Cloning and Characterization of the ∆6 Polyunsaturated Fatty Acid Elongase from the Green Microalga <i>Parietochloris incisa</i>

Iskandarov, Umidjon; Khozin‐Goldberg, Inna; Ofir, Rivka; Cohen, Zvi

doi:10.1007/s11745-009-3301-y

Cited by 28 publications

(14 citation statements)

References 43 publications

(55 reference statements)

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“…Based on the clone (SZ_07_83) screened from this cDNA library of M. incisa, a fatty acid elongase gene has been cloned [11] and confirmed to elongate γ-linolenic acid (18:36, GLA) and 18:43 [43]. The transcription of this gene was found to be enhanced prior to ArA content accumulation, suggesting it may be responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions [11], which is consistent with the results of a study conducted by Iskandarov et al [17]. Hence, a possible synthesis pathway of ArA in M. incisa was hypothesized to start from oleic acid via LA, GLA and 20:36 to the final ArA ( Figure 6).…”

Section: Biosynthesis Of Ara In M Incisasupporting

confidence: 89%

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Expressed sequence tags analysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green microalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta)

Ouyang

et al. 2012

Chin. Sci. Bull.

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cDNA library of Myrmecia incisa H4301 was constructed using λ phage vectors. The library consisted of 1.5×10 6 clones with a recombination rate of 90%, and 1942 clones were randomly sequenced. All 1854 readable expressed sequence tags (ESTs) were clustered into 596 non-redundant sequences (NRSs), among which 126 NRSs were from 1384 ESTs, showing a high redundancy. Among the 596 NRSs, 30 were ribosomal RNA, and 152 significantly matched with those available in NCBI database and JGI Genome Portal, the latter were divided into nine subcategories. Overall, 59 NRSs were involved in photosynthesis, the respiratory electron transport chain, ATP synthesis, oxidation reduction, fatty acid biosynthesis, glucose metabolism, protein metabolism, and small molecular metabolism, suggesting that these genes were abundantly transcribed during energy and substance metabolism. Acyl-carrier protein, ferrodoxin and fatty acid elongase genes obtained from this cDNA library enabled presumption of a possible biosynthesis pathway of ArA in M. incisa. Codon usage analysis of 142 NRSs with 17798 codons in the predicted coding regions showed that the average G+C content level of the total codons was 55.39%, and that of the third position in base trimers was 66.42%, indicating a strong bias toward cytosine and/or guanosine in this algal genome. Among all synonymous codons, NAG was most favored, while NUA was most avoided. Phylogenic trees inferred from ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes and the extra partial sequences of 18S rRNA obtained from this library demonstrated that M. incisa belonged to Trebouxiophyceae and was significantly clustered with M. incisa SAG 2007, Lobosphaera tirolensis, M. bisecta, and Parietochloris incisa, but was clearly distant from P. pseudoalveolaris and P. alveolar. Transmission electron microscopy revealed pyrenoids traversed by many parallel thylakoids membranes, while starch grains were only clearly observed when cells were grown under nitrogen stress. Based on combined investigation of the phylogeny and morphological characteristics, it is proposed that M. incisa be kept in the genus Myrmecia in which there might be two parallel groups, one living freely and another symbiotic species.

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Section: Biosynthesis Of Ara In M Incisasupporting

confidence: 89%

“…The latter is released from their lipid carriers and elongated to 20:3n-6, which is primarily reincorporated into phosphatidylethanolamine (PE) and PC and finally desaturated to ArA. However, there is little other information available about the metabolic mechanism of fatty acids in M. incisa, especially at the molecular level [11,16,17].…”

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confidence: 99%

Expressed sequence tags analysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green microalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta)

Ouyang

et al. 2012

Chin. Sci. Bull.

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“…Axenic cultures of Lobosphaera incisa (formerly Parietochloris incisa) (Trebouxiophyceae, Chlorophyta) (deposited in SAG collection under accession number 2468) were grown in an mBG-11 nutrient medium in 250-mL Erlenmeyer glass flasks in an incubator shaker as described previously (Iskandarov et al 2009). For gene expression analy s i s , L .…”

Section: Methodsmentioning

confidence: 99%

“…The culture aliquots (40 mL) were filtered through a glass fiber paper filter (GF-52, Schleicher & Schuell, Germany), and cells were collected by scraping and immediately flash-frozen in liquid nitrogen in 1.5-mL Eppendorf tubes and stored at −80°C for further use. For cloning, total RNA was isolated and complementary DNA (cDNA) synthetized using the procedures described in Iskandarov et al (2009). For real-time quantitative PCR (qPCR), total RNA was isolated using the SV Total RNA Isolation Kit (Promega, USA).…”

