The Experts below are selected from a list of 1464 Experts worldwide ranked by ideXlab platform
Sakayu Shimizu - One of the best experts on this subject based on the ideXlab platform.
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arachidonic acid production by the oleaginous fungus Mortierella alpina 1s 4 a review
Journal of Advanced Research, 2018Co-Authors: Eiji Sakuradani, Sakayu Shimizu, Hiroshi Kikukawa, Akinori Ando, Jun OgawaAbstract:Abstract The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4.
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Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review
Elsevier, 2018Co-Authors: Hiroshi Kikukawa, Eiji Sakuradani, Sakayu Shimizu, Akinori Ando, Jun OgawaAbstract:The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4. Keywords: Arachidonic acid, Mortierella alpina, Molecular breeding, Fatty acid desaturas
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Essential fatty acids for oleaginous fungus Mortierella alpina
Biocatalysis and Agricultural Biotechnology, 2016Co-Authors: Tomohiro Fujita, Eiji Sakuradani, Akinori Ando, Jun Ogawa, Hiroshi Kawashima, Takaiku Sakamoto, Sakayu ShimizuAbstract:Abstract Oleaginous fungus Mortierella alpina 1S-4 accumulates arachidonic acid (20:4ω6) as a major polyunsaturated fatty acid. However, 20:4ω6 is not essential for the growth of M. alpina 1S-4, because various mutants that produce no 20:4ω6 were isolated. M. alpina JT-180, a Δ12 desaturation-defective mutant that was derived from M. alpina 1S-4 by treatment with a chemical mutagen, accumulates Mead acid (20:3ω9) instead of 20:4ω6. p -Anisidine was found to be a Δ6 desaturation inhibitor in this study. The concentration of 0.1 mg/ml of p -anisidine had an inhibitory effect on the growth of M. alpina JT-180. The addition of p -anisidine to the medium caused M. alpina JT-180 to produce only monoenoic acids such as oleic acid (18:1ω9) and eicosenoic acid (20:1ω9) as unsaturated fatty acids. The effects of exogenous fatty acids were investigated when M. alpina JT-180 was cultivated in medium containing p -anisidine. The addition of linoleic acid (18:2ω6) and 6 Z ,9 Z -octadecadienoic acid (18:2ω9) restored the growth of M. alpina JT-180 cultivated on the medium containing p -anisidine, but palmitic acid (16:0), 18:1ω9, and vaccenic acid (18:1ω7) had no effect. For the growth of oleaginous fungus M. alpina , 18-carbon length fatty acids with more than two double bonds are considered to be essential.
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Transformation of Zygomycete Mortierella alpina Using Biolistic Particle Bombardment
Fungal Biology, 2014Co-Authors: Eiji Sakuradani, Sakayu Shimizu, Hiroshi Kikukawa, Seiki Takeno, Akinori Ando, Jun OgawaAbstract:Uracil auxotrophs derived from the Zygomycete Mortierella alpina through spontaneous mutation were transformed by biolistic particle bombardment. Effective biolistic transformation of intact spores was achieved using the Bio-Rad PDS-1000/He system. Transformation frequencies of up to several colonies per μg of circular DNA were obtained under optimized conditions.
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Polyunsaturated fatty acids production and transformation by Mortierella alpina and anaerobic bacteria
European Journal of Lipid Science and Technology, 2012Co-Authors: Jun Ogawa, Eiji Sakuradani, Akinori Ando, Shigenobu Kishino, Kenzo Yokozeki, Sakayu ShimizuAbstract:Microorganisms are promising as producers of various polyunsaturated fatty acids (PUFAs) and as catalysts transforming them into unique molecular species beyond common PUFAs. This article describes PUFA production through chemical mutant- and molecular-breeding of an oleaginous filamentous fungus Mortierella alpina 1S-4 and PUFA transformation by anaerobic bacteria. M. alpina 1S-4 and its mutants and transformants produce oils containing not only common n − 6 and n − 3 PUFAs but also rare PUFAs. Unique PUFA-transforming activities were found in anaerobic bacteria. They isomerized PUFA to conjugated fatty acids and further transformed to partially saturated fatty acids with hydroxyl fatty acids as intermediates. The functions of these unique PUFAs have been attracting much attention for improving our health and for developing new chemical materials.
Eiji Sakuradani - One of the best experts on this subject based on the ideXlab platform.
