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Role of the phenylalanine-hydroxylating system in aromatic substance degradation and lipid metabolism in the oleaginous fungus Mortierella alpina.

ABSTRACT: 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.

SUBMITTER: Wang H

PROVIDER: S-EPMC3685260 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Role of the phenylalanine-hydroxylating system in aromatic substance degradation and lipid metabolism in the oleaginous fungus Mortierella alpina.

Wang Hongchao H Chen Haiqin H Hao Guangfei G Yang Bo B Feng Yun Y Wang Yu Y Feng Lu L Zhao Jianxin J Song Yuanda Y Zhang Hao H Chen Yong Q YQ Wang Lei L Chen Wei W

Applied and environmental microbiology 20130315 10

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 bios ...[more]

PMID: 23503309

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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.

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Dataset Information

Role of the phenylalanine-hydroxylating system in aromatic substance degradation and lipid metabolism in the oleaginous fungus Mortierella alpina.

Publications

Role of the phenylalanine-hydroxylating system in aromatic substance degradation and lipid metabolism in the oleaginous fungus Mortierella alpina.

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