title: [Cloning of delta8-fatty acid desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae].
abstract: Delta8 desaturase pathway, different from common delta6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Delta8-fatty acid desaturase is one of the key enzymes in delta8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic acid and omega3-eicosatrienoic acid for the expression of delta8-fatty acid desaturase gene. The results indicated that high level expressed As-fatty acid desaturase could convert omega6-eicosadienoic acid and omega3-eicosatrienoic acid to dihomo-gamma-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin 300071, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.forename: Xiuyuan
authors.initials: X
authors.lastname: Ou
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Wei
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Yang
authors.forename: Dongquan
authors.initials: D
authors.lastname: Guo
authors.forename: Xueyan
authors.initials: X
authors.lastname: Yian
authors.forename: Laijun
authors.initials: L
authors.lastname: Xing
authors.forename: Mingchun
authors.initials: M
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005056:Euglena gracilis / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; C119257:delta-8 fatty acid desaturase