The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Sakayu Shimizu - One of the best experts on this subject based on the ideXlab platform.
-
Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review
Elsevier, 2018Co-Authors: Hiroshi Kikukawa, Sakayu Shimizu, Eiji Sakuradani, 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
-
establishment of agrobacterium tumefaciens mediated transformation of an oleaginous fungus Mortierella alpina 1s 4 and its application for eicosapentaenoic acid producer breeding
Applied and Environmental Microbiology, 2009Co-Authors: Akinori Ando, Eiji Sakuradani, Jun Ogawa, Yosuke Sumida, Hiroaki Negoro, Dian Anggraini Suroto, Sakayu ShimizuAbstract:Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5− strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ω3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ω3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.
-
Single cell oil production by Mortierella alpina.
Journal of biotechnology, 2009Co-Authors: Eiji Sakuradani, Sakayu ShimizuAbstract:A filamentous fungus, Mortierella alpina 1S-4, was obtained, through extensive screening, as an potential producer of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid. With this discovery as a starting point, we conducted employing methods from metabolic engineering and molecular biology for controlling cultures and breeding mutant strains. These parental and mutant strains are now used for large-scale production of a variety of PUFAs.
-
gene cloning and functional analysis of a second δ6 fatty acid desaturase from an arachidonic acid producing Mortierella fungus
Bioscience Biotechnology and Biochemistry, 2003Co-Authors: Eiji Sakuradani, Sakayu ShimizuAbstract:We demonstrated that Mortierella alpina 1S-4 has two Δ6-desaturases, which are involved in the desaturation of linoleic acid to γ-linolenic acid. For one of the two Δ6-desaturases, designated as Δ6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two Δ6-desaturases, designated as Δ6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella Δ6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast Δ9-desaturase, which has the corresponding domain at the C-terminus. The full-length Δ6II cDNA was expressed in A. oryzae, resulting in the accumulation of γ-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quan...
-
production of arachidonic acid by Mortierella fungi
Biotechnology and Bioprocess Engineering, 2002Co-Authors: Kenichi Higashiyama, Shigeaki Fujikawa, Enoch Y Park, Sakayu ShimizuAbstract:The growing interest in the application of arachidonic acid (ARA) in various fields of health and dietary requirements has elicited much attention on the industrial production of ARA-containing oil by the cultivation ofMortierella fungi. For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies including the investigation of morphology are important because ARA is accumulated in the mycelia, and thus cultivation with high biomass concentration is essential for obtaining a high ARA yield. Combining the results derived from various studies, a high ARA yield was attained in an industrial fermentor. These ARA production techniques are applicable to the production of other polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation technology especially in the field of fungal cultivation.
Eiji Sakuradani - One of the best experts on this subject based on the ideXlab platform.
-
Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S-4: A review
Elsevier, 2018Co-Authors: Hiroshi Kikukawa, Sakayu Shimizu, Eiji Sakuradani, 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
-
establishment of agrobacterium tumefaciens mediated transformation of an oleaginous fungus Mortierella alpina 1s 4 and its application for eicosapentaenoic acid producer breeding
Applied and Environmental Microbiology, 2009Co-Authors: Akinori Ando, Eiji Sakuradani, Jun Ogawa, Yosuke Sumida, Hiroaki Negoro, Dian Anggraini Suroto, Sakayu ShimizuAbstract:Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5− strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for ω3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/108 spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal ω3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.
-
Single cell oil production by Mortierella alpina.
Journal of biotechnology, 2009Co-Authors: Eiji Sakuradani, Sakayu ShimizuAbstract:A filamentous fungus, Mortierella alpina 1S-4, was obtained, through extensive screening, as an potential producer of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid. With this discovery as a starting point, we conducted employing methods from metabolic engineering and molecular biology for controlling cultures and breeding mutant strains. These parental and mutant strains are now used for large-scale production of a variety of PUFAs.
-
gene cloning and functional analysis of a second δ6 fatty acid desaturase from an arachidonic acid producing Mortierella fungus
Bioscience Biotechnology and Biochemistry, 2003Co-Authors: Eiji Sakuradani, Sakayu ShimizuAbstract:We demonstrated that Mortierella alpina 1S-4 has two Δ6-desaturases, which are involved in the desaturation of linoleic acid to γ-linolenic acid. For one of the two Δ6-desaturases, designated as Δ6I, gene cloning and its heterologous expression in a fungus, Aspergillus oryzae, has previously been reported. In addition, we indicated in this paper that there is an isozyme of the two Δ6-desaturases, designated as Δ6II, in M. alpina 1S-4. The predicted amino acid sequences of the Mortierella Δ6-desaturases were similar to those of ones from other organisms, i.e. borage and Caenorhabditis elegans, and had a cytochrome b5-like domain at the N-terminus, being different from the yeast Δ9-desaturase, which has the corresponding domain at the C-terminus. The full-length Δ6II cDNA was expressed in A. oryzae, resulting in the accumulation of γ-linolenic acid (which was not detected in the control Aspergillus) up to 37% of the total fatty acids. The analysis of real-time quantitative PCR (RTQ-PCR) showed that the quan...
