丙酮酸羧化酶基因的过量表达对富马酸代谢的影响  

Effect of Over-expressing of Pyruvate Carboxylase Gene on Production of Fumarate

作　　者：吴世根[1] 张婷[1] 邓利[1] 

机构地区：[1]北京化工大学生命科学与技术学院北京生物加工过程重点实验室,北京100029

出　　处：《生物技术通报》2011年第12期175-180,198,共7页Biotechnology Bulletin

摘　　要：旨在研究丙酮酸羧化酶胞质同工酶(PC)基因在毕赤酵母中的过量表达对TCA还原途径碳源分流及草酰乙酸、L-苹果酸生产的影响,构建了表达载体pPIC3.5K-PC,并转化了毕赤酵母GS115。转化子经过表型鉴定,PCR分析和G418浓度梯度筛选获得了高拷贝的重组子(pas-01)。甲醇诱导表达后,经SDS-PAGE分析及PC活性检测,发现酶活提高了3倍,说明PC在毕赤酵母中获得了成功表达。以pPIC3.5K转化菌为对照,对该重组子进行了草酰乙酸及苹果酸的发酵研究。结果显示,草酰乙酸产量(177.4 mg/L)提高了109.6%,L-苹果酸的产量(127.45 mg/L)提高33.7%,菌体生物量提高15.7%,表明PC的过量表达有助于L-苹果酸和草酰乙酸的积累,并且对菌体的生长有一定的促进作用。We overexpressed PC gene in Pichia pastoris GS115,and investigated its influence on the diversion of carbon to the reductive TCA pathway and the yields of the oxaloacetate and L-malic acid.The expression vector pPIC3.5K-PC was constructed,and was introduced in Pichia pastoris GS115.A high-copy recombinant（pas-01）was screened out from 30 positive transformants by phenotypic identification,PCR analysis and G418 concentration gradient selection.After methanol induction,recombinant protein was analysed by SDS-PAGE and PC activity was detected.Results showed that PC was overexpressed in the Pichia pastoris,and the activity was increased 3-folds.Compared to the control（pPIC3.5K transformant）,oxaloacetate production（177.4 mg/L）of the recombinant（pas-01）was increased by 109.6%,L-malic acid production（127.45 mg/L）increased by 33.7% and cell biomass increased by 15.7% through the fermentation.These results indicated that overexpression of PC is propitious to the accumulation of oxaloacetate and L-malic acid,which also play a significant role in promoting the growth of strain.

关 键 词：丙酮酸羧化酶 草酰乙酸 L-苹果酸 pPIC3.5K 毕赤酵母 

分 类 号：Q75[生物学—分子生物学]