Identification and characterization of a novel bifunctional Δ12/Δ15-fatty acid desaturase gene from Rhodosporidium kratochvilovae

Cui, Jinjin; He, Shiwu; Ji, Xiuling; Lü, Lin; Wei, Yunlin; Zhāng, Qí

doi:10.1007/s10529-016-2090-7

Cited by 35 publications

(31 citation statements)

References 37 publications

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“…Subsequently, GLA is synthesized by introducing a double bond into LA using a delta-6 desaturase. Thus, three desaturases, the delta-9, delta-12 and delta-6, accomplish the desaturation processes in GLA synthesis [5, 13, 14]. Genes coding for those desaturases have been cloned from diverse organisms ranging from prokaryotes to higher eukaryotes, and overexpressed in several hosts, including microalgae, yeasts and plants [8, 13–16].…”

Section: Introductionmentioning

confidence: 99%

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases

et al. 2017

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Backgroundγ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides.ResultsTo enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain.ConclusionThese findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0723-8) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases

et al. 2017

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“…The bifunctional FADS12 enzyme has been widely characterized as being responsible for converting the OA and LA fatty acid substrates to LA and ALA, respectively, in many species [17,20,21]: functional analysis results showed that FADS12 from Rhodosporidium kratochvilovae converted 11.4% of OA to LA, and 19.1% of LA to ALA; Wei et al [20] reported that FADS12 from Rhizopus arrhizus had a high conversion rate of 16.0% for OA when it was expressed in yeast; and the conversion rate of OA by FADS12 from Mortierella alpina 1S-4 was approximately 24.1%, and that of LA was 36.8%. FADS12 isolated from G. candidum in cheese in this study was also bifunctional for both OA and LA, and we found from our functional analysis that GcFADS12 should continually catalyze the product (LA) of OA to ALA.…”

Section: Discussionmentioning

confidence: 99%

“…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 ).…”

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confidence: 99%

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

Luo

Shi

et al. 2018

FEBS Open Bio

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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.

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Shi

Tian

et al. 2019

Preprint

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Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

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Identification and characterization of a novel bifunctional Δ12/Δ15-fatty acid desaturase gene from Rhodosporidium kratochvilovae

Abstract: RKD12 is a novel bifunctional ∆(12)/∆(15)-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.

Cited by 35 publications

References 37 publications

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases

Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases

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

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

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