Yin Yanchen, Mao Youzhi, Yin Xiaolie, Gao Bei, Wei Dongzhi
State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China.
J Microbiol Biotechnol. 2015 Jul;25(7):988-98. doi: 10.4014/jmb.1410.10022.
The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30°C. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.
丝状真菌米曲霉是一种著名的表达宿主，用于在许多工业应用中表达同源和异源蛋白质。为了提高目标蛋白质的产量，我们构建了pAsOP载体以在米曲霉中表达异源蛋白质。pAsOP携带一个来自构巢曲霉的选择标记pyrG，以及一个来自米曲霉的amyB基因的强启动子和终止子。pAsOP通过PEG-CaCl2介导的转化方法高效转化米曲霉。作为概念验证，绿色荧光蛋白（GFP）在由pAsOP-GFP转化的米曲霉中成功表达。此外，我们从青霉属中鉴定出一个新的真菌α-淀粉酶（PcAmy）基因，并将该基因克隆到载体中。用pAsOP-PcAmy转化后，来自青霉属的α-淀粉酶PcAmy首次在异源宿主系统中成功表达。与未转化的米曲霉对照相比，米曲霉转化体中的α-淀粉酶活性提高了62.3%。该系统中产生的PcAmy蛋白的最适pH为5.0，最适温度为30°C。作为一种低温适应酶，PcAmy由于其在低温下的高催化活性而在工业应用中显示出潜在价值。此外，本研究报道的表达载体为进一步的科学研究和生物技术应用提供了有前景的用途。
