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.

Rhizosphere Mortierella strain of alfalfa exerted weed growth inhibition by inducing expression of plant hormone-related genes.

Introduction: Weeds are significant factors that detrimentally affect crop health and hinder optimal herbage yield. Rhizosphere microorganisms play crucial roles in plant growth, development, and nutrient uptake. Therefore, research focusing on weed control through the lens of microorganisms has emerged as a prominent area of study. The oil-producing fungus Mortierella, which is known for its numerous agricultural benefits, has garnered significant attention in recent years. Methods: In this study, we conducted inoculation experiments in a controlled artificial culture climate chamber to investigate the effects of differential hormones and differentially expressed genes in the stems and leaves of Digitaria sanguinalis using Liquid Chromatography Tandem Mass Spectrometry and RNA-seq techniques, respectively. Additionally, Pearson's correlation analysis was used to establish correlations between differential hormones and growth indicators of Digitaria sanguinalis. Results and discussion: The results demonstrated that inoculation with Mortierella sp. MXBP304 effectively suppressed aboveground biomass and plant height in Digitaria sanguinalis. Furthermore, there was significant upregulation and downregulation in the expression of genes involved in the synthesis and metabolism of phenylalanine and L-phenylalanine. Conversely, the expression of genes related to tryptophan, L-tryptophan, and indole was significantly downregulated. The addition of Mortierella sp. MXBP304 can influence the gene expression associated with phenylalanine and tryptophan synthesis and metabolism during Digitaria sanguinalis growth, subsequently reducing the relative contents of phenylalanine and tryptophan, thereby directly inhibiting Digitaria sanguinalis growth.

Interrelationship among substrate utilization, metabolic productions, and housekeeping-related gene expression levels in Mortierella alpine CBS 754.68.

The impacts of Magnesium oxide nanoparticles (MgONPs) on the expression of 10 potential housekeeping genes of Mortierella alpine were assayed. Actin emerged as the good candidate when Mortierella alpine entered the death phase subsequent to the growth phase while Dihydropteridine reductase and 28 s were identified as suitable candidates when Mortierella alpine remained in the growth phase.

Gypsum Cave Biofilm Communities are Strongly Influenced by Bat- And Arthropod-Related Fungi.

The Gypsum Karst of Sorbas, Almeria, southeast Spain, includes a few caves whose entrances are open and allow the entry and roosting of numerous bats. Caves are characterized by their diversity of gypsum speleothems, such as stalactites, coralloids, gypsum crusts, etc. Colored biofilms can be observed on the walls of most caves, among which the Covadura and C3 caves were studied. The objective was to determine the influence that bat mycobiomes may have on the fungal communities of biofilms. The results indicate that the fungi retrieved from white and yellow biofilms in Covadura Cave (Ascomycota, Mortierellomycota, Basidiomycota) showed a wide diversity, depending on their location, and were highly influenced by the bat population, the guano and the arthropods that thrive in the guano, while C3 Cave was more strongly influenced by soil- and arthropod-related fungi (Ascomycota, Mortierellomycota), due to the absence of roosting bats.

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.

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.

Deciphering Biotic and Abiotic Mechanisms Underlying Straw Decomposition and Soil Organic Carbon Priming in Agriculture Soils Receiving Long-Term Fertilizers.

Straw-related carbon (C) dynamics are central for C accrual in agro-ecosystems and should be assessed by investigating their decomposition and soil organic carbon (SOC) priming effects. Our understanding of biotic and abiotic mechanisms underpinning these two C processes, however, is still not sufficiently profound. Soils that had received organic and mineral fertilizers for 26 years were sampled for a 28 day incubation experiment to assess 13C-labeled straw decomposition and SOC priming effects. On the basis of analyzing physicochemical properties, fungal taxonomic (MiSeq sequencing) and functional (metagenomics) guilds, we quantified the contributions of biotic and abiotic attributes to straw decomposition and SOC priming. Here, we propose two distinct mechanisms underlying straw decomposition and SOC priming in agriculture soils: (i) accelerated straw mineralization in manure-treated soils was mainly driven by biotic forces, while (ii) larger SOC priming in NPK-amended soils was through abiotic regulation.

The Beneficial Fungus Mortierella hyalina Modulates Amino Acid Homeostasis in Arabidopsis under Nitrogen Starvation.

Non-mycorrhizal but beneficial fungi often mitigate (a)biotic stress-related traits in host plants. The underlying molecular mechanisms are mostly still unknown, as in the interaction between the endophytic growth-promoting soil fungus Mortierella hyalina and Arabidopsis thaliana. Here, abiotic stress in the form of nitrogen (N) deficiency was used to investigate the effects of the fungus on colonized plants. In particular, the hypothesis was investigated that fungal infection could influence N deficiency via an interaction with the high-affinity nitrate transporter NRT2.4, which is induced by N deficiency. For this purpose, Arabidopsis wild-type nrt2.4 knock-out and NRT2.4 reporter lines were grown on media with different nitrate concentrations with or without M. hyalina colonization. We used chemical analysis methods to determine the amino acids and phytohormones. Experimental evidence suggests that the fungus does not modulate NRT2.4 expression under N starvation. Instead, M. hyalina alleviates N starvation in other ways: The fungus supplies nitrogen (15N) to the N-starved plant. The presence of the fungus restores the plants' amino acid homeostasis, which was out of balance due to N deficiency, and causes a strong accumulation of branched-chain amino acids. We conclude that the plant does not need to invest in defense and resources for growth are maintained, which in turn benefits the fungus, suggesting that this interaction should be considered a mutualistic symbiosis.

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.

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.

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.

Biocatalytic Production of Odor-Active Fatty Aldehydes from Fungal Lipids.

