Yang Zhe, Wei Dongsheng, Xing Laijun, Li Mingchun
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Department of Microbiology, Nankai University, Tianjin 300071, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Feb;25(2):195-9.
Delta5-fatty acid desaturase is the key enzyme in synthesis of arachidonic acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the delta5-fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 containerizing target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc 1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-gamma-linolenic acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic acid. The results indicated that high level expression of delta5-fatty acid desaturase, and the substrate conversion reached 45.9%.
Δ5-脂肪酸去饱和酶是花生四烯酸合成中的关键酶。根据已报道的序列设计引物,通过PCR分别从三角褐指藻的基因组DNA和总cDNA中克隆出两个特异性片段,分别为1520 bp和1410 bp。基因组序列和cDNA序列的比较显示,基因组DNA中的Δ5-脂肪酸去饱和酶基因有一个110 bp的内含子。将1.4 kb的片段亚克隆到酵母-大肠杆菌穿梭载体pYES2.0中,构建了一个包含目标基因的表达重组质粒pYPTD5。通过电转化法将质粒pYPTD5转化到酿酒酵母的缺陷型突变体INCSc 1中进行表达。以二高-γ-亚麻酸作为酵母培养物的外源底物,以半乳糖作为诱导剂。通过气相色谱检测,重组酿酒酵母产生了花生四烯酸。结果表明,Δ5-脂肪酸去饱和酶实现了高水平表达,底物转化率达到45.9%。