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.

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.

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.

Determination of allosteric and active sites responsible for catalytic activity of delta 12 fatty acid desaturase from Geotrichum candidum and Mortierella alpina by domain swapping.

Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

Ultra Performance Liquid Chromatography-Q Exactive Orbitrap/Mass Spectrometry-Based Lipidomics Reveals the Influence of Nitrogen Sources on Lipid Biosynthesis of Mortierella alpina.

The objective of the present study was to reveal the effects of four types of nitrogen sources (soymeal, yeast extract, KNO3, and ammonium tartrate) on the lipid metabolism of the oleaginous fungus Mortierella alpina using untargeted lipidomics, targeted fatty acid, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Our results showed clear differences in the contents and compositions of lipids between four types of nitrogen sources. Soymeal and ammonium tartrate supplementation favored the accumulation of triglycerides with arachidonic acid (ARA) and C16-18 fatty acids, respectively. These results were further validated by our targeted fatty acid analysis. RT-qPCR analysis of related genes in M. alpina between the four nitrogen source conditions found that soymeal supplementation dramatically increased the expression of GPAT, ELOVL, and Δ12/Δ6 desaturase. Our findings provided new insights into the regulation of lipid biosynthesis in M. alpina and potential avenues for genetic manipulation and highlighted the importance of an optimal nitrogen source for ARA-rich oil production.

Role of Adenosine Monophosphate Deaminase during Fatty Acid Accumulation in Oleaginous Fungus Mortierella alpina.

In oleaginous micro-organisms, nitrogen limitation activates adenosine monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of isocitrate dehydrogenase. We found that the overexpression of homologous AMPD in Mortierella alpina favored lipid synthesis over cell growth. Total fatty acid content in the recombinant strain was 15.0-34.3% higher than that in the control, even though their biomass was similar. During the early fermentation stage, the intracellular AMP level reduced by 40-60%, together with a 1.9-2.7-fold increase in citrate content compared with the control, therefore provided more precursors for fatty acid synthesis. Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by the blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid synthesis pathways. By coupling the energy balance with lipogenesis, this study provides new insights into cell metabolism under nitrogen-limited conditions and targets the regulation of fatty acid accumulation in oleaginous micro-organisms.

Structural Determinants of Substrate Specificity of Omega-3 Desaturases from Mortierella alpina and Rhizophagus irregularis by Domain-Swapping and Molecular Docking.

Although various ω-3 fatty acid desaturases (ω3Des) have been identified and well-studied regarding substrate preference and regiospecificity, the molecular mechanism of their substrate specificities remains to be investigated. Here we compared two ω3Des, FADS15 from Mortierella alpina and oRiFADS17 from Rhizophagus irregularis, which possessed a substrate preference for linoleic acid and arachidonic acid, respectively. Their sequences were divided into six sections and a domain-swapping strategy was used to test the role of each section in catalytic activity. Heterologous expression and fatty acid experiments of hybrid enzymes in Saccharomyces cerevisiae INVSc1 indicated that the sequences between his-boxes I and II played critical roles in influencing substrate preference. Based on site-directed mutagenesis and molecular docking, the amino acid substitutions W129T and T144W, located in the upper part of the hydrocarbon chain, were found to be involved in substrate specificity, while V137T and V152T were confirmed to interfere with substrate recognition. This study provides significant insight into the structure-function relationship of ω3Des.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

Molecular mechanism of substrate preference for ω-3 fatty acid desaturase from Mortierella alpina by mutational analysis and molecular docking.

The ω-3 fatty acid desaturase (ω3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.

A novel malic enzyme gene, Mime2, from Mortierella isabellina M6-22 contributes to lipid accumulation.

OBJECTIVE: This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation. METHODS: Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as Km and Vmax for NADP+ were determined. The effects of EDTA or metal ions (Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Zn2+) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively. RESULTS: The act ivity of MIME2 was significantly increased by Mg2+, Ca2+, or Mn2+ at 0.5 mM but inhibited by Cu2+ or Zn2+ (p < 0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the Km and Vmax for NADP+ are 0.703 mM and 156.25 µg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15 ± 0.24 vs. 2.17 ± 0.31 g/L, p < 0.01). CONCLUSIONS: The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

An Organic Solvent-Tolerant Lipase with Both Hydrolytic and Synthetic Activities from the Oleaginous Fungus Mortierella echinosphaera.

