glycerate-1, 3-biphosphate, a process accompanied by the production of NADH. Its role in the NADPH production system of the oleaginous filamentous fungus Mortierella alpina was explored. Two copies of genes encoding GAPDH were characterized, then endogenously overexpressed and silenced through Agrobacterium tumefaciens-mediated transformation methods. The results showed that the lipid content of the overexpression strain, MA-GAPDH1, increased by around 13%. RNA interference of GAPDH1 and GAPDH2 (MA-RGAPDH1 and MA-RGAPDH2) greatly reduced the biomass of the fungus. The lipid content of MA-RGAPDH2 was found to be about 23% higher than that of the control. Both of the lipid-increasing transformants showed a higher NADPH/NADP ratio. Analysis of metabolite and enzyme expression levels revealed that the increased lipid content of MA-GAPDH1 was due to enhanced flux of glyceraldehyde-3-phosphate to glycerate-1, 3-biphosphate. MA-RGAPDH2 was found to strengthen the metabolic flux of dihydroxyacetone phosphate to glycerol-3-phosphate. Thus, GAPDH1 contributes to NADPH supply and lipid accumulation in M. alpina, and has a distinct role from GAPDH2.
| S-EPMC7198782 | biostudies-literature
Project description:We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH₄) in the lipid-producing fungus Mortierella alpina. The BH₄ cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H₂-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H₂-NTP to 6-pyruvoyltetrahydropterin (PPH₄), and sepiapterin reductase (SR) for the conversion of PPH₄ to BH₄, were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH₄ in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH₄ and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH₄ biosynthesis pathway in a fungus.
| S-EPMC4811656 | biostudies-literature
Project description:The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi.
| S-EPMC3993310 | biostudies-literature
Project description:Tetrahydrobiopterin (BH4) is well-known as a cofactor of phenylalanine hydroxylase (PAH) and nitric oxide synthase (NOS), but its exact role in lipogenesis is unclear. In this study, the GTP cyclohydrolase I (GTPCH) gene was overexpressed to investigate the role of BH4 in lipogenesis in oleaginous fungus Mortierella alpina. Transcriptome data analysis reveal that GTPCH expression was upregulated when nitrogen was exhausted, resulting in lipid accumulation. Significant changes were also found in the fatty acid profile of M. alpina grown on medium that contained a GTPCH inhibitor relative to that of M. alpina grown on medium that lacked the inhibitor. GTPCH overexpression in M. alpina (the MA-GTPCH strain) led to a sevenfold increase in BH4 levels and enhanced cell fatty acid synthesis and poly-unsaturation. Increased levels of nicotinamide adenine dinucleotide phosphate (NADPH) and upregulated expression of NADPH-producing genes in response to enhanced BH4 levels were also observed, which indicate a novel aspect of the NADPH regulatory mechanism. Increased BH4 levels also enhanced phenylalanine hydroxylation and nitric oxide synthesis, and the addition of an NOS or a PAH inhibitor in the MA-GTPCH and control strain cultures decreased fatty acid accumulation, NADPH production, and the transcript levels of NADPH-producing genes. Our research suggests an important role of BH4 in lipogenesis and that the phenylalanine catabolism and arginine-nitric oxide pathways play an integrating role in translating the effects of BH4 on lipogenesis by regulating the cellular NADPH pool. Thus, our findings provide novel insights into the mechanisms of efficient lipid biosynthesis regulation in oleaginous microorganisms and lay a foundation for the genetic engineering of these organisms to optimize their dietary fat yield.
| S-EPMC7044132 | biostudies-literature
Project description:Background:Global resource reallocation is an established critical strategy through which organisms deal with environmental stress. The regulation of intracellular lipid storage or utilization is one of the most important strategies for maintaining energy homeostasis and optimizing growth. Oleaginous microorganisms respond to nitrogen deprivation by inducing lipid hyper accumulation; however, the associations between resource allocation and lipid accumulation are poorly understood. Results:Here, the time-resolved metabolomics, lipidomics, and proteomics data were generated in response to nutrient availability to examine how metabolic alternations induced by nitrogen deprivation drive the triacylglycerols (TAG) accumulation in M. alpina. The subsequent accumulation of TAG under nitrogen deprivation was a consequence of the reallocation of carbon, nitrogen sources, and lipids, rather than an up-regulation of TAG biosynthesis genes. On one hand, nitrogen deprivation induced the down-regulation of isocitrate dehydrogenase level in TCA cycle and redirected glycolytic flux of carbon from amino acid biosynthesis into fatty acids' synthesis; on the other hand, nitrogen deprivation induced the up-regulation of cell autophagy and ubiquitin-mediated protein proteolysis which resulted in a recycling of preformed protein nitrogen and carbon. Combining with the up-regulation of glutamate decarboxylase and succinic semialdehyde dehydrogenase in GABA shunt, and the phosphoenolpyruvate carboxykinase in the central hub involving pyruvate/phosphoenolpyruvate/oxaloacetate, the products from nitrogen-containing compounds degradation were recycled to be intermediates of TCA cycle and be shunted toward de novo biosynthesis of fatty acids. We found that nitrogen deprivation increased the protein level of phospholipase C/D that contributes to degradation of phosphatidylcholine and phosphatidylethanolamine, and supplied acyl chains for TAG biosynthesis pathway. In addition, ATP from substrate phosphorylation was presumed to be a critical factor regulation of the global resource allocation and fatty acids' synthesis rate. Conclusions:The present findings offer a panoramic view of resource allocation by M. alpina in response to nutrient stress and revealed a set of intriguing associations between resource reallocation and TAG accumulation. This system-level insight provides a rich resource with which to explore in-depth functional characterization and gain information about the strategic combination of strain development and process integration to achieve optimal lipid productivity under nutrient stress.
