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Genome characterization of the oleaginous fungus Mortierella alpina.

ABSTRACT: 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.

SUBMITTER: Wang L

PROVIDER: S-EPMC3234268 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Genome characterization of the oleaginous fungus Mortierella alpina.

Wang Lei L Chen Wei W Feng Yun Y Ren Yan Y Gu Zhennan Z Chen Haiqin H Wang Hongchao H Thomas Michael J MJ Zhang Baixi B Berquin Isabelle M IM Li Yang Y Wu Jiansheng J Zhang Huanxin H Song Yuanda Y Liu Xiang X Norris James S JS Wang Suriguga S Du Peng P Shen Junguo J Wang Na N Yang Yanlin Y Wang Wei W Feng Lu L Ratledge Colin C Zhang Hao H Chen Yong Q YQ

PloS one 20111208 12

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 m ...[more]

PMID: 22174787

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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.

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.

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.

Dataset Information

Genome characterization of the oleaginous fungus Mortierella alpina.

Publications

Genome characterization of the oleaginous fungus Mortierella alpina.

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