Evaluating the effects of mefenoxam on taxonomic and functional dynamics of nontarget fungal communities during carrot cultivation.

Ridomil Gold SL (45.3% a.i. mefenoxam) is a widely used chemical fungicide for the control of oomycetes. However, its impact on fungal communities remains unexplored. Therefore, the goal of this study was to examine the effects of mefenoxam on the temporal dynamics of fungal taxonomic and functional diversities during carrot cultivation under four treatment groups: mefenoxam application with and without Pythium inoculation, and untreated control groups with and without Pythium inoculation. Our in vitro sensitivity assay showed that the maximum recommended concentration of mefenoxam, 0.24 ppm, did not suppress the mycelial growth of P. irregulare. At 100 ppm, mycelial growth was only reduced by 11.4%, indicating that the isolate was resistant to mefenoxam. MiSeq sequencing data revealed transient taxonomic variations among treatments 2 weeks post-treatment. Mortierella dominated the fungal community in the mefenoxam-Pythium combination treatment, as confirmed through PCR using our newly designed Mortierella-specific primers. Conversely, mefenoxam-Pythium combination had adverse effects on Penicillium, Trichoderma, and Fusarium, and decrease the overall alpha diversity. However, these compositional changes gradually reverted to those observed in the control by the 12th week. The predicted ecological functions of fungal communities in all Pythium and mefenoxam treatments shifted, leading to a decrease in symbiotrophs and plant pathogen functional groups. Moreover, the community-level physiological profiling approach, utilizing 96-well Biolog FF microplates, showed discernible variations in the utilization of 95 diverse carbon sources among the treatments. Notably, arbutin, L-arabinose, Tween 80, and succinamic acid demonstrated a strong positive association with Mortierella. Our findings demonstrate that a single application of mefenoxam at its recommended rate triggers substantial taxonomic and functional shifts in the soil fungal community. Considering this impact, the conventional agricultural practice of repeated mefenoxam application is likely to exert considerable shifts on the soil ecosystem that may affect agricultural sustainability.

Characterization of potassium-solubilizing fungi, Mortierella spp., isolated from a poplar plantation rhizosphere soil.

Potassium-solubilizing microorganisms are capable of secreting acidic chemicals that dissolve and release potassium from soil minerals, thus facilitating potassium uptake by plants. In this study, three potassium-dissolving filamentous fungi were isolated from the rhizosphere soil of a poplar plantation in Jiangsu Province, China. Phylogenetic analyses based on ITS, 18 S, and 28 S showed that these three isolates were most similar to Mortierella. These strains also possessed spherical or ellipsoidal spores, produced sporangia at the hyphal tip, and formed petal-like colonies on PDA media resembling those of Mortierella species. These findings, along with further phenotypic observations, suggest that these isolates were Mortierella species. In addition, the potassium-dissolution experiment showed that strain 2K4 had a relatively high potassium-solubilizing capacity among these isolated fungi. By investigating the influences of different nutrient conditions (carbon source, nitrogen source, and inorganic salt) and initial pH values on the potassium-dissolving ability, the optimal potassium-solubilization conditions of the isolate were determined. When potassium feldspar powder was used as an insoluble potassium source, isolate 2K4 exhibited a significantly better polysaccharide aggregation ability on the formed mycelium-potassium feldspar complex. The composition and content of organic acids secreted by strain 2K4 were further detected, and the potassium-dissolution mechanism of the Mortierella species and its growth promotion effect were discussed, using maize as an example.

Modular Metabolic Engineering of Mortierella alpina by the 2A Peptide Platform to Improve Arachidonic Acid Production.

Arachidonic acid (ARA) is an essential fatty acid in human nutrition. Mortierella alpina, a filamentous fungus, has been widely used for the production of ARA. Here, we report a modular engineering approach that systematically eliminates metabolic bottlenecks in the multigene elongase/desaturase pathway and has led to significant improvements of the ARA titer. The elongase/desaturase pathway in Mortierella alpina was recast into two modules, namely, push and pull modules, based on its function in the ARA synthesis. Combinatorial optimization of these two modules has balanced the production and consumption of intermediate metabolites. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the push and pull modules in Mortierella alpina. In the shake-flask fermentation, the lipid and ARA contents of the engineered strain MA5 were increased by 1.2-fold and 77.6%, respectively, resulting in about fivefold increase of the ARA yield. The final ARA titer reached 4.4 g L-1 in shake-flask fermentation. The modular engineering strategies presented in this study demonstrate a generalized approach for the engineering of cell factories in the production of valuable metabolites.

Flocculation of oleaginous green algae with Mortierella alpina fungi.

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80-85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3-10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.

Overexpression of Citrate-Malate Carrier Promoted Lipid Accumulation in Oleaginous Filamentous Fungus Mortierella alpina.

The mitochondrial citrate-malate carrier is responsible for the transport of citrate and malate between the mitochondria and cytosol, ensuring citrate supply substrate for fatty acid synthesis. In this study, we investigated the overexpression of the citrate-malate carrier coded by three genes (MaCT1/MaCT2/MaTCT) in Mortierella alpina to enhance lipid accumulation. Our results showed that the overexpression of MaCT1, MaCT2, and MaTCT increased the fatty acid content by up to 21.7, 29.5, and 12.8%, respectively, compared with the control strain, but had no effect on the growth. Among them, the MaCT2-overexpressing strain performed the best, and its total fatty acid yield was increased by 51.6% compared to the control. Furthermore, the relative transcription level of MaCT2 indeed increased significantly in the recombinant strains. These findings are beneficial to understanding the citrate transport system and improve the industrial applications of the oleaginous filamentous fungus M. alpina.

