圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

Expression, purification and characterization of a novel fatty acid synthase from Rhodosporidium toruloides

摘要:

脂肪酸合酶 (Fatty acid synthase，FAS) 催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸，是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS，它含两个亚基，与其他物种的FAS相比，具有独特的结构域组成，尤其是含两个酰基载体蛋白 (ACP) 结构域。由于ACP在脂肪酸合成反应中起辅因子作用，推测多个ACP有利于提高FAS的催化活性，为研究该FAS的生物化学和结构特征，构建了表达FAS两个亚基的载体，并转化大肠杆菌Escherichia coli BL21(DE3)，含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基，经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化，得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究，且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。

Abstract:

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids, an important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21(DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.