Molecular cloning and characterization of the genes encoding a microsomal oleate Δ12 desaturase (CsFAD2) and linoleate Δ15 desaturase (CsFAD3) from Camelina sativa

Abstract

Camelina sativa produces oil that is rich in polyunsaturated linoleic (18:2 Δ9,12) and linolenic (18:3 Δ9,12,15) acids. These fatty acids are obtained by the successive desaturation of oleic (18:1 Δ9) acid, which is catalyzed in the endoplasmic reticulum by two different microsomal desaturases: oleate Δ 12 desaturase (CsFAD2) and linoleate Δ 15 desaturase (CsFAD3). The objective of the present study is to investigate the contribution of these two desaturases to the composition and properties of C. sativa seed oil, and look over the effect that temperature exerts on their activity. Three different copies of both these genes were identified, which when analysed contained three histidine rich motifs (HXCGHX, HRXHH and HVXHH) and six highly conserved transmembrane domains. Comparing their sequences, the CsFAD2 copies accommodated four conservative changes (Glu-36-Asp, Arg-48-His, Val-97-Ala and Ala-177-Pro) and two semi-conservative ones (Val-63-Ile and Leu-249-Met), whereas only one semi-conservative change (Ala-327-Ser) was detected in CsFAD3 but with two extra amino acids (His-147 and Gly-148). The CsFAD2 and CsFAD3 cDNAs were heterologously expressed in Saccharomyces cerevisiae to confirm that they were active enzymes and their dependence on temperature was investigated by growing the recombinant yeast cells at low (22 °C) and optimal (30 °C) temperatures. The conversion of 18:1 Δ9 to 18:2 Δ9,12 by CsFAD2 was slightly better at 30 °C than at 22 °C, and it was also able to desaturate palmitoleic acid (16:1 Δ9) to hexadecadienoic acid (16:2 Δ9,12). By contrast, the conversion driven by CsFAD3 was enhanced 5-fold at 22 °C compared to 30 °C, although it was not able to desaturate 16:2 Δ9,12. The distribution of these microsomal desaturases was also studied in C. sativa, which were expressed most strongly in expanding leaves and developing seeds.

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

“…GcFADS12 possessed typical characteristics of other FADS12 family members, including three histidine‐rich conserved motifs (HXXHHK, HWXVXXTXLQH and HLVHH) and six transmembrane domains (Fig. ), and these observations were in agreement with our previous reports on Δ6 fatty acid desaturase . The amino acid sequence of GcFADS12 was highly similar to the one that S. Casaregola submitted to the GenBank (protein id: CDO51572.1), which was in agreement with the observation from our phylogenetic analysis of FADS12 (Fig.…”

“…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 . The genome of G. candidum has been sequenced and submitted to the GenBank database (LOCUS: CCBN010000001) by Casaregola, and we found from its genome sequence that there was a sequence encoding Δ12 fatty acid desaturase of G. candidum ( GcFADS12 ).…”

Functional Characterization of Lysophosphatidylcholine: Acyl-CoA Acyltransferase Genes From Sunflower (Helianthus annuus L.)

“…The heterologous expression of HaLPCATs was carried out in yeast, a host that has been demonstrated to be appropriate for the characterization of other ER-associated enzymes involved in lipid metabolism (Zheng et al, 2012;Rodríguez-Rodríguez et al, 2016;González-Mellado et al, 2019). The first step in characterizing the recombinant sunflower LPCATs, produced in yeast, was to assess their functionality in vivo.…”

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

“…Delta 12 fatty acid desaturase ( FADS12 ) plays a key role in the synthesis of EFAs during fermentation. FADS12 is a key membrane-bound desaturase that converts oleic acid [OA, 18:1 Δ9 ] to linoleic acid [LA, 18:2 Δ9,12 ] by introducing a double bond between carbons 12 and 13 in the carboxyl end of the substrate in the biosynthesis of EFAs (Cui et al, 2016; Lee, 2016; Rodríguez-Rodríguez, 2016). As a rate-limiting enzyme, FADS12 plays an important role in the metabolism of PUFAs, and its activity directly affects the level and distribution of EFAs in cheese.…”

“…FADS12 has been studied as an important desaturase in fatty acid transformation for many years, with experiments based on gene cloning, heterologous expression, and analysis of its catalytic mechanism (Brandstetter & Ruther, 2016; Kaye et al, 2015; Kazuhiro et al, 2004; Kikukawa et al, 2013; Lamers et al, 2019; Rodríguez-Rodríguez, 2016; Sakamoto et al, 2017; ShanlinYu et al, 2008; Sun et al, 2016; Watanabe et al, 2004). These studies were mostly focused on the preparation of PUFAs, the regulation of the proportion of omega-3/omega-6 fatty acids and heterologous expression from EFAs, which could not be synthesized in the human body (Wang et al, 2016; Yan et al, 2013; Zhang et al, 2013; Zhang et al, 2017).…”