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arachidonic acid production by the oleaginous fungus Mortierella alpina 1s 4 a review
Journal of Advanced Research, 2018Co-Authors: Eiji Sakuradani, Sakayu Shimizu, Hiroshi Kikukawa, Akinori Ando, Jun OgawaAbstract:Abstract The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4.
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Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review
Elsevier, 2018Co-Authors: Hiroshi Kikukawa, Eiji Sakuradani, Sakayu Shimizu, Akinori Ando, Jun OgawaAbstract:The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4. Keywords: Arachidonic acid, Mortierella alpina, Molecular breeding, Fatty acid desaturas
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Essential fatty acids for oleaginous fungus Mortierella alpina
Biocatalysis and Agricultural Biotechnology, 2016Co-Authors: Tomohiro Fujita, Eiji Sakuradani, Akinori Ando, Jun Ogawa, Hiroshi Kawashima, Takaiku Sakamoto, Sakayu ShimizuAbstract:Abstract Oleaginous fungus Mortierella alpina 1S-4 accumulates arachidonic acid (20:4ω6) as a major polyunsaturated fatty acid. However, 20:4ω6 is not essential for the growth of M. alpina 1S-4, because various mutants that produce no 20:4ω6 were isolated. M. alpina JT-180, a Δ12 desaturation-defective mutant that was derived from M. alpina 1S-4 by treatment with a chemical mutagen, accumulates Mead acid (20:3ω9) instead of 20:4ω6. p -Anisidine was found to be a Δ6 desaturation inhibitor in this study. The concentration of 0.1 mg/ml of p -anisidine had an inhibitory effect on the growth of M. alpina JT-180. The addition of p -anisidine to the medium caused M. alpina JT-180 to produce only monoenoic acids such as oleic acid (18:1ω9) and eicosenoic acid (20:1ω9) as unsaturated fatty acids. The effects of exogenous fatty acids were investigated when M. alpina JT-180 was cultivated in medium containing p -anisidine. The addition of linoleic acid (18:2ω6) and 6 Z ,9 Z -octadecadienoic acid (18:2ω9) restored the growth of M. alpina JT-180 cultivated on the medium containing p -anisidine, but palmitic acid (16:0), 18:1ω9, and vaccenic acid (18:1ω7) had no effect. For the growth of oleaginous fungus M. alpina , 18-carbon length fatty acids with more than two double bonds are considered to be essential.
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Transformation of Zygomycete Mortierella alpina Using Biolistic Particle Bombardment
Fungal Biology, 2014Co-Authors: Eiji Sakuradani, Sakayu Shimizu, Hiroshi Kikukawa, Seiki Takeno, Akinori Ando, Jun OgawaAbstract:Uracil auxotrophs derived from the Zygomycete Mortierella alpina through spontaneous mutation were transformed by biolistic particle bombardment. Effective biolistic transformation of intact spores was achieved using the Bio-Rad PDS-1000/He system. Transformation frequencies of up to several colonies per μg of circular DNA were obtained under optimized conditions.
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Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4
Current Genetics, 2014Co-Authors: Tomoyo Okuda, Eiji Sakuradani, Hiroshi Kikukawa, Akinori Ando, Nozomu Kamada, Misa Ochiai, Jun Shima, Jun OgawaAbstract:An inducible promoter is a useful tool for the controlled expression of a given gene. In this report, we describe galactose-dependent promoters for potential use in an oleaginous fungus Mortierella alpina . We cloned the putative promoter regions of two genes encoding galactose metabolic enzymes, GAL1 and GAL10, from the genome of M. alpina 1S-4. The β-glucuronidase ( GUS ) reporter gene assay in M. alpina 1S-4 revealed that regulation of these promoters was dependent on the presence of galactose in the medium both with and without other sugars. With the GAL10 promoter, an approximately 50-fold increase of GUS activity was demonstrated by addition of galactose into the culture media at any cultivation phase. The 5′ deletion analysis of the GAL10 promoter revealed that a promoter region of over 2,000 bp length was required for its high-level activity and sufficient inducible response. Significantly, this is the first report of inducible promoters of zygomycetes. The GAL10 promoter will be a valuable tool for gene manipulation in M. alpina 1S-4.
Wei Chen - One of the best experts on this subject based on the ideXlab platform.
