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

Abstract

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.

Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

“…Each chimeric and wild-type desaturase gene was induced by 0.5 mM cis -OA at 28°C for 12 h under previously described conditions ( Shi et al, 2018a). Then, the expression level of each chimeric desaturase in Pichia pastoris was determined by western blotting analysis as described previously ( Shi et al, 2015); each sample loading volume was adjusted to the same level according to the western blot results to determine desaturase activity of each chimera.…”

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

“…Total RNA was isolated using the RNAprep Pure Plant Kit, and reverse‐transcribed with QuantScript RT Kit as described previously . RT‐qPCR was performed as described previously , and the transcript levels were calculated using the method.…”

“…Lipids from an equivalent weight of cells were extracted and methyl esterified, and fatty acid content was analyzed by gas chromatography as described previously .…”

“…In EFA biosynthesis, Δ12 fatty acid desaturase (FADS12), Δ15 fatty acid desaturase (FADS15) and Δ6 fatty acid desaturase (FADS6) play a key role in regulating the level of EFAs. In a previous study, the molecular mechanism, substrate specificity and catalytic activity for FADS6 were analyzed and applied to PUFA synthesis . FADS12 is a key bifunctional membrane‐bound desaturase that converts oleic acid (OA, 18:1 Δ9 ) to linoleic acid (LA, 18:2 Δ9,12 ) and LA to α‐linolenic acid (ALA, 18:3 Δ9,12,15 ) by introducing a double bond between the carbons 12 and 13 from the carboxyl end of the substrate in the biosynthesis of EFAs .…”

Pengimbuhan Minyak Jagung Terproteksi dengan Berbagai Level Protein Ransum Sapi Friesian Holstein Meningkatkan Kadar Asam Lemak Tidak Jenuh Susu

“…Enzim desaturase delta 4, 5, dan 6 termasuk enzim desaturase depan terakhir yang bertugas mempertemukan ikatan rangkap antara karboksilat sebuah molekul dan ikatan yang sudah ada sebelumnya (Lee et al, 2016). Beberapa mikrob penghasil enzim delta 6 desaturase antara lain Mortierella alpina, Pythium irregulare, Micromonas pusilla, Phaeodactylum tricornutum, Yarrowia lipolytica (Shi et al, 2016).…”