Identification and functional characterization of the delta 6-fatty acid desaturase gene from Thamnidium elegans.
A cDNA sequence was cloned from the filamentous fungus Thamnidium elegans As3.2806 using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends method (RACE). Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,380 bp, which encodes a 52.4 kDa peptide of 459 amino acids. The designated amino acid sequence has high similarity with that found in fungal delta 6-fatty acid desaturases: it shows three conserved histidine-rich motifs and two hydrophobic domains. A cytochrome b5-like domain was observed at the N-terminus. To elucidate the function of this novel putative desaturase, the open reading frame was cloned into the intracellular expression vector pPIC3.5K and the gene was expressed heterologously in Pichia pastoris. Accumulation of gamma-linolenic acid to the level of 6.83% in total fatty acid demonstrated that the deduced amino acid sequence possesses of delta 6-fatty acid desaturase activity.
Wang
Depei
D
Tianjin Key Laboratory of Microbial Functional Genomics, Department of Microbiology, Nankai University, 300071 Tianjin, China.
Li
Mingchun
M
Wei
Dongsheng
D
Cai
Yi
Y
Zhang
Yinghui
Y
Xing
Laijun
L
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D050602:Linoleoyl-CoA Desaturase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009090:Mucorales / Q000201:enzymology* / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013696:Temperature
D018076:DNA, Complementary; D011994:Recombinant Proteins; D050602:Linoleoyl-CoA Desaturase