Lipase enzymes of the oleaginous fungal group Mortierella are rarely studied. However, considering that most commercial lipases are derived from filamentous fungal sources, their investigation can contribute to the cost-effective development of new biotechnological processes. Here, an extracellular lipase with a molecular mass of 30 kDa was isolated from Mortierella echinosphaera CBS 575.75 and characterized. The purified lipase exhibited an optimal p-nitrophenyl palmitate (pNPP)-hydrolyzing activity at 25 °C and pH 6.6-7.0 and proved to be highly stable at temperatures up to 40 °C and under broad pH conditions. The enzyme was active under low temperatures, retaining 32.5% of its activity at 10 °C, and was significantly stable in polar and non-polar organic solvents. The Km, Vmax, and kcat for pNPP were 0.336 mM, 30.4 µM/min, and 45.7 1/min for pNPP and 0.333 mM, 36.9 µM/min, and 55.6 1/min for pNP-decanoate, respectively. The pNPP hydrolysis was inhibited by Hg2+, N-bromosuccinimide, and sodium dodecyl sulfate, while ethylenediaminetetraacetic acid and metal ions, such as Ca2+, Mg2+, Na⁺, and K⁺ enhanced the activity. The purified lipase had non-regioselective activity and wide substrate specificity, showing a clear preference for medium-chained p-nitrophenyl esters. Besides its good transesterification activity, the enzyme appeared as a suitable biocatalyst to operate selective esterification reactions to long-chained alkyl esters. Adsorption to Accurel MP1000 improved the storage stability of the enzyme at 5 °C. The immobilized lipase displayed tolerance to a non-aqueous environment and was reusable for up to five cycles without significant loss in its synthetic and hydrolytic activities. These findings confirm the applicability of both the free and the immobilized enzyme preparations in future research.

Substrate specificity and membrane topologies of the iron-containing ω3 and ω6 desaturases from Mortierella alpina.

Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ω6 and ω3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl-coenzyme A desaturase-1 (SCD1) and the related sphingolipid α-hydroxylase (Scs7p) that act upon different substrates.

Diacylglycerol acyltransferase 2 of Mortierella alpina with specificity on long-chain polyunsaturated fatty acids: A potential tool for reconstituting lipids with nutritional value.

Based on available genome sequences and bioinformatics tools, we searched for an uncharacterized open reading frame of Mortierella alpina (MaDGAT2) using diacylglycerol acyltransferase sequence (fungal DGAT type 2B) as a query. Functional characterization of the identified native and codon-optimized M. alpina genes were then performed by heterologous expression in Saccharomyces cerevisiae strain defective in synthesis of neutral lipid (NL). Lipid analysis of the yeast tranformant carrying MaDGAT2 showed that the NL biosynthesis and lipid particle formation were restored by the gene complementation. Substrate specificity study of the fungal enzyme by fatty acid supplementation in the transformant cultures showed that it had a broad specificity on saturated and unsaturated fatty acid substrates for esterification into triacylglycerol (TAG). The n-6 polyunsaturated fatty acids (PUFAs) with 18 and 20 carbon atoms, including linoleic acid, γ-linolenic acid, dihomo γ-linolenic and arachidonic acid could be incorporated into TAG fraction in the yeast cells. Interestingly, among n-3 PUFAs tested, the MaDGAT2 enzyme preferred eicosapentaenoic acid (EPA) substrate as its highly proportional constituent found in TAG fraction. This study provides a potential genetic tool for reconstituting oils rich in long-chain PUFAs with nutritional value.

Identification and characterization of a novel diacylglycerol acyltransferase gene from Mortierella alpina.

OBJECTIVES: To clone and express a diacylglycerol acyltransferase (DGAT) gene from Mortierella alpina in Saccharomyces cerevisiae and characterize oil production and fatty acid composition of the resulting recombinant RESULTS: A new, full-length cDNA, putatively encoding a DGAT, was cloned from M. alpina. We subsequently cloned the gene, except the transmembrane-encoding region, termed MaDGAT, its molecular mass was 31.3 kDa. MaDGAT shares 75% identity with a DGAT from Mortierella verticillata NRRL 6337. A recombinant vector expressing MaDGAT, pYES2-DGAT, was constructed and transformed into S. cerevisiae H1246, a neutral, lipid-deficient quadruple mutant. TLC analysis showed that the recombinant vector restored triacylglycerol biosynthesis and its content in the recombinant strain was 3.9%. CONCLUSION: MaDGAT is a novel DGAT gene and could increase TAG biosynthesis in M. alpina or other filamentous fungi, thereby promoting the synthesis of polyunsaturated fatty acids.

Differential expression of desaturase genes and changes in fatty acid composition of Mortierella sp. AGED in response to environmental factors.

BACKGROUND: Some oleaginous fungi can produce large amounts of polyunsaturated fatty acids (PUFAs) which serve many physiological functions. Numerous desaturases are critical for the synthesis of PUFAs. This study aimed to investigate the regulation of lipid production and desaturase gene expression in Mortierella sp. AGED in response to different environmental factors, and the relationships between lipid production and desaturase gene expression. RESULTS: The fatty acid composition and mRNA levels of desaturase genes were significantly changed under low temperatures. With the exception of Δ5-desaturase, the transcript levels of all desaturase genes increased at a temperature of 20 °C. Changes in content of lipid and PUFAs responding to low temperature were consistent with desaturase gene expression. Time course studies on gene expression showed that mRNA levels of four desaturase genes increased rapidly after transferring the cells to low temperature. Ethanol (1.5% v/v) increased the transcript levels of Δ9-, Δ6- and Δ5-desaturase genes significantly and of Δ12-desaturase gene slightly. Different metal ions such as Ca2+ , Zn2+ and Fe3+ could stimulate PUFA synthesis and up-regulate desaturase gene transcription, while Cu2+ inhibited desaturase gene expression and lipid accumulation. CONCLUSION: This study should enable us to understand the regulatory mechanism of desaturase gene expression and lipid synthesis. It is helpful to improve PUFA productivity in Mortierella sp. AGED. © 2016 Society of Chemical Industry.

