拟南芥硫酯酶基因在毕赤酵母中的表达

Expression of Arabidopsis thaliana thioesterase gene in Pichia pastoris

摘要:

硫酯酶在生物体内能催化水解脂酰酰基载体蛋白和饱和脂肪脂酰链，对中链脂肪酸 (Medium chain fatty acids，MCFAs) 的积累起着关键作用。为获得具有生产中链脂肪酸能力的工程菌，以拟南芥cDNA为模板，PCR扩增得到其脂酰-ACP硫酯酶基因AtFatA，经EcoRⅠ/XbaⅠ双酶切后连接至同样双酶切的质粒中，获得重组质粒pPICZαA-AtFat。将重组质粒电击转入毕赤酵母GS115中，通过Zeocin抗性筛选，挑选出阳性克隆子并摇瓶发酵诱导，SDS-聚丙烯酰胺凝胶电泳分析获得明显目的蛋白条带，首次成功构建AtFatA的真菌表达系统。气质联用法检测发酵产物胞外游离脂肪酸，发现毕赤酵母AtFatA重组菌株比起始GS115菌株胞外游离MCFAs (主要是辛酸) 产量增加51%，MCFAs产量占胞外总脂肪酸产量的28.7%，而野生菌中这一比例仅有18.1%，这为日后生产安全无毒害的MCFAs探索了一条新的途径。

Abstract:

Thioesterase catalyzes the hydrolysis of acyl-ACP and saturated fatty acyl chain. It plays a key role in the accumulation of medium chain fatty acids in vivo. In this study, to construct an engineering strain to produce MCFAs, the Arabidopsis acyl-ACP thioesterase gene AtFatA was amplified by PCR from cDNA of arabidopsis and double digested by EcoRⅠ/XbaⅠ, then linked to the plasmid digested with same enzymes to get the recombinant plasmid pPICZαA-AtFatA. We transformed the gene into Pichia pastoris GS115 by electroporation and screened positive colonies by YPD medium with Zeocin. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the recombinant enzyme had a molecular of 45 kDa band which was consistent with the predicted molecular mass and we constructed the expression system of gene AtFatA in fungus for the first time. Under shake-flask conditions, Gas Chromatograph-Mass Spectrometer-computer results indicated that recombinant strain produced 51% more extracellular free MCFAs than the wild and its yield reached 28.7% of all extracellular fatty acids. This figure is 10% higher than the control group. The result provides a new way to produce MCFAs.