毕赤酵母新启动子GCW14在细胞表面展示CALB中的应用  

Application of Pichia pastoris Original Constitutive Strong Promoter GCW14 in Candidn antarctic Lipase B Yeast Surface Display

作　　者：张轩薇[1] 叶燕锐[1] 刘晓肖[1] 鲁柳柳[1] 林影[1] 

机构地区：[1]华南理工大学生物科学与工程学院,广州510006

出　　处：《生物技术通报》2013年第4期129-135,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(20976062;31170031);广东省自然科学基金项目(S2011020001457)

摘　　要：以毕赤酵母内源壁蛋白Gcwl4p为锚定蛋白,分别以新的内源组成型启动子P_(GCW14)与诱导型启动子P_(AOXI)、组成型启动子P_(GAP)和P_(TEF1)4种启动子将南极假丝酵母脂肪酶B(CALB)展示于毕赤酵母细胞表面,通过检测4种重组细胞的CALB酶活比较这4个启动子在毕赤酵母中的转录活性。结果显示,P_(GCW14)的启动子活性与P_(AOX1)相当,最高菌体酶活力分别在发酵48 h和168h时达到694.8 U/g(Dry cell weight)和684.3 U/g(Dry cell weight),以P_(GCW14)为启动子的重组菌产酶周期大大缩短;组成型启动子P_(GAP)和P_(TEF1)的最高菌体酶活分别在发酵24 h和168 h达到266.7 U/g(Dry cell weight)和449.2 U/g(Dry cell weight),可见P_(GCW14)的启动子活力性要高于P_(GAP)和P_(TEF1)。此外,以P_(GCW14)为启动子,利用壁蛋白Gcw14p自身的信号肽代替载体上的α-信号肽使重组菌的菌体酶活力提高了约4%。To compare the transcription activity of original promoter P_GCW14 with other promoters, Candidn antarctic lipase B （ CALB ） was displayed on the cell surface of Pichia pastoris using P_GCW14, P_AOXI, P_GAP and P_TEFI as promoter respectively. Anchor protein was a cell wall protein named Gcw14p, which was obtained originally from P, pastoris. In shake-flask cultivation, the activity of P_GCW14 was equal to that of P_AOXI. Under control of P_GCW14 and P_AOXI, the highest lipase hydrolytic activities of recombinant yeasts reached 694.8 U/g （ Dry cell weight ） and 684.3 U/g （ Dry cell weight ） after cultivation for 48 h and 120 h, respectively. The fermentation period of recombinant yeast using P_GCW14 as promoter was shortened significantly. While the highest lipase hydrolytic activities of recombinant yeasts controlled by constitutive promoter P_GAP and P_TEFI roached 266.7 U/g （ Dry cell weight ） and 449.2 U/g （ Dry cell weight ） respectively after cultivation for 48 h. This means that the activity ofP_GCW14 was higher than that of P_GAP and P_TEFI. Furthermore, replacing the a-factor with signal peptide from protein Gcw14p, the lipase hydrolytic activity increased about 4%.

关 键 词：巴斯德毕赤酵母 启动子 南极假丝酵母脂肪酶B 酵母表面展示 

分 类 号：Q78[生物学—分子生物学]