Identification and Characterization of Δ12, Δ6, and Δ5 Desaturases from the Green Microalga <i>Parietochloris incisa</i>

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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“…A gas chromatographic analysis (GC-FID) of FAMEs was performed as described previously (Iskandarov et al 2010).…”

Section: Analysis Of Fatty Acid Content and Compositionmentioning

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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“…Recently, some genes encoding FADS6 have been cloned and sequenced from many organisms ( 13,(20)(21)(22)(23)(24)(25)(26), but little is known about their molecular mechanism of FADS6 substrate preference. This is mainly due to the lack of crystal structure information.…”

Section: Site-directed Mutagenesis Of Mpfads6mentioning

confidence: 99%

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

Shi

Chen

et al. 2015

Journal of Lipid Research

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

Cited by 46 publications

References 66 publications

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

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

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

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

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