Pan Yang, Wu Dan, Wu Jing
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Key Laboratory of Industrial Biotechnology Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2018 May 25;34(5):712-721. doi: 10.13345/j.cjb.170409.
Pichia pastoris is a versatile protein expression system. At present, the expression plasmids are integrated plasmids whereas the episomal plasmids are rarely used. In this study, the autonomously replicating sequence derived from yeast itself was ligated into the integrative expression vector pGAP to generate an autonomously replicative expression vector pGAPZαA-PARS. When the vector was used to express the xylanase (XynA) gene in P. pastoris, the highest enzyme activity reached 343 U/mL with glycerol as the carbon source, which was 45.9% higher than that of the integrative expression. At the same time, the specific enzyme activity of XynA was increased by 81.2%. We further studied the expression level of recombinant strains with different carbon sources such as glycerol, glucose, sucrose and mixed carbon source (sucrose︰glycerol=1︰2). The highest expression level was achieved with glycerol and the lowest with sucrose. The episomal expression vector pGAPZαA-PARS greatly promotes the application of P. pastoris in the food industry because GAP promoter does not require methanol as induction material. Meanwhile, the episomal vector can greatly improve the expression level, which lays the foundation for further research to improve the high expression of GAP promoter.
毕赤酵母是一种多功能的蛋白质表达系统。目前，表达质粒为整合质粒，而游离型质粒很少使用。在本研究中，将来源于酵母自身的自主复制序列连接到整合表达载体pGAP中，构建出自主复制表达载体pGAPZαA-PARS。当用该载体在毕赤酵母中表达木聚糖酶（XynA）基因时，以甘油作为碳源时酶活最高可达343 U/mL，比整合表达提高了45.9%。同时，XynA的比酶活提高了81.2%。我们进一步研究了以甘油、葡萄糖、蔗糖及混合碳源（蔗糖︰甘油 = 1︰2）为碳源时重组菌株的表达水平。结果表明，甘油为碳源时表达水平最高，蔗糖为碳源时最低。游离型表达载体pGAPZαA-PARS极大地推动了毕赤酵母在食品工业中的应用，因为GAP启动子不需要甲醇作为诱导物。同时，该游离型载体可显著提高表达水平，为进一步研究提高GAP启动子的高表达奠定了基础。