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The role of MTHFDL in mediating intracellular lipogenesis in oleaginous Mortierella alpina.
Microbiology (Reading England), 2020Co-Authors: Hongchao Wang, Yong Q. Chen, Haiqin Chen, Hao Zhang, Chen Zhang, Qi-zai Wang, Lu Wenwei, Jianxin Zhao, Wei ChenAbstract:The oleaginous fungus Mortierella alpina can synthesize a variety of polyunsaturated fatty acids, which are used extensively in industry for the production of arachidonic acid (AA). NADPH is the limiting factor and critical reducing agent in lipid biosynthesis. In the folate cycle, methylenetetrahydrofolate dehydrogenase (MTHFDL) catalyzes the conversion of methylene tetrahydrofolate into 10-formyl-tetrahydrofolate with the reduction of NADP+ to NADPH. MTHFDL RNAi was used to investigate the role of the folate cycle in lipogenesis. Gene knockdown decreased the transcript levels of MTHFDL by about 50 % and attenuated cell fatty acid synthesis. The observation of decreased NADPH levels and downregulated NADPH-producing genes in response to MTHFDL RNAi indicates a novel aspect of the NADPH regulatory mechanism. Thus, our study demonstrates that MTHFDL plays key role in the mediation of NADPH in lipogenesis in M. alpina.
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Application of the cbh1 promoter in Mortierella alpina and optimization of induction conditions.
Letters in applied microbiology, 2020Co-Authors: Yuzhuo Wang, Yong Q. Chen, Haiqin Chen, Hao Zhang, Xin Tang, Shunxian Wang, Wei ChenAbstract:Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.
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Effects of Agrobacterium tumefaciens strain types on the Agrobacterium-mediated transformation efficiency of filamentous fungus Mortierella alpina.
Letters in applied microbiology, 2020Co-Authors: Shunxian Wang, Haiqin Chen, Hao Zhang, Wei Chen, Xin Tang, Yuzhuo Wang, Caixing Pan, Yong Q. ChenAbstract:Four Agrobacterium tumefaciens strains AGL‐1, C58C1, EHA105 and LBA4404 were tested for the effects of strain types on the transformation efficiency in Mortierella alpina. Results showed that AGL‐1, C58C1 and EHA105 transformed M. alpina successfully. Among them, A. tumefaciens EHA105 was first proven successful transformation of M. alpina. AGL‐1 and EHA105 had the highest transformation efficiency among the four strains, while LBA4404 failed to transform M. alpina. The reason leading to the transformation efficiency difference among the four strains was explored by determining transcription levels of the virulence (vir) gene in the induction medium. Results showed that the expressions of virD1, virD2, virD4 and virE1 genes were obviously induced by acetosyringone in all the strains, and their transcriptional levels as well as virA’s of AGL‐1, C58C1 and EHA105 were higher than that of LBA4404, suggesting high transcriptional levels of vir genes were important for successful transformation. The study selected A. tumefaciens with high transformation efficiency of M. alpina, and would accelerate the genetic management of M. alpina. SIGNIFICANCE AND IMPACT OF THE STUDY: Oleaginous filamentous fungus Mortierella alpina is a commercial strain for the production of arachidonic acid. Genetic manipulation of M. alpina requires highly efficient transformation method. In this study, we explore the effect of Agrobacterium tumefaciens strain types on the transformation efficiency of M. alpina and select A. tumefaciens with the highest transformation efficiency, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of virulence genes in A. tumefaciens were proven to play an important role for successful transformation.
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Two Co-Expression Strategies for Eicosapentaenoic Acid Production in Mortierella alpina
American Journal of Biochemistry and Biotechnology, 2019Co-Authors: Xin Tang, Yong Q. Chen, Haiqin Chen, Hao Zhang, Xiaoke Zhang, Wei ChenAbstract:The omega-3 long-chain Polyunsaturated Fatty Acid (PUFA) Eicosapentaenoic Acid (EPA) has beneficial effects on human health, leading to its use in health products and foods. The oleaginous microorganism Mortierella alpina has been used in the industrial production of Arachidonic Acid (AA). The omega-3 desaturase oPpFADS17 is a key enzyme in the bioconversion of AA to EPA from Phytophthora parasitica and Glucose-6-Phosphate Dehydrogenase (G6PD2) is a critical enzyme that provide reducing power NADPH for lipid biosynthesis. In this study, we used a double expression cassette and 2A peptide strategies to co-express the these two critical enzymes. Subsequently, we investigated the effects of these strategies on total lipid and EPA accumulation. The recombination strains generated using the above-described strategies exhibited no differences in cell growth, compared with the control strain. Recombination strains generated using the double expression cassette exhibited an increase in total lipids to 43% of the cell dry weight, but did not accumulate EPA. Recombination strains generated using the 2A peptide strategy exhibited increased EPA accumulation, such that this PUFA accounted for 30% of the total lipid content. These co-expression strategies provide the improvements of multi-gene modification in PUFA accumulation in M. alpina.
