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Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina.

ABSTRACT: 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.

SUBMITTER: Hao G

PROVIDER: S-EPMC3993310 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina.

Hao Guangfei G Chen Haiqin H Wang Lei L Gu Zhennan Z Song Yuanda Y Zhang Hao H Chen Wei W Chen Yong Q YQ

Applied and environmental microbiology 20140214 9

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 incre ...[more]

PMID: 24532075

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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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Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina.

Publications

Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina.

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