Odor-active fatty aldehydes are important compounds for the flavor and fragrance industry. By a coupled enzymatic reaction using an α-dioxygenase (α-DOX) and an aldehyde dehydrogenase (FALDH), scarcely available aldehydes from the biotransformation of margaroleic acid [17:1(9Z)] were characterized and have shown highly interesting odor profiles, including citrus-like, soapy, herbaceous, and savory notes. In particular, (Z)-8-hexadecenal and (Z)-7-pentadecenal exhibited notable meaty odor characteristics. Submerged cultivation of Mortierella hyalina revealed the accumulation of the above-mentioned, naturally uncommon fatty acid 17:1(9Z). Its production was significantly increased by the modulation of culture conditions, whereas the highest accumulation was observed after 4 days at 24 °C and l-isoleucine supplementation. The lipase-, α-DOX-, and FALDH-mediated biotransformation of M. hyalina lipid extract resulted in a complex aldehyde mixture with a high aldehyde yield of ∼50%. The odor qualities of the formed aldehydes were assessed by means of gas chromatography-olfactometry, and several of the obtained fatty aldehydes have been sensorially described for the first time. To assess the aldehyde mixture's potential as a flavor ingredient, a sensory evaluation was conducted. The obtained product exhibited intense citrus-like, green, and soapy odor impressions.

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.

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.

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.

Bioremoval of copper by filamentous fungi isolated from contaminated soils of Puchuncaví-Ventanas Central Chile.

Pollution represents a high risk to plants, animals, and human beings, causing an imbalance and affecting the environment. Soil is considered a universal sink, containing the highest load of environmental pollution. Puchuncaví-Ventanas sector, decreed as a saturated contamination zone in 1993, is considered one of the most affected areas by industrial pollution and belongs to one of the 5 sacrifice zones of Chile. The localities of Puchuncaví and Ventanas have heavy metal pollution levels that exceed up to 99% of the limits allowed by Canadian standards. The objective of this study was to characterize heavy metal tolerance and removal potential of filamentous fungi isolated from polluted soils for their use in decontamination systems and in situ soil improvement. Six fungal strains were selected based on their tolerance and a high capability to accumulate heavy metals, achieving copper bioaccumulation of 84% (Mortierella sp. strain LG01), 49% (Clonostachys sp. strain CQ23) and 48-77.5% (Trichoderma sp. strain LM01A). Trichoderma sp. strain LM01A was able to remove 41% of copper from contaminated soil under ex situ conditions. Some fungal strains belong to beneficial fungal genera, which are used as bioproducts in agriculture. The results of this study highlighted the use of Trichoderma sp. in soils contaminated, which may be of special interest in agriculture due to the large amounts of copper sulfate still applied as a pesticide in Chile and the world.

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.

Phosphorus-Solubilizing Capacity of Mortierella Species Isolated from Rhizosphere Soil of a Poplar Plantation.

Phosphorus is one of the main nutrients necessary for plant growth and development. Phosphorus-dissolving microorganisms may convert insoluble phosphorus in soil into available phosphorus that plants can easily absorb and utilize. In this study, four phosphorus-solubilizing fungi (L3, L4, L5, and L12) were isolated from the rhizosphere soil of a poplar plantation in Dongtai, Jiangsu Province, China. Phylogenetic analysis based on the internal transcribed spacer (ITS) and large subunit (LSU) of the ribosomal DNA sequences showed that the ITS and 28S sequences of isolates were the most similar to those of Mortierella. Morphological observation showed that most colonies grew in concentric circles and produced spores under different culture conditions. These results and further microscopic observations showed that these isolated fungi belonged to the genus Mortierella. Pikovskaya (PKO) medium, in which tricalcium phosphate was the sole phosphorus source, was used to screen strain L4 with the best phosphorus-solubilizing effect for further study. When the carbon source was glucose, the nitrogen source was ammonium chloride, the pH was 5, and the available phosphorus content was the highest. By exploring the possible mechanism of phosphorus release by phosphorus-solubilizing fungi, it was found that strain L4 produces several organic acids, such as oxalic acid, lactic acid, acetic acid, succinic acid, tartaric acid, malic acid, and citric acid. At 24 h, the alkaline phosphatase and acid phosphatase activities reached 154.72 mol/(L·h) and 120.99 mol/(L·h), respectively.

Green synthesis of biogenetic Te(0) nanoparticles by high tellurite tolerance fungus Mortierella sp. AB1 with antibacterial activity.

Tellurite [Te(IV)] is a high-toxicity metalloid. In this study, a fungus with high Te(IV) resistance was isolated. Strain AB1 could efficiently reduce highly toxic Te(IV) to less toxic Te(0). The reduced products formed rod-shaped biogenetic Te(0) nanoparticles (Bio-TeNPs) intracellularly. Further TEM-element mapping, FTIR, and XPS analysis showed that the extracted Bio-TeNPs ranged from 100 to 500 nm and consisted of Te(0), proteins, lipids, aromatic compounds, and carbohydrates. Moreover, Bio-TeNPs exhibited excellent antibacterial ability against Shigella dysenteriae, Escherichia coli, Enterobacter sakazakii, and Salmonella typhimurium according to inhibition zone tests. Further growth and live/dead staining experiments showed that E. coli and S. typhimurium were significantly inhibited by Bio-TeNPs, and cells were broken or shriveled after treatment with Bio-TeNPs based on SEM observation. Additionally, the antioxidant and cytotoxicity tests showed that the Bio-TeNPs exhibited excellent antioxidant capacity with no cytotoxicity. All these results suggested that strain AB1 showed great potential in bioremediation and Bio-TeNPs were excellent antibacterial nanomaterials with no cytotoxicity.

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.