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Application of a ω-3 Desaturase with an Arachidonic Acid Preference to Eicosapentaenoic Acid Production in Mortierella alpina.
Frontiers in bioengineering and biotechnology, 2018Co-Authors: Haiqin Chen, Hao Zhang, Wei Chen, Mei Tiantian, Xin Tang, Chang Lulu, Yong Q. ChenAbstract:In the industrial oleaginous fungus Mortierella alpina, the arachidonic acid (AA; C20:4; ω-6) fraction can reach 50% of the total fatty acids in vivo. However, the eicosapentaenoic acid (EPA; C20:5; ω-3) fraction is less than 3% when this fungus is cultivated at a low temperature (12°C). Omega-3 fatty acid desaturase is a key enzyme in ω-3 long-chain polyunsaturated fatty acids (LC-PUFAs) biosynthesis pathways. To enhance EPA production, we transformed the ω-3 fatty acid desaturase (PaD17), which exhibits strong Δ-17 desaturase activity, into Mortierella alpina, thus increasing the AA to EPA conversion rate to 49.8%. This PaD17-harbouring Mortierella alpina reconstruction strain produced 617 mg L-1 of EPA at room temperature in broth medium, this yield was increased to 1.73 g L-1 after culture medium optimisation, (i.e., about three fold higher than that under original culture conditions), with concomitant respective increases in dry cell weight and total fatty acids content to 16.55 g L-1 and 6.46 g L-1. These findings suggest a new platform for the future industrial production of EPA.
Haiqin Chen - One of the best experts on this subject based on the ideXlab platform.
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The role of MTHFDL in mediating intracellular lipogenesis in oleaginous Mortierella alpina.
Microbiology (Reading England), 2020Co-Authors: Hongchao Wang, Yong Q. Chen, Haiqin Chen, Hao Zhang, Chen Zhang, Qi-zai Wang, Lu Wenwei, Jianxin Zhao, Wei ChenAbstract:The oleaginous fungus Mortierella alpina can synthesize a variety of polyunsaturated fatty acids, which are used extensively in industry for the production of arachidonic acid (AA). NADPH is the limiting factor and critical reducing agent in lipid biosynthesis. In the folate cycle, methylenetetrahydrofolate dehydrogenase (MTHFDL) catalyzes the conversion of methylene tetrahydrofolate into 10-formyl-tetrahydrofolate with the reduction of NADP+ to NADPH. MTHFDL RNAi was used to investigate the role of the folate cycle in lipogenesis. Gene knockdown decreased the transcript levels of MTHFDL by about 50 % and attenuated cell fatty acid synthesis. The observation of decreased NADPH levels and downregulated NADPH-producing genes in response to MTHFDL RNAi indicates a novel aspect of the NADPH regulatory mechanism. Thus, our study demonstrates that MTHFDL plays key role in the mediation of NADPH in lipogenesis in M. alpina.
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Application of the cbh1 promoter in Mortierella alpina and optimization of induction conditions.
Letters in applied microbiology, 2020Co-Authors: Yuzhuo Wang, Yong Q. Chen, Haiqin Chen, Hao Zhang, Xin Tang, Shunxian Wang, Wei ChenAbstract:Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.
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Effects of Agrobacterium tumefaciens strain types on the Agrobacterium-mediated transformation efficiency of filamentous fungus Mortierella alpina.
