Regulation of carbon metabolic fluxes to enhance lipid and succinate production in oleaginous fungus Mortierella alpina.

Mortierella alpina is popular for lipid production, but the low carbon conversion rate and lipid yield are major obstacles for its economic performance. Here, external addition of organic acids involved in tricarboxylic acid cycle was used to tune carbon flux and improve lipid production. Citrate was determined to be the best organic acid that can be used for enhancing lipid production. By the addition of citrate, the lipid titer and content were approximately 1.24 and 1.34 times higher, respectively. Meanwhile, citrate supplement also promoted the accumulation of succinate, an important value-added platform chemical. Owing to the improved lipid and succinate production through adding citrate, the carbon conversion rate of M. alpina reached up to 52.17%, much higher than that of the control group (14.11%). The addition of citrate could redistribute carbon flux by regulating the expression level of genes related to tricarboxylic acid cycle metabolism. More carbon fluxes flow to lipid and succinate synthesis, which greatly improved the carbon conversion efficiency of M. alpina. This study provides an effective and straightforward strategy with potential economic benefits to improve carbon conversion efficiency in M. alpina.

Identification and Biological Characteristics of Mortierella alpina Associated with Chinese Flowering Cherry (Cerasus serrulata) Leaf Blight in China.

The Chinese flowering cherry (Cerasus serrulata), an ornamental tree with established medicinal values, is observed to suffer from leaf blight within Xi'an's greenbelts. This disease threatens both the plant's growth and its ornamental appeal. In this study, 26 isolates were obtained from plants with typical leaf blight, and only 3 isolates (XA-10, XA-15, and XA-18) were found to be pathogenic, causing similar symptoms on the leaves of the host plant. Based on sequence alignment, the ITS and LSU sequences of the three selected isolates were consistent, respectively. Following morphological and molecular analyses, the three selected isolates were further identified as Mortierella alpina. The three selected isolates exhibited similar morphological characteristics, including wavy colonies with dense, milky-white aerial mycelia on PDA medium. Therefore, isolate XA-10 was used as a representative strain for subsequent experiments. The representative strain XA-10 was found to exhibit optimal growth at a temperature of 30 °C and a pH of 7.0. Host range infection tests further revealed that the representative strain XA-10 could also inflict comparable disease symptoms on both the leaves and fruits of three different Rosaceae species (Prunus persica, Pyrus bretschneideri, and Prunus salicina). This study reveals, for the first time, the causative agent of leaf blight disease affecting the Chinese flowering cherry. This provides a deeper understanding of the biology and etiology of M. alpina. This study lays a solid foundation for the sustainable control and management of leaf blight disease in the Chinese flowering cherry.

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.

Mead acid production by disruption of Δ12-desaturase gene in Mortierella alpina 1S-4.

Mead acid (MA; 20:3ω9) is one of the ω9 series of polyunsaturated fatty acids (PUFAs). MA is used to inhibit the inflammation of joints and is applied to the medicinal or health food field. We aimed to construct MA-producing strains with disruption of the Δ12-desaturase gene (Δ12ds) via an efficient gene-targeting system using the lig4-disrupted strain of Mortierella alpina 1S-4 as the host. The transformants showed a unique fatty acid composition that only comprised ω9-PUFAs and saturated fatty acids, while ω6-and ω3-PUFAs were not detected, and the total composition of ω9-PUFAs, including oleic acid (18:1ω9), 18:2ω9, 20:1ω9, 20:2ω9, and MA, was up to 68.4% of the total fatty acids. The MA production in the Δ12ds-disruptant reached 0.10 g/L (8.5%), which exceeded 0.050 g/L (4.6%) in the conventional Δ12ds-defective mutant JT-180.

Functions and substrate selectivity of diacylglycerol acyltransferases from Mortierella alpina.

