缺刻缘绿藻Δ15脂肪酸去饱和酶的功能分析

Functional analysis of Δ15 fatty acid desaturase from Myrmecia incisa

摘要:

缺刻缘绿藻（Myrmecia incisa Reisigl）是一种富含多不饱和脂肪酸的球形单细胞淡水绿藻。在脂肪酸合成途径中，脂肪酸去饱和酶与脂肪酸酰基结合载体结合进行脂肪酸去饱和反应。为探究与缺刻缘绿藻中Δ15脂肪酸去饱和酶（fatty acid desaturase，FAD）结合的脂肪酸酰基结合载体，选用酿酒酵母（Saccharomyces cerevisiae） INVSc1和聚球藻（Synechococcus sp.） PCC7942分别验证Δ15 FAD与酰基辅酶A （酰基-CoA）或酰基载体蛋白（酰基-ACP）结合的脂肪酸去饱和过程。根据缺刻缘绿藻的Δ15 FAD基因构建双源表达载体pYES2-Δ15 FAD和pCAMBIA1300-Δ15 FAD，通过电穿孔法将重组表达质粒分别转入酿酒酵母INVSc1和聚球藻PCC7942中，筛选得到转基因菌株。取相同细胞数的转基因酵母和转基因聚球藻，培养时分别添加等量的底物亚油酸（linoleic acid，18：2^Δ9，12，LA），以LA作为酰基受体，使其进入转基因酵母和转基因聚球藻的生物合成途径中，培养36~72 h。利用气相色谱-质谱联用系统对总脂肪酸的各组分进行分析，结果显示，在转基因实验组中检测到LA和α-亚麻酸（α-linolenic acid，18：3^{Δ9，12，15}，ALA）的存在，空白实验则未检测出相应的产物。通过计算，转基因酵母催化LA生成ALA的效率为29.31%，转基因聚球藻催化底物LA生成ALA的效率为30.86%。结果证明无论是酰基-ACP还是酰基-CoA，与缺刻缘绿藻中的Δ15 FAD结合进行反应的效率相近，不存在偏好性，由于Δ15 FAD是特异性蛋白，因此证明缺刻缘绿藻中的Δ15 FAD属于脂酰基结合蛋白。

Abstract:

Myrmecia incisa Reisigl is a green coccoid freshwater microalga which is rich in polyunsaturated fatty acid. In the fatty acid synthesis pathway, the fatty acid desaturase was combined with the fatty acid acyl binding carrier to perform fatty acid desaturation. In order to explore the fatty acid acyl binding carrier combined with Δ15 fatty acid desaturase (FAD) of M. incisa Reisigl, Saccharomyces cerevisiae INVSc1 and (Synechococcus sp.) PCC 7942 were selected for verification whether Δ15 FAD bound to acyl-CoA (acyl-CoA) or acyl carrier protein (acyl-ACP) in the desaturation process, respectively. The binary expression vectors of pYES2-Δ15 FAD and pCAMBIA1300-Δ15 FAD were constructed on the basis of the Δ15 FAD gene of M. incisa Reisigl. The recombinant expression plasmids were transferred into INVSc1 and PCC7942 respectively, and the transgenic strains were screened. The similar number of transgenic yeast and transgenic cyanobacteria cells were cultured with addition of the same volume of linoleic acid (LA). After 36-72 h culture, the components of the total fatty acid were analyzed using the gas chromatography-mass (GC-MS) spectrometry system. The results showed that LA and ALA were detected in the transgenic cells, but the corresponding products were not detected in the blank control groups. By calculation, the efficiencies of conversion of LA to ALA were 29.31% and 30.86% in the transgenic yeast and transgenic cyanobacteria, respectively. According to the results, similar conversion efficiencies were obtained whether Δ15 FAD of M. incisa Reisigl combined with acyl-ACP or acyl-CoA, and there was no preference. In conclusion, Δ15 FAD was a fatty acyl binding protein in M. incisa Reisigl.