Letters in applied microbiology, 2020Co-Authors: Shunxian Wang, Haiqin Chen, Hao Zhang, Wei Chen, Xin Tang, Yuzhuo Wang, Caixing Pan, Yong Q. ChenAbstract:Four Agrobacterium tumefaciens strains AGL‐1, C58C1, EHA105 and LBA4404 were tested for the effects of strain types on the transformation efficiency in Mortierella alpina. Results showed that AGL‐1, C58C1 and EHA105 transformed M. alpina successfully. Among them, A. tumefaciens EHA105 was first proven successful transformation of M. alpina. AGL‐1 and EHA105 had the highest transformation efficiency among the four strains, while LBA4404 failed to transform M. alpina. The reason leading to the transformation efficiency difference among the four strains was explored by determining transcription levels of the virulence (vir) gene in the induction medium. Results showed that the expressions of virD1, virD2, virD4 and virE1 genes were obviously induced by acetosyringone in all the strains, and their transcriptional levels as well as virA’s of AGL‐1, C58C1 and EHA105 were higher than that of LBA4404, suggesting high transcriptional levels of vir genes were important for successful transformation. The study selected A. tumefaciens with high transformation efficiency of M. alpina, and would accelerate the genetic management of M. alpina. SIGNIFICANCE AND IMPACT OF THE STUDY: Oleaginous filamentous fungus Mortierella alpina is a commercial strain for the production of arachidonic acid. Genetic manipulation of M. alpina requires highly efficient transformation method. In this study, we explore the effect of Agrobacterium tumefaciens strain types on the transformation efficiency of M. alpina and select A. tumefaciens with the highest transformation efficiency, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of virulence genes in A. tumefaciens were proven to play an important role for successful transformation.
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Two Co-Expression Strategies for Eicosapentaenoic Acid Production in Mortierella alpina
American Journal of Biochemistry and Biotechnology, 2019Co-Authors: Xin Tang, Yong Q. Chen, Haiqin Chen, Hao Zhang, Xiaoke Zhang, Wei ChenAbstract:The omega-3 long-chain Polyunsaturated Fatty Acid (PUFA) Eicosapentaenoic Acid (EPA) has beneficial effects on human health, leading to its use in health products and foods. The oleaginous microorganism Mortierella alpina has been used in the industrial production of Arachidonic Acid (AA). The omega-3 desaturase oPpFADS17 is a key enzyme in the bioconversion of AA to EPA from Phytophthora parasitica and Glucose-6-Phosphate Dehydrogenase (G6PD2) is a critical enzyme that provide reducing power NADPH for lipid biosynthesis. In this study, we used a double expression cassette and 2A peptide strategies to co-express the these two critical enzymes. Subsequently, we investigated the effects of these strategies on total lipid and EPA accumulation. The recombination strains generated using the above-described strategies exhibited no differences in cell growth, compared with the control strain. Recombination strains generated using the double expression cassette exhibited an increase in total lipids to 43% of the cell dry weight, but did not accumulate EPA. Recombination strains generated using the 2A peptide strategy exhibited increased EPA accumulation, such that this PUFA accounted for 30% of the total lipid content. These co-expression strategies provide the improvements of multi-gene modification in PUFA accumulation in M. alpina.
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Application of a ω-3 Desaturase with an Arachidonic Acid Preference to Eicosapentaenoic Acid Production in Mortierella alpina.
Frontiers in bioengineering and biotechnology, 2018Co-Authors: Haiqin Chen, Hao Zhang, Wei Chen, Mei Tiantian, Xin Tang, Chang Lulu, Yong Q. ChenAbstract:In the industrial oleaginous fungus Mortierella alpina, the arachidonic acid (AA; C20:4; ω-6) fraction can reach 50% of the total fatty acids in vivo. However, the eicosapentaenoic acid (EPA; C20:5; ω-3) fraction is less than 3% when this fungus is cultivated at a low temperature (12°C). Omega-3 fatty acid desaturase is a key enzyme in ω-3 long-chain polyunsaturated fatty acids (LC-PUFAs) biosynthesis pathways. To enhance EPA production, we transformed the ω-3 fatty acid desaturase (PaD17), which exhibits strong Δ-17 desaturase activity, into Mortierella alpina, thus increasing the AA to EPA conversion rate to 49.8%. This PaD17-harbouring Mortierella alpina reconstruction strain produced 617 mg L-1 of EPA at room temperature in broth medium, this yield was increased to 1.73 g L-1 after culture medium optimisation, (i.e., about three fold higher than that under original culture conditions), with concomitant respective increases in dry cell weight and total fatty acids content to 16.55 g L-1 and 6.46 g L-1. These findings suggest a new platform for the future industrial production of EPA.
