Identification of a Δ6 fatty acid elongase gene for arachidonic acid biosynthesis localized to the endoplasmic reticulum in the green microalga Myrmecia incisa Reisigl

Yu, Shuiyan; Li, H.; Tong, Min; Ouyang, Long‐Ling; Zhou, Zhigang

doi:10.1016/j.gene.2011.11.053

Cited by 22 publications

(13 citation statements)

References 45 publications

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“…The Δ6 FAE contains an ELO family elongase domain, which is involved in very‐long‐chain fatty acid elongation and sphingolipid formation (Oh et al ., ; Tvrdik et al ., ). Further evidence of a possible elongase function is provided by the presence of FLHXYHH and MYSYY motifs characteristic of Δ6 and Δ5 fatty acid elongases; the first is positioned with an upstream glutamine characteristic of PUFA elongases, and the latter is typically found in microalga Δ6 and Δ5 fatty acid elongases (Hashimoto et al ., ; Jiang et al ., ; Yu et al ., ) (Figure ).…”

Section: Resultsmentioning

confidence: 98%

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Poliner

Pulman

Zienkiewicz

et al. 2017

Plant Biotechnology Journal

121

105

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Summary Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long‐chain polyunsaturated fatty acids (LC‐PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC‐PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC‐PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC‐OX and pNOC‐stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase‐encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC‐PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.

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Section: Resultsmentioning

confidence: 98%

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Poliner

Pulman

Zienkiewicz

et al. 2017

Plant Biotechnology Journal

121

105

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“…4). A delta 6 elongase has been localized to the ER in the green alga Myrmecia incisa (Yu et al 2012). In T. pseudonana, the SFA elongase, Elo3, is localized to an unidentified intracellular organelle or vesicle (Fig.…”

Section: Discussionmentioning

confidence: 99%

Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes

Cook

Hildebrand

2015

J Appl Phycol

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The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10-20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.

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“…q-RT-PCR was carried out according to Yu et al [22]. The complementary DNA was synthesized using the Reverse Transcribed Kit II (TaKaRa, Dalian, China).…”

Section: Midgat Transcript Levels Under a Nitrogen Starvation Stressmentioning

confidence: 99%

Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl

et al. 2015

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Acyl-CoA: diacylglycerol acyltransferase (DGAT) is a key enzyme responsible for triacylglycerol (TAG) synthesis in eukaryotic organisms. The present work reported DGAT genes in the green alga Myrmecia incisa, a promising candidate for arachidonic acid (ArA) production. According to the results of homology search against a transcriptome database, we cloned three cDNAs encoding putative DGAT1 and DGAT2. The 2238-bp, 1056-bp and 1068-bp of open reading frame (ORF) of these three cDNAs, designated as MiDGAT1, MiDGAT2A and MiDGAT2B, were predicted to encode proteins composed of 745, 351 and 355 amino acids. They were separated by 14, 6 and 6 introns, respectively, as revealed by comparing their corresponding cDNA and DNA sequences. Multiple sequence alignment of amino acids indicated that MiDGAT1 had a pleckstrin homology (PH) domain, whilst MiDGAT2s contained a highly conserved HPHG, a characteristic motif of DGAT2 family. To determine the function, they were expressed heterologously in a Saccharomyces cerevisiae mutant strain with impaired TAG metabolism. Results of thin-layer chromatography and BODIPY staining indicated that both MiDGAT1 and MiDGAT2s were able to restore TAG synthesis and lipid body formation. GC-MS analysis indicated that palmitic acid and stearic acid were the major components of TAGs in yeast cells, and their ratio between wild type and the transformed yeasts was not significantly different. Quantitative RT-PCR results showed that the transcript level of MiDGAT2A was regulated by nitrogen starvation, which was consistent with TAG accumulation in M. incisa.

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Identification of a Δ6 fatty acid elongase gene for arachidonic acid biosynthesis localized to the endoplasmic reticulum in the green microalga Myrmecia incisa Reisigl

Cited by 22 publications

References 45 publications

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes

Identification and characterization of three genes encoding acyl-CoA: diacylglycerol acyltransferase (DGAT) from the microalga Myrmecia incisa Reisigl

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