-
delta6 fatty acid desaturase from an arachidonic acid producing Mortierella fungus gene cloning and its heterologous expression in a fungus aspergillus
Gene, 1999Co-Authors: Eiji Sakuradani, Michihiko Kobayashi, Sakayu ShimizuAbstract:A DNA fragment was cloned from the fungal strain, Mortierella alpina 1S-4 (which is used industrially to produce arachidonic acid), after PCR amplification with oligonucleotide primers designed based on the sequence information for delta6-desaturase genes (from borage and Caenorhabditis elegans), which are involved in the desaturation of linoleic acid (delta9, delta12-18:2) to gamma-linolenic acid (delta6, delta9, delta12-18:3). This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 457 amino acids from a M. calpina 1S-4 library. The predicted amino-acid sequence showed similarity to those of the above delta6-desaturases, and contained a cytochrome b5-like domain at the N-terminus, being different from the yeast delta9-desaturase which has the corresponding domain at the C-terminus. The full-length cDNA clone was expressed under the control of the amyB promoter in a filamentous fungus, Aspergillus oryzae, resulting in the accumulation of gamma-linolenic acid (which was not detected in the control Aspergillus) to the level of 25.2% of the total fatty acids. These findings revealed that the recombinant product has delta6-desaturase activity. The Mortierella delta6-desaturase is the first to be reported in fungi.
Haiqin Chen - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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.
-
Genome Characterization of the Oleaginous Fungus Mortierella alpina
2016Co-Authors: Wei Chen, Haiqin Chen, Hongchao Wang, Yun Feng, Yan Ren, Michael J. Thomas, Baixi Zhang, Isabelle M. BerquinAbstract:Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50 % of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50 % of its 12,796 gene models, and 60 % of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina’s fatt
-
Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.
Microbial Cell Factories, 2016Co-Authors: Haiqin Chen, Zhennan Gu, Hao Zhang, Wei Chen, Yong Q. ChenAbstract: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.
-
Reconstruction and analysis of a genome-scale metabolic model of the oleaginous fungus Mortierella alpina
BMC systems biology, 2015Co-Authors: Haiqin Chen, Yong Q. Chen, Wei Chen, Liming LiuAbstract:Background Mortierella alpina is an oleaginous fungus used in the industrial scale production of arachidonic acid (ARA). In order to investigate the metabolic characteristics at a systems level and to explore potential strategies for enhanced lipid production, a genome-scale metabolic model of M. alpina was reconstructed.
Wei Chen - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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.
-
Genome Characterization of the Oleaginous Fungus Mortierella alpina
2016Co-Authors: Wei Chen, Haiqin Chen, Hongchao Wang, Yun Feng, Yan Ren, Michael J. Thomas, Baixi Zhang, Isabelle M. BerquinAbstract:Mortierella alpina is an oleaginous fungus which can produce lipids accounting for up to 50 % of its dry weight in the form of triacylglycerols. It is used commercially for the production of arachidonic acid. Using a combination of high throughput sequencing and lipid profiling, we have assembled the M. alpina genome, mapped its lipogenesis pathway and determined its major lipid species. The 38.38 Mb M. alpina genome shows a high degree of gene duplications. Approximately 50 % of its 12,796 gene models, and 60 % of genes in the predicted lipogenesis pathway, belong to multigene families. Notably, M. alpina has 18 lipase genes, of which 11 contain the class 2 lipase domain and may share a similar function. M. alpina’s fatt
-
Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.
Microbial Cell Factories, 2016Co-Authors: Haiqin Chen, Zhennan Gu, Hao Zhang, Wei Chen, Yong Q. ChenAbstract: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.
-
Reconstruction and analysis of a genome-scale metabolic model of the oleaginous fungus Mortierella alpina
BMC systems biology, 2015Co-Authors: Haiqin Chen, Yong Q. Chen, Wei Chen, Liming LiuAbstract:Background Mortierella alpina is an oleaginous fungus used in the industrial scale production of arachidonic acid (ARA). In order to investigate the metabolic characteristics at a systems level and to explore potential strategies for enhanced lipid production, a genome-scale metabolic model of M. alpina was reconstructed.
Yong Q. Chen - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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.
-
Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.
Microbial Cell Factories, 2016Co-Authors: Haiqin Chen, Zhennan Gu, Hao Zhang, Wei Chen, Yong Q. ChenAbstract: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.
-
Reconstruction and analysis of a genome-scale metabolic model of the oleaginous fungus Mortierella alpina
BMC systems biology, 2015Co-Authors: Haiqin Chen, Yong Q. Chen, Wei Chen, Liming LiuAbstract:Background Mortierella alpina is an oleaginous fungus used in the industrial scale production of arachidonic acid (ARA). In order to investigate the metabolic characteristics at a systems level and to explore potential strategies for enhanced lipid production, a genome-scale metabolic model of M. alpina was reconstructed.