Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding δ12- and δ15-fatty acid desaturases in Hansenula polymorpha

Juthaporn Sangwallek, Yoshinobu Kaneko, Tomoya Tsukamoto, Makoto Marui, Minetaka Sugiyama, Hisayo Ono, Takeshi Bamba, Eiichiro Fukusaki, Satoshi Harashima

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha δ12-fatty acid desaturase (HpFad2) and δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, δ9, δ12) and α-linolenic acid (ALA, αC18:3, δ9, δ12, δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215. bp and 1239. bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode δ12- and δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.

Original languageEnglish
Pages (from-to)110-118
Number of pages9
JournalGene
Volume533
Issue number1
DOIs
Publication statusPublished - Jan 1 2014
Externally publishedYes

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Fatty Acid Desaturases
Pichia
Organism Cloning
Genes
Fatty Acids
Linoleic Acid
Oxygen
Saccharomyces cerevisiae
alpha-Linolenic Acid
Response Elements
Oleic Acid
Unsaturated Fatty Acids
Histidine
Open Reading Frames
Amino Acid Sequence
Yeasts
Amino Acids
Food

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Sangwallek, J., Kaneko, Y., Tsukamoto, T., Marui, M., Sugiyama, M., Ono, H., ... Harashima, S. (2014). Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding δ12- and δ15-fatty acid desaturases in Hansenula polymorpha. Gene, 533(1), 110-118. https://doi.org/10.1016/j.gene.2013.09.115

Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding δ12- and δ15-fatty acid desaturases in Hansenula polymorpha. / Sangwallek, Juthaporn; Kaneko, Yoshinobu; Tsukamoto, Tomoya; Marui, Makoto; Sugiyama, Minetaka; Ono, Hisayo; Bamba, Takeshi; Fukusaki, Eiichiro; Harashima, Satoshi.

In: Gene, Vol. 533, No. 1, 01.01.2014, p. 110-118.

Research output: Contribution to journalArticle

Sangwallek, J, Kaneko, Y, Tsukamoto, T, Marui, M, Sugiyama, M, Ono, H, Bamba, T, Fukusaki, E & Harashima, S 2014, 'Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding δ12- and δ15-fatty acid desaturases in Hansenula polymorpha', Gene, vol. 533, no. 1, pp. 110-118. https://doi.org/10.1016/j.gene.2013.09.115
Sangwallek, Juthaporn ; Kaneko, Yoshinobu ; Tsukamoto, Tomoya ; Marui, Makoto ; Sugiyama, Minetaka ; Ono, Hisayo ; Bamba, Takeshi ; Fukusaki, Eiichiro ; Harashima, Satoshi. / Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding δ12- and δ15-fatty acid desaturases in Hansenula polymorpha. In: Gene. 2014 ; Vol. 533, No. 1. pp. 110-118.
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abstract = "Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha δ12-fatty acid desaturase (HpFad2) and δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, δ9, δ12) and α-linolenic acid (ALA, αC18:3, δ9, δ12, δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215. bp and 1239. bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60{\%} similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode δ12- and δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.",
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AU - Sugiyama, Minetaka

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N2 - Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha δ12-fatty acid desaturase (HpFad2) and δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, δ9, δ12) and α-linolenic acid (ALA, αC18:3, δ9, δ12, δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215. bp and 1239. bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode δ12- and δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.

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