| S-EPMC7328260 | biostudies-literature
Project description:Mortierella alpina is a filamentous fungus commonly found in soil that is able to produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight. Analysis of the M. alpina genome suggests that there is a phenylalanine-hydroxylating system for the catabolism of phenylalanine, which has never been found in fungi before. We characterized the phenylalanine-hydroxylating system in M. alpina to explore its role in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant changes were found in the profile of fatty acids in M. alpina grown on medium containing an inhibitor of the phenylalanine-hydroxylating system compared to M. alpina grown on medium without inhibitor. Genes encoding enzymes involved in the phenylalanine-hydroxylating system (phenylalanine hydroxylase [PAH], pterin-4?-carbinolamine dehydratase, and dihydropteridine reductase) were expressed heterologously in Escherichia coli, and the resulting proteins were purified to homogeneity. Their enzymatic activity was investigated by high-performance liquid chromatography (HPLC) or visible (Vis)-UV spectroscopy. Two functional PAH enzymes were observed, encoded by distinct gene copies. A novel role for tetrahydrobiopterin in fungi as a cofactor for PAH, which is similar to its function in higher life forms, is suggested. This study establishes a novel scheme for the fungal degradation of an aromatic substance (phenylalanine) and suggests that the phenylalanine-hydroxylating system is functionally significant in lipid metabolism.
| S-EPMC3685260 | biostudies-literature
Project description:BACKGROUND: Isocitrate Dehydrogenases (IDHs) are important enzymes present in all living cells. Three subfamilies of functionally dimeric IDHs (subfamilies I, II, III) are known. Subfamily I are well-studied bacterial IDHs, like that of Escherischia coli. Subfamily II has predominantly eukaryotic members, but it also has several bacterial members, many being pathogens or endosymbionts. subfamily III IDHs are NAD-dependent. The eukaryotic-like subfamily II IDH from pathogenic bacteria such as Mycobacterium tuberculosis IDH1 are expected to have regulation similar to that of bacteria which use the glyoxylate bypass to survive starvation. Yet they are structurally different from IDHs of subfamily I, such as the E. coli IDH. RESULTS: We have used phylogeny, structural comparisons and molecular dynamics simulations to highlight the similarity and differences between NADP-dependent dimeric IDHs with an emphasis on regulation. Our phylogenetic study indicates that an additional subfamily (IV) may also be present. Variation in sequence and structure in an aligned region may indicate functional importance concerning regulation in bacterial subfamily I IDHs. Correlation in movement of prominent loops seen from molecular dynamics may explain the adaptability and diversity of the predominantly eukaryotic subfamily II IDHs. CONCLUSION: This study discusses possible regulatory mechanisms operating in various IDHs and implications for regulation of eukaryotic-like bacterial IDHs such as that of M. tuberculosis, which may provide avenues for intervention in disease.
| S-EPMC3521221 | biostudies-literature
Project description:Fungi are traditionally considered a reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early-diverging fungi such as Mortierella Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC 32222. The molecular structures of malpicyclins were elucidated by high-resolution tandem mass spectrometry (HR-MS/MS), Marfey's method, and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative quantitative real-time PCR (qRT-PCR) expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPSs), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays proved promiscuous substrate selection and confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered l-amino acids and the subsequent incorporation of d-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicated a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as-yet-unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as a novel and underrated resource of natural products.IMPORTANCE Fungal natural compounds are industrially produced, with application in antibiotic treatment, cancer medications, and crop plant protection. Traditionally, higher fungi have been intensively investigated concerning their metabolic potential, but reidentification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early-diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, designated malpicyclins and malpibaldins, respectively, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much more closely related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella Both enzymes were biochemically characterized and are involved in as-yet-unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early-diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.
| S-EPMC7848919 | biostudies-literature
Project description: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.
| S-EPMC8564746 | biostudies-literature