Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

Chen

et al. 2016

Microb Cell Fact

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0516-5) contains supplementary material, which is available to authorized users.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Chen

et al. 2016

Microb Cell Fact

“…We confirmed that it only catalyzed LA, and its conversion rate was low (24.3 ± 1.3%). In addition, TpFADS6 had no substrate specificity for the two substrates LA and ALA, but had a much higher ability to catalyze ALA and LA relative to that of FADS6 in Micromonas pusilla [ 12 ]. Therefore, we reasoned that transformation of the TpFADS6 gene into D. salina may promote ω3-PUFA accumulation.…”

Section: Discussionmentioning

confidence: 99%

“…This can be achieved by the transformation of an exogenous ALA-preferring delta 6 fatty acid desaturase ( FADS6 ) gene. Based on previous analysis of the substrate specificity of FADS6 from various species for LA and ALA [ 12 ], FADS6 of Thalassiosira pseudonana (TpFADS6), a marine diatom, has higher catalytic activity toward the ω3 substrate (ALA) than that of other algae, plants, and fungi. Its conversion efficiency is 68% in the ω-6 pool and 80% in the ω-3 pool [ 13 ], which is the highest ALA catalytic activity by far.…”

Section: Introductionmentioning

confidence: 99%

Production of eicosapentaenoic acid by application of a delta-6 desaturase with the highest ALA catalytic activity in algae

Luo

et al. 2018

Microb Cell Fact

Abstract:Dunaliella salina is a unicellular green alga with a high α-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% CO 2 aeration with glucose/KNO 3 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% CO 2 aeration with glucose/KNO 3 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 α-linolenic acid, was used to enhance EPA production by Dunaliella salina. Transformation of FADS6 from Thalassiosira pseudonana into Dunaliella salina with myoinositol, CO 2 , 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.

Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Luo

et al. 2018

FEBS Open Bio

Soft cheese with white rind lacks essential fatty acids ( EFA s), and as a result its long‐term consumption may lead to various kinds of cardiovascular and cerebrovascular diseases, such as hyperlipidemia, hypertension, and atherosclerosis. Geotrichum candidum is a dimorphic yeast that plays an important role in the ripening of mold cheese. A gene coding for Δ12 fatty acid desaturase, a critical bifunctional enzyme desaturating oleic acid ( OA ) and linoleic acid ( LA ) to produce LA and α‐linolenic acid ( ALA ), respectively, was isolated from G. candidum , and then cloned and heterologously expressed in Saccharomyces cerevisiae . This gene, named Gc FADS 12 , had an open reading frame of 1257 bp and codes for a protein of 419 amino acids with a predicted molecular mass of 47.5 kDa. Characterization showed that Gc FADS 12 had the ability to convert OA to LA and LA to ALA , and the conversion rates for OA and LA were 20.40 ± 0.66% and 6.40 ± 0.57%, respectively. We also found that the protein product of Gc FADS 12 catalyzes the conversion of the intermediate product ( LA ) to ALA by addition of OA as the sole substrate. The catalytic activity of Gc FADS 12 on OA and LA was unaffected by fatty acid concentrations. Kinetic analysis revealed that Gc FADS 12 had stronger affinity for the OA than for the LA substrate. This study offers a solid basis for improving the production of EFA s by G. candidum in cheese.