Section: Rna Extraction and Cdna Synthesismentioning

confidence: 99%

Cloning and characterization of a GPAT-like gene from the microalga Lobosphaera incisa (Trebouxiophyceae): overexpression in Chlamydomonas reinhardtii enhances TAG production

et al. 2015

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The oleaginous green microalga Lobosphaera (formerly Parietochloris) incisa accumulates high amounts of arachidonic-acid-rich triacylglycerols (TAG), in particular under conditions of nitrogen starvation. This photosynthetic organism is of great interest for studying the mechanisms responsible for storage lipid biosynthesis and the deposition of long-chain polyunsaturated fatty acids (LC-PUFA) in TAG. In this work, we report on cloning a complementary DNA (cDNA) for the putative L. incisa glycerol-3-phosphate acyltransferase (GPAT), whose deduced amino acid sequence features distinctive motives found in those mammalian and Arabidopsis GPAT isoforms that have been implicated in TAG biosynthesis. Temporal analysis of LiGPAT expression in the course of nitrogen starvation showed a positive relationship between changes in the transcript level and patterns of fatty acid production. When expressed in Arabidopsis leaf mesophyll protoplasts, the green fluorescent protein (GFP) fused to the C-terminus of LiGPAT localized outside the chloroplasts in agreement with its predicted extraplastidial localization. Based on an in silico analysis of a deduced amino acid sequence and on similarity to other GPATs participating in TAG biosynthesis, LiGPAT was expressed in the green model microalga Chlamydomonas reinhardtii in order to confirm the predicted function in a heterologous microalgal system. Overexpression of LiGPAT resulted in an up to 50 % increase in the content of TAG on a cell dry weight basis as compared to the control in the stationary phase culture without negative impact on growth parameters. Total fatty acids and TAG of the transformant lines featured an elevated level of oleic acid (18:1 n-9) and a concurrent decrease in C18 PUFA. Additional studies on the acyl substrate preference of LiGPAT are required.

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“…Primer pairs were validated as described by Iskandarov et al [43]. The nucleotide sequences of primer pairs and the amplicon sizes are presented in Table 2.…”

Section: Primer Design and Validation For Real-time Quantitative Pcrmentioning

confidence: 99%

Identification and Characterization of Δ12, Δ6, and Δ5 Desaturases from the Green Microalga Parietochloris incisa

Iskandarov

Khozin‐Goldberg

Cohen

2010

Lipids

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The freshwater microalga Parietochloris incisa accumulates, under nitrogen starvation, large amounts of triacylglycerols containing approximately 60% of the omega6 very long-chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. Based on sequence homology, we isolated three cDNA sequences from P. incisa, designated PiDesD12, PiDesD6, PiDesD5. The deduced amino acid sequences of the three genes contained three conserved histidine motifs; the front-end desaturases, PiDes6 and PiDes5, contained a fused N-terminal cytochrome b5 domain. By functional characterization in the yeast Saccharomyces cerevisiae, we confirmed that PiDesD6, PiDesD5 cDNA encode membrane bound desaturases with Delta6, and Delta5 activity, respectively. Both PiDes6 and PiDes5 can indiscriminately desaturate both omega6 and omega3 substrates. A phylogenetic analysis showed that the three genes were homologous to the corresponding desaturases from green microalgae and lower plants that were functionally characterized. Quantitative real-time PCR revealed the concerted expression pattern of all three genes in P. incisa cells subjected to nitrogen starvation, featuring maximum expression level on day 3 of starvation, corresponding to the sharpest increase in the share of arachidonic acid.

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Cloning and Characterization of the ∆6 Polyunsaturated Fatty Acid Elongase from the Green Microalga Parietochloris incisa

Cited by 28 publications

References 43 publications

Expressed sequence tags analysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green microalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta)

Expressed sequence tags analysis revealing the taxonomic position and fatty acid biosynthesis in an oleaginous green microalga, Myrmecia incisa Reisigl (Trebouxiophyceae, Chlorophyta)

Cloning and characterization of a GPAT-like gene from the microalga Lobosphaera incisa (Trebouxiophyceae): overexpression in Chlamydomonas reinhardtii enhances TAG production

Identification and Characterization of Δ12, Δ6, and Δ5 Desaturases from the Green Microalga Parietochloris incisa

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