Mortierella alpina produces various polyunsaturated fatty acids in the form of triacylglycerols (TAG). Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form TAG and is the key enzyme involved in TAG synthesis. A variety of DGATs are present in M. alpina; however, comparative analysis of the functional properties and substrate selectivity of these DGATs is insufficient. In this study, DGAT1 (MaDGAT1A/1B/1C) and DGAT2 (MaDGAT2A/2B) isoforms from M. alpina were analyzed and heterologously expressed in S. cerevisiae H1246. The results showed that MaDGAT1A/1B/2A/2B were able to restore TAG synthesis, and the corresponding TAG content in recombinant yeasts was 2.92 ± 0.42%, 3.62 ± 0.22%, 0.86 ± 0.34%, and 0.18 ± 0.09%, respectively. In S. cerevisiae H1246, MaDGAT1A preferred C16:1 among monounsaturated fatty acids, MaDGAT1B preferred C16:0 among saturated fatty acids (SFAs), and MaDGAT2A/2B preferred C18:0 among SFAs. Under exogenous addition of polyunsaturated fatty acids (PUFAs), MaDGAT1A and 2A preferentially assembled linoleic acid into TAG, and MaDGAT2B had substrate selectivity for eicosapentaenoic and linoleic acids in ω-6 PUFAs. In vitro, MaDGAT1A showed no obvious acyl-CoA selectivity and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred C18:1/C18:1-DAG compared with C20:4/C20:4-DAG. This study indicates that MaDGATs have the potential to be used in the production of LA/EPA-rich TAG and provide a reference for improving the production of TAGs in oleaginous fungi. KEY POINTS: • MaDGAT1A preferred C16:1 among MUFAs, MaDGAT1B and MaDGAT2A/2B preferred C16:0 and C18:0 among SFAs, respectively • MaDGAT1A/2A preferentially assembled linoleic acid into TAG, and MaDGAT2B has substrate selectivity for eicosapentaenoic acid and linoleic acid in ω-6 PUFAs • MaDGAT1A showed no obvious acyl-CoA selectivity, and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred to select C18:1/C18:1-DAG compared with C20:4/C20:4-DAG.

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.

Applications of Diacylglycerol Acyltransferase for Triacylglycerol Production in Mortierella alpina.

Triacylglycerol (TG) with high-value long-chain polyunsaturated fatty acids is beneficial to human health; consequently, there is an urgent need to broaden its sources due to the current growing demand. Mortierella alpina, one of the most representative oleaginous fungi, is the only certificated source of dietary arachidonic acid-rich oil supplied in infant formula. This study was conducted to improve TG production in M. alpina by homologous overexpression of diacylglycerol acyltransferase (DGAT) and linseed oil (LSO) supplementation. Our results showed that the homologous overexpression of MaDGAT1B and MaDGAT2A strengthened TG biosynthesis and significantly increased the TG content compared to the wild-type by 12.24% and 14.63%, respectively. The supplementation with an LSO concentration of 0.5 g/L elevated the TG content to 83.74% and total lipid yield to 4.26 ± 0.38 g/L in the M. alpina-MaDGAT2A overexpression strain. Our findings provide an effective strategy for enhancing TG production and highlight the role of DGAT in TG biosynthesis in M. alpina.

NADPH-Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina.

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH-cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH-cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min-1 mg-1 and 10.0 ± 0.5 nmol min-1 mg-1, respectively. The highest kcat/Km value of ω3 and ω6 desaturase was 0.41 µM-1 min-1 and 0.09 µM-1 min-1 when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH-cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.

Regulating Root Fungal Community Using Mortierella alpina for Fusarium oxysporum Resistance in Panax ginseng.

Plant-associated microbes play important roles in plant health and disease. Mortierella is often found in the plant rhizosphere, and its possible functions are not well known, especially in medical plants. Mortierella alpina isolated from ginseng soil was used to investigate its effects on plant disease. The promoting properties and interactions with rhizospheric microorganisms were investigated in a medium. Further, a pot experiment was conducted to explore its effects on ginseng root rot disease. Physicochemical properties, high-throughput sequencing, network co-occurrence, distance-based redundancy analysis (db-RDA), and correlation analysis were used to evaluate their effects on the root rot pathogen. The results showed that Mortierella alpina YW25 had a high indoleacetic acid production capacity, and the maximum yield was 141.37 mg/L at 4 days. The growth of M. alpina YW25 was inhibited by some probiotics (Bacillus, Streptomyces, Brevibacterium, Trichoderma, etc.) and potential pathogens (Cladosporium, Aspergillus, etc.), but it did not show sensitivity to the soil-borne pathogen Fusarium oxysporum. Pot experiments showed that M. alpina could significantly alleviate the diseases caused by F. oxysporum, and increased the available nitrogen and phosphorus content in rhizosphere soil. In addition, it enhanced the activities of soil sucrase and acid phosphatase. High-throughput results showed that the inoculation of M. alpina with F. oxysporum changed the microbial community structure of ginseng, stimulated the plant to recruit more plant growth-promoting bacteria, and constructed a more stable microbial network of ginseng root. In this study, we found and proved the potential of M. alpina as a biocontrol agent against F. oxysporum, providing a new idea for controlling soil-borne diseases of ginseng by regulating rhizosphere microorganisms.

Investigation on Cell Disruption Techniques and Supercritical Carbon Dioxide Extraction of Mortierella alpina Lipid.