Jun Ogawa - One of the best experts on this subject based on the ideXlab platform.
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arachidonic acid production by the oleaginous fungus Mortierella alpina 1s 4 a review
Journal of Advanced Research, 2018Co-Authors: Eiji Sakuradani, Sakayu Shimizu, Hiroshi Kikukawa, Akinori Ando, Jun OgawaAbstract:Abstract The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4.
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Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review
Elsevier, 2018Co-Authors: Hiroshi Kikukawa, Eiji Sakuradani, Sakayu Shimizu, Akinori Ando, Jun OgawaAbstract:The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%–70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4. Keywords: Arachidonic acid, Mortierella alpina, Molecular breeding, Fatty acid desaturas
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Essential fatty acids for oleaginous fungus Mortierella alpina
Biocatalysis and Agricultural Biotechnology, 2016Co-Authors: Tomohiro Fujita, Eiji Sakuradani, Akinori Ando, Jun Ogawa, Hiroshi Kawashima, Takaiku Sakamoto, Sakayu ShimizuAbstract:Abstract Oleaginous fungus Mortierella alpina 1S-4 accumulates arachidonic acid (20:4ω6) as a major polyunsaturated fatty acid. However, 20:4ω6 is not essential for the growth of M. alpina 1S-4, because various mutants that produce no 20:4ω6 were isolated. M. alpina JT-180, a Δ12 desaturation-defective mutant that was derived from M. alpina 1S-4 by treatment with a chemical mutagen, accumulates Mead acid (20:3ω9) instead of 20:4ω6. p -Anisidine was found to be a Δ6 desaturation inhibitor in this study. The concentration of 0.1 mg/ml of p -anisidine had an inhibitory effect on the growth of M. alpina JT-180. The addition of p -anisidine to the medium caused M. alpina JT-180 to produce only monoenoic acids such as oleic acid (18:1ω9) and eicosenoic acid (20:1ω9) as unsaturated fatty acids. The effects of exogenous fatty acids were investigated when M. alpina JT-180 was cultivated in medium containing p -anisidine. The addition of linoleic acid (18:2ω6) and 6 Z ,9 Z -octadecadienoic acid (18:2ω9) restored the growth of M. alpina JT-180 cultivated on the medium containing p -anisidine, but palmitic acid (16:0), 18:1ω9, and vaccenic acid (18:1ω7) had no effect. For the growth of oleaginous fungus M. alpina , 18-carbon length fatty acids with more than two double bonds are considered to be essential.
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Transformation of Zygomycete Mortierella alpina Using Biolistic Particle Bombardment
Fungal Biology, 2014Co-Authors: Eiji Sakuradani, Sakayu Shimizu, Hiroshi Kikukawa, Seiki Takeno, Akinori Ando, Jun OgawaAbstract:Uracil auxotrophs derived from the Zygomycete Mortierella alpina through spontaneous mutation were transformed by biolistic particle bombardment. Effective biolistic transformation of intact spores was achieved using the Bio-Rad PDS-1000/He system. Transformation frequencies of up to several colonies per μg of circular DNA were obtained under optimized conditions.
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Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4
Current Genetics, 2014Co-Authors: Tomoyo Okuda, Eiji Sakuradani, Hiroshi Kikukawa, Akinori Ando, Nozomu Kamada, Misa Ochiai, Jun Shima, Jun OgawaAbstract:An inducible promoter is a useful tool for the controlled expression of a given gene. In this report, we describe galactose-dependent promoters for potential use in an oleaginous fungus Mortierella alpina . We cloned the putative promoter regions of two genes encoding galactose metabolic enzymes, GAL1 and GAL10, from the genome of M. alpina 1S-4. The β-glucuronidase ( GUS ) reporter gene assay in M. alpina 1S-4 revealed that regulation of these promoters was dependent on the presence of galactose in the medium both with and without other sugars. With the GAL10 promoter, an approximately 50-fold increase of GUS activity was demonstrated by addition of galactose into the culture media at any cultivation phase. The 5′ deletion analysis of the GAL10 promoter revealed that a promoter region of over 2,000 bp length was required for its high-level activity and sufficient inducible response. Significantly, this is the first report of inducible promoters of zygomycetes. The GAL10 promoter will be a valuable tool for gene manipulation in M. alpina 1S-4.