小眼虫藻Δ8-脂肪酸脱氢酶基因的克隆及其在酿酒酵母中的表达

Cloning of Δ8-fatty acid desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae

摘要:

Δ8途径是合成多不饱和脂肪酸的替代途径，Δ8-脂肪酸脱氢酶是该途径的关键酶之一。根据已报道的Δ8-脂肪酸脱氢酶基因设计引物，分别从小眼虫藻基因组DNA和cDNA中扩增得到该基因片段，序列分析表明：结构基因长1 266 bp，编码421个氨基酸；该基因没有内含子，比已经报道的Δ8-脂肪酸脱氢酶基因长6 bp，并且N末端序列也有所不同。利用酿酒酵母的载体pYES2.0构建Δ8-脂肪酸脱氢酶表达载体pYEFD，并转化到营养缺陷型酿酒酵母菌株INVSc1中，在选择培养基中筛选得到酿酒酵母转化菌株YD8。YD8在合适的培养条件下，添加外源底物二十碳二烯酸和二十碳三烯酸并诱导基因表达。脂肪酸甲酯气相色谱分析表明小眼虫藻Δ8-脂肪酸脱氢酶基因在酿酒酵母中获得了高效表达，将二十碳二烯酸和二十碳三烯酸分别转化成二高-γ-亚麻酸和二十碳四烯酸，其底物转化率分别达到了31.2％和46.3％。

Abstract:

Δ8 desaturase pathway, different from common Δ6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Δ8-fatty acid desaturase is one of the key enzymes in Δ8 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 Δ8-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 Δ8-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 ω6-eicosadienoic acid and ω3-eicosatrienoic acid for the expression of Δ8-fatty acid desaturase gene. The results indicated that high level expressed Δ8-fatty acid desaturase could convert ω6-eicosadienoic acid and ω3-eicosatrienoic acid to dihomo-γ-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.