Mortierella alpina, an oleaginous fungus, has been shown to be a potential source for arachidonic acid (ARA) production. The recovery of intracellular lipids from M. alpina is an important step for the downstream bioprocessing, and green extraction techniques with a focus on being efficient and eco-friendly have drawn much attention. In this study, different cell disruption techniques (mechanical: high-speed homogenization 10,000 rpm, ultrasonication 20 kHz, high-pressure process (HPP) 200-600 MPa; non- mechanical: acid treatment HCl) were investigated for lipid recovery from M. alpina, and process parameters (A. temperature, B. pressure, C. cosolvent ratio) of supercritical carbon dioxide (SC-CO2) lipid extraction were studied by applying response surface methodology (RSM). Compared with Soxhlet extraction as a control group (100%), high-speed homogenization has the highest lipid recovery (115.40%) among mechanical disruption techniques. Besides, there was no significant difference between high-speed homogenization and 1 M HCl treatment (115.55%) in lipid recovery. However, lipid recovery decreased to 107.36% as the concentration of acid was increased to 3 M, and acid treatment showed a negative effect on the ARA ratio. In HPP treatment, the highest lipid recovery (104.81%) was obtained at 400 MPa, 1 time of treatment and water medium. In the response surface model of SC-CO2 extraction, results showed the major influence of the process parameters to lipid recovery was pressure, and there are interaction effects of AC (temperature and cosolvent ratio) and BC (pressure and cosolvent ratio). Lipid recovery of SC-CO2 extraction reached 92.86% at 201 bar, 58.9 °C and cosolvent ratio 1:15. The microbial lipid recovery process of this study could be used as a reference and an eco-friendly alternative for the future downstream bioprocessing of ARA production by M. alpina.

Statistical optimization of arachidonic acid synthesis by Mortierella alpina CBS 754.68 in a solid-state fermenter.

Arachidonic acid (ARA) is an omega-6 fatty acid that plays a major role in human health. The present study optimizes the production of ARA by the soil fungus Mortierella alpina CBS 754.68 on oil cakes. In the first step, the best substrate was chosen from four oil cakes, namely soybean, sunflower, olive, and colza oil cakes, of which sunflower oil cake showed the highest yield. In the next step, screening tests were performed using the Plackett-Burman design. Seven variables (substrate particle size, moisture content, time, temperature, yeast extract, glucose, and glutamate) were investigated (each taking values of +1 and -1). Among these variables, time, temperature, and substrate particle size significantly affected ARA production (p < .05), so they were further investigated in the optimization step. The optimal fermentation time, temperature, and substrate particle size calculated by response surface methodology were 8.75 days, 18.5°C, and 1.3 mm-1.7 mm, respectively. Under these conditions, M. alpina was predicted to produce 4.19 mg of ARA/g dry weight of substrate (DWS). The actual yield, determined in evaluation tests, was 4.48 ± 0.16 mg ARA/g DWS, which shows the accuracy of the model. In the final step, the effect of the aeration rate on producing ARA was investigated in a packed-bed solid-state fermenter under the determined optimal conditions. In this stage, the highest ARA yield was 10.13 ± 0.26 mg/g DWS, approximately double that of the optimization step, and this confirms that aeration increases ARA production by M. alpina.

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.

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.

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.

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.

Characterization of NAD+/NADP+-Specific Isocitrate Dehydrogenases From Oleaginous Fungus Mortierella alpina Involved in Lipid Accumulation.

Mortierella alpina has a strong capacity for lipid accumulation. Isocitrate dehydrogenase (IDH) plays an important role in affecting the flow of intracellular carbon sources and reducing power NADPH for lipid biosynthesis. In this study, the effect of various IDHs (NAD+- and NADP+-specific) in M. alpina on the lipid accumulation was investigated through homologous overexpression. The results showed that the transcription level and enzyme activity of the IDHs from M. alpina (MaIDHs) in homologous overexpressing strains were higher than those of the control strain, but that their biomass was not significantly different. Among the various NAD+-specific MaIDH1/2/3 overexpression, NAD+-MaIDH3 reduced total lipid content by 12.5%, whereas overexpression NAD+-MaIDH1 and NAD+-MaIDH2 had no effect on fatty acid content. Intracellular metabolites analysis indicated that the overexpression NAD+-MaIDH3 strain had reduced the fatty acid accumulation, due to its greater carbon flux with the tricarboxylic acid cycle and less carbon flux with fatty acid biosynthesis. For the NADP+-MaIDH4/5/6 recombinant strains overexpressing only NADP+-MaIDH4 enhanced the total fatty acid content by 8.2%. NADPH analysis suggested that this increase in lipid accumulation may have been due to the great reducing power NADPH is produced in this recombinant strain. This study provides theoretical basis and guidance for the analysis of the mechanism of IDH function and the potential to improve lipid production in M. alpina.

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.