Role of dihydrofolate reductase in tetrahydrobiopterin biosynthesis and lipid metabolism in the oleaginous fungus Mortierella alpina.

Mortierella alpina is a well-known polyunsaturated fatty acid-producing oleaginous fungus. Analysis of the Mort. alpina genome suggests that there is a putative dihydrofolate reductase (DHFR) gene playing a role in the salvage pathway of tetrahydrobiopterin (BH4), which has never been explored in fungi before. DHFR is the sole source of tetrahydrofolate and plays a key role in maintaining BH4 levels. Transcriptome data analysis revealed that DHFR was up-regulated by nitrogen exhaustion, when Mort. alpina starts to accumulate lipids. Significant changes were found in the fatty acid profile in Mort. alpina grown on medium containing DHFR inhibitors compared to Mort. alpina grown on medium without inhibitors. To explore the role of DHFR in folate/BH4 metabolism and its relationship to lipid biosynthesis, we expressed heterologously the gene encoding DHFR from Mort. alpina in Escherichia coli and we purified the recombinant enzyme to homogeneity. The enzymatic activity was investigated by liquid chromatography and MS and VIS-UV spectroscopy. The kinetic parameters and the effects of temperature, pH, metal ions and inhibitors on the activity of DHFR were also investigated. The transcript level of cytosolic NADPH-producing gene involved in folate metabolism is down-regulated by DHFR inhibitors, which highlights the functional significance of DHFR in lipid biosynthesis. The relationship between DHFR and lipid metabolism is thus of major importance, and folate metabolism may be an alternative NADPH source in fatty acid synthesis. To our knowledge, this study is the first to report the comprehensive characterization of a BH4salvage pathway in a fungus.

Biochemical characterization of an isoform of GDP-D-mannose-4,6-dehydratase from Mortierella alpina.

OBJECTIVE: To clarify the molecular mechanism of GDP-L-fucose biosynthesis in Mortierella alpina. RESULTS: Analysis of the M. alpina genome suggests that there were two isofunctional GDP-D-mannose-4,6-dehydratase genes (GMD1 and GMD2) that have never been found in a microorganism before. GMD2 was expressed heterologously in Escherichia coli and purified to homogeneity. The addition of exogenous NAD(+) or NADP(+) was not essential for GMD2 activity. GMD2 may have considerable importance for GDP-L-fucose biosynthesis under nitrogen starvation. The transcriptional regulation of GMD1 may be more susceptible to GDP and GTP than that of GMD2. Significant changes were observed in the concentration of GDP-L-fucose (30 and 36 % inhibition respectively) and total fatty acids (18 and 12 % inhibition respectively) in M. alpina grown on GMD inhibitors medium, which suggests that GDP-L-fucose is functionally significant in lipid metabolism. CONCLUSIONS: This is the first time that an isofunctional GDP-D-mannose-4,6-dehydratase has been characterized in a microorganism.

Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.

BACKGROUND: Delta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels. RESULT: To enhance EPA production in M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate. CONCLUSION: An ALA-preferring FADS6 from M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.

Characterization of an fungal l-fucokinase involved in Mortierella alpina GDP-l-fucose salvage pathway.

GDP-l-fucose functions as a biological donor for fucosyltransferases, which are required for the catalysis of l-fucose to various acceptor molecules including oligosaccharides, glycoproteins and glycolipids. Mortierella alpina is one of the highest lipid-producing fungi and can biosynthesis GDP-l-fucose in the de novo pathway. Analysis of the M. alpina genome suggests that there is a gene encoding l-fucokinase (FUK) for the conversion of fucose to l-fucose-1-phosphate in the GDP-l-fucose salvage pathway, which has never been found in fungi before. This gene was characterized to explore its role in GDP-l-fucose synthesis. The yield of GDP-l-fucose is relatively higher in lipid accumulation phase (0.096 mg per g cell) than that in cell multiplication phase (0.074 mg per g cell) of M. alpina Additionally, the transcript level of FUK is up regulated by nitrogen exhaustion when M. alpina starts to accumulate lipid, highlights the functional significance of FUK in the GDP-l-fucose biosynthesis in M. alpina Gene encoding FUK was expressed heterologously in Escherichia coli and the resulting protein was purified to homogeneity. The product of FUK reaction was analyzed by liquid chromatography and mass spectrometry. Kinetic parameters and other properties of FUK were investigated. Comparative analyses between the FUK protein and other homologous proteins were performed. To our knowledge, this study is the first to report a comprehensive characterization of FUK in a fungus. Mortierella alpina could be used as an alternative source for the production of GDP-l-fucose.

Microbial production of dihomo-γ-linolenic acid by Δ5-desaturase gene-disruptants of Mortierella alpina 1S-4.

We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase (Δ5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds. The isolation of nine Δ5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4.