Dataset Information

Characterization of an Omega-3 Desaturase From Phytophthora parasitica and Application for Eicosapentaenoic Acid Production in Mortierella alpina.

ABSTRACT: Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have important therapeutic and nutritional benefits in humans. In the biosynthesis pathways of these LC-PUFAs, omega-3 desaturase plays a critical role. In this study, we report a new omega-3 desaturase (PPD17) from Phytophthora parasitica. This desaturase shares high similarities with the known omega-3 desaturases and was expressed in Saccharomyces cerevisiae for the activity and substrate specificity research. The desaturase has a wide omega-6 fatty acid substrate, containing both 18C and 20C fatty acids, and exhibits a strong activity of delta-17 desaturase but a weak activity of delta-15 desaturase. The new desaturase converted the omega-6 arachidonic acid (AA, C20:4) to EPA (an omega-3 LC-PUFA, C20:5) with a substrate conversion rate of 70%. To obtain a high EPA-producing strain, we transformed PPD17 into Mortierella alpina, an AA-producing filamentous fungus. The EPA content of the total fatty acids in reconstruction strains reached 31.5% and was followed by the fermentation optimization of the EPA yield of up to 1.9 g/L. This research characterized a new omega-3 desaturase and provides a possibility of industrially producing EPA using M. alpina.

SUBMITTER: Tang X

PROVIDER: S-EPMC6102326 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Characterization of an Omega-3 Desaturase From Phytophthora parasitica and Application for Eicosapentaenoic Acid Production in Mortierella alpina.

Tang Xin X Chen Haiqin H Mei Tiantian T Ge Chengfeng C Gu Zhennan Z Zhang Hao H Chen Yong Q YQ Chen Wei W

Frontiers in microbiology 20180814

Omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) have important therapeutic and nutritional benefits in humans. In the biosynthesis pathways of these LC-PUFAs, omega-3 desaturase plays a critical role. In this study, we report a new omega-3 desaturase (PPD17) from Phytophthora parasitica. This desaturase shares high similarities with the known omega-3 desaturases and was expressed in Saccharomyces cerevisiae for the activity and substrate specificity research. The desatura ...[more]

PMID: 30154780

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Project description:BackgroundDelta-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.ResultTo 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.ConclusionAn 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.

Project description:Dunaliella salina is a unicellular green alga with a high ?-linolenic acid (ALA) level, but a low eicosapentaenoic acid (EPA) level. In a previous analysis of the catalytic activity of delta 6 fatty acid desaturase (FADS6) from various species, FADS6 from Thalassiosira pseudonana (TpFADS6), a marine diatom, showed the highest catalytic activity for ALA. In this study, to enhance EPA production in D. salina, FADS6 from D. salina (DsFADS6) was identified, and substrate specificities for DsFADS6 and TpFADS6 were characterized. Furthermore, a plasmid harboring the TpFADS6 gene was constructed and overexpressed in D. salina. Our results revealed that EPA production reached 21.3 ± 1.5 mg/L in D. salina transformants. To further increase EPA production, myoinositol (MI) was used as a growth-promoting agent; it increased the dry cell weight of D. salina transformants, and EPA production reached 91.3 ± 11.6 mg/L. The combination of 12% CO2 aeration with glucose/KNO3 in the medium improved EPA production to 192.9 ± 25.7 mg/L in the Ds-TpFADS6 transformant. We confirmed that the increase in ALA was optimal at 8 °C; the EPA percentage reached 41.12 ± 4.78%. The EPA yield was further increased to 554.3 ± 95.6 mg/L by supplementation with 4 g/L perilla seed meal (PeSM), 500 mg/L MI, and 12% CO2 aeration with glucose/KNO3 at varying temperatures. EPA production and the percentage of EPA in D. salina were 343.8-fold and 25-fold higher than those in wild-type D. salina, respectively. IMPORTANCE:FADS6 from Thalassiosira pseudonana, which demonstrates high catalytic activity toward ?-linolenic acid, was used to enhance EPA production by Dunaliella salina. Transformation of FADS6 from Thalassiosira pseudonana into Dunaliella salina with myoinositol, CO2, low temperatures, and perilla seed meal supplementation substantially increased EPA production in Dunaliella salina to 554.3 ± 95.6 mg/L. Accordingly, D. salina could be a potential alternative source of EPA and is suitable for its large-scale production.

Dataset Information

Characterization of an Omega-3 Desaturase From Phytophthora parasitica and Application for Eicosapentaenoic Acid Production in Mortierella alpina.

Publications

Characterization of an Omega-3 Desaturase From <i>Phytophthora parasitica</i> and Application for Eicosapentaenoic Acid Production in <i>Mortierella alpina</i>.

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