Li Song, Sing Suren, Wang Zhengxiang
Research Center of Bioresource and Bioenergy, School of Biotechnology, Jiangnan University, Wuxi, China.
Protein Expr Purif. 2011 Sep;79(1):142-8. doi: 10.1016/j.pep.2011.05.007. Epub 2011 May 17.
In our previous study, the α-amylase from Rhizopus oryzae (RoAmy) was expressed in Escherichia coli and Saccharomyces cerevisiae but the obtained recombinant RoAmy (rRoAmy) yields were too low. The aim of the present research was to obtain high-level expressions of RoAmy in the methylotrophic yeast Pichia pastoris. To this end, we constructed P. pastoris strains with the capability to express recombinant α-amylase under the control of constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) and methanol-inducible alcohol oxidase 1 promoters. The levels of inducibly expressed rRoAmy were higher than those of constitutively expressed. The maximal inducible rRoAmy expression levels for the Mut(+) strains (41.1mg/l) were approximately eight times higher than those for the Mut(s) strains and 24 times higher than those expressed under the control of the GAP promoter. For both inducible and constitutive expressions, the S. cerevisiae α-prepro sequence and the native signal sequence of RoAmy were used separately to direct the secretion of rRoAmy into the culture medium of P. pastoris. Low levels of intracellular amylase activities that had been detected after shake-flask fermentation indicated that both signal sequences could effectively direct the secretion of rRoAmy under all studied conditions. In addition, the secretion levels of rRoAmy directed with its own signal peptide were 7-10% higher than those directed by the α-prepro sequence.
在我们之前的研究中，米根霉α淀粉酶（RoAmy）在大肠杆菌和酿酒酵母中表达，但获得的重组RoAmy（rRoAmy）产量过低。本研究的目的是在甲基营养型酵母巴斯德毕赤酵母中实现RoAmy的高水平表达。为此，我们构建了巴斯德毕赤酵母菌株，使其能够在组成型甘油醛-3-磷酸脱氢酶（GAP）和甲醇诱导型醇氧化酶1启动子的控制下表达重组α淀粉酶。诱导表达的rRoAmy水平高于组成型表达的水平。Mut(+)菌株的最大诱导rRoAmy表达水平（41.1mg/l）比Mut(s)菌株高约8倍，比在GAP启动子控制下表达的水平高24倍。对于诱导型和组成型表达，分别使用酿酒酵母α-前导序列和RoAmy的天然信号序列将rRoAmy分泌到巴斯德毕赤酵母的培养基中。摇瓶发酵后检测到的细胞内淀粉酶活性水平较低，表明在所有研究条件下，这两种信号序列都能有效地指导rRoAmy的分泌。此外，用其自身信号肽指导的rRoAmy分泌水平比α-前导序列指导的分泌水平高7-10%。