Potential of Mortierellaceae for polyunsaturated fatty acids production: mini review.

The health benefits of polyunsaturated fatty acids (PUFAs) have encouraged the search for rich sources of these compounds. However, the supply chain of PUFAs from animals and plants presents environmental concerns, such as water pollution, deforestation, animal exploitation and interference in the trophic chain. In this way, a viable alternative has been found in microbial sources, mainly in single cell oil (SCO) production by yeast and filamentous fungi. Mortierellaceae is a filamentous fungal family world-renowned for PUFA-producing strains. For example, Mortierella alpina can be highlighted due to be industrially applied to produce arachidonic acid (20:4 n6), an important component of infant supplement formulas. Thus, the state of the art of strategies to increase PUFAs production by Mortierellaceae strains is presented in this review. Firstly, we have discussed main phylogenetic and biochemical characteristics of these strains for lipid production. Next, strategies based on physiological manipulation, using different carbon and nitrogen sources, temperature, pH and cultivation methods, which can increase PUFA production by optimizing process parameters are presented. Furthermore, it is possible to use metabolic engineering tools, controlling the supply of NADPH and co-factors, and directing the activity of desaturases and elongase to the target PUFA. Thus, this review aims to discuss the functionality and applicability of each of these strategies, in order to support future research for PUFA production by Mortierellaceae species.

Redistributing Carbon Flux by Impairing Saccharide Synthesis to Enhance Lipid Yield in Oleaginous Fungus Mortierella alpina.

Increasing carbon flux toward target metabolites is important in improving microbial productivity and economic value. To improve the efficiency of lipid production in Mortierella alpina, we knocked down genes for trehalose-6-phosphate synthetase (Matps) and phosphoenolpyruvate carboxykinase (Mapepck) in the major pathways for saccharide synthesis. The knockdown of Matps reduced trehalose content by an average of 31.87%, while the knockdown of Mapepck reduced the total saccharide content by 28.6%, and both recombinant strains showed more than 20% increased lipid yield. Trehalose plays a vital role in stress resistance, but a higher polyunsaturated fatty acid-rich lipid content was found to partly compensate for the loss of trehalose after Matps knockdown. As compared with Matps knockdown, the knockdown of Mapepck gave better lipid production by bringing forward the time to maximum lipid yield by three days in a scale-up test. The arachidonic acid yield after the Mapepck knockdown reached 1.23 g/L, which was 39.9% higher than that of the original strain. The present research provided an efficient strategy for redistributing carbon flux among different metabolites and therefore promoted microbial lipid yield in a shorter fermentation period.

Autophagy Improves ARA-Rich TAG Accumulation in Mortierella alpina by Regulating Resource Allocation.

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.

Lipid metabolism research in oleaginous fungus Mortierella alpina: Current progress and future prospects.

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.

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina.

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.

SNF1ß-Modulated Glucose Uptake and the Balance between Polyunsaturated Fatty Acids and Carbohydrates in Mortierella alpina.

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.

Molecular characterization and genetic diversity of four undescribed novel oleaginous Mortierella alpina strains from Libya.

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.

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina.

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.

Advances in improving the biotechnological application of oleaginous fungus Mortierella alpina.

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.

Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina.

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.

Role of the mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina.

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.

Characterization of ω3 fatty acid desaturases from oomycetes and their application toward eicosapentaenoic acid production in Mortierella alpina.

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.

Research advances on arachidonic acid production by fermentation and genetic modification of Mortierella alpina.

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.

Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina.

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.

Improved Lipogenesis in Mortierella alpina by Abolishing the Snf4-Mediated Energy-Saving Mode under Low Glucose.

Sensing nutrient levels and coordinating metabolism are requisites for all living organisms. In eukaryotes, heterotrimeric adenosine monophosphate-activated protein kinase/sucrose nonfermenting 1 (SNF1) is an energy monitor that primarily functions by regulating cell metabolism with its γ-subunit being responsible for energy sensing. Because of its strong lipogenesis capacity and dependence on nutrient availability, Mortierella alpina is an ideal model to investigate the SNF1 role. Knockdown of the M. alpina SNF1-γ-subunit (MaSnf4) abolished the energy preservation mode. In a low glucose medium (15 g/L), the fatty acid content in the MaSnf4-knockdown strain was similar to that in a high glucose medium (50 g/L), comprising 16 ± 1.17% of the dry cell weight after 96 h of culture (1.59 g/L), together with 1.41 ± 0.13 and 4.15 ± 0.19 fold increased acetyl-CoA carboxylase 1 and ATP-citrate lyase enzymatic activities, respectively. Metabolite analysis confirmed that knocking down MaSnf4 enhanced amino acid recycling and repressed the tricarboxylic acid cycle. In this case, more carbon skeleton acetyl-CoA and reductive nicotinamide adenine dinucleotide phosphate were rerouted into the fatty acid synthesis pathway. These findings provide new insight into the correlation between energy preservation and MaSnf4-regulated lipogenesis, which may enhance further development of cost-effective strategies to enhance lipid productivity in M. alpina.